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Supplementary Figure Legends 1-6 from Increased Expression of Androgen Receptor Sensitizes Prostate Cancer Cells to Low Levels of Androgens

Supplementary Tables 1-9 from Increased Expression of Androgen Receptor Sensitizes Prostate Cancer Cells to Low Levels of Androgens

Supplementary Figures 1-6 from Increased Expression of Androgen Receptor Sensitizes Prostate Cancer Cells to Low Levels of Androgens

Activating gene mutations, gene amplifications and overexpressed proteins may be useful as targets for novel therapies. Alterations at chromosome locus 4q12 are associated with gliomas and the region harbors the receptor tyrosine kinase gene KIT, which is frequently amplified in gliomas, and also overexpressed in a subset of gliomas. KIT and its ligand stem cell factor are widely expressed in embryonic and adult mouse brain, and they play a role in many signal transduction pathways involved in cellular proliferation, differentiation and cancer cell metastasis. However, the function of KIT in gliomagenesis or disease progression remains unresolved as well as its role in neural and brain tumor development. In this study, we utilized lentivirus-mediated gene transfer to deliver the KIT gene into mouse astrocytes. The growth properties of KIT overexpressing cells were analyzed using several in vitro functional assays. The effect of receptor tyrosine kinase inhibitor imatinib on astrocyte growth was also investigated. Our results indicate that overexpression of KIT in mouse astrocytes promotes cell proliferation, and the increased proliferation is partly inhibited by imatinib treatment. Furthermore, KIT overexpression induces phenotypic changes in the cells suggesting that KIT may play a role in astrocyte growth regulation.

Purpose: The androgen receptor (AR)-mediated signaling pathway seems to be essentially involved in the development and progression of prostate cancer. In vitro studies have shown that altered expression of AR coregulators may significantly modify transcriptional activity of AR, suggesting that these coregulators could also contribute to the progression of prostate cancer. Here, our goal was to assess alterations in the expression of the AR coregulators in prostate cancer in vivo. Experimental Design: The expression of 16 AR coactivators and corepressors (SRC1, β-catenin, TIF2, PIAS1, PIASx, ARIP4, BRCA1, AIB1, AIB3, CBP, STAT1, NCoR1, AES, cyclin D1, p300, and ARA24) was measured in prostate cancer cell lines, xenografts, and clinical prostate tumor specimens by using real-time quantitative reverse transcription-PCR. In addition, gene copy number of SRC1 was analyzed by fluorescence in situ hybridization. Results: Both AR-positive and AR-negative cell lines and xenografts expressed the coregulators. Most of the coregulators studied were expressed at equal levels in benign prostatic hyperplasia and untreated and hormone-refractory carcinomas. However, the expression of PIAS1 and SRC1 was significantly (P = 0.048 and 0.017, respectively) lower in hormone-refractory prostate tumors than in untreated prostate tumors. No overexpression of the coregulators was found in the clinical material. Paradoxically, the SRC1 gene was found to be amplified and highly expressed in a LuCaP 70 prostate cancer xenograft. Conclusions: These findings suggest that the decreased expression of PIAS1 and SRC1 could be involved in the progression of prostate cancer. In addition, gene amplification of SRC1 in one of the xenografts implies that, in some tumors, genetic alteration of SRC1 may provide a growth advantage.

The long arm of chromosome 8 is one of the most common regions of amplification in cancers of several organs, especially carcinomas of the breast and prostate. TRPS1, MYC and EIF3S3 genes are located in one of the minimal regions of amplification, 8q23-q24, and have been suggested to be the target genes of the amplification. Here, our goal was to study copy number and expression of the three genes in order to investigate the significance of the genes in breast and prostate cancer. By using fluorescence in situ hybridisation (FISH), we first found that TRPS1 and EIF3S3 were amplified together in about one-third of hormone-refractory prostate carcinomas. Next, we analysed the mRNA expression of the three genes by real-time quantitative RT-PCR and the gene copy number by FISH in six breast and five prostate cancer cell lines. Breast cancer cell line, SK-Br-3, which contained the highest copy number of all three genes, showed overexpression of only EIF3S3. Finally, the expression levels of TRPS1, EIF3S3 and MYC were measured in freshly frozen clinical samples of benign prostate hyperplasia (BPH), as well as untreated and hormone-refractory prostate carcinoma. The TRPS1 and MYC expression levels were similar in all prostate tumour groups, whereas EIF3S3 expression was higher (P=0.029) in prostate carcinomas compared to BPH. The data suggest that the expression of EIF3S3 is increased in prostate cancer, and that one of the mechanisms underlying the overexpression is the amplification of the gene.

BACKGROUND It has been suggested that estrogens and their receptors (ERs) may be involved in the development and progression of prostate cancer. To elucidate the significance of these receptors, expression of both ERα and ERβ was measured in benign and malignant prostate tumors, as well as in cell lines. METHODS Expression of ERα and ERβ was measured in prostate hyperplasia (BPH, n = 7), androgen-dependent (n = 30) as well as hormone-refractory (n = 12) prostate carcinomas, and in four prostate cancer cell lines (LNCaP, DU145, PC-3, and 22Rv1) using real-time quantitative RT-PCR. RESULTS Only low-level expression of ERα was found in all tumor types and cell lines. The level of expression was similar to that observed in breast carcinomas found to be negative for ERα by immunohistochemistry. All cell lines showed low, but detectable, levels of ERβ expression. The mean expression of ERβ in the hormone-refractory carcinomas was about half that seen in BPH or the androgen-dependent carcinomas; however, the difference was not statistically significant. CONCLUSIONS The data suggest it is unlikely that alterations in the expression of either ER are commonly involved in the progression of prostate cancer. Prostate 55: 180–186, 2003. © 2003 Wiley-Liss, Inc.

