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Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

S100P protein regulates calcium signal transduction and mediates cytoskeletal interaction, protein phosphorylation and transcriptional control. We have previously shown how elevated S100P levels in prostate cancer strongly correlate with progression to metastatic disease. In our study, we evaluated the functional significance of S100P expression on prostate tumor growth in vitro and in vivo. S100P levels were modulated by overexpressing S100P in PC3 prostate cancer cells and by silencing S100P levels in 22Rv1 prostate cancer cells. Overexpression of S100P in PC3 cells promoted cell growth, increased the percentage of S-phase cells, decreased basal apoptosis rate and promoted anchorage independent growth in soft agar. Furthermore, prostate cancer cells overexpressing S100P were protected against camptothecin-induced apoptosis. Conversely, silencing of S100P in 22Rv1 cells using siRNA resulted in a prominent cytostatic effect. The influence of S100P on tumor growth and metastases were assessed in vivo. S100P-overexpressing PC3 cells had a dramatically increased tumor formation compared to controls. Microarray analysis showed the involvement of growth pathways including increased androgen receptor expression in S100P-overexpressing cells. These results provide the first functional proof that S100P overexpression can upregulate androgen receptor expression and thereby promote prostate cancer progression by increasing cell growth. Moreover, the results confirm the oncogenic nature of S100P in prostate cancer and suggest that the protein may directly confer resistance to chemotherapy. Hence, S100P could be considered a potential drug target or a chemosensitization target, and could also serve as a biomarker for aggressive, hormone-refractory and metastatic prostate cancer. © 2008 Wiley-Liss, Inc.

Transient gene silencing in mammalian cells can be mediated by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs). Naturally occurring siRNAs in lower eukaryotes have characteristic structural elements; however, little is known about what features are critical for an exogenous siRNA to mediate RNAi in mammals. We have recently determined some of the critical parameters that influence the efficiency of siRNA-mediated RNAi in mammalian cells and have been considering the use of RNAi as a functional genomics tool, particularly for high throughput analysis, and the potential use of RNAi as a therapeutic tool.

We have shown recently that inhibition of androgen receptor (AR) expression with an antisense AR oligonucleotide (ODN) inhibits LNCaP prostate tumor cells in vitro as well as in vivo. In this study, we investigated gene expression changes that occur after AR signaling blockade, either through AR elimination by antisense treatment or through complete androgen receptor inhibition by androgen deprivation combined with the antiandrogen bicalutamide, in order to search for genes that are directly or indirectly regulated through the AR. Gene expression changes were investigated with cDNA NIH 10K gene microarrays in response to treatment over 48 h. Expression of selected genes was further analyzed by real-time reverse transcriptase (RT)-polymerase chain reaction (PCR), Western blotting, and radioimmunoassay. A comparison of antisense-treated and androgen-deprived cells revealed several concordances such as significant downregulation of prostate-specific genes, cell-cycle regulatory genes, genes of the cholesterol biosynthesis pathway, and several cytoskeletal genes. However, there were also several genes that were differentially regulated. Among the genes that were exclusively changed by treatment with the antisense AR ODN were the insulin-like growth factor binding protein 2 (IGFBP2) and the phosphatidylinositol-4-phosphate 5-kinase type I alpha (PIP5KIA). On the other hand, complete androgen receptor blockade induced changes in the expression of the prostate overexpressed gene 1 and the S100 calcium binding protein P. In summary, we identified a cohort of interesting genes whose expression was highly affected by elimination of the AR in LNCaP prostate cancer cells. Further investigations are warranted to clarify their role in the AR signaling pathway and their susceptibility as a target for the treatment of prostate cancer.

BACKGROUND In some malignancies, p53 mutations are associated with tumor progression. To address the role of p53 mutations in the development and progression of osteosarcoma, the authors analyzed specimens from 247 patients with primary localized osteosarcomas and 25 patients with osteosarcomas that were metastatic at the time of diagnosis. The group included 27 matched biopsy-resection specimens and 21 biopsy-metastasis paired specimens. METHODS The authors examined the nature and location of p53 mutations (exons 4–10) by polymerase chain reaction–single-strand conformation polymorphism and confirmed mutations by direct DNA sequencing. RESULTS The overall frequency of p53 mutations was 22% (60 of 272 specimens), with 13 of 60 mutations located in exons 4 or 10. A similar proportion of localized osteosarcomas had alterations of the p53 gene (55 of 247 specimens; 22.3%) compared with tumors from patients who had metastases at the time of diagnosis (5 of 25 specimens; 20%; P = 0.96). Patients who had p53 missense mutations were older compared with patients who had nonsense alterations or a wild type gene (P = 0.01). Examination of paired biopsy-resection and biopsy-metastasis specimens revealed that the p53 status was concordant between the biopsy and later tumor specimens in all patients. CONCLUSIONS The p53 mutation status did not differentiate between patients who presented with a localized osteosarcoma and those who presented with metastases at the time of diagnosis. The current data indicate that p53 mutations are not late events in osteosarcoma tumor progression, because they are evident before the development of metastases. The inclusion of exons 4 and 10 increased the sensitivity of the analysis. Cancer 2001;92:2181–9. © 2001 American Cancer Society.