Endocrine Related Cancer recent issues

Novel roles of prolactin and estrogens in breast cancer: resistance to chemotherapy

LaPensee, E. W, Ben-Jonathan, N. — Mon, 01 Mar 2010 04:10:41 PST

Resistance to chemotherapy is a major complication in the treatment of advanced breast cancer. Estrogens and prolactin (PRL) are implicated in the pathogenesis of breast cancer but their roles in chemoresistance have been overlooked. A common feature to the two hormones is activation of their receptors by diverse compounds, which mimic or antagonize their actions. The PRL receptor is activated by lactogens (PRL, GH, or placental lactogen) originating from the pituitary, breast, adipose tissue, or the placenta. Estrogen receptors exist in multiple membrane-associated and cytoplasmic forms that can be activated by endogenous estrogens, man-made chemicals, and phytoestrogens. Here, we review evidence that low doses of PRL, estradiol (E₂), and bisphenol A (BPA) antagonize multiple anticancer drugs that induce cell death by different mechanisms. Focusing on cisplatin, a DNA-damaging drug which is effective in the treatment of many cancer types but not breast cancer, we compare the abilities of PRL, E₂, and BPA to antagonize its cytotoxicity. Whereas PRL acts by activating the glutathione-S-transferase detoxification enzyme, E₂ and BPA act by inducing the antiapoptotic protein Bcl-2. The implications of these findings to patients undergoing chemotherapy are discussed.

Familial testicular germ cell tumors in adults: 2010 summary of genetic risk factors and clinical phenotype

Mon, 08 Mar 2010 02:41:26 PST

Familial aggregations of testicular germ cell tumor (FTGCT) have been well described, suggesting the existence of a hereditary TGCT subset. Approximately 1.4% of newly diagnosed TGCT patients report a positive family history of TGCT. Sons and siblings of TGCT patients have four- to sixfold and eight- to tenfold increases in TGCT risk respectively. Segregation analyses suggest an autosomal recessive mode of inheritance. Linkage analyses have identified several genomic regions of modest interest, although no high-penetrance cancer susceptibility gene has been mapped yet. These data suggest that the combined effects of multiple common alleles, each conferring modest risk, might underlie familial testicular cancer. Families display a mild phenotype: the most common number of affected families is 2. Age at diagnosis is 2–3 years younger for familial versus sporadic cases. The ratio of familial seminoma to nonseminoma is 1.0. FTGCT is more likely to be bilateral than sporadic TGCT. This syndrome is cancer site specific. Testicular microlithiasis is a newly recognized FTGCT component. Candidate gene-association studies have implicated the Y chromosome gr/gr deletion and PDE11A gene mutations as genetic modifiers of FTGCT risk. Two genomewide association studies of predominantly sporadic but also familial cases of TGCT have implicated the KIT-ligand, SPRY4, and BAK1 genes as TGCT risk modifiers. All five loci are involved in normal testicular development and/or male infertility. These genetic data provide a novel insight into the genetic basis of FTGCT, and an invaluable guide to future TGCT research.

Nuclear export and mitochondrial and endoplasmic reticulum localization of IGF-binding protein 3 regulate its apoptotic properties

Paharkova-Vatchkova, V., Lee, K.-W. — Mon, 08 Mar 2010 02:41:25 PST

Tumor suppression by IGF-binding protein 3 (IGFBP3) may occur in an IGF-independent manner, in addition to its role as a regulator of IGF bioavailability. After secretion, IGFBP3 is internalized, rapidly localized to the nucleus, and is later detected in the cytoplasm. We identified a putative nuclear export sequence (NES) in IGFBP3 between amino acids 217 and 228, analogous to the leucine-rich NES sequence of p53 and HIV Rev. Mutation of the NES prevents nucleocytoplasmic shuttling of IGFBP3 and blocks its ability to induce apoptosis. Targeting of IGFBP3 to the mitochondria and endoplasmic reticulum (ER) was confirmed by co-localization with organelle markers using fluorescence confocal microscopy and subcellular fractionation. Mitochondrial targeting was also demonstrated in vivo in IGFBP3-treated prostate cancer xenografts. These results show that IGFBP3 shuttles from the nucleus to the mitochondria and ER, and that nuclear export is essential for its effects on prostate cancer apoptosis.

Hypoxia stimulates CXCR4 signalling in ileal carcinoids

Mon, 08 Mar 2010 02:41:25 PST

Tumour hypoxia is associated with increased metastatic potential and resistance to radiotherapy and chemotherapy. Ileal carcinoids are usually metastatic at the time of diagnosis and respond poorly to chemotherapy. The aim of this study was to investigate the extent of hypoxia in ileal carcinoids and the response of tumour cells to induced hypoxia. Vascular endothelial growth factor (VEGF), carbonic anhydrase (CA-IX), hypoxia-inducible factor (HIF)-1 and HIF-2 were studied by immunohistochemistry in biopsies from 24 patients with ileal carcinoids. All hypoxic markers were shown to be highly expressed in localized areas of the tumours irrespective of tumour location or stage. However, HIF-2 expression was significantly higher in distant metastases compared to primary tumours in the same patient. Global gene expression profiling of GOT1 carcinoid cells revealed a marked response to hypoxia. Expression of genes related to epithelial-to-mesenchymal transition and development was altered including increased expression of the C-X-C chemokine receptor type 4 (CXCR4), an important regulator of invasive growth and metastasis formation. High expression of CXCR4 was confirmed by immunohistochemistry in tumour biopsies. Stimulation of GOT1 cells by the CXCR4 ligand, CXCL12 (stromal cell-derived factor 1 (SDF-1)), activated the mitogen-activated protein kinase (MAPK) p42/44 signalling pathway and increased tumour cell migration. We conclude that ileal carcinoids contain hypoxic areas expressing HIF-1, HIF-2 and CXCR4. Signalling through the CXCL12–CXCR4 axis may contribute to the metastatic potential of ileal carcinoids. Targeting of HIFs and/or the CXCR4 signalling pathway may offer new therapeutic strategies for carcinoid tumour disease.

Allelic variant at -79 (C>T) in CDKN1B (p27Kip1) confers an increased risk of thyroid cancer and alters mRNA levels

Mon, 08 Mar 2010 02:41:25 PST

The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27^Kip1, are involved in thyroid cancer susceptibility. Based on the literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (–79C>T) and rs36228499 (–838C>A)) to perform the first case–control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (–79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09–4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27^Kip1 promoter (P value <0.001). This effect was observed in –79TT genotype control carriers, who showed a tendency towards lower CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the –79C>T (rs34330) variant as a novel mechanism underlying p27^Kip1 downregulation.

Histology-specific risks in testicular cancer in immigrants to Sweden

Hemminki, K., Mousavi, S. M., Brandt, A., Ji, J., Sundquist, J. — Mon, 08 Mar 2010 02:41:25 PST

The changes of cancer incidence upon immigration have been used as an estimator of environmental influence on cancer risk. The previous immigrant studies have indicated that the origins of testicular cancer are at an early age in life, probably in the intrauterine period. We wanted to reexamine the critical periods on histology-specific testicular cancer in sons of immigrants to Sweden. We used the nationwide Swedish Family-Cancer Database to calculate standardized incidence ratios (SIRs) for testicular cancer in sons of parents immigrating to Sweden from low- and high-risk countries compared with the native Swedes. Among the large immigrant groups, the SIRs for sons of two Finnish and Asian parents were decreased if the sons were born outside Sweden. The sons of a Danish immigrant couple showed an increased risk of testicular cancer. The changes in SIR were most systematic for seminoma. The present patterns of testicular cancer risk among sons of immigrants point to the early environmental risk factors, which influence the risk probably after the intrauterine period. These factors appear to influence seminoma risk in a more enduring way than they influence non-seminoma.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase C{delta}

Mertens-Walker, I., Bolitho, C., Baxter, R. C, Marsh, D. J — Mon, 08 Mar 2010 02:41:25 PST

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKC inhibitor rottlerin, and downregulation of PKC was inhibited by small interfering RNA (siRNA), highlighting the importance of PKC in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKC siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKC-dependent manner.

Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase

Algire, C., Amrein, L., Zakikhani, M., Panasci, L., Pollak, M. — Tue, 16 Mar 2010 09:14:29 PDT

The molecular mechanisms responsible for the association of obesity with adverse colon cancer outcomes are poorly understood. We investigated the effects of a high-energy diet on growth of an in vivo colon cancer model. Seventeen days following the injection of 5x10⁵ MC38 colon carcinoma cells, tumors from mice on the high-energy diet were approximately twice the volume of those of mice on the control diet. These findings were correlated with the observation that the high-energy diet led to elevated insulin levels, phosphorylated AKT, and increased expression of fatty acid synthase (FASN) by the tumor cells. Metformin, an antidiabetic drug, leads to the activation of AMPK and is currently under investigation for its antineoplastic activity. We observed that metformin blocked the effect of the high-energy diet on tumor growth, reduced insulin levels, and attenuated the effect of diet on phosphorylation of AKT and expression of FASN. Furthermore, the administration of metformin led to the activation of AMPK, the inhibitory phosphorylation of acetyl-CoA carboxylase, the upregulation of BNIP3 and increased apoptosis as estimated by poly (ADP-ribose) polymerase (PARP) cleavage. Prior work showed that activating mutations of PI3K are associated with increased AKT activation and adverse outcome in colon cancer; our results demonstrate that the aggressive tumor behavior associated with a high-energy diet has similar effects on this signaling pathway. Furthermore, metformin is demonstrated to reverse the effects of the high-energy diet, thus suggesting a potential role for this agent in the management of a metabolically defined subset of colon cancers.

{beta}-catenin activation is not involved in sporadic parathyroid carcinomas and adenomas

Sun, 31 Jan 2010 18:09:16 PST

Aberrant accumulation of β-catenin has been found in various types of human tumors. The aim of this study was to evaluate whether Wnt/β-catenin signaling is activated in parathyroid carcinomas and adenomas. We studied 154 parathyroid tumors (18 carcinomas (13 with distant metastases), six atypical adenomas, and 130 adenomas). Three normal parathyroid tissues were used as control. Direct sequencing of exon 3 of the CTNNB1 gene showed absence of stabilizing mutations in all the tumors. Immunostaining of β-catenin was performed in all carcinomas and in 66 adenomas (including three atypical). Normal parathyroid showed a homogeneous distinct outer cell membrane staining in the majority of cells and no nuclear staining. A weak cytoplasmic staining was observed in one case. All tumors showed negative nuclear staining. With the exception of one carcinoma, which had a negative membrane staining, all other samples showed a membrane staining which was similar to that of the normal parathyroid. β-Catenin expression was heterogeneous with a range of positive cells between 5 and 80%, independently of tumor type. Our results suggest that the Wnt/β-catenin signaling pathway is not involved in the development of parathyroid carcinomas and adenomas.

To remain current, and to do so briefly...

Fagin, J. A — Sun, 31 Jan 2010 18:09:17 PST

MicroRNAs and prostate cancer

Coppola, V., De Maria, R., Bonci, D. — Sun, 31 Jan 2010 18:09:17 PST

Despite much progress in prostate cancer management, new diagnostic, prognostic and therapeutic tools are needed to predict disease severity, choose among the available treatments and establish more effective therapies for advanced prostate cancer. In the last few years, compelling evidence has documented the role of microRNAs as new broad-spectrum oncogenes or tumour suppressor genes, thus their use as diagnostic, prognostic and therapeutic biomolecules is envisaged. This review extensively and critically summarizes the current knowledge about microRNA deregulation in prostate cancer disease, underlining present limits and future perspectives.

The clusterin paradigm in prostate and breast carcinogenesis

Rizzi, F., Bettuzzi, S. — Sun, 31 Jan 2010 18:09:17 PST

The biological functions of clusterin (CLU, also known as ApoJ, SGP2, TRPM-2, CLI) have been puzzling the researchers since its first discovery in the early 80's. We know that CLU is a single 9-exons gene expressing three protein forms with different sub-cellular localisations and diverse biological functions. Despite the many reports from many research teams on CLU action and its relation to tumourigenesis, contradictions in the data and alternative hypothesis still exist. Understanding the role of CLU in tumourigenesis is complicated not only by the existence of different protein forms but also by the changes of tumours over time and the selection pressures imposed by treatments such as hormone ablation or chemotherapy. This review focuses on recent discoveries concerning the role of CLU in prostate and breast cancer onset and progression. Although CLU acts primarily as a tumour suppressor in the early stages of carcinogenesis, consistent with its role in the involution of the prostate following castration, late stage cancer may overexpress CLU following chemotherapeutic drugs or hormonal ablation therapy. High expression of secreted or cytoplasmic CLU may represent a pro-survival stimulus because it confers increased resistance to killing by anti-cancer drugs or enhances tumour cell survival in specific niches.

MicroRNAs: a great challenge for the diagnosis and therapy of endocrine cancers

Fusco, A. — Sun, 31 Jan 2010 18:09:17 PST

Overexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis

Sun, 31 Jan 2010 18:09:16 PST

Therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) are limited due to lack of effective treatments. Thus, there is a need to thoroughly characterize the pathways of molecular pathogenesis and to identify potential targets for therapy in MTC. Since epidermal growth factor receptor (EGFR) seems to play a crucial role for RET activation, a key feature of MTCs, and several promising EGFR/vascular endothelial growth factor receptor 2 (VEGFR2)-targeted drugs have been developed, the present study was designed to investigate whether these proteins are altered in MTCs. We used a well-characterized series of 153 MTCs to evaluate EGFR activation by sequencing and FISH analysis, and to perform EGFR and VEGFR2 immunohistochemistry. EGFR tyrosine kinase domain mutations were not a feature of MTCs; however, EGFR polysomy and a strong EGFR expression were detected in 15 and 13% of the tumors respectively. Interestingly, EGFR was significantly overexpressed in metastases compared with primary tumors (35 vs 9%, P=0.002). We also studied whether specific RET mutations were associated with EGFR status, and found a decrease in EGFR polysomies (P=0.006) and a tendency towards lower EGFR expression for the most aggressive RET mutations (918, 883). Concerning VEGFR2, metastasis showed a higher expression than primary tumors (P=2.8x10^–8). In this first study investigating the relationship between EGFR, RET, and VEGFR2 in a large MTC series, we found an activation of EGFR and VEGFR2 in metastasis, using both independent and matched primary/metastasis samples. This suggests that some MTC patients may benefit from existing anti-EGFR/VEFGR2 therapies, although additional preclinical and clinical evidence is needed.

Association of testicular germ cell tumor with polymorphisms in estrogen receptor and steroid metabolism genes

Ferlin, A., Ganz, F., Pengo, M., Selice, R., Frigo, A. C., Foresta, C. — Sun, 31 Jan 2010 18:09:16 PST

It is generally assumed that the development of testicular germ cell tumor (TGCT) is under endocrine control. In particular, unbalanced androgen/estrogen levels and/or activity are believed to represent the key events for TGCT development and progression. Furthermore, recent evidence has suggested a strong genetic component for TGCT. In this study, we analyzed whether a genetic variation in estrogen receptor (ESR) genes and steroid hormone metabolism genes is associated with TGCT. We genotyped for 17 polymorphic markers in 11 genes in 234 TGCT cases and 218 controls: ESR (ESR1 and ESR2); CYP19A1 (aromatase); 17β-hydroxysteroid dehydrogenase types 1 and 4 (HSD17B1 and HSD17B4) dehydrogenases that convert potent androgens and estrogens to weak hormones; cytochrome P450 hydroxylating enzymes CYP1A1, CYP1A2, and CYP1B1; and the metabolic enzymes COMT, SULT1A1, and SULT1E1. We observed a significant association of rs11205 in HSD17B4 with TGCT. TGCT risk was increased twofold per copy of the minor A allele at this locus (odds ratios (OR)=2.273, 95% confidence interval (CI)=1.737–2.973). Homozygous carriage of the minor A allele was associated with an over fourfold increased risk of TGCT (OR=4.561, 95% CI=2.615–7.955) compared with homozygous carriage of the major G allele. The risk was increased both for seminoma (OR=5.327, 95% CI=2.857–9.931) and for nonseminoma (OR=3.222, 95% CI=1.471–7.059). We found for the first time an association of polymorphisms in HSD17B4 gene with TGCT. Our findings expand the current knowledge on the role of genetic contribution in testicular cancer susceptibility, and support the hypothesis that variations in hormone metabolism genes might change the hormonal environment implicated in testicular carcinogenesis.

