afimoxifene and Prostatic-Neoplasms

SRY is a sex-determining gene that encodes a transcription factor, which triggers male development in most mammals. The molecular mechanism of SRY action in testis determination is, however, poorly understood. In this study, we demonstrate that WDR5, which encodes a WD-40 repeat protein, is a direct target of SRY. EMSA experiments and ChIP assays showed that SRY could bind to the WDR5 gene promoter directly. Overexpression of SRY in LNCaP cells significantly increased WDR5 expression concurrent with histone H3K4 methylation on the WDR5 promoter. To specifically address whether SRY contributes to WDR5 regulation, we introduced a 4-hydroxy-tamoxifen-inducible SRY allele into LNCaP cells. Conditional SRY expression triggered enrichment of SRY on the WDR5 promoter resulting in induction of WDR5 transcription. We found that WDR5 was self regulating through a positive feedback loop. WDR5 and SRY interacted and were colocalized in cells. In addition, the interaction of WDR5 with SRY resulted in activation of Sox9 while repressing the expression of β-catenin. These results suggest that, in conjunction with SRY, WDR5 plays an important role in sex determination.

Topics: Animals; beta Catenin; Binding Sites; Cell Line, Tumor; Histone-Lysine N-Methyltransferase; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Promoter Regions, Genetic; Prostatic Neoplasms; Proteins; Sex-Determining Region Y Protein; SOX9 Transcription Factor; Tamoxifen; Testis

Bicalutamide (BIC) is an alternative treatment to castration for advanced prostate cancer. Breast events are common adverse effects which can be effectively prevented by the concurrent administration of tamoxifen, a selective estrogen receptor modulator.. We investigated the effects of BIC, 4-hydroxy Tamoxifen (4OHT), the active metabolite of tamoxifen, and their combination on the expression of a panel of genes implicated in prostate cancer development and progression in LNCaP cells stimulated with dihydrotestosterone.. Our findings confirm the anti-proliferative activity of BIC on LNCaP cell growth but also show the down-regulating function of this anti-androgen on the expression of genes involved in tumor proliferation and invasion [cyclins, caspases, epidermal growth factor (EGF)]. The combination with 4OHT exerts a synergistic effect on the downregulation of some genes involved in prostate cancer progression.. The observation that the expression of several genes [such as B-cell lymphoma-2 (BCL2), myelocytomatosis oncogene (MYC), caspases] is modulated midly-to-moderately, after 4OHT addition suggests that this combined approach in the clinical setting should be further investigated through appropriate trials.

Topics: Androgens; Anilides; Antineoplastic Combined Chemotherapy Protocols; Cell Growth Processes; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Kallikreins; Male; Neoplasms, Hormone-Dependent; Nitriles; Prostate-Specific Antigen; Prostatic Neoplasms; Tamoxifen; Tosyl Compounds

Bicalutamide (BIC) is widely used in prostate cancer therapy. The dose and schedule employed are well tolerated, but about 50% of patients develop gynecomastia. Several studies have shown a significant reduction of the troublesome effects when Tamoxifen is concomitantly administered with BIC. However, the results reported in the literature seem to be preliminary and possible interferences could be present. In order to clarify the molecular mechanisms of the combination of the two drugs, we have investigated whether the expression of the proteins belonging to nuclear matrix (NM), one modulator of hormone action, is altered by BIC and/or 4-hydroxy-tamoxifen (4OHT) in LNCaP cells. We focused above all on heterogeneous nuclear ribonucleoprotein K (hnRNP K) a NM protein with a key role in prostate carcinoma.. NM proteins were analyzed by two-dimensional gel electrophoresis. Modulation and compartmentalization of the androgen receptor and the hnRNP K were studied by Western blotting, confocal microscopy, and immunoprecipitation.. Proteomic analysis revealed that there is a similarity in the changes of the NM proteins elicited by drugs alone but that their combination does not result in a simple additive effect. Moreover, we found that in the nucleoplasm the androgen receptor and the hnRNP K colocalize in a complex that is highly proximal to DNA and that both proteins were synchronously modulated by BIC and/or 4OHT treatment.. This study confirm the pivotal role of hnRNP K in prostate carcinoma and suggest that this role might be played by the interaction with the androgen receptor.

Topics: Androgen Antagonists; Anilides; Cell Line, Tumor; Cell Nucleus; Cell Survival; Estrogen Antagonists; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Male; Nitriles; Phosphorylation; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Tamoxifen; Tosyl Compounds

Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16-20 wks post-tamoxifen exposure, to overtly malignant lesions by approximately 1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10-12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development.

Topics: Androgen-Binding Protein; Animals; Apoptosis; Arrestins; beta-Arrestins; Cell Proliferation; Crosses, Genetic; Disease Progression; Epithelium; Female; Gene Deletion; Genes, Tumor Suppressor; Humans; Integrases; Male; Mice; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Precancerous Conditions; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; PTEN Phosphohydrolase; Rats; Ribosomal Protein S6; Tamoxifen; Time Factors; Up-Regulation

Sex steroid hormone receptors play a central role in all stages of prostate cancer. Here, we tested whether estrogen receptor (ER) signaling contributes to telomerase activation, an early event in prostate tumorigenesis. Following 17beta-estradiol (E(2)) treatment, both mRNA encoding the catalytic subunit of human telomerase (hTERT) and telomerase activity were promptly induced in human prostate normal epithelial cells, fresh explants from benign prostate hyperplasia, and prostate cancer explants and cell lines. Reporter expression studies and in vivo chromatin immunoprecipitation assays revealed E(2)-dependent hTERT promoter induction and showed that both ERalpha and ERbeta bound this sequence. Crucially, addition of the anti-estrogen 4-hydroxytamoxifen caused a differential recruitment in vivo of ERalpha and ERbeta onto the hTERT promoter and inhibited telomerase activity. Treatment with the aromatase inhibitor letrozole, which prevented testosterone-mediated interaction between ER and the hTERT estrogen response element, resulted in a negative regulation of telomerase activity. Thus, intracellular conversion of androgens to estrogens may contribute to the etiopathogenesis of prostate cancer. Given the present evidence for direct control of hTERT gene expression and telomerase activity in the prostate by the ER, we suggest that this transcriptional regulator represents a possible therapeutic target in prostate cancer.

