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Department of Urology, Heinrich-Heine University, Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
1 Department of Cellular and Molecular Pathology, German Cancer Research Centre, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
2 Department of Pathology, Heinrich-Heine University, Düsseldorf, Germany
(Requests for offprints should be addressed to W A Schulz; Email: wolfgang.schulz{at}uni-duesseldorf.de)
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Modulation of growth factor signalling by SPRY proteins is particularly important during fetal development in shaping tissues and organs, including those of the urinary tract (Chi et al. 2004). In fact, the designation ‘sprouty’ derives from a Drosophila mutant displaying deficiencies in organ development (Hacohen et al. 1998). In humans, four SPRY genes are known (SPRY1–SPRY4), which are expressed in a tissue-specific fashion. In many adult tissues, SPRY1 and SPRY2 are deemed most important. Recently, a novel alternative SPRY1 mRNA has been reported to represent a fetal isoform (Wang et al. 2003). It differs from the standard transcript by an alternative non-coding exon 1 arising through alternative promoter use. We suggest the name SPRY1b to distinguish it from the standard transcript SPRY1a.
SPRY1 and SPRY2 proteins modulate signalling from tyrosine receptor kinases through RAS and MAP kinases and exert overall similar effects on FGF signalling, but differ in their influence on EGF signalling (Gross et al. 2001, Egan et al. 2002, Wong et al. 2002, Yusoff et al. 2002, Li et al. 2004). They disrupt the interaction between FGF receptors and the adaptor protein GRB2 and block activatory phosphorylation of RAF. However, SPRY2 in particular is not a pure feedback inhibitor, but also prolongs signalling by the EGF receptor under some circumstances.
Enhanced signalling by EGF and FGF family members in cancer ought to lead to an accordingly strong induction of MAPK pathway feedback inhibitors including SPRY mRNAs and proteins. Since this induction should impede cancer growth, one would expect selection to favour SPRY down-regulation, thereby exacerbating the tumourigenic effects of enhanced growth factor signalling. In contrast, oncogenic mutations of RAS or BRAF activate MAPK signalling downstream of SPRY action. Accordingly, SPRY2 has been reported as becoming induced in melanoma cell lines carrying such mutations (Bloethner et al. 2005). In prostate and breast cancers RAS mutations are rare and, indeed, evidence for down-regulation of SPRY expression has been published (Kwabi-Addo et al. 2004b, Lo et al. 2004, McKie et al. 2005). However, several issues are unresolved.
SPRY1 and SPRY2 were reported to be down-regulated in prostate cancer in two separate publications. The relation between the two genes has not been determined, i.e. are they down-regulated co-ordinately, as reported in breast cancer, or independently of each other? The degree of down-regulation reported was only moderate. Therefore, independent confirmation seems warranted. The alternative splice form of SPRY1 mRNA (SPRY1b) has not been investigated in cancers. Specifically, it is not known whether down-regulation of the presumed adult mRNA form is accompanied by up-regulation of the fetal isoform. Additionally, the role of epigenetic mechanisms such as DNA hypermethylation in SPRY down-regulation is unclear. In breast cancer, down-regulation did not appear to be associated with altered methylation, whereas in prostate cancer partial hypermethylation of SPRY2, but none of SPRY1, was reported. These questions were therefore addressed in the present study.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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RNase-free glass slides were prepared by baking at 200 °C for 4 h followed by covering with a PEN membrane (P.A.L.M. GmbH, Bernried, Germany). Frozen tissues were cut into 8 µm sections, mounted onto the PEN membrane and haematoxylin and eosin stained. Laser-controlled microdissection on up to 30 000 epithelial cells each from histological hyperplasia, prostatic intraepithelial neoplasia and cancerous samples was performed using the P.A.L.M. microlaser technology (P.A.L.M. GmbH) according to the manufacturer’s protocol. Total RNA was extracted from microdissected cells according to a standard method (Chomczynski & Sacchi 1987) and RNA quality was checked using the RNA6000 nanoassay on the Bioanalyzer 2100 Lab-On-A-Chip system (Agilent Technologies, Palo Alto, CA, USA). RNA yields were usually > 200 ng per sample.