The role of let-7 in cell differentiation and cancer

Boyerinas, B., Park, S.-M., Hau, A., Murmann, A. E, Peter, M. E — Sun, 31 Jan 2010 18:09:17 PST

MicroRNAs (miRNAs or miRs) are small noncoding RNAs capable of regulating gene expression at the translational level. Current evidence suggests that a significant portion of the human genome is regulated by microRNAs, and many reports have demonstrated that microRNA expression is deregulated in human cancer. The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans. The human let-7 family contains 13 members located on nine different chromosomes, and many human cancers have deregulated let-7 expression. A growing body of evidence suggests that restoration of let-7 expression may be a useful therapeutic option in cancers, where its expression has been lost. In this review, we discuss the role of let-7 in normal development and differentiation, and provide an overview of the relationship between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the relationship between let-7 and drug sensitivity are highlighted.

Molecular pathways mediating the anti-inflammatory effects of calcitriol: implications for prostate cancer chemoprevention and treatment

Krishnan, A. V, Feldman, D. — Sun, 31 Jan 2010 18:09:17 PST

Calcitriol, the hormonally active form of vitamin D, exerts multiple anti-proliferative and pro-differentiating actions including cell cycle arrest and induction of apoptosis in many malignant cells, and the hormone is currently being evaluated in clinical trials as an anti-cancer agent. Recent research reveals that calcitriol also exhibits multiple anti-inflammatory effects. First, calcitriol inhibits the synthesis and biological actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: i) suppression of the expression of cyclooxygenase-2, the enzyme that synthesizes PGs; ii) up-regulation of the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme that inactivates PGs; and iii) down-regulation of the expression of PG receptors that are essential for PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs results in a synergistic inhibition of the growth of prostate cancer (PCa) cells and offers a potential therapeutic strategy for PCa. Second, calcitriol increases the expression of mitogen-activated protein kinase phosphatase 5 in prostate cells resulting in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines. Third, calcitriol also exerts anti-inflammatory activity in PCa through the inhibition of nuclear factor-B signaling that results in potent anti-inflammatory and anti-angiogenic effects. Other important direct effects of calcitriol as well as the consequences of its anti-inflammatory effects include the inhibition of tumor angiogenesis, invasion, and metastasis. We hypothesize that these anti-inflammatory actions, in addition to the other known anti-cancer effects of calcitriol, play an important role in its potential use as a therapeutic agent for PCa. Calcitriol or its analogs may have utility as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early or precancerous disease.

Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

Sun, 31 Jan 2010 18:09:16 PST

NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22^phox protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4–p22^phox localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H₂O₂ production. Increased NOX4–p22^phox in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.

Apoptomirs: small molecules have gained the license to kill

Vecchione, A., Croce, C. M — Sun, 31 Jan 2010 18:09:17 PST

Apoptosis is a tightly regulated form of cell death and represents an important process during normal development. In the past years, the scientific community has produced remarkable advances in our understanding of cancer biology, realizing that apoptosis and the genes that control it have a profound effect on the malignant phenotype. Recently, a new class of non-coding RNA genes, known as microRNA (miRNA or miR), have been demonstrated to play important roles in diverse biological processes, including development, cell differentiation, proliferation, and apoptosis. This suggests that other oncogenic mechanisms are needed to produce selective pressure to override apoptosis during multistage carcinogenesis. Intriguingly, since most cytotoxic anticancer agents induce apoptosis, it is possible that defects in apoptotic programs may contribute to treatment failure. Several studies strongly suggest a role for microRNAs in modulating sensitive/resistant phenotypes to cytotoxic therapy, calling for further investigation and validation of microRNA functions and targets in order to improve sensitivity to cancer treatments, thus ultimately improving prognosis and survival. Here, we review the current findings about microRNAs focusing on their involvement in the apoptotic process.

Dexamethasone enhances cell resistance to chemotherapy by increasing adhesion to extracellular matrix in human ovarian cancer cells

Sun, 31 Jan 2010 18:09:16 PST

Glucocorticoids (GCs) are widely used as co-medication in the therapy of solid malignant tumors to relieve some of the side effects of chemotherapeutic drugs. However, recent studies have shown that GCs could render cancer cells more resistant to cytotoxic drug-induced apoptosis, but the mechanism is largely unknown. In the present study, we found that the treatment of human ovarian cancer cell lines HO-8910 and SKOV3 with synthetic GCs dexamethasone (Dex) significantly increased their adhesion to extracellular matrix (ECM) and their resistance to apoptosis induced by cytotoxic drugs cisplatin and paclitaxel. Dex also increased the protein levels of adhesion molecules integrins β1, 4, and 5 in HO-8910 cells. The neutralizing antibody against integrin β1 prevented Dex-induced adhesion and significantly abrogated the protective effect of Dex toward cytotoxic agents. We further found that transforming growth factor-β1 (TGF-β1) alone not only increased cell adhesion and cell survival of HO-8910 cells in the presence of cisplatin, but also had synergistic pro-adhesion and pro-survival effects with Dex. Moreover, TGF-β1-neutralizing antibody that could block TGF-β1-induced cell adhesion and apoptosis resistance markedly abrogated the synergistic pro-adhesion and pro-survival effects of Dex and TGF-β1. Finally, we further demonstrated that Dex could up-regulate the expression of TGF-β receptor type II and enhance the responsiveness of cells to TGF-β1. In conclusion, our results indicate that increased adhesion to ECM through the enhancement of integrin β1 signaling and TGF-β1 signaling plays an important role in chemoresistance induced by GCs in ovarian cancer cells.

Apoptosis by dietary agents for prevention and treatment of prostate cancer

Khan, N., Adhami, V. M., Mukhtar, H. — Sun, 31 Jan 2010 18:09:17 PST

Accumulating data clearly indicate that induction of apoptosis is an important event for chemoprevention of cancer by naturally occurring dietary agents. In mammalian cells, apoptosis has been divided into two major pathways: the extrinsic pathway, activated by pro-apoptotic receptor signals at the cellular surface; and the intrinsic pathway, which involves the disruption of mitochondrial membrane integrity. This process is strictly controlled in response to integrity of pro-death signaling and plays critical roles in development, maintenance of homeostasis, and host defense in multicellular organisms. For chemoprevention studies, prostate cancer (PCa) represents an ideal disease due to its long latency, its high incidence, tumor marker availability, and identifiable preneoplastic lesions and risk groups. In this article, we highlight the studies of various apoptosis-inducing dietary compounds for prevention of PCa in vitro in cell culture, in preclinical studies in animals, and in human clinical trials.

Racial differences in the association between body mass index and serum IGF1, IGF2, and IGFBP3

Sun, 31 Jan 2010 18:09:16 PST

African–American (AA) race/ethnicity, lower body mass index (BMI), and higher IGF1 levels are associated with premenopausal breast cancer risk. This cross-sectional analysis investigated whether BMI or BMI at age 21 years contributes to racial differences in IGF1, IGF2, IGF-binding protein 3 (IGFBP3), or free IGF1. Participants included 816 white and 821 AA women between ages 40 and 79 years across a wide BMI range (18.5–40 kg/m²). Compared with white women, AA women had higher mean IGF1 (146.3 vs 134.4 ng/ml) and free IGF1 (0.145 vs 0.127) levels, and lower IGF2 (1633.0 vs 1769.3 ng/ml) and IGFBP3 (3663.3 vs 3842.5 ng/ml) levels (all P<0.01; adjusted for age, height, BMI, BMI at age 21 years, and menopausal status). Regardless of race, IGF1 and free IGF1 levels rose sharply as BMI increased to 22–24 kg/m², and then declined thereafter, while IGF2 and IGFBP3 levels tended to rise with BMI. In contrast, BMI at age 21 years was inversely associated with all IGF levels, but only among white women (P-interaction=0.01). With the decline in IGF1 with BMI at age 21 years among whites, racial differences in IGF1 significantly increased among women who were obese in early adulthood. In summary, BMI was associated with IGF1 levels regardless of race/ethnicity, while obesity during childhood or young adulthood may have a greater impact on IGF1 levels among white women. The effects of obesity throughout life on the IGF axis and racial differences in breast cancer risk require study.

MicroRNAs: a complex regulatory network drives the acquisition of malignant cell phenotype

Santarpia, L., Nicoloso, M., Calin, G. A — Sun, 31 Jan 2010 18:09:17 PST

Several lines of evidence indicate that tumorigenesis is a complex multistep process, and that most, if not all, cancers acquire the same set of functional capabilities during development and progression, albeit through various mechanistic strategies. Increasing data show an important role of microRNAs (miRNAs or miRs) in regulating various aspects of cancer biology. This review describes the role of microRNAs during the multiple steps that drive the progressive transformation of normal cells into highly malignant derivatives, outlining the role of microRNAs in regulating the common hallmarks of tumorigenesis: self-sufficiency in growth signals, insensitivity to antigrowth signals, abnormal apoptosis, limitless replicative potential, induction and sustained angiogenesis, and tissue invasion and metastasis. Recent evidence suggests an important role of microRNAs in the regulation of the expression of most genes regulating and coordinating a wide variety of processes in endocrine glands. We will highlight microRNAs of potential relevance to endocrine tumors and hormone-dependent cancers. Through this overview of how microRNAs regulate multiple targets and entire pathways, we will provide insight into the potential to develop new molecular microRNA-targeted therapies for endocrine tumors.

Somatostatin receptor-based imaging and therapy of gastroenteropancreatic neuroendocrine tumors

Sun, 31 Jan 2010 18:09:17 PST

Somatostatin receptor imaging (SRI) with [¹¹¹In-DTPA⁰]octreotide has proven its role in the diagnosis and staging of gastroenteropancreatic neuroendocrine tumors (GEPNETs). Newer radiolabeled somatostatin analogs which can be used in positron emission tomography (PET) imaging, and which have a higher affinity for the somatostatin receptor, especially receptor subtype-2, have been developed. It would be desirable, however, if one radiolabeled analog became the new standard for PET imaging, because the current application of a multitude of analogs implies a fragmented knowledge on the interpretation of the images that are obtained in clinical practice. In our view, the most likely candidates for such a universal PET tracer for SRI are [⁶⁸Ga-DOTA⁰,Tyr³]octreotate or [⁶⁸Ga-DOTA⁰,Tyr³]octreotide. Treatment with radiolabeled somatostatin analogs is a promising new tool in the management of patients with inoperable or metastasized neuroendocrine tumors. Symptomatic improvement may occur with all ¹¹¹In-, ⁹⁰Y-, or ¹⁷⁷Lu-labeled somatostatin analogs that have been used for peptide receptor radionuclide therapy (PRRT). The results that were obtained with [⁹⁰Y-DOTA⁰,Tyr³]octreotide and [¹⁷⁷Lu-DOTA⁰,Tyr³]octreotate are very encouraging in terms of tumor regression. Also, if kidney protective agents are used, the side effects of this therapy are few and mild, and the median duration of the therapy response for these radiopharmaceuticals is 30 and 40 months respectively. The patients' self-assessed quality of life increases significantly after treatment with [¹⁷⁷Lu-DOTA⁰,Tyr³]octreotate. Lastly, compared to historical controls, there is a benefit in overall survival of several years from the time of diagnosis in patients treated with [¹⁷⁷Lu-DOTA⁰,Tyr³]octreotate. These data compare favorably with the limited number of alternative treatment approaches. If more widespread use of PRRT can be guaranteed, such therapy may well become the therapy of first choice in patients with metastasized or inoperable GEPNETs.

Expression of hypoxia-inducible factor 1{alpha} in thyroid carcinomas

Sun, 31 Jan 2010 18:09:16 PST

Hypoxia-inducible factor 1 (HIF-1) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1 and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1 was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1 staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1 pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O₂, anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1 in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1 and HIF-1 targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1 with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.

The Pit-1/Pou1f1 transcription factor regulates and correlates with prolactin expression in human breast cell lines and tumors

Sun, 31 Jan 2010 18:09:16 PST

The transcription factor Pit-1/Pou1f1 regulates GH and prolactin (PRL) secretion in the pituitary gland. Pit-1 expression and GH regulation by Pit-1 have also been demonstrated in mammary gland. However, no data are available on the role of Pit-1 on breast PRL. To evaluate this role, several human breast cancer cell lines were transfected with either the Pit-1 expression vector or a Pit-1 small interference RNA construct, followed by PRL mRNA and protein evaluation. In addition, transient transfection of MCF-7 cells by a reporter construct containing the proximal PRL promoter, and ChIP assays were performed. Our data indicate that Pit-1 regulates mammary PRL at transcriptional level by binding to the proximal PRL promoter. We also found that Pit-1 raises cyclin D1 expression before increasing PRL levels, suggesting a PRL-independent effect of Pit-1 on cell proliferation. By using immunohistochemistry, we found a significant correlation between Pit-1 and PRL expression in 94 human breast invasive ductal carcinomas. Considering the possible role of PRL in breast cancer disorders, the function of Pit-1 in breast should be the focus of further research.

Systemic therapy for neuroendocrine tumours of gastroenteropancreatic origin

Basu, B., Sirohi, B., Corrie, P. — Sun, 31 Jan 2010 18:09:17 PST

Systemic therapy is one of a number of treatment options routinely used in the management of advanced, unresectable neuroendocrine tumours (NETs). In contrast to many of the other NET treatment modalities, there is at least some evidence base to justify its use. Even so, well-designed clinical trials are limited, since conducting clinical research in this complex group of rare cancers is challenging. The remit of this review article is to summarise the oncology literature and explain the role of systemic therapy in treating NETs of gastroenteropancreatic origin, identifying benefits and limitations. The molecular biology of NETs is now being unravelled, which affords new opportunities for development of mechanism-driven therapies. The rationale for some of the newer systemic targeted therapies that are showing promise in the clinic is discussed.

MicroRNAs in ovarian carcinomas

Dahiya, N., Morin, P. J — Sun, 31 Jan 2010 18:09:17 PST

The molecular mechanisms involved in epithelial ovarian cancer initiation and progression are just beginning to be elucidated. In particular, it has become evident that microRNAs (miRNAs or miRs), a class of molecules that post-transcriptionally regulate gene expression, play a major role in ovarian tumorigenesis. Several microRNA profiling studies have identified changes in microRNA patterns that take place during ovarian cancer development. While most deregulated microRNAs are down-regulated in cancer, and may therefore act as tumor suppressors, others are elevated and may represent novel oncogenes in this disease. A number of microRNAs identified as aberrantly expressed in ovarian carcinoma have been shown to have important functional roles in cancer development and may therefore represent targets for therapy. In addition, some of the microRNA patterns may have prognostic significance. The identification of functional targets represents a major hurdle in our understanding of microRNA function in ovarian carcinoma, but significant progress is being made. It is hoped that a better understanding of the microRNA expression and roles in ovarian cancer may provide new avenues for the detection, diagnosis, and therapy of this deadly disease.

Novel gene rearrangements in transformed breast cells identified by high-resolution breakpoint analysis of chromosomal aberrations

Sun, 31 Jan 2010 18:09:17 PST

Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer, we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1 Mb bacterial artificial chromosome (BAC) arrays. The breakpoints were further narrowed down by fluorescence in situ hybridisation (FISH) with clones from the 32 k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only, it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers.

Deregulation of microRNA expression in follicular cell-derived human thyroid carcinomas

Pallante, P., Visone, R., Croce, C. M., Fusco, A. — Sun, 31 Jan 2010 18:09:17 PST

Carcinoma of the thyroid gland is an uncommon cancer, but one of the most frequent malignancies of the endocrine system. Most thyroid cancers are derived from the follicular cells. Follicular carcinoma is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Even though several genetic lesions have been already described in human thyroid cancer, particularly in the papillary histotype, the mechanisms underlying the development of these neoplasias are still far from being completely elucidated. Some years ago, several studies were undertaken to analyze the expression of microRNAs (miRNAs or miRs) in thyroid carcinoma to evaluate a possible role of their deregulation in the process of carcinogenesis. These studies showed an aberrant microRNA expression profile that distinguishes unequivocally among PTC, ATC, and normal thyroid tissue. Here, other than summarizing the current findings on microRNA expression in human thyroid carcinomas, we discuss the mechanisms by which microRNA deregulation may play a role in thyroid carcinogenesis, and the possible use of microRNA knowledge in the diagnosis and therapy of thyroid neoplasms.

Positive regulation of spondin 2 by thyroid hormone is associated with cell migration and invasion

Sun, 31 Jan 2010 18:09:17 PST

The thyroid hormone 3,3',5-triiodo-l-thyronine (T₃) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T₃ treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T₃. However, the underlying mechanism and the physiological role of T₃ in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T₃ treatment in the HepG2–TR1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T₃ response element on the spondin 2 promoter was localized in the –1104/–1034 or –984/–925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T₃, has an important role in cell invasion, cell migration, and tumor progression.

The Ca2+-calmodulin-dependent kinase II is activated in papillary thyroid carcinoma (PTC) and mediates cell proliferation stimulated by RET/PTC

Sun, 31 Jan 2010 18:09:17 PST

RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRaf^V600E, CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRaf^V600E, or Ras^V12 induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca²⁺/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRaf^V600E, oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.

Plasma sex hormone concentrations and breast cancer risk in an ethnically diverse population of postmenopausal women: the Multiethnic Cohort Study

Thu, 18 Feb 2010 09:17:26 PST

To add to the existing evidence that comes mostly from White populations, we conducted a nested case–control study to examine the association between sex hormones and breast cancer risk within the Multiethnic Cohort that includes Japanese American, White, Native Hawaiian, African American, and Latina women. Of the postmenopausal women for whom we had a plasma sample, 132 developed breast cancer during follow-up. Two controls per case, matched on study area (Hawaii, Los Angeles), ethnicity/race, birth year, date and time of blood draw and time fasting, were randomly selected from the women who had not developed breast cancer. Levels of estradiol (E₂), estrone (E₁), androstenedione, dehydroepiandrosterone (DHEA), and testosterone were quantified by RIA after organic extraction and Celite column partition chromatography. E₁ sulfate, DHEA sulfate (DHEAS), and sex hormone-binding globulin (SHBG) were quantified by direct immunoassays. Based on conditional logistic regression, the sex hormones were positively associated and SHBG was negatively associated with breast cancer risk. All associations, except those with DHEAS and testosterone showed a significant linear trend. The odds ratio (OR) associated with a doubling of E₂ levels was 2.26 (95% confidence interval (CI) 1.58–3.25), and the OR associated with a doubling of testosterone levels was 1.34 (95% CI 0.98–1.82). The associations in Japanese American women, who constituted 54% of our sample, were similar to or nonsignificantly stronger than in the overall group. This study provides the best evidence to date that the association between sex hormones and breast cancer risk is generalizable to an ethnically diverse population.

Differential expression of microRNAs in human parathyroid carcinomas compared with normal parathyroid tissue

Thu, 18 Feb 2010 09:17:26 PST

Parathyroid carcinoma (PaC) is a rare cause of primary hyperparathyroidism. Though the loss of the oncosuppressor CDC73/HRPT2 gene product, parafibromin, has been involved in the hyperparathyroidism–jaw tumor syndrome and in a consistent set of sporadic PaCs, parathyroid carcinogenesis remains obscure. MicroRNAs are a new class of small, non-coding RNAs implicated in development of cancer, since their deregulation can induce aberrant expression of several target genes. The aim of the present study was to identify differentially expressed microRNAs in parathyroid cancers compared with normal tissues. We performed a TaqMan low-density array profiling of four parathyroid cancers harboring CDC73 inactivating mutations and negative for parafibromin immunostaining. Their microRNA profiling was compared with that of two normal parathyroid biopsies. Out of 362 human microRNAs assayed, 279 (77%) were successfully amplified. Fourteen and three microRNAs were significantly down- and over-expressed in parathyroid cancers respectively. Of these, miR-296 and miR-139 were down-regulated, and miR-503 and miR-222 were over-expressed with a null false discovery rate. Carcinomas could be discriminated from parathyroid adenomas by a computed score based on the expression levels of miR-296, miR-222, and miR-503 as miR-139 was similarly down-regulated in both cancers and adenomas. Finally, miR-296 and miR-222 levels negatively correlated with mRNA levels of the hepatocyte growth factor receptor-regulated tyrosine kinase substrate and p27/kip1 levels respectively. These results suggest the existence of an altered microRNA expression pattern in PaCs together with a potential role of miR-296 as novel oncosuppressor gene in these neoplasia.

Knockdown of ICB-1 gene enhanced estrogen responsiveness of ovarian and breast cancer cells

Thu, 18 Feb 2010 09:17:26 PST

ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor (ER) expression and a significant decrease of ERβ expression on the mRNA level. Expression of ER-dependent genes progesterone receptor, pS2, fibulin 1c, and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ER transcript levels but sustaining high levels of ERβ, reducing both activation of ER target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy.

Thyroid cancer and renal transplantation: a meta-analysis

Karamchandani, D., Arias-Amaya, R., Donaldson, N., Gilbert, J., Schulte, K.-M. — Thu, 18 Feb 2010 09:17:26 PST

Kidney transplantation and the associated immune suppression are associated with a significantly increased risk of developing cancer during long-term follow-up. Thyroid cancer has been recognised as a potential post-transplant risk but has not yet been subject of a focused review. We therefore performed a meta-analysis on data of 50 861 patients with a total follow-up of 198 595 patient-years and identified a 6.9-fold higher standardised incidence ratio (95% confidence interval 5.6–8.7, P<0.001) of thyroid cancer post renal transplantation as compared with a non-transplant group. All such cancers were of papillary type as far as histopathology was known. The mean time to discovery was 6.0 years post transplantation. This puts thyroid cancer into the group of high cancer risk following solid organ transplantation which already includes cervical cancer, non-melanoma skin cancer, oral and lip cancer and haematological malignancies. It is unclear what causes the increased cancer incidence. Inclusion of thyroid ultrasound in long-term post-transplant evaluation may help to ensure timely recognition of this condition.

Rosiglitazone impairs proliferation of human adrenocortical cancer: preclinical study in a xenograft mouse model

Thu, 18 Feb 2010 09:17:26 PST

Adrenocortical carcinoma (ACC) is a rare aggressive tumor with a poor prognosis. The lack of a specific and effective medical treatment is due to the poor knowledge of the mechanisms underlying tumor growth. Research on potential drugs able to specifically interfere with tumor proliferation is essential to develop more efficacious therapies. We evaluated for the first time the in vivo effect of rosiglitazone (RGZ), an anti-diabetic drug with in vitro anti-tumor properties, on ACC proliferation in a xenograft model obtained by s.c. injection of human ACC H295R cells in athymic mice. When the tumor size reached 5 mm, animals were allocated to 5 mg/kg RGZ- or water-treated groups. Tumor volume was measured twice a week. A significant reduction of tumor growth in RGZ versus control (control) group was observed and was already maximal following 17 day treatment (1–T/C=75.4% (43.7–93.8%)). After 31 days of treatment, mice were killed and tumor analyzed. Tumor histological evaluation revealed characteristics of invasiveness, richness in small vessels and mitotic figures in control group, while RGZ group tumors presented non infiltrating borders, few vessels, and many apoptotic bodies. Tumor immunohistochemistry showed that Ki-67 was reduced in RGZ versus control group. Quantitative real-time RT-PCR demonstrated a significant reduction in the expression of angiogenic (VEGF), vascular (CD31), proliferation (BMI-1), and anti-apoptotic (Bcl-2) genes in RGZ versus control group tumors. The same inhibitory effects were confirmed in in vitro RGZ-treated H295R. Our findings support and expand the role of RGZ in controlling ACC proliferation and angiogenesis in vivo and in vitro.

Leptin stimulates the migration of colon carcinoma cells by multiple signaling pathways

Ratke, J., Entschladen, F., Niggemann, B., Zanker, K. S, Lang, K. — Thu, 18 Feb 2010 09:17:26 PST

Active migration of tumor cells is a prerequisite for the development of metastasis and tumor progression, and is regulated by a variety of extracellular ligands. Epidemiological studies have shown that obesity increases the risk of colon cancer by 1.5- to 2-fold with obesity-associated colon cancer accounting for 14–35% of total incidence. In obese individuals, serum levels of leptin are markedly increased, and therefore, we have investigated the impact of this adipocytokine on the migration of various human colon carcinoma cell lines such as SW480, SW620, and HCT116. Leptin significantly enhanced the migratory activity of all three cell lines, and the strongest effect was observed in SW480 cells, which increased their locomotor activity from 28% spontaneously locomoting cells to 50%. The intracellular signal transduction regulating this pro-migratory effect involves the activation of the transcription factor signal transducer and activator of transcription-3 via Janus kinases, but also the activity of src tyrosine kinases, focal adhesion kinase, exclusively protein kinase C, and the phosphatidyl-inositol-3-kinase, as proven by the use of particular inhibitors and target-specific small interfering RNAs. Herein, we deliver new evidence for a modulatory role of leptin in the regulation of colon cancer progression by stimulating tumor cell migration. Thus, our findings have potential clinical implications, because understanding the impact of leptin on tumor cell migration and the underlying signal transduction mechanisms is mandatory for future development of novel therapeutics to treat obesity-associated colorectal cancer.

Leptin-R and its association with PI3K/AKT signaling pathway in papillary thyroid carcinoma

Thu, 18 Feb 2010 09:17:27 PST

The putative role of leptin and its receptor (Ob-R) in the pathogenesis of various primary human malignancies has been reported; however, their role in papillary thyroid cancer (PTC) has not yet been evaluated. We investigated the role of Ob-R in a large tissue microarray cohort of PTC followed by in vitro studies using a panel of PTC cell lines. Ob-R overexpression was seen in 80% PTCs and was significantly associated with poor disease-free survival (P=0.0235). PTCs that overexpressed Ob-R showed a aggressive phenotype characterized by older age, extrathyroid extension, larger tumor size, nodal metastasis, advanced stage, tall cell variant histological subtype, and a poor disease-free survival (P=0.0005, P=0.0006, P=0.0398, P=0.0004, P=0.0111, P=0.0003, and P=0.0235 respectively). However, Ob-R expression was not an independent prognostic marker to predict disease-free survival in multivariate analysis. PTCs with overexpression of Ob-R showed a significant direct association with overexpression of XIAP (P<0.0001) and Bcl-XL (P<0.0001). In vitro analysis showed that leptin stimulated cell proliferation and inhibited apoptosis via activation of phosphatidylinisitol 3' kinase (PI3K)/protein kinase B (AKT) signaling pathway. Inhibition of PI3K activity by its inhibitor LY294002 abrogated leptin-mediated PI3K/AKT signaling. Gene silencing of Ob-R in PTC cells resulted in downregulation of phospho-AKT, Bcl-XL, and XIAP expression suggesting that leptin-mediated pathogenesis of PTC occurs via involvement of these downstream targets. Altogether, these data show that leptin plays an important role in PTC pathogenesis through PI3K/AKT pathway via Ob-R and is a potential prognostic marker associated with an aggressive phenotype and poor disease-free survival.

Glucocorticoid receptor over-expression promotes human small cell lung cancer apoptosis in vivo and thereby slows tumor growth

Thu, 18 Feb 2010 09:17:27 PST

Small cell lung cancer (SCLC) is an aggressive tumor, associated with ectopic ACTH syndrome. We have shown that SCLC cells are glucocorticoid receptor (GR) deficient, and that restoration of GR expression confers glucocorticoid sensitivity and induces apoptosis in vitro. To determine the effects of GR expression in vivo, we characterized a mouse SCLC xenograft model that secretes ACTH precursor peptides, and so drives high circulating corticosterone concentrations (analogous to the ectopic ACTH syndrome). Infection of SCLC xenografts with GR-expressing adenovirus significantly slowed tumor growth compared with control virus infection. Time to fourfold initial tumor volume increased from a median of 9 days to 16 days (P=0.05; n=7 per group). Post-mortem analysis of GR-expressing tumors revealed a threefold increase in apoptotic (TUNEL positive) cells (P<0.01). Infection with the GR-expressing adenovirus caused a significant reduction in Bcl-2 and Bcl-xL transcripts. Furthermore, in both the GR-expressing adenovirus-infected cells and tumors, a significant number of uninfected cells underwent apoptosis, supporting a bystander cell killing effect. Therefore, GR expression is pro-apoptotic for human SCLCs in vivo, as well as in vitro, suggesting that loss of GR confers a survival advantage to SCLCs.

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Thu, 18 Feb 2010 09:17:27 PST

Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.

L-thyroxine-treated patients with nodular goiter have lower serum TSH and lower frequency of papillary thyroid cancer: results of a cross-sectional study on 27 914 patients

Thu, 18 Feb 2010 09:17:27 PST

The risk of papillary thyroid cancer (PTC) is related to serum TSH, and the development of thyroid autonomy by reducing TSH levels decreases the frequency of PTC in patients with nodular goiter. Our aim was to investigate the effect of l-thyroxine (l-T₄) on the frequency of PTC diagnosed by cytology in a large series of patients with nodular goiter untreated (n=20 055) or treated with l-T₄ (n=7859). l-T₄-treated patients with respect to untreated patients presented significantly lower serum TSH (median, interquartile range: 0.30 µU/ml, 0.08–0.62 µU/ml versus 0.70 µU/ml, 0.38–1.14 µU/ml; P<0.0001) and prevalence of PTC (3.2 vs 5.1%; P<0.0001). The frequency of PTC was closely related to serum TSH, with it being lowest in patients with TSH below the normal range (<0.4 µU/ml; 189/10 059, 1.9%) and highest in patients with TSH above the normal range (>3.4 µU/ml; 21/127, 16.5%), also showing a progressive increase from the lower to the upper quartile of normal range. A significantly higher proportion of l-T₄-treated patients (6650/7859, 84.6%) had serum TSH below the median (0.90 µU/ml) with respect to untreated patients (12 599/20 055, 62.8%; ² P value <0.0001), with it being included in the range of TSH associated with a lower frequency of PTC. The relationship between serum TSH and frequency of PTC was unrelated to the type of nodularity (solitary versus multinodular) and was not age dependent. In conclusion, patients with nodular goiter, treatment with l-T₄ is responsible for the reduction of serum TSH and is associated with a decreased frequency of PTC.