Topics: Aromatase Inhibitors; Cell Line; DNA-Binding Proteins; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Humans; Male; Prostatic Neoplasms; Receptors, Estrogen; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tamoxifen; Telomerase; Tumor Cells, Cultured

Published data indicate that antiprogestins and antiestrogens could inhibit prostate cancer cell growth in vitro and in vivo. The main objective of the present studies was to explore the role of bcl(2) and TGFbeta(1) for induction of apoptosis in LNCaP prostate cancer cells growing in culture as a treatment response to the antiprogestin, mifepristone, and the antiestrogen, 4-hydroxytamoxifen.. In vitro cell viability (cytotoxicity), DNA fragmentation, and changes in the expression of bcl(2) and TGFbeta(1) proteins were assessed using the sulforhodamine B protein dye-binding assay, specific ELISA, and competitive inhibition assays.. Both steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of viable cells was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl(2), and induction of TGFbeta(1) protein. Abrogation of the mifepristone- and 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta(1)-neutralizing antibody and by the addition of mannose-6-phosphate confirmed the correlation between induction of active TGFbeta(1) and subsequent prostate cancer cell death. The effect of mifepristone was not significantly reduced or prevented by occupying the progesterone or glucocorticoid receptors by their corresponding high-affinity native ligands. On the contrary, the effect of a combination of mifepristone with progesterone or hydrocortisone on the increase in DNA fragmentation, bcl(2) downregulation, and induction of TGFbeta(1) protein was additive and significantly different (P < 0.05) from the effect of mifepristone monotherapy.. Our data suggest that mifepristone and tamoxifen are effective inducers of apoptosis and may represent nonandrogen-ablation, novel therapeutic approaches to overcome a potential intrinsic apoptosis resistance of androgen-independent prostate cancer cells.

Topics: Animals; Antibodies; Apoptosis; Cell Division; Cell Survival; DNA Fragmentation; Estrogen Antagonists; Hormone Antagonists; Humans; Male; Mannosephosphates; Mice; Mice, Nude; Mifepristone; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Transforming Growth Factor beta; Tumor Cells, Cultured

The aim of the current study is to demonstrate normal and malignant prostatic epithelial cells (PrECs) as targets for receptor-mediated estrogenic and antiestrogenic action. Using an improved protocol, we have successfully isolated and maintained highly enriched populations of normal PrECs from ultrasound-guided peripheral zone biopsies, individually determined to be morphologically normal. Semiquantitative reverse transcription-PCR analyses were used to determine whether transcripts of estrogen receptor (ER)-alpha and those of ER-beta were expressed in our normal PrEC primary cultures, in a commercially available PrEC preparation (PrEC; Clontech), in an immortalized PrEC line established from a benign prostatic hyperplasia specimen (BPH-1), and in three prostatic cancer cell lines (LNCaP, PC-3, and DU145). Expression levels of ER-alpha and ER-beta transcripts were related to those of two estrogen-responsive genes [progesterone receptor (PR) and pS2], at the message levels, to gain insights into the functionality of the ER subtypes in PrECs. Interestingly, only transcripts of ER-beta, but not those of ER-alpha, were found in our primary cultures of normal PrECs, along with both PR and pS2 mRNA. These data strongly suggest that estrogen action was signaled exclusively via ER-beta in normal human PrECs. In contrast, PrEC (Clontech) and BPH-1 cells expressed both ER-alpha and ER-beta transcripts and no PR nor pS2 mRNA in PrEC and only a minimal level of PR mRNA in BPH-1. Among the three prostate cancer cell lines, LNCaP expressed ER-beta mRNA along with transcripts of PR and pS2, DU145 expressed messages of ER-beta and PR, and PC-3 cells exhibited ER-alpha, ER-beta, and pS2 mRNA. Thus, unlike normal PrECs, expression patterns of these genes in malignant PrECs are more variable. Treatment of prostate cancer cells with demethylation agents effectively reactivated the expression of ER-alpha mRNA in LNCaP and DU145 and that of pS2 message in DU145. These findings provide experimental evidence that ER-alpha gene silencing in prostate cancer cells, and perhaps also in normal PrECs, are caused by DNA hypermethylation. To evaluate the potential of using antiestrogens as prostate cancer therapies, we have assessed the growth-inhibitory action of estrogens (estradiol and diethylstilbestrol) and antiestrogens (4-hydroxy-tamoxifen and ICI-182,780) on PC-3 and DU-145 cells. In PC-3 cells, which express both ER subtypes, estrogens as well as antiestrogens are effective in

Topics: Cell Culture Techniques; Cell Division; Cells, Cultured; Diethylstilbestrol; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens, Non-Steroidal; Fulvestrant; Humans; Male; Methylation; Oligonucleotides, Antisense; Prostate; Prostatic Neoplasms; Protein Biosynthesis; Proteins; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Tamoxifen; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins