Estrogen and Antiestrogen Actions on Human Prostate Cancer: A Dissertation

Prostate cancer increases its incidence with age after men in their fifth decade as the ratio of estrogen to androgen rises. Epidemiological studies indicated that high levels of estrogens are associated with the high-risk ethnic groups for prostate cancer. Therefore, estrogens may be involved in prostatic carcinogenesis. It is widely believed that the actions of estrogens are mediated by estrogen receptors. However, expression of estrogen receptor in normal prostate and lesions of the gland was controversial. With the recent discovery of second estrogen receptor (ER-β), this issue became more complicated and it needs to be readdressed. In addition, the biological involvement of ER-β in human prostate remains to be investigated. In this study, we demonstrated that human normal prostate epithelial cells express ER-β but not ER-α, suggesting that estrogens act directly on these epithelial cells via ER-β. Using RT-PCR analysis, the transcripts of ER-β were detected in our primary human prostatic epithelial cell cultures that were derived from the ultrasound-guided peripheral zone biopsies and the cells express two estrogen-regulated genes such as progesterone receptor (PR) and pS2. Moreover, we had developed an ER-β antibody with fully characterizations and used it for immunohistochemistry. Results indicated that ER-p protein is expressed in the basal compartment of prostatic epithelium of the gland. Our findings lead to a new hypothesis that estrogens directly act on human prostatic epithelial cells to modulate its biological functions. To investigate expression of ERs in prostate cancer, RT-PCR analysis was used. We found that all three human prostate metastatic cancer cell lines, DU145, PC-3 and LNCaP, express ER-β transcripts while ER-α mRNA expression only in PC-3 cells. Expressions of PR and pS2 in these cell lines are various. LNCaP cells express both PR and pS2 mRNAs but DU145 cells with only PR and PC-3 cells with only pS2. Our immunohistochemical results on prostatic lesions revealed down-regulation of ER-β expression in high-grade of dysplasia and carcinoma of peripheral zone of the prostate compared to their low-grade lesions. This down-regulation in high-grade carcinoma was verified in transcriptional level by RT-PCR analysis on micro dissected normal epithelium and lesion samples of the gland. In the metastasis, ER-β was found to be reactivated as we observed ER-β mRNA expression in prostate cancer cell lines. Recent evidence suggests that ER-β may be antiproliferative factor for a protective effect against the mitogenic activity of estrogens in breast and androgens in prostate. Activation of the receptor may exhibit cell growth inhibition. We demonstrated that antiestrogens [ICI-182,780 (ICI) and 4-hydroxytamoxifen], raloxifene and phytoestrogen (resveratrol), but not estrogens (17β-estradiol and diethylstilbestrol), inhibit growth of DU145 cells which express only ER-β while PC-3 cells with both ERs showed growth inhibition in response to estrogen and antiestrogen treatments. In DU145 cells, the ICI-induced cell growth inhibition was prevented by blockade of ER-β expression using antisense oligonucleotide. It indicated that the inhibition is mediated via ER-p associated pathway. Using flow cytometry, we found that ICI-treatment could induce accumulation of cells at GO-G1 phase of cell cycle. Similarly, this GO-G1 cell accumulation was also induced by raloxifene in DU145 cells. For resveratrol, the treatment exhibited dual effects on cell cycle distribution in DU145 cells. In the early treatment, resveratrol induced cell cycle arrests at GO-G1phase. The prolonged treatment leads to S-phase cell cycle arrest. To study the molecular mechanism of this ER-p associated cell growth inhibition, real-time RT-PCR analysis was used to semi-quantitate the transcript levels of tentative ER-β regulated genes such as telomerase reverse transcriptase (TERT), survivin and thymidylate synthase (TS) in the treated cells compared to those in control. Results demonstrated that the treatment of ICI could down-regulate TERT and survivin mRNA expressions with dose-dependent fashion. As the ICI-treatment, resveratrol downregulated expression levels of TERT, survivin and TS in DU145 cells. Down-regulation of TS may be related to the S-phase cell cycle arrest observed in the prolonged treatment of resveratrol. Taken together, our findings support the concept that ER-β participates in cell cycle regulation in normal and malignant prostatic epithelial cells. Presence of ER-β in basal cells of the prostate acini indicates that the direct actions of estrogens may be involved in the normal physiology of the gland. Loss of this receptor in primary prostate cancer and its re-expression in metastasis suggests the roles of ER-β in the cancer progression. Activation of the receptor by antiestrogen and phytoestrogen induced cell growth inhibition in prostate cancer cells. The mechanism may be mediated by reduction of cell survival factors and eventually decrease in cell viability and induction of cell cycle arrests

Overexpression of cytochrome P450 1A1 and its novel spliced variant in ovarian cancer cells: alternative subcellular enzyme compartmentation may contribute to carcinogenesis