Interleukin-6 trans-signalling differentially regulates proliferation, migration, adhesion and maspin expression in human prostate cancer cells

Santer, F. R, Malinowska, K., Culig, Z., Cavarretta, I. T — Thu, 18 Feb 2010 09:17:27 PST

Interleukin-6 (IL-6) is suggested to have a pathogenic role in the progression of prostate cancer (PC), therefore representing an attractive target for new therapies. However, due to the pleiotropy of this cytokine, targeting IL-6 results in different and unpredictable responses. In order to better understand the mechanisms underlying the different responses to the cytokine, we focused our attention on IL-6 receptors (IL-6Rs) that represent the first element in the cascade of cytokine-activated signalling pathways. IL-6 signal transduction may indeed occur through the membrane IL-6R (classical signalling) and/or through the less studied soluble IL-6R (sIL-6R; IL-6 trans-signalling (IL-6TS)). We provide the first evidence how responses to IL-6 may depend on the different content of IL-6Rs in PC. In particular, the studies of ³H-thymidine incorporation and exploitation of different approaches (i.e. activation or inhibition of IL-6TS in sIL-6R-negative and -positive cell lines and transfection of IL-6R siRNA) allowed us to demonstrate that IL-6TS specifically accounts for an anti-proliferative effect of the cytokine in three PC cell lines that are known to respond differently to IL-6. Additionally, by applying migration-, scratch- and adhesion assays, we show that IL-6TS increases motility and migration and decreases adhesion of prostate cells facilitating thereby processes that determine metastasis initiation and spread. Finally, by western analyses, we uncovered an IL-6- and sIL-6R-dependent downregulation of the tumour suppressor maspin. Collectively, these data suggest that selective targeting of IL-6TS might allow to refine the currently available experimental anti-IL-6 therapies against PC.

The role of oestrogen receptor {alpha} in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2

Thu, 18 Feb 2010 09:17:27 PST

Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ER, ERβ, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ER was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ER and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan–Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.

The long-term survival in adrenocortical carcinoma with active surgical management and use of monitored mitotane

Thu, 18 Feb 2010 09:17:27 PST

Adrenocortical carcinoma (ACC) is a rare tumour disease with sinister prognosis also after attempts to radical surgery; better prognosis is seen for low-stage tumours. Adjuvant treatment with the adrenolytic drug mitotane has been attempted, but not proven to prevent from recurrence. The drug may offer survival advantage in case of recurrence. The aim of this single-centre study (1979–2007) of 43 consecutive patients was to evaluate the long-term survival after active surgical treatment combined with monitored mitotane (to reduce side effects of the drug). The series is unique, since all patients were offered a period of mitotane as adjuvant or palliative treatment; six patients refused mitotane. Despite a high proportion of high-stage tumours (67%), the complete resection rate was high (77%). The disease-specific 5-year survival was high (64.1%); very high for patients with low-stage tumours without evident relation to mitotane levels. Patients with high-stage tumours had a clear survival advantage with mitotane levels above a threshold of 14 mg/l in serum. The hazard ratio for patients with high mitotane levels versus all patients indicates a significant effect of the drug. The results indicate that adjuvant mitotane may be the standard of care for patients with high-stage ACC after complete resection.

CDC73/HRPT2 CpG island hypermethylation and mutation of 5'-untranslated sequence are uncommon mechanisms of silencing parafibromin in parathyroid tumors

Thu, 18 Feb 2010 09:17:27 PST

The tumor suppressor HRPT2/CDC73 is mutated in constitutive DNA from patients with the familial disorder hyperparathyroidism–jaw tumor syndrome and in ~70% of all parathyroid carcinomas. In a number of HRPT2 mutant tumors however, expression of the encoded protein parafibromin is lost in the absence of a clear second event such as HRPT2 allelic loss or the presence of a second mutation in this tumor suppressor gene. We sought to determine whether hypermethylation of a 713 bp CpG island extending 648 nucleotides upstream of the HRPT2 translational start site and 65 nucleotides into exon 1 might be a mechanism contributing to the loss of expression of parafibromin in parathyroid tumors. Furthermore, we asked whether mutations might be present in the 5'-untranslated region (5'-UTR) of HRPT2. We investigated a pool of tissue from 3 normal parathyroid glands, as well as 15 individual parathyroid tumor samples including 6 tumors with known HRPT2 mutations, for hypermethylation of the HRPT2 CpG island. Methylation was not identified in any specimens despite complete loss of parafibromin expression in two parathyroid carcinomas with a single detectable HRPT2 mutation and retention of the wild-type HRPT2 allele. Furthermore, no mutations of a likely pathogenic nature were identified in the 5'-UTR of HRPT2. These data strongly suggest that alternative mechanisms such as mutation in HRPT2 intronic regions, additional epigenetic regulation such as histone modifications, or other regulatory inactivation mechanisms such as targeting by microRNAs may play a role in the loss of parafibromin expression.

The ETS oncogene family transcription factor FEV identifies serotonin-producing cells in normal and neoplastic small intestine

Thu, 18 Feb 2010 09:17:27 PST

Neuroendocrine (NE) or carcinoid tumors of the small intestine (SI) frequently metastasize and produce the hormone serotonin, causing significant morbidity and mortality. A member of the ETS oncogene family of transcription factors, Fev, acts with the homeodomain transcription factor Nkx2.2 in the development of serotonin neurons in mice. In this study, we investigated the role of Fev in normal and neoplastic SI. In NE tumors (NETs) of the SI, serotonin stimulates tumor growth and causes debilitating symptoms, such as diarrhea, flushing, wheezing, and right-sided valvular heart disease (i.e. carcinoid syndrome). Compared with those in the matched normal human SI, FEV expression levels were significantly elevated in primary NETs (20-fold, P<0.0001), lymph node metastases (35-fold, P=0.004), and NET liver metastases (22-fold, P<0.0001) resected from patients with serotonin excess. Fev is expressed in the wild type but not in Nkx2.2 (–/–) mouse SI, in which cells producing serotonin are absent. Using recombination-based cell lineage tracing, we found that FEV-positive cells give rise to serotonin-producing cells in the SI. In Fev (–/–) mouse SI, we observed no difference in the number of cells producing serotonin or other hormones. We conclude that FEV expression identifies serotonin-producing cells in normal and neoplastic SI and is a novel target for diagnosis of patients with NETs of the SI.

The association between serum TSH concentration and thyroid cancer

Boelaert, K. — Mon, 30 Nov 2009 04:38:48 PST

There is mounting evidence that the serum concentration of TSH is an independent predictor for the diagnosis of thyroid malignancy in patients with nodular thyroid disease. Furthermore, preoperative serum TSH concentrations are higher in patients with more aggressive tumours, suggesting a potential role for TSH in the progression of differentiated thyroid cancer. Based on these observations, patients with higher serum TSH concentrations and borderline cytological results may require more aggressive investigation and treatment when compared with those with lower baseline TSH levels. The mechanisms underlying the finding of higher serum TSH in patients with thyroid cancer remain unexplained. In this issue of Endocrine-Related Cancer, Fiore et al. have analysed the relationship between serum TSH and diagnosis of papillary thyroid cancer in 10 178 patients with nodular thyroid disease who were investigated by fine-needle aspiration biopsy. They found significantly higher TSH concentrations in patients who were subsequently diagnosed with thyroid cancer compared with those with benign disease. In addition, they found that the development of autonomous thyroid function (TSH<0.4 µU/ml) was associated with a reduction in the risk of papillary thyroid carcinoma. In this commentary, the evidence regarding the association between serum TSH and thyroid cancer is discussed placing these new findings into context.

Genomic actions of estrogen receptor {alpha}: what are the targets and how are they regulated?

Welboren, W.-J., Sweep, F. C G J, Span, P. N, Stunnenberg, H. G — Mon, 30 Nov 2009 04:38:48 PST

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates a large number of genes in many different target tissues and is important in the development and progression of breast cancer. ER-mediated transcription is a complex process regulated at many different levels. The interplay between ligand, receptor, DNA sequence, cofactors, chromatin context, and post-translational modifications culminates in transcriptional regulation by ER. Recent technological advances have allowed the identification of ER target genes on a genome-wide scale. In this review, we provide an overview of the progress made in our understanding of the different levels of regulation mediated by ER. We discuss the recent advances in the identification of the ER-binding sites and target gene network and their clinical applications.

Hormone receptor-positive early breast cancer: controversies in the use of adjuvant chemotherapy

Montemurro, F., Aglietta, M. — Mon, 30 Nov 2009 04:38:48 PST

Current adjuvant treatments for operable breast cancer include chemotherapy, endocrine therapy in hormone receptor-positive tumors, and trastuzumab for HER2-positive tumors. Metanalyses of randomized trials show that in patients with hormone receptor-positive breast cancer, the effects of endocrine therapy and chemotherapy on survival are non-mutually exclusive. Most of these patients are therefore considered candidates to combined treatment. Recently, however, the endocrine responsiveness of tumors has been redefined on clinical, histopathological, and molecular bases. An emerging concept is that as endocrine responsiveness increases, chemoresponsiveness decreases. In the adjuvant setting, therapeutic choices are often based on small projected improvements in clinical outcomes. As a consequence, the role of chemotherapy and traditional management algorithms in patients with hormone receptor positive is being challenged. This review will address the current controversy regarding the role of adjuvant chemotherapy, including the newer anthracycline and taxane-based programs, in these patients.

Diabetes and cancer

Vigneri, P., Frasca, F., Sciacca, L., Pandini, G., Vigneri, R. — Mon, 30 Nov 2009 04:38:48 PST

Diabetes and cancer are two heterogeneous, multifactorial, severe, and chronic diseases. Because of their frequency, reciprocal influences – even minor influences – may have a major impact. Epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract, and female reproductive organs) is increased in diabetic patients. Mortality is also moderately increased. Several confounding factors, having general or site-specific relevance, make it difficult to accurately assess cancer risk in diabetic patients. These factors include diabetes duration, varying levels of metabolic control, different drugs used for therapy, and the possible presence of chronic complications. Hyperinsulinemia most likely favors cancer in diabetic patients as insulin is a growth factor with pre-eminent metabolic but also mitogenic effects, and its action in malignant cells is favored by mechanisms acting at both the receptor and post-receptor level. Obesity, hyperglycemia, and increased oxidative stress may also contribute to increased cancer risk in diabetes. While anti-diabetic drugs have a minor influence on cancer risk (except perhaps the biguanide metformin that apparently reduces the risk), drugs used to treat cancer may either cause diabetes or worsen a pre-existing diabetes. In addition to the well-known diabetogenic effect of glucocorticoids and anti-androgens, an increasing number of targeted anti-cancer molecules may interfere with glucose metabolism acting at different levels on the signaling substrates shared by IGF-I and insulin receptors. In conclusion, diabetes and cancer have a complex relationship that requires more clinical attention and better-designed studies.

Reproductive disturbances in multiple neuroendocrine tumor syndromes

Lytras, A., Tolis, G. — Mon, 30 Nov 2009 04:38:48 PST

In the context of multiple neuroendocrine tumor syndromes, reproductive abnormalities may occur via a number of different mechanisms, such as hyperprolactinemia, increased GH/IGF-1 levels, hypogonadotropic hypogonadism, hypercortisolism, hyperandrogenism, hyperthyroidism, gonadotropin hypersecretion, as well as, tumorigenesis or functional disturbances in gonads or other reproductive organs. Precocious puberty and/or male feminization is a feature of McCune–Albright syndrome (MAS), neurofibromatosis type 1 (NF1), Carney complex (CNC), and Peutz–Jeghers syndrome (PJS), while sperm maturation and ovulation defects have been described in MAS and CNC. Although tumorigenesis of reproductive organs due to a multiple neuroendocrine tumor syndrome is very rare, certain lesions are characteristic and very unusual in the general population. Awareness leading to their recognition is important especially when other endocrine abnormalities coexist, as occasionally they may even be the first manifestation of a syndrome. Lesions such as certain types of ovarian cysts (MAS, CNC), pseudogynecomastia due to neurofibromas of the nipple–areola area (NF1), breast disease (CNC and Cowden disease (CD)), cysts and ‘hypernephroid’ tumors of the epididymis or bilateral papillary cystadenomas (mesosalpinx cysts) and endometrioid cystadenomas of the broad ligament (von Hippel–Lindau disease), testicular Sertoli calcifying tumors (CNC, PJS) monolateral or bilateral macroochidism and microlithiasis (MAS) may offer diagnostic clues. In addition, multiple neuroendocrine tumor syndromes may be complicated by reproductive malignancies including ovarian cancer in CNC, breast and endometrial cancer in CD, breast malignancies in NF1, and malignant sex-cord stromal tumors in PJS.

Prostate cancer stromal cells and LNCaP cells coordinately activate the androgen receptor through synthesis of testosterone and dihydrotestosterone from dehydroepiandrosterone

Mon, 30 Nov 2009 04:38:48 PST

One of the mechanisms through which advanced prostate cancer (PCa) usually relapses after androgen deprivation therapy (ADT) is the adaptation to residual androgens in PCa tissue. It has been observed that androgen biosynthesis in PCa tissue plays an important role in this adaptation. In the present study, we investigated how stromal cells affect adrenal androgen dehydroepiandrosterone (DHEA) metabolism in androgen-sensitive PCa LNCaP cells. DHEA alone had little effect on prostate-specific antigen (PSA) promoter activity and the proliferation of LNCaP cells. However, the addition of prostate stromal cells or PCa-derived stromal cells (PCaSC) increased DHEA-induced PSA promoter activity via androgen receptor activation in the LNCaP cells. Moreover, PCaSC stimulated the proliferation of LNCaP cells under physiological concentrations of DHEA. Biosynthesis of testosterone or dihydrotestosterone from DHEA in stromal cells and LNCaP cells was involved in this stimulation of LNCaP cell proliferation. Androgen biosynthesis from DHEA depended upon the activity of various steroidogenic enzymes present in stromal cells. Finally, the dual 5-reductase inhibitor dutasteride appears to function not only as a 5-reductase inhibitor but also as a 3β-hydroxysteroid dehydrogenase inhibitor in LNCaP cells. Taken together, this coculture assay system provides new insights of coordinate androgen biosynthesis under the microenvironment of PCa cells before and after ADT, and offers a model system for the identification of important steroidogenic enzymes involved in PCa progression and for the development of the corresponding inhibitors of androgen biosynthesis.

Manipulating prohibitin levels provides evidence for an in vivo role in androgen regulation of prostate tumours

Dart, D A., Spencer-Dene, B., Gamble, S. C, Waxman, J., Bevan, C. L — Mon, 30 Nov 2009 04:38:48 PST

Current hormonal therapies for prostate cancer are effective initially, but inevitably tumours progress to an advanced, metastatic stage, often referred to as ‘androgen independent’. However, the androgen receptor (AR) signalling pathway is still key for their growth. It is speculated that tumours escape hormonal control via reduction in corepressor proteins. Manipulating such proteins is thus a potential therapeutic strategy to halt or even reverse tumour progression. We aimed to elucidate the effects of altering levels of the AR corepressor and androgen-target protein prohibitin (PHB) on prostate tumour growth. Prostate cancer cells incorporating an integrated androgen-responsive reporter gene and stably expressing vectors to inducibly overexpress or knockdown PHB were generated and used to assess effects on androgen signalling (by real time imaging) and tumour growth both in culture and in vivo. PHB overexpression inhibited AR activity and prostate-specific antigen (PSA) expression as well as androgen-dependent growth of cells, inducing rapid accumulation in G₀/G₁. Conversely, reduction in PHB increased AR activity, PSA expression, androgen-mediated growth and S-phase entry. In vivo, doxycycline-induced PHB regulation resulted in marked changes in AR activity, and showed significant effects upon tumour growth. Overexpression led to tumour growth arrest and protection from hormonal starvation, whereas RNAi knockdown resulted in accelerated tumour growth, even in castrated mice. This study provides proof of principle that i) reduction in PHB promotes both androgen-dependent and ‘androgen-independent’ tumour growth, and ii) altering AR activity via increasing levels or activity of corepressors is a valid therapeutic strategy for advanced prostate cancer.

