Tina Bianco-Miotto

Androgen receptor inhibits estrogen receptor-alpha activity and is prognostic in breast cancer

There is emerging evidence that the balance between estrogen receptor-alpha (ER(alpha)) and androgen receptor (AR) signaling is a critical determinant of growth in the normal and malignant breast. In this study, we assessed AR status in a cohort of 215 invasive ductal breast carcinomas. AR and (ER(alpha)) were coexpressed in the majority (80-90%) of breast tumor cells. Kaplan-Meier product limit analysis and multivariate Cox regression showed that AR is an independent prognostic factor in (ER(alpha))-positive disease, with a low level of AR (less than median of 75% positive cells) conferring a 4.6-fold increased risk of cancer-related death (P = 0.002). Consistent with a role for AR in breast cancer outcome, AR potently inhibited (ER(alpha))transactivation activity and 17beta-estradiol-stimulated growth of breast cancer cells. Transfection of MDA-MB-231 breast cancer cells with either functionally impaired AR variants or the DNA-binding domain of the AR indicated that the latter is both necessary and sufficient for inhibition of (ER(alpha)) signaling. Consistent with molecular modeling, electrophoretic mobility shift assays showed binding of the AR to an estrogen-responsive element (ERE). Evidence for a functional interaction of the AR with an ERE in vivo was provided by chromatin immunoprecipitation data, revealing recruitment of the AR to the progesterone receptor promoter in T-47D breast cancer cells. We conclude that, by binding to a subset of EREs, the AR can prevent activation of target genes that mediate the stimulatory effects of 17beta-estradiol on breast cancer cells.

Suberoylanilide hydroxamic acid (vorinostat) represses androgen receptor expression and acts synergistically with an androgen receptor antagonist to inhibit prostate cancer cell proliferation

Growth of prostate cancer cells is initially dependent on androgens, and androgen ablation therapy is used to control tumor growth. Unfortunately, resistance to androgen ablation therapy inevitably occurs, and there is an urgent need for better treatments for advanced prostate cancer. Histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid (SAHA; vorinostat), are promising agents for the treatment of a range of malignancies, including prostate cancer. SAHA inhibited growth of the androgen-responsive LNCaP prostate cancer cell line at low micromolar concentrations and induced caspase-dependent apoptosis associated with chromatin condensation, DNA fragmentation, and mitochondrial membrane depolarization at higher concentrations (≥5 μmol/L). Gene profiling and immunoblot analyses showed a decrease in androgen receptor (AR) mRNA and protein in LNCaP cells cultured with SAHA compared with control cells, with a corresponding decrease in levels of the AR-regulated gene, prostate-specific antigen. Culture of LNCaP cells in steroid-free medium markedly sensitized the cells to SAHA. Moreover, a combination of low, subeffective doses of SAHA and the AR antagonist bicalutamide resulted in a synergistic reduction in cell proliferation and increase in caspase-dependent cell death. Addition of exogenous androgen prevented the induction of cell death, indicating that suppression of androgen signaling was required for synergy. At the subeffective concentrations, these agents had no effect, alone or in combination, on proliferation or death of AR-negative PC-3 prostate cancer cells. Our findings indicate that SAHA is effective in targeting the AR signaling axis and that androgen deprivation sensitizes prostate cancer cells to SAHA. Consequently, combinatorial treatments that target different components of the AR pathway may afford a more effective strategy to control the growth of prostate cancer cells. [Mol Cancer Ther 2007;6(1):51–60]

Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Background: Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis. Methods: Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions. Results: H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort. Conclusions: This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis. Impact: Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies. Cancer Epidemiol Biomarkers Prev; 19(10); 2611–22. ©2010 AACR.

Decreased androgen receptor levels and receptor function in breast cancer contribute to the failure of response to medroxyprogesterone acetate

Previously, we reported that androgen receptor (AR), but not estrogen receptor (ER) or progesterone receptor (PR), is predictive of response to the synthetic progestin, medroxyprogesterone acetate (MPA), in a cohort of 83 patients with metastatic breast cancer. To further investigate the role of AR in determining response to MPA in this cohort, we analyzed AR levels by immunohistochemistry with two discrete antisera directed at either the NH2 or the COOH termini of the receptor. Compared with tumors that responded to MPA (n = 31), there was a significant decrease in the intensity and extent of AR immunoreactivity with both AR antisera in tumors from nonresponders (n = 52). Whereas only a single AR immunostaining pattern was detected in responders to MPA, reflecting concordance of immunoreactivity with the two AR antisera, tumors from nonresponders exhibited four distinct AR immunostaining patterns: (a) concordance with the two antibodies (31%), (b) staining only with the COOH-terminal antibody (33%), (c) staining only with the NH2-terminal antibody (22%), or (d) no immunoreactivity with either NH2- or COOH-terminal antibody (14%). DNA sequencing and functional analysis identified inactivating missense gene mutations in the ligand-binding domain of the AR in tumors from two of nine nonresponders positive with the NH2-terminal AR antisera but negative for COOH-terminal immunoreactivity and lacking specific, high-affinity dihydrotestosterone binding in tumor cytosol fractions. Tumors with more AR than the median level (37 fmol/mg protein) had significantly lower levels of PR (30 fmol/mg protein) than tumors with low AR (PR; 127 fmol/mg protein) despite comparable levels of ER. Ligand-dependent activation of the AR in human T47D and MCF-7 breast cancer cells resulted in inhibition of estradiol-stimulated cell proliferation and a reduction in the capacity of the ER to induce expression of the PR. These effects could be reversed using a specific AR antisense oligonucleotide. Increasing the ratio of AR to ER resulted in a greater androgen-dependent inhibition of ER function. Collectively, these data suggest that reduced levels of AR or impaired AR function contribute to the failure of MPA therapy potentially due to abrogation of the inhibitory effect of AR on ER signaling.