RNA amplification and microarray analysis
Linear T7 polymerase-based amplification of total RNA from microdissected samples was performed essentially as previously described by Kenzelmann et al.(2004). RNA samples were then hybridized to HG U133A GeneChips (Affymetrix, Berlin, Germany) according to the manufacturer’s instructions. Statistical analysis and data mining were performed with the software package MicroArray Solution, version 1.0, from SAS (SAS Institute, Minneapolis, MN, USA). GeneChip hybridization quality was controlled by correlation analysis; normalization of data and data mining were performed using log-linear mixed models with Bonferroni corrections that were fitted for values of perfect-matches, and by mixed model ANOVA (Cui & Churchill 2003).
Tissues for RT-PCR and methylation analyses
Prostate carcinoma (PCa) specimens were obtained between 1997 and 2002 by radical prostatectomy. Cancerous and morphologically normal areas of the prostate were identified and specimens collected by a pathologist, rapidly frozen in liquid nitrogen and stored at –80 °C. Since several micrograms of high molecular weight DNA as well as sufficient amounts of RNA were prepared from the same sample, no microdissection was performed. Representative samples of 3 mm maximal diameter of tumour and tumour-free tissue specimens were collected, immediately snap frozen in liquid nitrogen and stored at –80 °C. Non-cancerous tissue samples were taken from areas of the transition zone as far away as possible from the grossly apparent tumour (i.e. in general, from the transition zone of the contra-lateral lobe). Tumour and matched tumour-free specimens were only collected first when tumours were grossly apparent in the peripheral zone and could be unequivocally identified by their characteristic yellow or orange-yellow colour and secondly when the transition zone was macroscopically free of tumour. Separation between tumour and non-tumourous tissues was histologically verified by analysing tissue specimens immediately adjacent to the specimens collected for analysis. Tumour node metastases (TNM) classification was performed according to the guidelines of the International Union Against Cancer from 1997. Clinical data are summarized in Table 1
. Of 49 prostate carcinoma tissues, 23 were staged as pT2, 24 as pT3, and 2 as pT4. Lymph node metastases were present in 10 patients. None of the patients had detectable distant metastases at the time of surgery. Fifteen tumours had Gleason scores 
6, 26 tumours scores of 7, and 8 tumours scores of 8–10. Median patient age was 68 years, ranging from 55 to 76 years. The median follow-up period was 66 months (range 44–130). The study was approved by the Ethics Committee of the Heinrich Heine University medical faculty.
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The prostate carcinoma cell lines 22Rv1, LNCaP, PC3 and Du145 were cultured in RPMI-1640 (Gibco Life Technologies), supplemented with 10% fetal calf serum and 100 µg/ml penicillin/streptomycin. Normal uroepithelial cells (UC) were cultured as described (Swiatkowski et al. 2003). For experiments using 5-aza-2'-deoxycytidine (5-aza-dC), the compound (Sigma) was used at a concentration of 2 µM every 24 h for 3 days. Treated and untreated cells, which received solvent only, were cultured in parallel until RNA extraction.
RNA isolation and RT-PCR
Total mRNA was isolated from cell cultures grown to 80% confluence, using the RNeasy Midi Kit (Qiagen). For tissues the same kit was used following guanidinium/acid phenol/chloroform extraction (peqGOLD TriFast, peqLab, Erlangen, Germany). Following photometric quantification, 2 µg mRNA was transcribed into first strand cDNA using SuperscriptII (Invitrogen) according to the manufacturer’s protocol with oligo-dT primers. Real-time PCR assays were performed using a fluorescence-detecting temperature cycler (LightCycler; Roche Diagnostics, Mannheim, Germany). The amplification mixture consisted of 1 x reaction mix (LightCycler-FastStart DNA Master PLUS SYBR Green I; Roche), 10 pmoles of each primer (see Table 2 for sequences and annealing temperatures) and 20 ng cDNA in a final volume of 10 µl. The generation of target amplicons for each sample was monitored between the annealing and elongation steps at 640 nm. After the final cycle, melting-point analysis of the samples was performed over the range of 69–99 °C. Turning-point values for the specific genes were related to those for ß-actin or CK18.