Strong humoral response elicited by a DNA vaccine targeting gastrin-releasing peptide with optimized adjuvants inhibits murine prostate carcinoma growth in vivo

Mon, 30 Nov 2009 04:38:48 PST

Previous studies demonstrated that the elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. DNA vaccine for hormone/growth factor immune deprivation represents a feasible and attractive approach for cancer treatment; nevertheless, there is still a need to increase the potency of the DNA vaccine. Here, based on six copies of the B cell epitope GRP_18–27 in a linear alignment as an immunogen, we designed several anti-GRP DNA vaccines containing different combinations of immunoadjuvants, such as HSP65, tetanus toxoid_830–844 (T), pan HLA-DR-binding epitope (PADRE) (P), and mycobacterial HSP70_407–426 (M), on a backbone of pCR3.1 plasmid vector with eight 5'-GACGTT-3' CpG motifs and the VEGF183 signal peptide (VS). The effects of these immunoadjuvants in enhancing GRP-specific humoral immune response were then evaluated by comparing the respective immunogenicity and antitumor effects. Immunization of mice with pCR3.1-VS-HSP65-TP-GRP6-M2 elicited much higher levels of specific anti-GRP antibodies and more effectively inhibited the growth of a GRP-dependent tumor RM-1 in vivo. Interestingly, plasmids encoding for 2HSP70_407–426, but not the one with 1 or 3HSP70_407–426 showed stronger immune stimulatory potential as well as impressive antitumor activity, suggesting that 2HSP70_407–426 is an efficient molecular adjuvant for developing self-epitope vaccines. The highly immunogenic, potent anti-tumorigenic and antiangiogenesis activities of the anti-GRP DNA vaccine offered a novel immunotherapeutic approach in the treatment of GRP-dependent tumors and their complications.

VP-128, a novel oestradiol-platinum(II) hybrid with selective anti-tumour activity towards hormone-dependent breast cancer cells in vivo

Mon, 30 Nov 2009 04:38:48 PST

We have previously reported the synthesis of VP-128, a new 17β-oestradiol (E₂)-linked platinum(II) hybrid with high affinity for oestrogen receptor (ER). In the present study, we have investigated the anti-tumour activity of VP-128 towards breast cancer cells in vitro and in vivo. We used human ER-positive (MCF-7) and -negative (MDA-MB-468) cells as a model for treatment with increasing doses of VP-128, cisplatin or E₂ in vitro and for xenograft experiments in nude mice in vivo. Compared with cisplatin, VP-128 showed markedly improved in vitro and in vivo anti-tumour activity towards ER-positive MCF-7 breast cancer cells, without increased systemic toxicity. In these caspase-3-deficient cells, treatment with VP-128 overcame weak cellular sensitivity to cisplatin in vitro and in vivo. In these cells, only the hybrid induced apoptosis in an ER-dependent manner, inactivated both X-linked inhibitor of apoptosis protein and Akt, and induced selective nuclear accumulation of ER and the expression of ER-regulated genes c-myc and tff1, which was blocked by ER-specific antagonist ICI 282 780. In the case of ER-negative MDA-MB-468 cells, VP-128, but not cisplatin, induced nuclear accumulation of apoptosis-inducing factor and inhibited c-myc expression. However, VP-128 did not show enhanced in vivo anti-tumour activity compared with cisplatin. These results reveal two different modes of action for VP-128 in ER-positive and -negative breast cancer cells, and highlight the promising therapeutic value of this unique E₂-platinum hybrid for selective targeting of hormone-dependent cancers.

Involvement of adiponectin and leptin in breast cancer: clinical and in vitro studies

Mon, 30 Nov 2009 04:38:48 PST

Obesity is a risk factor for breast cancer development. A recent hypothesis suggests that the adipokines, adiponectin and leptin, are involved in breast cancer development. This prompted us to investigate the role of adiponectin and leptin in mammary carcinogenesis. Adiponectin receptors (AdipoR1 and AdipoR2) and leptin receptor (Ob-Rt, representing all the isoforms of Ob-R) proteins were detected by immunohistochemistry in in situ ductal carcinoma, invasive ductal malignancy, and healthy adjacent tissue. In addition, mRNA expression of adiponectin, AdipoR1, AdipoR2, leptin, Ob-Rt, and Ob-Rl (the long isoform of Ob-R) was observed in MCF-7 breast cancer cells. Interestingly, leptin mRNA expression was 34.7-fold higher than adiponectin mRNA expression in the MCF-7 cell line. Moreover, adiponectin (10 µg/ml) tended to decrease the mRNA expression of leptin (–36%) and Ob-Rl (–28%) and significantly decreased Ob-Rt mRNA level (–26%). In contrast, leptin treatment (1 µg/ml) significantly decreased AdipoR1 mRNA (–23%). Adiponectin treatment (10 µg/ml) inhibited the proliferation of MCF-7 cells, whereas leptin (1 µg/ml) stimulated the growth of cancer cells. In addition, adiponectin inhibited leptin-induced cell proliferation (both 1 µg/ml). Using microarray analysis, we found that adiponectin reduced the mRNA levels of genes involved in cell cycle regulation (mitogen-activated protein kinase 3 and ATM), apoptosis (BAG1, BAG3, and TP53), and potential diagnosis/prognosis markers (ACADS, CYP19A1, DEGS1, and EVL), whereas leptin induced progesterone receptor mRNA expression. In conclusion, the current study indicates an interaction of leptin- and adiponectin-signaling pathways in MCF-7 cancer cells whose proliferation is stimulated by leptin and suppressed by adiponectin.

High frequency of SNAIL-expressing cells confirms and predicts metastatic potential of phaeochromocytoma

Hayry, V, Salmenkivi, K, Arola, J, Heikkila, P, Haglund, C, Sariola, H — Mon, 30 Nov 2009 04:38:48 PST

Phaeochromocytomas are uncommon tumours of adrenal or extra-adrenal chromaffin tissue. About 2–26% of these have been reported to metastasize, but, on histological criteria, it is virtually impossible to predict malignant behaviour of the tumour. Using immunohistochemistry, we analysed the protein expression of SNAIL, a zinc-finger transcription factor, in a series of 50 phaeochromocytoma specimens from 42 patients. We found that SNAIL-expressing cells are frequent in metastatic primary tumours and their metastases, whereas in tumours without metastases, SNAIL expression is commonly absent. We conclude that the expression of SNAIL may be of use in predicting the metastatic potential of phaeochromocytoma.

VHL inactivation is an important pathway for the development of malignant sporadic pancreatic endocrine tumors

Mon, 30 Nov 2009 04:38:48 PST

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel–Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1 (HIF1-), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1- downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1- (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

Estrogen receptor 1 mRNA is a prognostic factor in ovarian carcinoma: determination by kinetic PCR in formalin-fixed paraffin-embedded tissue

Mon, 30 Nov 2009 04:38:48 PST

Epidemiological and cell culture studies indicate that ovarian carcinoma growth is dependent on estrogen stimulation. However, possibly due to the lack of a reliable biomarker that helps to select patients according to prognostically relevant estrogen receptor (ER) levels, clinical trials using anti-estrogenic therapeutics in ovarian carcinoma have had inconsistent results. Therefore, we tested if ER expression analysis by a quantitative method might be useful in this regard in formalin-fixed paraffin-embedded (FFPE) tissue. In a study group of 114 primary ovarian carcinomas expression of estrogen receptor 1 (ESR1) mRNA was analyzed using a new method for RNA extraction from FFPE tissue that is based on magnetic beads, followed by kinetic PCR. The prognostic impact of ESR1 mRNA expression was investigated and compared to ER protein expression as determined by immunohistochemistry. In univariate survival analysis the expression level of ESR1 mRNA was a significant positive prognostic factor for patient survival (hazard ratio (HR) 0.230 (confidence interval (CI) 0.102–0.516), P=0.002). ER protein expression was correlated to ESR1 mRNA expression (P=0.0001); however, ER protein expression did not provide statistically significant prognostic information. In multivariate analysis, ESR1 mRNA expression emerged as a prognostic factor, independent of stage, grade, residual tumor mass, age, and ER protein expression (HR 0.227 (CI 0.078–0.656), P=0.006). Our results indicate that the determination of ESR1 levels by kinetic PCR may be superior to immunohistochemical methods in assessment of biologically relevant levels of ER expression in ovarian carcinoma, and is feasible in routinely used FFPE tissue.

Oestrogen receptor 1 mRNA is a prognostic factor in ovarian cancer patients treated with neo-adjuvant chemotherapy: determination by array and kinetic PCR in fresh tissue biopsies

Mon, 30 Nov 2009 04:38:48 PST

Oestrogen receptors (ESRs) regulate the growth and differentiation of normal ovarian epithelia. However, to date their role as biomarkers in the clinical setting of ovarian cancer remains unclear. In view of potential endocrine treatment options, we tested the role of ESR1 mRNA expression in ovarian cancer in the context of a neo-adjuvant chemotherapy trial. Study participants had epithelial ovarian or peritoneal carcinoma unsuitable for optimal upfront surgery and were treated with neo-adjuvant platinum-based chemotherapy before surgery. RNA was isolated from frozen tumour biopsies before treatment. RNA expression of ESR1 was determined by microarray and reverse transcriptase kinetic PCR technologies. The prognostic value of ESR1 was tested using univariate and multivariate Cox proportional hazards models, Kaplan–Meier survival statistics and the log-rank test. ESR1 positively correlates with proliferation markers and histopathological grading. ESR1 was a significant predictor of survival as a continuous variable in the univariate Cox regression analysis. In multivariate analysis, elevated baseline ESR1 mRNA levels predicted prolonged progression-free survival (P=0.041) and overall survival (P=0.01) after neo-adjuvant chemotherapy, independently of pathological grade and age. We conclude that pretreatment ESR1 mRNA is associated with tumour growth and is a strong prognostic factor in ovarian cancer, independent of the strongest clinical parameters used in clinical routine. We suggest that ESR1 mRNA status should be considered in order to minimize possible confounding effects in ovarian cancer clinical trials, and that early treatment with anti-hormonal agents based on reliable hormone receptor status determination is worth investigating.

Lower levels of TSH are associated with a lower risk of papillary thyroid cancer in patients with thyroid nodular disease: thyroid autonomy may play a protective role

Mon, 30 Nov 2009 04:38:48 PST

Higher TSH values, even within normal ranges, have been associated with a greater risk of thyroid malignancy. The relationship between TSH and papillary thyroid cancer (PTC) has been analyzed in 10 178 patients submitted to fine needle aspiration of thyroid nodules with a cytology of PTC (n=497) or benign thyroid nodular disease (BTND, n=9681). In 942 patients, submitted to surgery (521 from BTND and 421 from PTC), the histological diagnosis confirmed an elevated specificity (99.6%) and sensitivity (98.1%) of cytology. TSH levels were significantly higher in PTC than in BTND both in the cytological and histological series and also in patients with a clinical diagnosis of multinodular goiter (MNG) and single/isolate nodule (S/I). A significant age-dependent development of thyroid autonomy (TSH <0.4 µU/ml) was observed in patients with benign thyroid disease, but not in those with PTC, diagnosed both on cytology and histology. In patients with MNG, the frequency of thyroid autonomy was higher and the risk of PTC was lower compared to those with S/I. In all patients, the presence of thyroid auto-antibodies (TAb) was associated with a significant increase of TSH. However, both in TAb positive and TAb negative patients TSH levels were significantly higher in PTC than in BTND. Our data confirm a direct relationship between TSH levels and risk of PTC in patients with nodular thyroid diseases. Thyroid autonomy conceivably protects against the risk of PTC, while thyroid autoimmunity does not play a significant role.

Aggressive inherited and sporadic medullary thyroid carcinomas display similar oncogenic pathways

Mon, 30 Nov 2009 04:38:48 PST

RET oncogene mutations are found in familial medullary thyroid carcinomas (MTC) and in one-third of sporadic cases. Oncogenic mechanisms involved in non-RET mutated sporadic MTC remain unclear. To study alterations associated with the development of both inherited and sporadic MTC, pangenomic DNA microarrays were used to analyze the transcriptome of 13 MTCs (four familial and nine sporadic). By using an ANOVA test, a list of 173 gene sequences with at least a twofold change expression was obtained. A subset of differentially expressed genes was controlled by real-time quantitative PCR and immunohistochemistry on a larger collection of MTCs. The expression pattern of those genes allowed us to distinguish two groups of sporadic tumors. The first group displays an expression profile similar to that expressed by inherited RET634 tumors. The second presents an expression profile close to that displayed by inherited RET918 tumors and includes tumors from patients with distant metastases. It is characterized by the overexpression of genes involved in proliferation and invasion (PTN, ESM1, and CEACAM6) or matrix remodeling (COL1A1, COL1A2, and FAP). Interestingly, RET918 tumors showed overexpression of the PTN gene, encoding pleiotrophin, a protein associated with metastasis. Using a MTC cell line, silencing of RET induced the inhibition of PTN gene expression. Overall, our results suggest that familial MTC and sporadic MTC could activate similar oncogenic pathways.

Gene expression profiles of thymic neuroendocrine tumors (carcinoids) with ectopic ACTH syndrome reveal novel molecular mechanism

Mon, 30 Nov 2009 04:38:48 PST

Although there has been increased knowledge about the molecular biology of neuroendocrine tumors (NETs), little is known about thymic carcinoids and even less about those with excessive hormone disorders, such as ectopic ACTH syndrome. This study was designed to gain insights into the molecular networks underlying the tumorigenesis of thymic carcinoids with ACTH secretion. By an approach integrating cDNA microarray and methods of computational biology, we compare gene expression profile between ACTH-producing thymic carcinoids and the normal thymus. In total, there are 63 biological categories increased and 108 decreased in thymic carcinoids. Cell proliferation was stimulated, which may explain the relatively uncontrolled cell growth of the tumor. Dysregulation of the Notch-signaling pathway was likely to be underlying the neuroendocrine features of this type of tumors. Moreover, inhibition of immunity and increased neuropeptide signaling molecules (POMC and its sorting molecule CPE) made the clinical manifestation reasonable and thus validated the array data. In conclusion, thymic carcinoids have a distinct gene expression pattern from the normal thymus, and they are characterized by deregulations of a series of biofunctions, which may be involved in the development of NETs. Hence, this study has provided not only a detailed comprehension of the molecular pathogenesis of thymic carcinoids with ectopic ACTH syndrome, but also a road map to approach thymic NETs at the system level.

Blood dosimetry from a single measurement of the whole body radioiodine retention in patients with differentiated thyroid carcinoma

Hanscheid, H., Lassmann, M., Luster, M., Kloos, R. T, Reiners, C. — Mon, 30 Nov 2009 04:38:48 PST

A simple method is presented to estimate the radiation-absorbed dose to the blood after radioiodine administration from a single external measurement of the whole-body retention in patients suffering from differentiated thyroid cancer. The blood dose is calculated applying the formalism of the Medical International Radiation Dose Committee under the assumptions that whole-body activity decays exponentially and that 14% of the whole-body residence time can be attributed to the blood. Accuracy and applicability of the method were tested based on data from 29 assessments, 18 pre-therapeutic tracer studies, and 11 ablation therapies, with whole-body and blood-retention measurements over at least 4 days. The mean of the absolute deviations between estimates and actual blood doses was found to be 14%, if external whole-body counting was performed on day 1 or 2 after radioiodine administration. This simple formalism is: 1) applicable to pre-therapeutic dosimetry for remnant ablation or treatment of metastases in a blood dose-based treatment concept and 2) applicable to blood-dose estimates after radioiodine therapy to determine radiation exposure. When combined with a measurement of the whole body retention 1 or 2 days after radioiodine administration this single time-point method closely approximates the classic, yet much more labor intensive multi-day dosimetry that measures both blood and whole-body activities.

