Myosin VI is a modulator of androgen-dependent gene expression.

Myosin VI, one of the so-called unconventional myosins, is an actin-based molecular motor involved in intracellular vesicle and organelle transport. In human prostate, myosin VI is expressed in prostate epithelium. We examined the effect of myosin VI downregulation in the LNCaP human prostate cancer cell line using an RNA interference approach. Further, the expression of myosin VI in human prostate tissue was examined using immunohistochemistry. The expression of androgen receptor (AR) and E-cadherin was examined in myosin VI knocked-down cells and control cells. We determined 3H-testosterone uptake in the myosin knocked-down LNCaP cells. Next, we cotransfected LNCaP cells with the myosin VI-specific small interfering RNA (siRNA) duplex and an androgen-responsive luciferase reporter construct and then measured luciferase activity after androgen induction. To clarify whether myosin VI and the AR are interacting proteins, we performed immunoprecipitation studies using myosin VI and AR polyclonal antibodies in androgen-induced LNCaP cells. We confirmed previous results of myosin VI overexpression in human prostate cancer tissue, as in some cases malignant epithelium was more intensively stained than benign epithelium. We found that the expression of AR decreased as a result of myosin VI knock-down. Decreased myosin VI levels did not significantly influence the testosterone uptake of the LNCaP cell line. Instead, we noted a decreased activity of the androgen-regulated mouse mammary tumor virus promoter-reporter vector construct in LNCaP cells cotransfected with myosin VI siRNA duplexes. Finally, we detected the interaction between AR and myosin VI by immunoprecipitation. We propose that myosin VI is a modulator of androgen-dependent gene transcription via interaction with the AR. Thus, myosin VI is a potential therapeutic target for prostate cancer as it could be used as a modulator of AR-dependent gene expression.

Increased BTB-Kelch type substrate adaptor protein immunoreactivity associates with advanced stage and poor differentiation in renal cell carcinoma.

Renal cell carcinoma (RCC) is a malignancy with increasing incidence. Despite the well-known prognostic factors - the stage, grade and histological subtype - the clinical course of RCC can seem quite random. The aim of this study was to evaluate markers of the oxidative system as candidate prognostic factors for RCC. Our study population consisted of 152 patients who underwent operation for RCC between 1990 and 1999. The tumours were examined with three immunohistochemical markers of the oxidative system, thioredoxin (Trx), NF-E2-related factor (Nfr2) and BTB-Kelch type substrate adaptor protein (Keap1). Cytoplasmic Keap1 staining was related to poorer prognosis in renal cancer-specific survival. The difference was statistically significant (P=0.02). Keap1 staining was associated with a more advanced stage and a higher nuclear grade. Cytoplasmic Trx staining was associated with a trend of better prognosis in renal cancer-specific survival. Nfr2 staining was not a prognostic factor in renal cancer-specific survival. In RCC, Keap1 is associated with a more advanced disease, a higher grade and a poorer prognosis.

The interaction of CYP3A5 polymorphisms along the androgen metabolism pathway in prostate cancer.

Prostate cancer is a leading solid tumor among men in the Western world. Androgens play an important role in the carcinogenesis and treatment of prostate cancer. CYP3A5 is a cytochrome P450 superfamily member which also has activity in testosterone metabolism. In this study, we looked for two-gene interactions associated with clinical characteristics of prostate cancer in the Finnish population. We used multifactor-dimensionality reduction for the identification of the two-gene interactions in androgen metabolism pathway genes together with clinical characteristics of prostate cancer among 754 genotyped prostate cancer patients. The CYP3A5*3/*3 and SRD5A2 A49T GG genotype interaction was associated with the clinical tumor stage T2-T4 (T-stage, TNM classification) with odds ratio (OR) 2.14, 95% confidence interval (CI) 1.35-3.40. Patients with CYP3A5*3/*3 and KLK3 I179T CC/TC genotypes had increased OR 2.30, 95% CI 1.16-4.58 for metastatic disease. Further, two-gene interaction CYP3A5*3/*3 and KLK3 -252A > G AA was associated with Gleason scores >or=7 with OR 1.52, 95% CI 1.11-2.09. Prostate cancer patients with CYP3A5*3/*3 and KLK -252A > G GG/AG genotypes had decreased OR of 0.70 with 95% CI 0.50-0.98 for high prostate-specific antigen levels at diagnosis. For prostate cancer patients aged below 65 years, the OR for interaction of CYP3A5*1/*3 or *1/*1 and AKR1C3 Q5H CC genotypes was 1.84 with 95% CI 1.03-3.28. For prostate cancer, the best two-gene interaction included genotypes SRD5A2 V89L GG and AKR1C3 Q5H CC with OR 1.30, 95% CI 1.01-1.66. It remains to be clarified whether these polymorphism associations identified here are also present in other populations.

The androgen receptor CAG repeat length polymorphism associates with prostate volume in Finnish men with benign prostatic hyperplasia.

