Low Levels Of Androgen Receptor Research Articles

Targeting androgen receptor (AR) with a synthetic peptide increases apoptosis in triple negative breast cancer and AR-expressing prostate cancer cell lines.

The androgen receptor (AR) has been studied as an approach to cancer therapy. We used human breast cancer-derived cells with high, low, and very low expression levels of AR, in addition to prostate cancer-derived LNCaP and DU-145 cells as a positive and negative controls to examine apoptosis caused by a synthetic peptide that targets ARs. The peptide was produced to inhibit AR transactivation in breast cancer cell lines. We then measured cell viability, caspase-3 activity, and the ratio of Bax/Bcl-2. The findings indicated that the peptide (100-500 nM) in the presence of dihydrotestosterone (DHT) reduced cell growth in cells with high and low expression level of AR (p < .001), but not in cells with very low levels of AR. Treatment with 100-500 nM of peptide activated caspase-3 and increased the ratio of Bax/Bcl-2 in cells with high and low expression levels of AR. Also, increasing concentrations of the peptide (100-500 nM) reduced BrdU incorporation in the presence of DHT and promoted apoptosis in cells with high and low expression levels of AR (p < .001). The findings indicate the peptide significantly increased apoptosis in cancer cells.

Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination

While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.

Abstract 967: Real-world transcriptomic biomarkers as replacement for immunohistochemistry and FISH studies in breast cancer

Abstract Background: Human epidermal growth factor receptor-2 (HER2) and hormone receptors are typically used as binary biomarkers for selecting breast cancer therapy. There is a need to explore the clinical relevance of these biomarkers as continuous variables. This is particularly relevant for the new class of antibody-drug conjugates (ADC), in which a relatively low HER2 expression level is adequate for targeting tumor cells. We explored the potential of RNA profiling, determined by next generation sequencing (NGS), to provide more flexible clinical biomarkers as compared with immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH). Methods: Clinical and laboratory data from 57 breast cancers collected by the COTA real-world data company were used to study biomarker levels as detected by routine clinical transcriptomic tests. HER2 (ERBB2), estrogen receptor alpha (ESR1), and androgen receptor (AR) mRNA levels were compared with reported HER2 and estrogen receptor (ER) IHC and FISH results. Results: RNA levels accurately reflected and predicted HER2 amplification (see table below). Importantly, RNA data showed significant variation and overlap in the levels of ERBB2 mRNA between cases scored by IHC as zero, 1+, and 2+. This variation correlated with progression-free survival (PFS). Similarly, the ESR1 RNA levels accurately reflected ER status and demonstrated significant variation between positive cases. RNA data also showed significant variations in the AR levels. Patients wssith high AR mRNA levels had significantly better PFS (P=0.05). Patients expressing high ER and AR levels had significantly better PFS than those expressing low ESR1 and AR levels (P=0.03). Conclusions: These findings suggest that RNA analysis using NGS is an alternative to IHC and FISH. RNA provides continuous data that can determine cut-off points predicting response to therapy and should be explored in predicting ADC response. ERBB2 mRNA levels (FPKM) in various HER2 IHC groupss IHC Score Valid N Mean Median Minimum Maximum Quartile Range Std.Dev. Zero 19 231 243 63 537 170 110 Zero vs one 3 151 155 45 253 208 104 Zero to one vs one 18 397 302 172 845 365 222 One Vs two 13 495 423 159 1094 273 284 Two vs three 4 7523 7649 937 13859 7183 5310 Citation Format: Maher Albitar, Andre Goy, Andrew Pecora, Deena Graham, Donna Donna McNamara, Ahmad Ahmad Charifa, Andrew IP, Wanlong Ma, Stanley Waintraub. Real-world transcriptomic biomarkers as replacement for immunohistochemistry and FISH studies in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 967.

Abstract PD1-03: Estrogen receptor is a target of enzalutamide in ER+ breast cancer