Importance of gender-specific calcitonin thresholds in screening for occult sporadic medullary thyroid cancer

Machens, A., Hoffmann, F., Sekulla, C., Dralle, H. — Mon, 30 Nov 2009 04:38:49 PST

Men and women differ in thyroidal C-cell mass and calcitonin secretion. This difference may have implications for the definition of calcitonin thresholds to distinguish sporadic C-cell hyperplasia from occult medullary thyroid cancer. This retrospective study examined the hypothesis that gender-specific calcitonin thresholds predict occult medullary thyroid cancer more accurately among patients with increased basal calcitonin levels than unisex thresholds. A total of 100 consecutive patients were evaluated with occult sporadic C-cell disease no larger than 10 mm who were referred for increased basal calcitonin levels and underwent pentagastrin stimulation preoperatively at this institution. Altogether, gender-specific calcitonin thresholds predicted medullary thyroid cancer better than unisex thresholds. At lower (≤50 pg/ml basally; ≤500 pg/ml after stimulation), but not higher, calcitonin serum levels, women revealed medullary thyroid cancer four to eight times more often than men. Most discriminatory between C-cell hyperplasia and medullary thyroid cancer was a basal calcitonin threshold of 15 pg/ml (corrected 20 pg/ml) for women and 80 pg/ml (corrected 100 pg/ml) for men, based on the greatest accuracy at the lowest possible calcitonin level. The respective gender-specific stimulated peak calcitonin thresholds were 80 pg/ml (corrected 100 pg/ml) and 500 pg/ml. Corresponding positive predictive values for medullary thyroid cancer at these calcitonin thresholds were 89 and 90% for women, as opposed to 100% for men. To increase the positive predictive value for women to 100%, the respective calcitonin thresholds would have to be raised to 40 pg/ml (corrected 50 pg/ml) and 250 pg/ml. These findings indicate that gender-specific calcitonin thresholds predict sporadic occult medullary thyroid cancer better than unisex thresholds.

Dysregulation of secretion of CXC {alpha}-chemokine CXCL10 in papillary thyroid cancer: modulation by peroxisome proliferator-activated receptor-{gamma} agonists

Mon, 30 Nov 2009 04:38:49 PST

In papillary thyroid carcinomas (PTCs), oncogenes activate a transcriptional program including the upregulation of CXCL10 chemokine, which stimulates proliferation and invasion. Furthermore, peroxisome proliferator-activated receptor- (PPAR) activators thiazolidinediones (TZDs) modulate CXCL10 secretion in normal thyroid follicular cells (TFC), and inhibit PTC growth. Until now, no study has evaluated the effect of cytokines on CXCL10 secretion in PTCs, nor the effect of PPAR activation. The combined effects of interferon (IFN) and tumor necrosis factor (TNF) stimulation on CXCL10 secretion in primary cells from PTCs and TFC were tested. Furthermore, the effect of PPAR activation by TZDs, on CXCL10 secretion and proliferation in these cell types was studied. In primary cultures of TFC and PTCs CXCL10 production was absent under basal conditions; a similar dose-dependent secretion of CXCL10 was induced by IFN in both cell types. TNF alone induced a slight but significant CXCL10 secretion only in PTCs. The stimulation with IFN+TNF induced a synergistic CXCL10 release in both cell types; however, a secretion more than ten times higher was induced in PTCs. Treatment of TFC with TZDs dose-dependently suppressed IFN+TNF-induced CXCL10 release, while TZDs stimulated CXCL10 secretion in PTCs. A significant antiproliferative effect by TZDs was observed only in PTCs. In conclusion, a dysregulation of CXCL10 secretion has been shown in PTCs. In fact, a CXCL10 secretion more than ten times higher has been induced by IFN+TNF in PTCs with respect to TFC. Moreover, TZDs inhibited CXCL10 secretion in TFC and stimulated it in PTCs. The effect of TZDs on CXCL10 was unrelated to the significant antiproliferative effect in PTCs.

Multiple endocrine neoplasia type 1 knockout mice develop parathyroid, pancreatic, pituitary and adrenal tumours with hypercalcaemia, hypophosphataemia and hypercorticosteronaemia

Mon, 30 Nov 2009 04:38:49 PST

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized in man by parathyroid, pancreatic, pituitary and adrenal tumours. The MEN1 gene encodes a 610-amino acid protein (menin) which is a tumour suppressor. To investigate the in vivo role of menin, we developed a mouse model, by deleting Men1 exons 1 and 2 and investigated this for MEN1-associated tumours and serum abnormalities. Men1^+/– mice were viable and fertile, and 220 Men1^+/– and 94 Men1^+/+ mice were studied between the ages of 3 and 21 months. Survival in Men1^+/– mice was significantly lower than in Men1^+/+ mice (<68% vs >85%, P<0.01). Men1^+/– mice developed, by 9 months of age, parathyroid hyperplasia, pancreatic tumours which were mostly insulinomas, by 12 months of age, pituitary tumours which were mostly prolactinomas, and by 15 months parathyroid adenomas and adrenal cortical tumours. Loss of heterozygosity and menin expression was demonstrated in the tumours, consistent with a tumour suppressor role for the Men1 gene. Men1^+/– mice with parathyroid neoplasms were hypercalcaemic and hypophosphataemic, with inappropriately normal serum parathyroid hormone concentrations. Pancreatic and pituitary tumours expressed chromogranin A (CgA), somatostatin receptor type 2 and vascular endothelial growth factor-A. Serum CgA concentrations in Men1^+/– mice were not elevated. Adrenocortical tumours, which immunostained for 3-β-hydroxysteroid dehydrogenase, developed in seven Men1^+/– mice, but resulted in hypercorticosteronaemia in one out of the four mice that were investigated. Thus, these Men1^+/– mice are representative of MEN1 in man, and will help in investigating molecular mechanisms and treatments for endocrine tumours.

Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors

Mon, 30 Nov 2009 04:38:49 PST

Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

Curcumin acts as anti-tumorigenic and hormone-suppressive agent in murine and human pituitary tumour cells in vitro and in vivo

Mon, 30 Nov 2009 04:38:49 PST

Curcumin (diferuloylmethane) is the active ingredient of the spice plant Curcuma longa and has been shown to act anti-tumorigenic in different types of tumours. Therefore, we have studied its effect in pituitary tumour cell lines and adenomas. Proliferation of lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells as well as of corticotroph AtT20 mouse pituitary cells was inhibited by curcumin in monolayer cell culture and in colony formation assay in soft agar. Fluorescence-activated cell sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M. Analysis of cell cycle proteins by immunoblotting showed reduction in cyclin D₁, cyclin-dependent kinase 4 and no change in p27^kip. FACS analysis with Annexin V-FITC/7-aminoactinomycin D staining demonstrated curcumin-induced early apoptosis after 3, 6, 12 and 24 h treatment and nearly no necrosis. Induction of DNA fragmentation, reduction of Bcl-2 and enhancement of cleaved caspase-3 further confirmed induction of apoptosis by curcumin. Growth of GH3 tumours in athymic nude mice was suppressed by curcumin in vivo. In endocrine pituitary tumour cell lines, GH, ACTH and prolactin production were inhibited by curcumin. Studies in 25 human pituitary adenoma cell cultures have confirmed the anti-tumorigenic and hormone-suppressive effects of curcumin. Altogether, the results described in this report suggest this natural compound as a good candidate for therapeutic use on pituitary tumours.

Phase III trial of chemotherapy using 5-fluorouracil and streptozotocin compared with interferon {alpha} for advanced carcinoid tumors: FNCLCC-FFCD 9710

Mon, 30 Nov 2009 04:38:49 PST

The aim of this randomized multicenter phase III trial was to compare chemotherapy and interferon (IFN) in patients with metastatic carcinoid tumors. Patients with documented progressive, unresectable, metastatic carcinoid tumors were randomized between 5-fluorouracil plus streptozotocin (day 1–5) and recombinant IFN--2a (3 MUx3 per week). Primary endpoint was progression-free survival (PFS). From February 1998 to June 2004, 64 patients were included. The two arms were well matched for median age, sex ratio, PS 0–1, previous chemotherapy, surgery, or radiotherapy. The median PFS for chemotherapy was 5.5 months versus 14.1 for IFN (hazard ratio=0.75 (0.41–1.36)). Overall survival (OS), tolerance, and effects on carcinoid symptoms were not significantly different. Despite a trend in favor of IFN, there was no difference in PFS and OS in advanced metastatic carcinoid tumors and therapeutic effect of both treatments was mild.

Emerging roles of chemokines in prostate cancer

Vindrieux, D., Escobar, P., Lazennec, G. — Thu, 20 Aug 2009 07:48:39 PDT

Prostate cancer (PCa) represents the second leading cause of death among all cancer types in men in Europe and North America. Among the factors suspected to control PCa, incidence and progression, chemokines, and their receptors are now intensively studied. Chemokines are produced by tumor cells and also by the stromal microenvironment, both in the primary tumor site and in distant metastatic locations. The wide and differential distribution of chemokines and their receptors account for the pleiotropic actions of chemokines in PCa, including the modulation of growth, angiogenesis, invasion, metastasis, and hormone escape. This review will focus on the roles and the mechanisms of action and regulation of chemokines in the different steps of PCa development and will discuss the novel strategies that are currently envisioned to target chemokines in PCa.

Target-based therapies in breast cancer: current status and future perspectives

Thu, 20 Aug 2009 07:48:39 PDT

Identification of molecular alterations in key proteins involved in breast cancer cell proliferation and survival resulted in the development of a new treatment strategy with target-based agents. The anti-ErbB-2 monoclonal antibody (mAb) trastuzumab and the dual epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor lapatinib are effective in patients with breast cancer that overexpresses ErbB-2. The anti-vascular endothelial growth factor-A mAb bevacizumab is approved in combination with taxanes for treatment of unselected patients with metastatic breast cancer. In addition, preclinical data suggest that signaling inhibitors can prevent or overcome resistance to endocrine therapy in estrogen receptor positive (ER+) breast cancer. However, the majority of signaling inhibitors explored in breast cancer patients has shown little activity, at least when used as monotherapy; and the results of clinical trials in ER+ breast cancer of combinations of signaling inhibitors and endocrine therapies are rather disappointing. Negative findings are likely due to mechanisms of intrinsic or acquired resistance to target-based agents. Breast carcinoma is a complex and heterogeneous disease and several different molecular alterations are involved in its pathogenesis and progression. The redundancy of oncogenic pathways activated in cancer cells, the heterogeneity of the mechanisms of resistance, and the plasticity of tumor cells that are capable to adapt to different growth conditions, significantly hamper the efficacy of each signaling inhibitor in breast cancer. Therefore, a comprehensive approach that takes into account the complexity of the disease is definitely required to improve the efficacy of target-based therapy in breast cancer.

Mechanisms of bone metastases of breast cancer

Suva, L. J, Griffin, R. J, Makhoul, I. — Thu, 20 Aug 2009 07:48:39 PDT

Cancer development is a multi-step process driven by genetic alterations that elicit the progressive transformation of normal human cells into highly malignant derivatives. The altered cell proliferation phenotype of cancer involves a poorly characterized sequence of molecular events, which often result in the development of distant metastasis. In the case of breast cancer, the skeleton is among the most common of metastatic sites. In spite of its clinical importance, the underlying cellular and molecular mechanisms driving bone metastasis remain elusive. Despite advances in our understanding of the phenotype of cancer cells, the increased focus on the contribution of the tumor microenvironment and the recent revival of interest in the role of tumor-propagating cells (so called cancer stem cells) that may originate or be related to normal stem cells produced in the bone marrow, many important questions remain unanswered. As such, a more complete understanding of the influences of both the microenvironment and the tumor phenotype, which impact the entire multi-step metastatic cascade, is required. In this review, the importance of tumor heterogeneity, tumor-propagating cells, the microenvironment of breast cancer metastasis to bone as well as many current endocrine therapies for the prevention and treatment of metastatic breast cancer is discussed.

New therapeutic advances in the management of progressive thyroid cancer

Woyach, J. A, Shah, M. H — Thu, 20 Aug 2009 07:48:39 PDT

The spectrum of thyroid cancers ranges from one of the most indolent to one of the most aggressive solid tumors identified. Conventional therapies for thyroid cancers are based on the histologic type of thyroid cancers such as papillary or follicular thyroid cancer (differentiated thyroid cancer (DTC)), medullary thyroid cancer (MTC), or anaplastic thyroid cancer (ATC). While surgery is one of the key treatments for all such types of thyroid cancers, additional therapies vary. Effective targeted therapy for DTC is a decades-old practice with systemic therapies of thyroid stimulating hormone suppression and radioactive iodine therapy. However, for the iodine-refractory DTC, MTC, and ATC there is no effective systemic standard of care treatment. Recent advances in understanding pathogenesis of DTC and development of molecular targeted therapy have dramatically transformed the field of clinical research in thyroid cancer. Over the last five years, incredible progress has been made and phases I–III clinical trials have been conducted in various types of thyroid cancers with some remarkable results that has made an impact on lives of patients with thyroid cancer. Such history-making events have boosted enthusiasm and interest among researchers, clinicians, patients, and sponsors and we anticipate ongoing efforts to develop more effective and safe therapies for thyroid cancer.

Radiation-induced hypopituitarism

Fernandez, A., Brada, M., Zabuliene, L., Karavitaki, N., Wass, J. A H — Thu, 20 Aug 2009 07:48:39 PDT

The hypothalamic–pituitary unit is a particularly radiosensitive region in the central nervous system. As a consequence, hypopituitarism commonly develops after radiation treatments for sellar and parasellar neoplasms, extrasellar brain tumours, head and neck tumours, and following whole body irradiation for systemic malignancies. Increasing tumour-related survival rates provide an expanding population at risk of developing hypopituitarism. In this population, long-term monitoring tailored to the individual risk profile is required to avoid the sequelae of untreated pituitary hormonal deficiencies and resultant decrease in the quality of life. This review analyses the pathogenesis, prevalence and consequences of radiation-induced hypopituitarism (RIH) in diverse subgroups at risk. Also discussed is the impact of modern radiotherapy techniques in the prevalence of RIH, the spectrum of endocrine disorders and radiation-induced brain conditions that also occur in patients with RIH.

Mouse models of altered protein kinase A signaling

Kirschner, L. S, Yin, Z., Jones, G. N, Mahoney, E. — Thu, 20 Aug 2009 07:48:40 PDT

Protein kinase A (PKA) is an evolutionarily conserved protein which has been studied in model organisms from yeast to man. Although the cAMP–PKA signaling system was the first mammalian second messenger system to be characterized, many aspects of this pathway are still not well understood. Owing to findings over the past decade implicating PKA signaling in endocrine (and other) tumorigenesis, there has been renewed interest in understanding the role of this pathway in physiology, particularly as it pertains to the endocrine system. Because of the availability of genetic tools, mouse modeling has become the pre-eminent system for studying the physiological role of specific genes and gene families as a means to understanding their relationship to human diseases. In this review, we will summarize the current data regarding mouse models that have targeted the PKA signaling system. These data have led to a better understanding of both the complexity and the subtlety of PKA signaling, and point the way for future studies, which may help to modulate this pathway for therapeutic effect.