The development of benign prostatic hyperplasia requires the presence of testicular androgens during prostate development, puberty, and ageing. We thus examined the association of three polymorphisms, namely, CYP3A5 6986A>G, CYP19A1 1531C>T, and androgen receptor (AR) gene CAG repeat length, which have previously been linked to the androgen pathway and with clinical characteristics of benign prostatic hyperplasia. Tissue samples from 262 consecutive prostate operations were used for genotyping. Prostate volumes and prostate-specific antigen values were collected from patient records. Linear regression analysis was performed to study the polymorphisms in an age-adjusted model. We did not find any association between the CYP3A5 6986A>G polymorphism and clinical characteristics of benign prostatic hyperplasia. Further, the previously published CYP19A1 1531C>T polymorphism association with an enlarged prostate could not be confirmed with this material. However, we detected an association between short AR gene CAG repeat length and a small prostate volume, which confirms a previous finding in the Finnish population. The data presented suggest a negligible role for the CYP3A5 6986A>G polymorphism in benign prostate enlargement in the Finnish population. However, the results presented do provide further evidence for potentially different genetic mechanisms behind benign prostatic hyperplasia in Finnish and other Caucasian populations. This is based on the conflicting results for AR gene CAG repeat length associations with benign prostatic hyperplasia found in published works.

Identification of androgen-regulated genes in human prostate.

Androgens are essential for the development of the prostate and prostate cancer. We examined androgen-regulated gene expression in the human prostate. Samples from benign and malignant prostate tissue and samples containing prostate tissue obtained from prostate cancer patients three days after surgical castration were further processed as probes for a GeneChip array. The comparison of gene expression profiles in castrated samples and in benign or malignant prostate tissue samples revealed androgen-regulated genes. We further evaluated the genes which were differentially expressed in benign and malignant prostate samples. The androgen-regulated expression of dual specificity phosphatase 1 (DUSP1) was confirmed in the LNCaP prostate cancer cell line, as the expression of DUSP1 increased with androgen treatment over the course of time. The expression of the genes CRISP3, PCA3, OR51E2, HOXC6, AGR3, AMACR and SLC14A1 was affected by castration in addition to differential expression in the benign and malignant prostate. These sample results require further investigation for the role of AGR3 and SLC14A1 in prostate cancer as these associations have not been reported previously.

Absent Toll-like receptor-9 expression predicts poor prognosis in renal cell carcinoma.

BACKGROUND: Toll-like receptor 9 (TLR9) is a cellular DNA-receptor whose activation with cognate ligands triggers an immune reaction, with increased production of inflammatory cytokines. The aim of this study was to examine the expression of TLR9 in renal cell carcinoma (RCC), which is generally renowned of its immunogenic nature. We also evaluated the prognostic value of TLR9 in RCC. METHODS: TLR9 expression in RCC was characterized with immunohistochemistry in a retrospective study population of 152 RCC patients who underwent renal surgery. The TLR9 staining intensity was compared with clinical parameters. RESULTS: Of the studied tumours, 112 (81%) exhibited cytoplasmic TLR9 immunostaining. No association was detected between cytoplasmic TLR9 immunoexpression intensity and stage, nuclear grade, histological subtype or tumour necrosis. Cytoplasmic TLR9 immunoexpression was, however, a marker of favourable RCC specific survival both in univariate analysis and in multivariate regression model. CONCLUSIONS: We conclude that TLR9 expression is an independent prognostic marker of RCC and the absence of TLR9 expression is related to poorer prognosis in RCC.

Sorbitol dehydrogenase expression is regulated by androgens in the human prostate.

Sorbitol is an intermediate in the polyol pathway, which converts from glucose to fructose by sorbitol dehydrogenase (SORD). Androgens are essential for the development of prostate cancer. We studied castration-induced gene expression changes in the human prostate using the GeneChip array, and identified SORD as being androgen-regulated in the human prostate. A putative androgen-responsive regulatory region at the SORD 5' promoter was identified using promoter deletion constructs in a luciferase reporter assay in COS-7 cells. Chromatin immunoprecipitation assay was used to assess the binding of androgen receptor to suggested androgen responsive regulatory region. Finally, the expression of SORD in the human prostate was evaluated in 29 prostate tissue samples by immunohistochemistry. The expression of SORD decreased after castration. Androgen supplementation to the LNCaP prostate cancer cell line led to a 7.5-fold increase in SORD mRNA expression. Furthermore, a chromatin immunoprecipitation assay proved that the androgen receptor can bind to this putative androgen-responsive regulatory region. Finally, the expression of SORD in the human prostate was localised to epithelial cells of both benign and malignant prostate tissue by immunohistochemistry. In prostate cancer, increased immunostaining was associated with high Gleason patterns and high serum prostate-specific antigen concentrations. These results show that SORD is a novel androgen-regulated gene in the human prostate and suggest the need for more detailed analysis of the physiological role of SORD in the prostate.

Characterization of androgen-regulated expression of CYP3A5 in human prostate.

Testosterone is needed for the growth and development of the prostate. Androgen deprivation therapy is used for the treatment of prostate cancer. CYP3A5 is a human drug-metabolizing cytochrome P450 enzyme that metabolizes testosterone to the inactive 6beta-hydroxylated metabolite. We identified CYP3A5 as a novel androgen-regulated gene in human prostate by GeneChip analysis of human prostate tissues obtained from patients 3 days after therapeutic castration and from control patients. We further showed androgen induction of CYP3A5 messenger RNA (mRNA) in LNCaP prostate cancer cell line. Immunoblotting studies revealed CYP3A5 protein expression in all prostate samples studied. Immunohistochemistry and in situ hybridization was used for localization of CYP3A5 expression in prostate tissue. CYP3A5 was detected both in luminal and in basal epithelial cells of human prostate. Androgen response element was identified in the CYP3A5 proximal promoter and in electrophoretic mobility shift assay androgen receptor was found to bind this element. Androgen induction was abolished by mutation of the response element. We suggest that CYP3A5 is a part of an autoregulatory feedback loop controlling prostate cell exposure to androgens.