An anti-ERBB2 antibody, trastuzumab, has been shown to be highly efficient in the treatment of metastatic breast cancers overexpressing the ERBB2 gene. It has been suggested that overexpression and even amplification of ERBB2 may play a role in the development of prostate cancer. Here, we have analyzed gene copy number and expression of the ERBB2 gene in both androgen-dependent primary and metastatic tumors, as well as recurrent hormone-refractory tumors. The expression levels were compared to the expression of ERBB2 in breast cancers with or without ERBB2 gene amplification. Of 126 prostate tumors, chromogenic in situ hybridization (CISH) revealed only 1 case containing borderline (six to eight copies) amplifications of ERBB2. This hormone-refractory tumor, however, did not express ERBB2 protein. Immunohistochemical staining of ERBB2 protein was negative (0 or 1+ intensity) in all prostate samples (n = 124) analyzed. To quantitate the level of ERBB2 mRNA expression in prostate tumors (n = 34) and cell lines (n = 3), as well as in breast tumors (n = 30) and cell lines (n = 16), real-time reverse transcriptase-polymerase chain reaction (LightCycler) methodology was used. The expression level was similar in all prostate tumor types and corresponded to the level of expression in breast carcinomas without ERBB2 amplification. Breast tumors with ERBB2 amplification expressed, on average, approximately 20 times (P0.001) higher mRNA levels than prostate tumors or breast carcinomas without the gene amplification. In conclusion, the expression of ERBB2 in prostate cancer is relatively low, and is not altered during disease progression. Thus, it is unlikely that treatment modalities relying on the overexpression of ERBB2 gene will be useful in treating prostate cancer.

The significance of androgens in the development of prostate cancer has been known for more than half century. During the last decade, a lot of effort has been put to study the significance of the specific nuclear receptor of the hormone, androgen receptor (AR). It has been suggested that polymorphisms, especially the length of CAG repeat in exon 1 of the gene, are associated with the risk of prostate cancer. However, not all studies have confirmed the association. Most surprisingly, it has now become clear that prostate carcinomas emerging during the androgen withdrawal therapy (i.e. hormone-refractory tumors) are capable of reactivating the AR-mediated signalling despite of the low levels of androgens. In addition, it has been shown that AR gene itself is genetically targeted. One-third of the hormone-refractory prostate carcinomas contains amplification of the gene. In addition, 10-30% of prostate carcinomas treated by antiandrogens acquire point mutation in the AR gene. The genetic alterations in AR indicate that receptor should be considered as putative treatment target. Evidently, the currently available antiandrogens are not capable to abolish the AR-mediated signalling efficiently enough.

It has been suggested that estrogens and their receptors (ERs) may be involved in the development and progression of prostate cancer. To elucidate the significance of these receptors, expression of both ERalpha and ERbeta was measured in benign and malignant prostate tumors, as well as in cell lines.Expression of ERalpha and ERbeta was measured in prostate hyperplasia (BPH, n = 7), androgen-dependent (n = 30) as well as hormone-refractory (n = 12) prostate carcinomas, and in four prostate cancer cell lines (LNCaP, DU145, PC-3, and 22Rv1) using real-time quantitative RT-PCR.Only low-level expression of ERalpha was found in all tumor types and cell lines. The level of expression was similar to that observed in breast carcinomas found to be negative for ERalpha by immunohistochemistry. All cell lines showed low, but detectable, levels of ERbeta expression. The mean expression of ERbeta in the hormone-refractory carcinomas was about half that seen in BPH or the androgen-dependent carcinomas; however, the difference was not statistically significant.The data suggest it is unlikely that alterations in the expression of either ER are commonly involved in the progression of prostate cancer.

Prostate cancer is considered to be one of the most hormone-dependent human malignancies. As a key mediator of hormonal response, the androgen receptor (AR) is believed to have an important role in the progression of prostate cancer. Mutations in the coding region of the AR gene have been found in both untreated and hormone-refractory prostate cancer, but the frequency of such mutations at different stages of the disease is poorly documented and even contradictory results have been published. In the present study, the frequency of AR gene mutations was determined in 30 locally recurrent and two metastatic hormone-refractory prostate tumours using the polymerase chain reaction (PCR), non-radioactive single strand conformation polymorphism (SSCP), and sequencing. The length of the polymorphic CAG repeat, which is inversely correlated with the ability of the AR to activate transcription, was also analysed as well as the GGC repeat. Twelve samples were known to contain an AR gene amplification. Altogether, one point mutation (Gly674Ala) and one microsatellite mutation (CAG20CAG18) were found, both in cancers containing the AR gene amplification. The mean lengths of the polymorphic CAG and GGC repeats were similar to those observed in the normal population. These results favour the view that mutations in the AR gene are rare in hormone-refractory prostate cancer and do not play an important role, at least, in local relapse. Instead, the amplification and consequent overexpression of the wild-type AR gene seem to be the most common alteration involving the AR in hormone-refractory prostate cancer. Copyright © 1999 John Wiley & Sons, Ltd.