Integrative transcriptome meta-analysis reveals widespread sex-biased gene expression at the human fetal–maternal interface

As males and females share highly similar genomes, the regulation of many sexually dimorphic traits is constrained to occur through sex-biased gene regulation. There is strong evidence that human males and females differ in terms of growth and development in utero and that these divergent growth strategies appear to place males at increased risk when in sub-optimal conditions. Since the placenta is the interface of maternal–fetal exchange throughout pregnancy, these developmental differences are most likely orchestrated by differential placental function. To date, progress in this field has been hampered by a lack of genome-wide information on sex differences in placental gene expression. Therefore, our motivation in this study was to characterize sex-biased gene expression in the human placenta. We obtained gene expression data for >300 non-pathological placenta samples from 11 microarray datasets and applied mapping-based array probe re-annotation and inverse-variance meta-analysis methods which showed that >140 genes (false discovery rate (FDR) <0.05) are differentially expressed between male and female placentae. A majority of these genes (>60%) are autosomal, many of which are involved in high-level regulatory processes such as gene transcription, cell growth and proliferation and hormonal function. Of particular interest, we detected higher female expression from all seven genes in the LHB-CGB cluster, which includes genes involved in placental development, the maintenance of pregnancy and maternal immune tolerance of the conceptus. These results demonstrate that sex-biased gene expression in the normal human placenta occurs across the genome and includes genes that are central to growth, development and the maintenance of pregnancy.

Epigenetic biomarkers in prostate cancer: Current and future uses

Epigenome alterations are characteristic of nearly all human malignancies and include changes in DNA methylation, histone modifications and microRNAs (miRNAs). However, what induces these epigenetic alterations in cancer is largely unknown and their mechanistic role in prostate tumorigenesis is just beginning to be evaluated. Identification of the epigenetic modifications involved in the development and progression of prostate cancer will not only identify novel therapeutic targets but also prognostic and diagnostic markers. This review will focus on the use of epigenetic modifications as biomarkers for prostate cancer.

Antiproliferative actions of the synthetic androgen, mibolerone, in breast cancer cells are mediated by both androgen and progesterone receptors.

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.

GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells.

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2′-deoxycytidine (5-aza-CdR) either with a single high dose (5–20 µM), every alternate day (0.1–10 µM) or daily (0.005–2.5 µM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 µM or greater (p<0.0001) and induction of cell death from 0.5 µM (p<0.0001). In contrast, treatment with a single high dose of 20 µM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥0.05 µM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥0.5 µM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥50 µM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.

DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

Background Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. Methodology/Principal Findings To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). Conclusions/Significance We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.

A gene signature identified using a mouse model of androgen receptor‐dependent prostate cancer predicts biochemical relapse in human disease

Mutations in the androgen receptor (AR) have been detected in experimental and clinical prostate tumors. Mice with enforced prostate‐specific expression of one such receptor variant, AR‐E231G, invariably develop prostatic intraepithelial neoplasia by 12 weeks and metastatic prostate cancer by 52 weeks. The aim of this study was to identify genes with altered expression in the prostates of AR‐E231G mice at an early stage of disease that may act as drivers of AR‐mediated tumorigenesis. The gene expression profile of AR‐E231G prostate tissue from 12‐week‐old mice was compared to an equivalent profile from mice expressing the AR‐T857A receptor variant (analogous to the AR‐T877A variant in LNCaP cells), which do not develop prostate tumors. One hundred and thirty‐two genes were differentially expressed in AR‐E231G prostates. Classification of these genes revealed enrichment for cellular pathways known to be involved in prostate cancer, including cell cycle and lipid metabolism. Suppression of two genes upregulated in the AR‐E231G model, ADM and CITED1, increased cell death and reduced proliferation of human prostate cancer cells. Many genes differentially expressed in AR‐E231G prostates are also deregulated in human tumors. Three of these genes, ID4, NR2F1 and PTGDS, which were expressed at consistently lower levels in clinical prostate cancer compared to nonmalignant tissues, formed a signature that predicted biochemical relapse (hazard ratio 2.2, p = 0.038). We believe that our findings support the value of this novel mouse model of prostate cancer to identify candidate therapeutic targets and/or biomarkers of human disease.