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High molecular weight genomic DNA from tissue, cell lines and whole blood was isolated using the blood and cell culture DNA kit (Qiagen) with additional proteinase K treatment. Following bisulphite treatment using the CpGenome DNA Modification Kit (Q-Biogene, Eschwege, Germany), 80 ng of bisulphite-treated DNA were used for amplification by HotStar Taq polymerase (Qiagen) in a 50 µl reaction with 20 pmoles of each primer indicated in Table 2
. Following 15 min initial denaturation at 95 °C, 36 cycles of 30 s at 95 °C, 30 s at the annealing temperature and 45 s at 72 °C were performed, with a final 10 min elongation. Q-solution (Qiagen) was added for SPRY2 amplification. PCR products were separated by 2.5% agarose gel electrophoresis and cloned into the TA-vector of the TOPO TA Cloning Kit (Invitrogen). Plasmid DNA was prepared from several clones for each cell line and sequenced by standard methods.
Immunohistochemistry
Immunohistochemistry using the avidin-biotinylated enzyme complex (ABC) method was performed essentially as described by Cohen et al.(2002) using a rabbit polyclonal antibody against SPRY2 (07-524, Upstate/Biomol, Hamburg, Germany).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In order to study the expression patterns of SPRY1 and SPRY2 along with prostate cancer development, HG U133A Affymetrix GeneChip microarray-based analyses were performed on histopathologically confirmed laser-controlled microdissected epithelial cells of 7 human hyperplastic tissues, 6 tissues showing severe prostatic intraepithelial neoplasia (PIN III; a pre-cancerous lesion), and 7 tissues of prostate cancer, respectively. Microarray results for SPRY1 and SPRY2 revealed a gradual and co-ordinated down-regulation of both mRNAs from hyperplasia to PIN and prostate cancer, with SPRY1 being more abundantly expressed (Fig. 1). However, due to tissue-inherent individual differences in the expression intensity of both genes the down-regulation was not statistically significant (P> 0.05). Since the microarray oligonucleotide probe set for SPRY1 does not distinguish between the two SPRY1 isoforms (1a and 1b), it cannot be decided whether the expression signal belongs to only one isoform or to a cumulative detection of both isoforms.
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). KRT18 (CK18) was used as a reference gene to adjust for the epithelial content in normal and cancerous prostate samples (Kwabi-Addo et al. 2004b). SPRY1a expression was not significantly lower in cancerous than in non-cancerous tissues, whereas the SPRY2/CK18 expression ratio was significantly decreased in the cancer tissues (P = 0.017). Importantly, expression of SPRY1a and SPRY2 in the carcinoma tissues was positively correlated (r2 = 0.658, P<0.001) moderately strongly, i.e. cancers with low expression of SPRY2 also displayed low expression of SPRY1. This indicates that down-regulation typically affects both SPRY genes in parallel, although in the case of SPRY1 it is typically modest.
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). Expression of both genes tended to be lower in advanced stage cancers and in cases with lymph node metastases, although the difference did not reach statistical significance (Fig. 3). No association with pre-operative serum levels of prostate specific antigen and Gleason score (data not shown) was found.
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To further ensure that the observed changes were specific to the epithelial component of the prostate, SPRY protein expression was investigated by immunohistochemistry. After testing commercially available antibodies in Western blot analyses we found only one antibody against SPRY2 (see Methods and materials) that was sufficiently specific, as it yielded a single band of the expected 35 kDa molecular mass (data not shown). Using this antibody, SPRY2 was found localized in prostate sections predominantly in the cytoplasm of basal cells of the glands (Fig. 4A, arrows), while secretory cells showed a weaker and more heterogeneous staining pattern and the mesenchymal compartment remained almost unstained (Fig. 4A). Prostate cancer specimens retained staining in the epithelial compartment, at a staining intensity similar to that in normal glands (Fig. 4B). This finding supports the RT-PCR data, since the moderate decreases found at the mRNA level are unlikely to result in protein changes detectable by immunohistochemistry.
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The expression of SPRY1a and SPRY2 and, in addition, that of the alternative splice form SPRY1b was also determined in prostate cancer cell lines (Fig. 5). Normal urothelial cells proliferating in primary culture were used as a control. All three messages were detectable in the prostate cancer cell lines. Whereas SPRY1b and SPRY2 mRNA were detectable at similar or higher levels compared to normal cells, the level of SPRY1a mRNA was somewhat lower in three prostate carcinoma lines (except PC3) than in normal cells.
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Although the ratio of the two alternative SPRY1 mRNAs differed among the prostate carcinoma cell lines, both forms were detected. We further compared the expression of the two SPRY1 mRNA splice forms in various normal genitourinary tissues and cells. Their expression correlated positively and moderately well (r2 = 0.60) with each other (data not shown).
