Somatic mutation analysis of <Emphasis Type="Italic">MYH11</Emphasis>in breast and prostate cancer

Background  

MYH11 (also known as SMMHC) encodes the smooth-muscle myosin heavy chain, which has a key role in smooth muscle contraction. Inversion at the MYH11 locus is one of the most frequent chromosomal aberrations found in acute myeloid leukemia. We have previously shown that MYH11 mutations occur in human colorectal cancer, and may also be associated with Peutz-Jeghers syndrome. The mutations found in human intestinal neoplasia result in unregulated proteins with constitutive motor activity, similar to the mutant myh11 underlying the zebrafish meltdown phenotype characterized by disrupted intestinal architecture. Recently, MYH1 and MYH9 have been identified as candidate breast cancer genes in a systematic analysis of the breast cancer genome.     

Mutation screening of the androgen receptor promoter and untranslated regions in prostate cancer

BACKGROUND: Mechanisms, other than gene amplification, leading to overexpression of AR in androgen ablation-resistant prostate cancer remain unknown and could include genetic alterations in the promoter or untranslated regions (UTR) of the AR gene. MATERIALS AND METHODS: DNAs from five prostate cancer cell lines, 19 LuCaP xenografts, 44 clinical tumors, and 36 non-malignant controls were used for screening mutations in the upstream regulatory region, promoter and the 5'- and 3'-UTRs of the AR gene with denaturating high performance liquid chromatography (DHPLC) and sequencing. RESULTS: Ten different sequence variations were found in prostate cancer cell lines and xenografts. However, none of them were recurrent or were found in clinical prostate cancer specimens or in normal controls. CONCLUSIONS: Recurrent mutations in the promoter or UTRs of AR seem to be rare, and thus not likely mechanisms for the increased expression of the gene in the androgen ablation-resistant prostate cancer.     

Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer

The androgen receptor (AR) signaling pathway is involved in the emergence of castration-resistant prostate cancer (CRPC). Here, we identified several androgen-regulated microRNAs (miRNAs) that may contribute to the development of CRPC. Seven miRNAs, miR-21, miR-32, miR-99a, miR-99b, miR-148a, miR-221 and miR-590-5p, were found to be differentially expressed in CRPC compared with benign prostate hyperplasia (BPH) according to microarray analyses. Significant growth advantage for LNCaP cells transfected with pre-miR-32 and pre-miR-148a was found. miR-32 was demonstrated to reduce apoptosis, whereas miR-148a enhanced proliferation. Androgen regulation of miR-32 and miR-148a was confirmed by androgen stimulation of the LNCaP cells followed by expression analyses. The AR-binding sites in proximity of these miRNAs were demonstrated with chromatin immunoprecipitation (ChIP). To identify target genes for the miRNAs, mRNA microarray analyses were performed with LNCaP cells transfected with pre-miR-32 and pre-miR-148a. Expression of BTG2 and PIK3IP1 was reduced in the cells transfected with pre-miR-32 and pre-miR-148a, respectively. Also, the protein expression was reduced according to western blot analysis. BTG2 and PIK3IP1 were confirmed to be targets by 3'UTR-luciferase assays. Finally, immunostainings showed a statistically significant (P<0.0001) reduction of BTG2 protein in CRPCs compared with untreated prostate cancer (PC). The lack of BTG2 staining was also associated (P<0.01) with a short progression-free time in patients who underwent prostatectomy. In conclusion, androgen-regulated miR-32 is overexpressed in CRPC, leading to reduced expression of BTG2. Thus, miR-32 is a potential marker for aggressive disease and is a putative drug target in PC.     

Bcl-2 antisense oligonucleotides (G3139) inhibit Merkel cell carcinoma growth in SCID mice

Merkel cell carcinoma was first described in 1972 by Toker and is an aggressive neuroendocrine skin tumor with a high metastatic potential. Merkel cell carcinoma is thought to derive from the neuroendocrine (Merkel) cells of the skin, although in contrast to fetal and especially adult Merkel cells, Merkel cell carcinomas express high levels of the Bcl-2 oncoprotein. Bcl-2 is capable of blocking programmed cell death and has been shown to play an important role in normal cell turnover, tumor biology, and chemoresistance. High Bcl-2 expression leading to prolonged survival of cells may therefore be of importance in the biological and clinical characteristics of Merkel cell carcinoma. In a SCID mouse xenotransplantation model for human Merkel cell carcinoma, we investigated the influence of the bcl-2 antisense oligonucleotide G3139 (Genta) on tumor growth in comparison with control oligonucleotides or cisplatin. Bcl-2 antisense treatment, targeting the first six codons of the bcl-2 mRNA, resulted in either a dramatic reduction of tumor growth or complete remission, whereas reverse sequence and two-base mismatch control oligonucleotides or cisplatin had no significant antitumor effects compared with saline-treated controls. Apoptosis was enhanced 2.4-fold in the bcl-2 antisense treated tumors compared with the saline-treated group, and no other treatment showed a comparable increase in apoptosis. Our findings suggest that bcl-2 antisense treatment may be a novel approach to improve treatment outcome of human Merkel cell carcinoma.     