Novel mechanism of IGF-binding protein-3 action on prostate cancer cells: inhibition of proliferation, adhesion, and motility

Thu, 20 Aug 2009 07:48:40 PDT

IGF-binding protein-3 (IGFBP-3) is a modulator of the IGF-signaling pathway and was described as an anti-cancer agent in prostate cancer. The molecular mechanisms underlying these effects remained, however, largely undefined. We analyzed the influence of recombinant IGFBP-3 on cell proliferation of PC3, Du145, and LNCaP prostate cancer cells. As expected, IGFBP-3 inhibited IGF-stimulated cell proliferation by blocking IGF-mediated proliferation signals, but we observed an IGF-independent inhibitory effect of IGFBP-3 on prostate cancer cell proliferation in long-term cultures. We further investigated the influence of IGFBP-3 on adhesion, motility, and invasion of prostate cancer cells using adhesion assays, live-cell imaging techniques, and matrigel invasion measurements. There was a clear inhibitory effect of IGFBP-3 on tumor cell adhesion to extracellular matrix components in the presence and absence of IGF, whereas cell–cell adhesion was not affected. The same inhibitory effect of IGFBP-3 was determined on cell motility when real-time cell movements were followed. In addition, IGFBP-3 was able to inhibit tumor cell invasion through matrigel. In summary, we show that IGFBP-3 inhibits proliferation, adhesion, migration, and invasion processes of prostate tumor cells. These newly described mechanisms of IGFBP-3 can be of importance for tumor progression and support a role of IGFBP-3 in therapeutic settings.

Regulation of thrombospondin-1 by natural and synthetic progestins in human breast cancer cells

Hyder, S. M, Liang, Y., Wu, J., Welbern, V. — Thu, 20 Aug 2009 07:48:40 PDT

Our recent studies show that progestins induce vascular endothelial growth factor (VEGF) in breast cancer cells that express mutant p53 protein. Here, we show that natural and synthetic progestins also induce thrombospondin-1 (TSP-1) mRNA and protein in T47-D and BT-474 breast cancer cells. Antiprogestin RU-486 inhibits the induction of VEGF and TSP-1 by progestins, suggesting that this effect of progestin is mediated by the progesterone receptor (PR). Actinomycin-D, but not puromycin, also blocks progestin-dependent induction of TSP-1. A putative progestin-response element was identified in the human TSP-1 promoter, which is consistent with the hypothesis that a progestin–PR complex might directly regulate transcription of the TSP-1 gene in human cells. Conditioned medium from progestin-treated breast cancer cells stimulates endothelial cell proliferation in the absence though not in the presence of antibody to TSP-1, indicating that TSP-1 secreted by breast cancer cells could be pro-angiogenic. Since tumor cell-derived TSP-1 has the potential to promote angiogenesis in the tumor microenvironment, it could be a potential target for breast cancer therapy.

Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor {alpha} and c-Myc

Thu, 20 Aug 2009 07:48:40 PDT

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor (ER) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ER and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

The aryl hydrocarbon receptor as a target for estrogen receptor-negative breast cancer chemotherapy

Thu, 20 Aug 2009 07:48:40 PDT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3',4,4',5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines. MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.

Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ

Thu, 20 Aug 2009 07:48:40 PDT

DNA double-strand breaks (DSBs) are considered as one of the primary causes of cancer but their induction by hydrogen peroxide (H₂O₂) is still controversial. In this work, we studied whether the high levels of H₂O₂ produced in the thyroid to oxidize iodide could induce DNA modifications. Scores of DNA damage, in terms of strand breaks, were obtained by comet assay (alkaline condition for single-strand breaks (SSBs) and neutral condition for DSBs). We demonstrated that in a rat thyroid cell line (PCCl3), non-lethal concentrations of H₂O₂ (0.1–0.5 mmol/l) as well as irradiation (1–10 Gy) provoked a large number of SSBs (~2–3 times control DNA damage values) but also high levels of DSBs (1.2–2.3 times control DNA damage values). We confirmed the generation of DSBs in this cell line and also in human thyroid in primary culture and in pig thyroid slices by measuring phosphorylation of histone H2AX. l-Buthionine-sulfoximine, an agent that depletes cells of glutathione, decreased the threshold to observe H₂O₂-induced DNA damage. Moreover, we showed that DNA breaks induced by H₂O₂ were more slowly repaired than those induced by irradiation. In conclusion, H₂O₂ causes SSBs and DSBs in thyroid cells. DSBs are produced in amounts comparable with those observed after irradiation but with a slower repair. These data support the hypothesis that the generation of H₂O₂ in thyroid could also play a role in mutagenesis particularly in the case of antioxidant defense deficiency.

The role of AUF1 in thyroid carcinoma progression

Thu, 20 Aug 2009 07:48:40 PDT

AUF1/heterogeneous nuclear ribonucleoprotein D is an adenylate–uridylate-rich elements (AREs) -binding protein, which regulates the mRNA stability of many genes related to growth regulation, such as proto-oncogenes, growth factors, cytokines, and cell cycle-regulatory genes. Several studies demonstrated AUF1 involvement in the processes of apoptosis, tumorigenesis, and development by its interactions with ARE-bearing mRNAs. We report here that AUF1 may be involved in thyroid carcinoma progression. Investigations on thyroid tissues revealed that cytoplasmic expression of AUF1 in malignant tissues was increased when compared with benign thyroid tissues. In thyroid carcinoma cell lines, AUF1 was mostly detectable in the nucleus; however, in dividing cells, its increased production was also observed in the cytoplasm. We found AUF1 in complexes with ARE-bearing mRNAs, previously described to be crucial for proliferation and cell cycle of thyroid carcinoma. Total or exon-selective knockdown of AUF1 led to growth inhibition accompanied by induction of cell cycle inhibitors and decreased levels of cell cycle promoters. Our data demonstrate the existence of a complex network between AUF1 and mRNAs encoding proteins related to cell proliferation. AUF1 may control the balance between stabilizing and destabilizing effects, both of which are exerted on cell cycle machinery in thyroid carcinoma. Although we cannot exclude participation of other factors, thyroid carcinoma may recruit cytoplasmic AUF1 to disturb the stability of mRNAs encoding cyclin-dependent kinase inhibitors, leading to uncontrolled growth and progression of tumor cells. Thus, AUF1 may be considered as a new, additional marker for thyroid carcinoma.

Impact of connexin32 deletion on E7 or RET/PTC3 oncogene-driven growth and neoplastic transformation of the thyroid gland

Prost, G., Bernier-Valentin, F., Croset, M., Rousset, B. — Thu, 20 Aug 2009 07:48:40 PDT

Connexins (Cx) form gap junctions and allow direct cell-to-cell communication. Cx through gap junctions or by themselves play regulatory roles on cell growth and differentiation. Using genetically modified mice, we previously found that Cx32 acts as a down-regulator of growth in normal thyroid gland. In this study, we examined the impact of Cx32 ablation on oncogene-driven thyroid growth and neoplastic transformation. Cx32 knockout (Cx32-KO) mice were crossed with transgenic mice expressing, selectively in the thyroid gland, either the E7 or RET/PTC3 (RP3) oncogene. As already described, Cx32-KO mice had no detectable thyroid alteration in physiological conditions and mice expressing E7 or RP3 exhibited time-dependent thyroid hypertrophy and variable changes in expression of differentiation. The thyroid of E7 mice evolved towards a large colloid goitre whereas RP3 mice developed a hyperplastic thyroid of variable size, and the largest glands (about 40% of total) represented a profound tissue remodeling with proliferative papillary formations. E7-induced thyroid hypertrophy was reduced by about 40% in Cx32-KO mice as compared with wild-type (WT) littermates. On the contrary, thyroid hypertrophy induced by thyrotropin stimulation (in response to goitrogen treatment) was enhanced by about 40% in Cx32-KO mice as compared with WT mice. Thyroid hypertrophy of RP3 mice and the proportion of glands showing extensive tissue remodeling were drastically reduced in mice devoid of Cx32. Our data show that Cx32, which negatively controls thyroid growth activated by thyrotropin via the cAMP pathway, would act as a positive effector of thyroid growth triggered by oncogenes acting through other signaling cascades.

Midgut neuroendocrine tumours with liver metastases: results of the UKINETS study

Thu, 20 Aug 2009 07:48:40 PDT

We intended to identify the prognostic factors and the results of interventions on patients with liver metastatic midgut carcinoids. Five institutions that are part of United Kingdom and Ireland neuroendocrine tumour (NET) group took part in this study. Patients were included if they had histology proven NET of midgut origin and liver metastases at the time of the study. Clinical and biochemical data were collected retrospectively from hospital charts, pathology reports, radiology reports and biochemistry records for each patient. Three hundred and sixty patients were included in the study. The median survival from date of diagnosis was 7.69 years (confidence interval (CI) 6.40–8.99) and 5.95 years (CI 5.02–6.88) from date of diagnosis of liver metastases. On univariate analysis, increasing age at diagnosis, increasing urinary hydroxyindole acetic acid levels, increasing plasma chromogranin A levels, high Ki67, high tumour volume and treatment with chemotherapy were identified as factors associated with a significantly poorer outcome. Resection of liver metastases, resection of small bowel primary, treatment with somatostatin analogue therapy and treatment with peptide receptor therapy were associated with improved prognosis. Multivariate analysis revealed that age at diagnosis (P=0.014), Ki67 level (P=0.039) and resection of primary (P=0.015) were independent predictors of survival. This is the largest study to our knowledge looking specifically at the prognosis and clinical course of patients with liver metastatic midgut NETs. For the first time, we have shown that Ki67 and resection of primary are independent predictors of survival for this group of patients.

Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis

Thu, 20 Aug 2009 07:48:40 PDT

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR–mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT_miR-511 and dCT_miR-503 (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Expression of excision repair cross complementing group 1 and prognosis in adrenocortical carcinoma patients treated with platinum-based chemotherapy

Thu, 20 Aug 2009 07:48:40 PDT

Therapeutic progress in adrenocortical carcinoma (ACC) is severely hampered by its low incidence. Platinum-based chemotherapies are the most effective cytotoxic treatment regimens in ACC but response rates remain <50%. In other tumor entities, expression of excision repair cross complementing group 1 (ERCC1) predicts resistance to platinum compounds. Therefore, we correlated ERCC1 protein expression and clinical outcome. We have retrolectively established adrenal tissue microarrays and analyzed prospectively samples from 163 ACCs, 15 benign adrenal adenomas, and 8 normal adrenal glands by immunohistochemistry for ERCC1 protein expression. Detailed clinical data were available by the German ACC Registry. ERCC1 protein was highly expressed in all normal adrenal glands, 14 benign tumors (93%) and in 75 ACCs (47%). In ACC, no differences in baseline parameters were found between patients with and without ERCC1 expression. Detection of ERCC1 was not correlated with survival in patients who never received platinum-based chemotherapy. In platinum-treated patients (n=45), objective response to platinum compounds was observed in 3/21 patients (14.3%) with high ERCC1 expression and in 7/24 patients (29.2%) with low ERCC1 expression (P=0.23). ERCC1 expression was strongly correlated with overall survival after platinum treatment (median: eight months in patients with high ERCC1 versus 24 months in low ERCC1 expression, hazard ratio (HR) 2.95 (95% confidence interval (CI) 1.4–6.2), P=0.004). Multivariate analysis confirmed that high ERCC1 expression was a predictive factor for poor prognosis in platinum treated patients (HR 2.2, 95% CI 1.0–4.5, P=0.038). Our findings suggest that ERCC1 expression is the first factor for predicting survival in ACC patients treated with platinum-based chemotherapy.

Glucose transporter GLUT1 expression is an stage-independent predictor of clinical outcome in adrenocortical carcinoma

Thu, 20 Aug 2009 07:48:40 PDT

Owing to the rarity of adrenocortical carcinoma (ACC) no prognostic markers have been established beyond stage and resection status. Accelerated glycolysis is a characteristic feature of cancer cells and in a variety of tumour entities key factors in glucose metabolism like glucose transporter 1 and 3 (GLUT1 and -3), transketolase like-1 enzyme (TKTL1) and pyruvate kinase type M2 (M2-PK) are overexpressed and of prognostic value. Therefore, we investigated the role of these factors in ACC. Immunohistochemical analysis was performed on tissue microarrays of paraffin-embedded tissue samples from 167 ACCs, 15 adrenal adenomas and 4 normal adrenal glands. Expression was correlated with baseline parameters and clinical outcome. GLUT1 and -3 were expressed in 33 and 17% of ACC samples respectively, but in none of the benign tumours or normal adrenals glands. By contrast, TKTL1 and M2-PK were detectable in all benign tissues and the vast majority of ACCs. GLUT1 expression was strongly associated with prognosis in univariate and multivariate analysis (P<0.01), whereas GLUT3, TKTL1 and M2-PK did not correlate with clinical outcome. Patients with strong GLUT1 staining showed a considerably higher overall mortality (hazard ratio (HR) 6.34 (95% confidence interval 3.10–12.90) compared with patients with no GLUT1 staining. When analysing patients in their early stages and advanced disease separately, similar results were obtained. HR for survival was 5.31 (1.80–15.62) in patients with metatastic ACC and in patients after radical resection the HR for disease-free survival was 6.10 (2.16–16.94). In conclusion, GLUT1 is a highly promising stage-independent, prognostic marker in ACC.

Molecular characterization of novel germline deletions affecting SDHD and SDHC in pheochromocytoma and paraganglioma patients

Thu, 20 Aug 2009 07:48:40 PDT

A major cause of paraganglioma and pheochromocytoma is germline mutation of the tumor suppressor genes SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH). While many SDH missense/nonsense mutations have been identified, few large deletions have been described. We performed multiplex ligation-dependent probe amplification deletion analysis in 126 point mutation-negative patients, and here we describe four novel deletions of SDHD and SDHC. Long-range PCR was used for the fine mapping of deletions. One patient had a 10 kb AluSg–AluSx-mediated deletion including SDHD exons 1 and 2, the entire TIMM8B gene, and deletion of exons of C11orf57. A second patient had a deletion of SDHD exons 1 and 2 and exon 1 of the TIMM8B gene. A third patient showed a deletion of exon 2 of SDHD, together with a 235 bp MIRb–Tensin gene insertion. In a fourth patient, a deletion of exons 5 and 6 of the SDHC gene was found, only the second SDHC deletion currently known. The deletions of the TIMM8B and C11orf57 genes are the first to be described, but do not appear to result in an additional phenotype in these patients. Four of the eight breakpoints occurred in Alu sequences and all three SDHD deletions showed an intron 2 breakpoint. This study underlines the fact that clinically relevant deletions may encompass neighboring genes, with the potential to modify phenotype. Gene deletions of SDHD and SDHC represent a substantial proportion of all mutations, and must be considered in paraganglioma patients shown to be negative for mutations by sequencing.

Hypermethylation of the IGF2 differentially methylated region 2 is a specific event in insulinomas leading to loss-of-imprinting and overexpression

Thu, 20 Aug 2009 07:48:40 PDT

Prediction of the evolution of endocrine pancreatic tumors remains difficult based on histological criteria alone. We have previously demonstrated that epigenetic changes are an early event in a mouse model developing insulinomas. Particularly, overexpression of the imprinted IGF2 was caused by the hypermethylation of CpGs in the differentially methylated region 2 (DMR2). Here, we investigated whether IGF2 hypermethylation is also observed in human insulinomas and whether this alteration is common to other human endocrine tumors of the pancreas and the digestive tract. We analyzed the methylation status of 40 CpGs located in the DMR0 and DMR2 of the IGF2 as well as in the H19 DMR by pyrosequencing in a cohort of 62 patients with pancreatic or small intestine endocrine tumors. Altered methylation patterns were observed in all tumor types for the different regions of IGF2, but not for H19. However, hypermethylation of the IGF2 DMR2 was specific for insulinomas and did not occur in any of the other types of tumors which were characterized by a loss of methylation in this region. Gain of methylation in the IGF2 DMR2 in insulinomas correlated with loss-of-imprinting and promoter 4 mediated overexpression of IGF2 at the RNA and protein level. Furthermore, a decreasing degree of methylation in the different regions of IGF2 correlated well with increasing degree of malignancy according to the WHO classification of pancreatic endocrine tumors (PETs), suggesting that methylation of IGF2 might be a useful biomarker for classification and staging of PETs.