Epithelial ovarian cancer derived from the human ovarian surface epithelium (HOSE) is the leading cause of death from gynecologic malignancies among American women. Metabolic activation of endogenous and exogenous chemicals by cytochrome P450 (CYP) class I enzymes has been implicated in its etiology. In this study, we showed overexpression of CYP1A1 mRNA, but not CYP1B1 transcripts, in ovarian cancer cell lines when compared with primary cultures or immortalized HOSE cell lines. Importantly, we identified a novel, enzymatically active, spliced variant of CYP1A1 (CYP1A1v) formed by excision of an 84-bp cryptic intron in exon 2. CYP1A1v is overexpressed in ovarian cancer cell lines and exhibits a unique subcellular distribution restricted to the nucleus and mitochondria, contrary to the endoplasmic reticulum localization of the wild-type enzyme. In concordance, total CYP1A1 activity, as measured by the ethoxyresorufin O-deethylase assay, was detected in mitochondrial, nuclear, and microsomal fractions of ovarian cancer cells but was notably absent in all subcellular fractions of HOSE cells. Immunocytochemistry studies in 30 clinical specimens revealed overexpression of CYP1A1 in various types of ovarian cancers compared with benign epithelia and frequent localization of the enzyme to cancer cell nuclei. Forced expression of CYP1A1wt or CYP1A1v in HOSE cells resulted in nuclear localization of the enzyme and acquisition of anchorage-independent growth, which was further exacerbated following exposure to benzo(a)pyrene or 17beta-estradiol. Collectively, these data provided the first evidence that CYP1A1 overexpression and alternative splicing could contribute to ovarian cancer initiation and progression

Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation

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 inhibitors. In contrast, in DU145 cells, which express only ER-beta, antiestrogens, but not estrogens, are growth inhibitors. By comparison, ICI 182,780 is the more effective cell growth inhibitor. Importantly, the ICI 182,780-induced antiproliferative effects were reversed by cotreatment of DU145 cells with an ER-beta antisense oligonucleotide, hence lending additional support to a central role played by ER-beta in mediating growth-inhibitory action of antiestrogens

Hsa-miRNA-765 as a key mediator for inhibiting growth, migration and invasion in fulvestrant-treated prostate cancer

Fulvestrant (ICI-182,780) has recently been shown to effectively suppress prostate cancer cell growth in vitro and in vivo. But it is unclear whether microRNAs play a role in regulating oncogene expression in fulvestrant-treated prostate cancer. Here, this study reports hsa-miR-765 as the first fulvestrant-driven, ERβ-regulated miRNA exhibiting significant tumor suppressor activities like fulvestrant, against prostate cancer cell growth via blockage of cell-cycle progression at the G2/M transition, and cell migration and invasion possibly via reduction of filopodia/intense stress-fiber formation. Fulvestrant was shown to upregulate hsa-miR-765 expression through recruitment of ERβ to the 5′-regulatory-region of hsa-miR-765. HMGA1, an oncogenic protein in prostate cancer, was identified as a downstream target of hsa-miR-765 and fulvestrant in cell-based experiments and a clinical study. Both the antiestrogen and the hsa-miR-765 mimic suppressed HMGA1 protein expression. In a neo-adjuvant study, levels of hsa-miR-765 were increased and HMGA1 expression was almost completely lost in prostate cancer specimens from patients treated with a single dose (250 mg) of fulvestrant 28 days before prostatectomy. These findings reveal a novel fulvestrant signaling cascade involving ERβ-mediated transcriptional upregulation of hsa-miR-765 that suppresses HMGA1 protein expression as part of the mechanism underlying the tumor suppressor action of fulvestrant in prostate cancer. © 2014 Leung et al

Constitutive and Treatment-Induced CXCL8-Signalling Selectively Modulates the Efficacy of Anti-Metabolite Therapeutics in Metastatic Prostate Cancer

<div><h3>Background</h3><p>The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent.</p> <h3>Methods</h3><p>The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression.</p> <h3>Results</h3><p>Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P<0.001), and increased 5-FU-induced apoptosis in PC3 cells (P<0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU.</p> <h3>Conclusions</h3><p>CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis.</p> </div