SPRY promoter methylation shows few changes in prostate cancer
Since genes hypermethylated in cancer can often be induced by DNA methyltransferase inhibitors, the prostate carcinoma cell lines were treated with 5-aza-dC at an active but non-toxic concentration (Hoffmann et al. 2005). This treatment led only to small increases, none of them more than 2.5-fold (Fig. 5). In particular, the strongest increase in SPRY1a expression was observed in PC3, which had the strongest expression before treatment.
Additionally, DNA methylation was investigated by bisulphite sequencing in a region near the transcriptional start sites of SPRY1a and SPRY2 each. Three prostate carcinoma cell lines were compared to normal leukocyte samples and normal prostate tissue. The SPRY1a promoter contains only a single CpG site located close to the transcriptional start site, but twelve sites in the first exon (Fig. 6). These were found to be largely, but not completely, methylated in normal leukocytes and prostate tissue. In three cell lines differing in expression levels (cf. Fig. 5), moderate differences in methylation were discernible. In particular, DU145 presented the lowest level of methylation, while the sequence was fully methylated in LNCaP. Intermediate levels were found in the PC3 line, which exhibits the highest mRNA level (cf. Fig. 5).
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). Surprisingly, a series of 5 prostate carcinoma tissues with low SPRY2 expression levels lacked significant methylation as well (Fig. 6) Importantly, with one exception, these specimens had been observed to exhibit significant hypermethylation in GSTP1, RARB2, APC and RASSF1A (Florl et al. 2004). |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A factor potentially confounding expression analyses of SPRY1 is the presence of the alternative splice form SPRY1b, which shares the coding exon 2 (Wang et al. 2003). It was postulated to represent a fetal isoform of the transcript, but our analyses show it to be expressed in several adult tissues and cell lines generally in parallel with the standard RNA form. In prostate carcinoma cell lines, SPRY1b expression also remained detectable. If the alternative mRNA is translated at the same efficiency as the standard transcript, its presence would tend to further buffer any changes in SPRY1 expression. Since SPRY1b is not simply the fetal isoform of SPRY1a, its physiological function deserves further investigation, e.g. whether the two promoters respond differently to growth factor stimulation.
In prostate cancer, DNA hypermethylation of promoter CpG-islands is a particularly frequent mechanism responsible for the down-regulation of tumour suppressors and other genes (Florl et al. 2004, Kang et al. 2004, Yegnasubramanian et al. 2004). Accordingly, each of the prostate carcinoma cell lines and cancer tissues investigated here contains multiple aberrantly methylated CpG-islands in genes like GSTP1, RARB2, APC and RASSF1A. Nevertheless, no indication was obtained for a major function of DNA methylation in down-regulation of SPRY genes. SPRY1 does not contain CpG-islands at its promoters. Several CpG-sites in exon 1a were predominantly methylated in normal tissue and leukocytes as well as in two prostate carcinoma cell lines. Interestingly, the sequence was almost completely methylated in LNCaP displaying the lowest expression level. Increased methylation in this cell line could be a consequence, rather than a cause, of decreased expression, as observed in a number of similar cases (Chen & Riggs 2005). Increased methylation of the SPRY2 CpG-island had been reported in prostate carcinomas (McKie et al. 2005). In contrast, we could neither detect any methylation of this sequence in prostate carcinoma cell lines by bisulphite sequencing nor any significant induction by the DNA methylation inhibitor 5-aza-dC by quantitative RT-PCR. According to our data, down-regulation of SPRY2 in prostate cancers occurs independently of DNA methylation, as previously reported for breast cancers (Lo et al. 2004). The discrepancies may relate to the tendency of techniques used for bisulphite treatment and methylation detection to yield false positive results. Moreover, most of the sites found hypermethylated by McKie et al. are located at the 3'-end of the CpG-island in a segment extending into intron 1.
In conclusion, the present study confirms and extends previous reports on down-regulation of SPRY genes demonstrating concomitant down-regulation and a significant association with recurrence underlining the importance of these changes. However, it should be cautioned that the decreases in expression might be quite modest and appear not to be accompanied by promoter hypermethylation. Therefore, it remains to be investigated to what extent down-regulation is mediated by changes in chromatin structure, e.g. histone methylation, and which signals regulate the SPRY gene promoters in normal and cancerous prostate epithelial cells.
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Acknowledgements |
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References |
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