High-resolution genomic profiling reveals gain of chromosome 14 as a predictor of poor outcome in ileal carcinoids

Andersson, E., Sward, C., Stenman, G., Ahlman, H., Nilsson, O. — Thu, 20 Aug 2009 07:48:40 PDT

Ileal carcinoids are malignant neuroendocrine tumours of the small intestine. The aim of this study was to obtain a high-resolution genomic profile of ileal carcinoids in order to define genetic changes important for tumour initiation, progression and survival. Forty-three patients with ileal carcinoids were investigated by high-resolution array-based comparative genomic hybridization. The average number of copy number alterations (CNAs) per tumour was 7.1 (range 1–22), with losses being more common than gains (ratio 1.4). The most frequent CNA was loss of chromosome 18 (74%). Other frequent CNAs were gain of chromosome 4, 5, 14 and 20, and loss of 11q22.1–q22.2, 11q22.3–q23.1 and 11q23.3, and loss of 16q12.2–q22.1 and 16q23.2-qter. Two distinct patterns of CNAs were found; the majority of tumours was characterized by loss of chromosome 18 while a subgroup of tumours had intact chromosome 18, but gain of chromosome 14. Survival analysis, using a series of Poisson regressions including recurrent CNAs, demonstrated that gain of chromosome 14 was a strong predictor of poor survival. In conclusion, high-resolution profiling demonstrated two separate patterns of CNAs in ileal carcinoids. The majority of tumours showed loss of chromosome 18, which most likely represents a primary event in the development and pathogenesis of tumours. A different genetic pathway is operative in a subgroup of tumours; this is characterized by gain of chromosome 14 and is strongly associated with poor prognosis. Predictive fluorescence in situ hybridization analysis of chromosome 14 status in patients with ileal carcinoids is suggested.

Circulating angiopoietin-2 is elevated in patients with neuroendocrine tumours and correlates with disease burden and prognosis

Srirajaskanthan, R, Dancey, G, Hackshaw, A, Luong, T, Caplin, M E, Meyer, T — Thu, 20 Aug 2009 07:48:40 PDT

Angiogenesis is an essential process in the development and growth of tumours. There are a large number of angiogenic mediators including the angiopoietin (Ang) family and vascular endothelial growth factor, which play an important role in both physiological and pathological angiogenesis. This study examines serum levels of Ang-1 and Ang-2 in patients with neuroendocrine tumour (NET) compared healthy controls. ELISA for Ang-1 and Ang-2 was performed in 47 patients with histologically proven NETs and 44 healthy controls. Immunohistochemical staining for Ang-2 was performed in patients to demonstrate cellular location of Ang-2. Serum Ang-2 levels were significantly elevated in patients compared controls (median 4756 vs 2495 pg/ml, P<0.001), while there was no significant difference in Ang-1 levels. The ratio of Ang-2:Ang-1 was significantly elevated in patients compared controls (0.13 vs 0.066, P<0.001). Serum Ang-2 levels were significantly elevated in patients with distant metastases compared with those without metastasis (median 5080 vs 3360 pg/ml, P=0.01). There was also a significant increase between Ang-2 levels and volume of liver metastases (P=0.014). Time to disease progression was worse in patients with serum Ang-2 levels >4756 pg/ml (P=0.04). Serum Ang-2 but not Ang-1 is elevated in NET patients. Ang-2 may be a useful serum marker for monitoring and assessment of prognosis in patients with NETs.

Clinical and imaging follow-up after exhaustive liver resection of endocrine metastases: a 15-year monocentric experience

Thu, 20 Aug 2009 07:48:40 PDT

Liver metastases are common in gastroenteropancreatic neuroendocrine tumors and significantly impair survival. Hepatic resection is the only potential curative treatment. The records of 41 consecutive patients undergoing exhaustive resection of liver-only endocrine metastases and followed between 1992 and 2006 were reviewed. Patient's outcome and diagnostic accuracy of somatostatin receptor scintigraphy (SRS) and morphological imaging (MI) for detection of recurrences during post-operative follow-up were assessed. All identified primary had been resected. MI studies including abdominal computed tomography (CT) and/or liver magnetic resonance imaging and thoracic CT if indicated were performed every 6 months; SRS timing was decided by referring clinician. Tumor recurrences were confirmed by pathology or subsequent imaging studies. The results of 136 MI and SRS examinations performed within a 30-day interval from each other were retrospectively compared. Median post-operative follow-up was 51 months (7–165). Recurrences developed in 32 patients (78%), mainly in the liver (n=24) after a median of 19 months (2–79). Five-year overall and disease-free survival rates were 79 and 3% respectively. For recurrence detection, sensitivity, specificity, and accuracy were 89, 94, and 91% for SRS, 68, 91, and 74% for MI respectively. In 11 out of 32 patients (34%), abdominal or extra-abdominal metastases were detected 15.5 months earlier by SRS than MI. In conclusion, despite exhaustive liver surgery for endocrine metastases, hepatic or extra-hepatic recurrences are frequent and develop early. SRS is highly accurate for the detection of recurrences during post-operative follow-up and permitted early diagnosis in one third of patients; therapeutic implications of this early diagnosis remain to be determined.

Activation of a prometastatic gene expression program in hypoxic neuroblastoma cells

Poomthavorn, P., Wong, S. H X, Higgins, S., Werther, G. A, Russo, V. C — Thu, 20 Aug 2009 07:48:40 PDT

The hypoxia inducible factor-1 (HIF1) is a key regulator of oxygen homeostasis, modulating cell survival, and growth in cells exposed to hypoxia. In this study, neuroblastoma (NB) cells SH-SY5Y and SK-N-MC were employed to determine the mechanisms regulating adaptation to hypoxia. NB cells were cultured in a serum-free medium in the presence or absence of CoCl₂ (100 µM, hypoxia mimic) for up to 48 h. SH-SY5Y and SK-N-MC cell numbers were not affected by CoCl₂ treatment, while mitochondrial activity was reduced by ~50% in SH-SY5Y cells and by ~70% in SK-N-MC cells. Intracellular accumulation of HIF1 protein was detected as early as 30 min of post-hypoxia, followed by the increase of mRNA for vascular endothelial growth factor (VEGF) and nuclear accumulation of the ID1–2 transcription factors by 4 h. In hypoxic SH-SY5Y NB cells, real-time PCR analysis showed that the genes involved in maintenance of cell–cell and cell–matrix interactions (i.e. adenomatosis polyposis coli, E-cadherin, catenin, EphB2, fibronectin-1, HTATIP2, tissue inhibitor of metalloprotease-4) were down-regulated by up to 90%, while genes involved in enhancement of metastatic behavior (integrin a7b1, hepatocyte growth factor receptor, transforming growth factor-β1, VEGF, kisspeptin, interleukin-1β) were dramatically up-regulated above 200%. These changes were all consistent with the induction of epithelial–mesenchymal transition. We have thus demonstrated that NB cell adaptation to hypoxia, in addition to the modulation of HIF1 and VEGF expression and nuclear translocation of ID1 and ID2 transcription factors, involve in the activation of a gene expression program consistent with the pro-metastatic events. These processes are probably responsible for the NB cell transition from an adherent phenotype to a highly migratory, invasive and aggressive NB cell type.

Androgen, progesterone, and FSH receptor polymorphisms in ovarian cancer risk and outcome

Ludwig, A. H., Murawska, M., Panek, G., Timorek, A., Kupryjanczyk, J. — Thu, 20 Aug 2009 07:48:40 PDT

Genes encoding hormone receptors are among candidate genes modulating the risk of ovarian cancer. We aimed to assess a frequency of PGRG+331A, FSHRAla307Thr, and FSHRSer680Asn polymorphic variants, and the length of (CAG)n and (GGN)n repeat tracts in the androgen receptor gene (AR) with respect to ovarian cancer risk and outcome. We genotyped 215 ovarian cancer patients and 352 unaffected control subjects. Statistical analysis was performed with the logistic regression model with adjustment for age. Clinical importance of the polymorphic variants was evaluated in multivariate models on 69 patients treated with taxane–platinum chemotherapy, with respect to TP53 status. Longer AR (GGN)n and (CAG)n repeat tracts decreased the risk of ovarian cancer. For (GGN)n, each additional repeat decreased the risk by 17% (P=0.011) or 27% (P=0.002), while the presence of at least 23 repeats decreased the risk by 41% (P=0.032) or 68% (P=0.008), for the shorter or longer allele respectively. The risk of disease was also decreased by 11% with each additional (CAG)n repeat (P=0.006 for the longer allele). FSHRAla307Ala or FSHRSer680Ser polymorphisms increased ovarian cancer risk by 1.8 times (P=0.042). In all 69 patients, longer AR (CAG)n repeats decreased the risk of recurrence (P=0.031). In the group with TP53 accumulation, longer AR (CAG)n repeats decreased the risk of recurrence (P=0.003) and death (P=0.03), while the FSHRSer680Ser polymorphism increased the risk of recurrence (P=0.037). Progesterone receptor polymorphisms analyzed did not show any associations. Our results support both the androgen and gonadotropin hypotheses of ovarian cancer development.

Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro

Thu, 20 Aug 2009 07:48:40 PDT

The effect of mammalian target of rapamycin (mTOR) inhibitors on pituitary tumors is unknown. Akt overexpression was demonstrated in pituitary adenomas, which may render them sensitive to the anti-proliferative effects of these drugs. The objective of the study was to evaluate the anti-proliferative efficacy of the mTOR inhibitor, rapamycin, and its orally bioavailable analog RAD001 on the GH-secreting pituitary tumor GH3 and MtT/S cells and in human GH-secreting pituitary adenomas (GH-omas) in primary cell cultures. Treatment with rapamycin or RAD001 significantly decreased the number of viable cells and cell proliferation in a dose- and time-dependent manner. This was reflected by decreased phosphorylation levels of the downstream mTOR target p70S6K. Rapamycin treatment of GH3 cells induced G0/G1 cell cycle arrest. In other tumor cell types, this was attributed to a decrease in cyclin D1 levels. However, rapamycin did not affect cyclin D1 protein levels in GH3 cells. By contrast, it decreased cyclin D3 and p21/CIP, which stabilizes cyclin D/cyclin-dependent kinase 4 (cdk4) complexes. Rapamycin inhibited FCS-induced retinoblastoma phosphorylation and subsequent E2F-transcriptional activity. In response to decreased E2F activity, the expression of the E2F-regulated genes cyclin E and cdk2 was reduced. Our results showed that mTOR inhibitors potently inhibit pituitary cell proliferation, suggesting that mTOR inhibition may be a promising anti-proliferative therapy for pituitary adenomas. This therapeutic manipulation may have beneficial effects particularly for patients harboring invasive pituitary tumors resistant to current treatments.

Expression of aryl hydrocarbon receptor (AHR) and AHR-interacting protein in pituitary adenomas: pathological and clinical implications

Thu, 20 Aug 2009 07:48:40 PDT

Germline mutations of the aryl hydrocarbon receptor (AHR)-interacting protein (AIP) gene confer a predisposition to pituitary adenomas (PA), usually in the setting of familial isolated PA. To provide further insights into the possible role of AIP in pituitary tumour pathogenesis, the expression of AIP and AHR was determined by real-time RT-PCR and/or immunohistochemistry (IHC) in a large series of PA (n=103), including 17 with AIP mutations (AIP^mut). Variable levels of AIP and AHR transcripts were detected in all PA, with a low AHR expression (P<0.0001 versus AIP). Cytoplasmic AIP and AHR were detected by IHC in 84.0 and 38.6% of PA respectively, and significantly correlated with each other (P=0.006). Nuclear AHR was detected in a minority of PA (19.7%). The highest AIP expression was observed in somatotrophinomas and non-secreting (NS) PA, and multivariate analysis in somatotrophinomas showed a significantly lower AIP immunostaining in invasive versus non-invasive cases (P=0.019). AIP expression was commonly low in other secreting PA. AIP immunostaining was abolished in a minority of AIP^mut PA, with a frequent loss of cytoplasmic AHR and no evidence of nuclear AHR. In contrast, AIP overexpression in a subset of NS PA could be accompanied by nuclear AHR immunopositivity. We conclude that down-regulation of AIP and AHR may be involved in the aggressiveness of somatotrophinomas. Overall, IHC is a poorly sensitive tool for the screening of AIP mutations. Data obtained on AHR expression suggest that AHR signalling may be differentially affected according to PA phenotype.

Lanreotide promotes apoptosis and is not radioprotective in GH3 cells

Ning, S., Knox, S. J, Harsh, G. R, Culler, M. D, Katznelson, L. — Thu, 20 Aug 2009 07:48:40 PDT

Somatostatin analogs are a mainstay of medical therapy in patients with GH producing human pituitary tumors, and it has been suggested that somatostatin analogs may be radioprotective. We utilized GH secreting rat GH3 cells to investigate whether a somatostatin analog may limit the effects of radiation on proliferation and apoptosis in vitro and on tumor growth in vivo. Treatment with lanreotide alone at doses of either 100 or 1000 nM for 48 h reduced clonogenic survival by 5–10%. Radiation alone produced a dose-dependent survival curve with a SF2 of 48–55%, and lanreotide had no effect on this curve. The addition of lanreotide resulted in a 23% increase in the proportion of apoptotic sub-G1 cells following irradiation (P<0.01). In a mouse GH3 tumor xenograft model, lanreotide 10 mg/kg moderately inhibited the growth of GH3 tumors, with a 4x tumor growth delay (TGD) time that ranged from 4.5 to 8.3 days. Fractionated local tumor radiation alone significantly inhibited tumor growth and produced a TGD of 35.1±5.7 days for 250 cGy fractions. The combination of lanreotide, either antecedent to or concurrent, with radiation of 250, 200 or 150 cGy/fraction for 5 days inhibited tumor growth and produced the TGD times that were similar to radiation alone (P>0.05). Pretreatment with lanreotide had the most significant radiosensitizing effect. These studies demonstrate that the somatostatin analog lanreotide is not radioprotective in GH3 cells, and further studies are necessary to determine the impact of lanreotide on apoptosis.

Genome-wide scan identifies novel modifier loci of acromegalic phenotypes for isolated familial somatotropinoma

Thu, 20 Aug 2009 07:48:40 PDT

Isolated familial somatotropinoma (IFS) accounts for 18% of familial isolated pituitary adenoma (FIPA) cases. Recently, germline mutations of the aryl hydrocarbon receptor-interacting protein gene (AIP) have been found in families with pituitary adenoma predisposition, FIPA, and IFS. In this study, we investigate the AIP mutation status and perform a genome-wide scan to search for the modifier regions of acromegalic phenotypes in an IFS family of 31 aborigines from Borneo. Complete endocrine diagnosis and data could not be collected due to logistical and cultural reasons. AIP mutation screening was carried out by direct sequencing and the genome-wide scan was performed using 400 microsatellites. Non-parametric linkage analysis was performed to obtain the logarithm of odds (LOD) scores. A novel AIP frameshift mutation in exon 4 (c.500delC) (p.P167HfsX3) was identified in all members with acromegalic features, as well as in 15 members without acromegalic features, revealing incomplete penetrance of AIP. The data showed that patients with the same mutation may express acromegalic features of differing severity, suggesting the existence of modifier genes. The highest LOD score of 2.2 was obtained near D19S571 (19q13.41). We also found weak linkages on chromosomes 3q28, 8q12.1, and 21q22.13, with LOD scores of 1.1, 1.8, and 1.4 respectively. Our results show the first genome-wide scan that identifies novel modifier loci for acromegalic phenotypes in an IFS family. Identification of modifier loci may provide further insight into the disease mechanism and explain the clinical variability observed in its patients.