Prostate Cancer Induced by Loss of Apc Is Restrained by TGFβ Signaling

Recent work with mouse models of prostate cancer (CaP) has shown that inactivation of TGFβ signaling in prostate epithelium can cooperate with deletion of the Pten tumor suppressor to drive locally aggressive cancer and metastatic disease. Here, we show that inactivating the TGFβ pathway by deleting the gene encoding the TGFβ type II receptor (Tgfbr2) in combination with a deletion of the Apc tumor suppressor gene specifically in mouse prostate epithelium, results in the rapid onset of invasive CaP. Micro-metastases were observed in the lymph nodes and lungs of a proportion of the double mutant mice, whereas no metastases were observed in Apc single mutant mice. Prostate-specific Apc;Tgfbr2 mutants had a lower frequency of metastasis and survived significantly longer than Pten;Tgfbr2 double mutants. However, all Apc;Tgfbr2 mutants developed invasive cancer by 30 weeks of age, whereas invasive cancer was rarely observed in Apc single mutant animals, even by one year of age. Further comparison of the Pten and Apc models of CaP revealed additional differences, including adenosquamous carcinoma in the Apc;Tgfbr2 mutants that was not seen in the Pten model, and a lack of robust induction of the TGFβ pathway in Apc null prostate. In addition to causing high-grade prostate intra-epithelial neoplasia (HGPIN), deletion of either Pten or Apc induced senescence in affected prostate ducts, and this restraint was overcome by loss of Tgfbr2. In summary, this work demonstrates that TGFβ signaling restrains the progression of CaP induced by different tumor suppressor mutations, suggesting that TGFβ signaling exerts a general tumor suppressive effect in prostate.This work was supported by a Program Project Grant from the National Cancer Institute (2P01CA104106 to B. Paschal and D. Wotton), and by a pilot grant from the UVA Cancer Center (funded from the CCSG P30 CA44579, the James and Rebecca CraigFoundation, and UVA Women's Oncology fund) to D. Wotton. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Sharon Birdsall for technical assistance, Anindya Dutta and Dan Gioeli for helpful discussions, and Chun-Song Yang for advice and reagent

Importance of Estrogenic Signaling and Its Mediated Receptors in Prostate Cancer

Prostate cancer (PCa) treatment was first established by Huggins and Hodges in 1941, primarily described as androgen deprivation via interference of testicular androgen production. The disease remains incurable with relapse of hormone-refractory cancer after treatments. Epidemiological and clinical studies disclosed the importance of estrogens in PCa. Discovery of estrogen receptor ERβ prompted direct estrogenic actions, in conjunction with ERα, on PCa cells. Mechanistically, ERs upon ligand binding transactivate target genes at consensus genomic sites via interactions with various transcriptional co-regulators to mold estrogenic signaling. With animal models, Noble revealed estrogen dependencies of PCa, providing insight into potential uses of antiestrogens in the treatment. Subsequently, various clinical trials were conducted and molecular and functional consequences of antiestrogen treatment in PCa were delineated. Besides, estrogens can also trigger rapid non-genomic signaling responses initiated at the plasma membrane, at least partially via an orphan G-protein-coupled receptor GPR30. Activation of GPR30 significantly inhibited in vitro and in vivo PCa cell growth and the underlying mechanism was elucidated. Currently, molecular networks of estrogenic and antiestrogenic signaling via ERα, ERβ and GPR30 in PCa have not been fully deciphered. This crucial information could be beneficial to further developments of effective estrogen- and antiestrogen-based therapy for PCa patients

DNA microarrays in prostate cancer

DNA microarray technology provides a means to examine large numbers of molecular changes related to a biological process in a high throughput manner. This review discusses plausible utilities of this technology in prostate cancer research, including definition of prostate cancer predisposition, global profiling of gene expression patterns associated with cancer initiation and progression, identification of new diagnostic and prognostic markers, and discovery of novel patient classification schemes. The technology, at present, has only been explored in a limited fashion in prostate cancer research. Some hurdles to be overcome are the high cost of the technology, insufficient sample size and repeated experiments, and the inadequate use of bioinformatics. With the completion of the Human Genome Project and the advance of several highly complementary technologies, such as laser capture microdissection, unbiased RNA amplification, customized functional arrays (eg, single-nucleotide polymorphism chips), and amenable bioinformatics software, this technology will become widely used by investigators in the field. The large amount of novel, unbiased hypotheses and insights generated by this technology is expected to have a significant impact on the diagnosis, treatment, and prevention of prostate cancer. Finally, this review emphasizes existing, but currently underutilized, data-mining tools, such as multivariate statistical analyses, neural networking, and machine learning techniques, to stimulate wider usage

Rat estrogen receptor-alpha and -beta, and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats

Semiquantitative RT-PCR was used to determine if transcripts of the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and the progesterone receptor (PR) are differentially expressed and/or regulated in the various normal lobes of the Noble (NBL) rat prostate. We found that ER beta mRNA was present at comparable, high levels in all three major prostatic lobes: dorsal (DP), lateral (LP) and ventral (VP) prostate. ER alpha mRNA was, however, expressed at low levels among the various lobes in the following descending order of abundance: LP\u3eDP\u3eVP. Expression of PR transcript was low and paralleled the expression pattern of ER alpha mRNA. Treatments of rats with testosterone (T) plus estradiol-17beta (E2) (T+E2) or T alone induced no discernible alterations in ER alpha, ER beta, and PR mRNA levels in the VP, DP and LP, while those with E2 caused a general decline in the expression of all three transcripts. We then studied the expression of the three receptors in the normal and dysplastic epithelium of the dorsolateral prostates (DLPs) of rats treated with T+E2. Comparable levels of ER beta mRNA were found in microdissected dysplastic and normal epithelia. In contrast, significantly higher levels of PR mRNA were present in epithelial samples from dysplastic acini. ER alpha mRNA was not detected in any of the microdissected epithelial samples. Results from this study suggest that upregulation of PR mRNA expression, likely mediated via ER beta action, is involved in the genesis of T+E2-induced dysplasia in this animal model