Differences of bcl-2 protein expression between Merkel cells and Merkel cell carcinomas

The bcl-2 gene, originally identified in B-cell lymphomas, encodes for proteins which may assume oncogenic functions by blocking apoptosis. Bcl-2 proteins are broadly distributed among various tissues, including epithelial ones. Within the skin, bcl-2 is strongly expressed in melanocytes, but its further distribution is yet unclear. The Merkel cells, neuroendocrine-epithelial cells of the skin, are present within the epidermis and hair follicles, mostly nerve-associated, and are believed to be postmitotic and long lived. Possibly they give rise to the malignant Merkel cell carcinomas. In the present study we investigated the bcl-2 expression on the protein level by means of immunohistochemical techniques including double confocal laser scanning microscopy, as well as on the RNA level by RT-PCR techniques, in Merkel cells, Merkel cell carcinomas, and cell lines. Merkel cells were identified by double staining for cytokeratins 20 or 8/18. We demonstrate that fetal epidermal and dermal Merkel cells are immunostained for bcl-2 protein, most of them clearly weaker than melanocytes. Adult Merkel cells also express bcl-2 protein very heterogeneously, mostly weak. In contrast, Merkel cell carcinomas are visually strongly positive for bcl-2 protein with some degree of heterogeneity. This is different from malignant melanomas in which bcl-2 expression is reduced as compared to normal melanocytes. Bcl-2 gene expression was also shown for Merkel cell carcinoma cell lines on both the mRNA and the protein level. Possibly bcl-2 protein expression is downregulated during the life span of Merkel cells, arguing that they may succumb to a certain cell turnover. The comparably high bcl-2 protein level in Merkel cell carcinomas may reflect peculiar biological and clinical characteristics.     

BRCA2 mutations in 154 finnish male breast cancer patients

The etiology and pathogenesis of male breast cancer (MBC) are poorly known. This is due to the fact that the disease is rare, and large-scale genetic epidemiologic studies have been difficult to carry out. Here, we studied the frequency of eight recurrent Finnish BRCA2 founder mutations in a large cohort of 154 MBC patients (65% diagnosed in Finland from 1967 to 1996). Founder mutations were detected in 10 patients (6.5%), eight of whom carried the 9346(-2) A>G mutation. Two novel mutations (4075 delGT and 5808 del5) were discovered in a screening of the entire BRCA2 coding region in 34 samples. However, these mutations were not found in the rest of the 120 patients studied. Patients with positive family history of breast and/or ovarian cancer were often BRCA2 mutation carriers (44%), whereas those with no family history showed a low frequency of involvement (3.6%; P < .0001). Finally, we found only one Finnish MBC patient with 999 del5, the most common founder mutation in Finnish female breast cancer (FBC) patients, and one that explains most of the hereditary FBC and MBC cases in Iceland. The variation in BRCA2 mutation spectrum between Finnish MBC patients and FBC patients in Finland and breast cancer patients in Iceland suggests that modifying genetic and environmental factors may significantly influence the penetrance of MBC and FBC in individuals carrying germline BRCA2 mutations in some populations.     

Screening of genetic and expression alterations of SRC1 gene in prostate cancer

BACKGROUND: Genetic alterations of the SRC1 gene have not been thoroughly studied in prostate cancer. MATERIALS AND METHODS: Five prostate cancer cell lines and 32 xenografts were screened for mutations and gene copy number alterations. Subsequently, frequencies of detected sequence variations were further analyzed in 44 clinical prostate cancers, 6 benign prostate hyperplasias, and 48 normal controls. Finally, the protein expression of SRC1 in 254 clinical prostate tumors was investigated. RESULTS: Three non-recurrent sequence variations, and one single nucleotide polymorphism in the coding region of SRC1, as well as one case of SRC1 gene amplification were found. The protein expression of SRC1 was higher in androgen ablation resistant than untreated prostate carcinomas, but the difference was not statistically significant (P = 0.0796). CONCLUSIONS: Genetic alterations of SRC1 are rare in prostate cancer. The nuclear protein accumulation of SRC1 seems to be mildly increased in androgen ablation resistant prostate cancers. .     

Androgen receptor (AR) aberrations in castration-resistant prostate cancer

Genetic aberrations affecting the androgen receptor (AR) are rare in untreated prostate cancers (PCs) but have been found in castration-resistant prostate cancers (CRPCs). Further, successful treatment with novel endocrine therapies indicates that CRPCs remain androgen-sensitive. Known AR aberrations include amplification of the AR gene leading to the overexpression of the receptor, point mutations of AR resulting in promiscuous ligand usage, and constitutively active AR splice variants. Gain, or amplification, of the AR gene is one of the most frequent genetic alterations observed in CRPCs. Up to 80% of CRPCs have been reported to carry an elevated AR gene copy number, and about 30% have a high-level amplification of the gene. AR mutations are also commonly observed and have been found in approximately 10-30% of the CRPC treated with antiandrogens; however, the frequency and significance of AR splice variants is still unclear. Because AR aberrations are found almost exclusively in CRPC, these alterations must have been selected for during therapy. Interestingly, these aberrations lead to activation of the receptor, despite treatment-induced emergence of therapy-resistant tumor clones. Therefore, future novel treatment strategies should focus on suppressing AR activity in CRPC.