Abstract Background: Androgen receptor (AR) is widely expressed in ER+ breast cancers. Accumulated evidence showed that AR is a tumor suppressor in ERα+ breast cancers. Enzalutamide (ENZ), an AR antagonist, blocks AR signalling, and was reported to inhibit estrogen-driven tumor growth as effectively as tamoxifen. Hence, this study was performed to further examine the mechanism of action of ENZ in ERα+ breast cancers. Methods: The AR gene was knocked out using the CRISPR-Cas9 system. Four crRNAs targeting AR and trans-activating crRNA were co-transfected to Cas9-positive MCF7 and T47D cells, two ERα+ breast cancer cell lines with relatively low AR levels based on RNA seq. ESR1 knock down was performed using a lentivirus system. MCF7 and T47D cells were infected with the ESR1-shRNA lentivirus and selected by puromycin for 2 weeks. Single clones of knockdown or knockout cells were tested using western blot. MCF7 and T47D cells were collected for RNA-seq and ChIP-seq after treated with DMSO or 10µM ENZ for 6 hours in complete medium. All sequences were aligned to the Human Reference Genome (hg38, UCSC). Differential gene expression was performed using limma package and upstream analysis was done in Ingenuity Pathway Analysis software. ChIP peaks were called using MACS2 and overlapping was analyzed using ChIPpeakAnno package in R. Tritium labeled ENZ or estradiol were incubated with ERα overnight. The bound radioactivity was measured using a Beckmann LS 6500 liquidscintillation counter. Results: We found the cell growth inhibition effect of ENZ was little impaired by AR gene knock out in MCF7 and T47D cells as was expected. As ENZ has been shown to inhibit estrogen-driven tumor growth, we performed studies to understand the mechanism of ENZ.We performed RNA-seq and identified 193 differential expressed genes (76 up and 117 down) between DMSO and ENZ-treated MCF7 cells (FC&amp;gt;2 or &amp;lt;0.5, FDR&amp;lt;0.05). Upstream analysis showed that ESR1 might be the upstream regulator of those differentially expressed genes and ESR1 signaling was predicted to be inhibited after ENZ treatment (P=1.69×10-10). To examine the alteration of ERα genomic binding, we performed global ERα ChIP-seq and found 39.58% (8,681/21,933) of ERα-bound sites were abolished by ENZ and the binding intensity was significantly decreased upon ENZ treatment in 12,868 shared peaks. Moreover, radiolabled binding assays showed that ENZ displaced estrogen binding to ERα and directly bound to ERα with a KD= 399.60nM. We further observed a reduction of proliferation inhibition of ENZ in ESR1-KD cells compared with control cells. This suggests that ENZ inhibits ER+ cell growth by displacing E2 from ERα binding sites and subsequently diminishes ERα genomic binding and blocks ERα signaling in ER+ cancers. Conclusion: We found that enzalutamide inhibits cell growth not through AR but by antagonizing ERα activity in MCF7 and T47D ER+ cells. These results reveal ER is the main factor affecting enzalutamide efficacy in these two ERα+ breast cancer cell lines. Further clinical trials are needed to investigate AR antagonists in ERα+ breast cancers. Citation Format: Lixuan Wei, Jia Yu, Huanyao Gao, Huan Zhang, Thanh Nguyen, Yayun Gu, Richard M. Weinshilboum, James N. Ingle, Liewei Wang. Estrogen receptor is a target of enzalutamide in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-03.

Menin Enhances Androgen Receptor-Independent Proliferation and Migration of Prostate Cancer Cells.

The androgen receptor (AR) is an important therapeutic target for treating prostate cancer (PCa). Moreover, there is an increasing need for understanding the AR-independent progression of tumor cells such as neuroendocrine prostate cancer (NEPC). Menin, which is encoded by multiple endocrine neoplasia type 1 (MEN1), serves as a direct link between AR and the mixed-lineage leukemia (MLL) complex in PCa development by activating AR target genes through histone H3 lysine 4 methylation. Although menin is a critical component of AR signaling, its tumorigenic role in AR-independent PCa cells remains unknown. Here, we compared the role of menin in AR-positive and AR-negative PCa cells via RNAi-mediated or pharmacological inhibition of menin. We demonstrated that menin was involved in tumor cell growth and metastasis in PCa cells with low or deficient levels of AR. The inhibition of menin significantly diminished the growth of PCa cells and induced apoptosis, regardless of the presence of AR. Additionally, transcriptome analysis showed that the expression of many metastasis-associated genes was perturbed by menin inhibition in AR-negative DU145 cells. Furthermore, wound-healing assay results showed that menin promoted cell migration in AR-independent cellular contexts. Overall, these findings suggest a critical function of menin in tumorigenesis and provide a rationale for drug development against menin toward targeting high-risk metastatic PCa, especially those independent of AR.

Sustained androgen receptor signaling is a determinant of melanoma cell growth potential and tumorigenesis.

Melanoma susceptibility differs significantly in male versus female populations. Low levels of androgen receptor (AR) in melanocytes of the two sexes are accompanied by heterogeneous expression at various stages of the disease. Irrespective of expression levels, genetic and pharmacological suppression of AR activity in melanoma cells blunts proliferation and induces senescence, while increased AR expression or activation exert opposite effects. AR down-modulation elicits a shared gene expression signature associated with better patient survival, related to interferon and cytokine signaling and DNA damage/repair. AR loss leads to dsDNA breakage, cytoplasmic leakage, and STING activation, with AR anchoring the DNA repair proteins Ku70/Ku80 to RNA Pol II and preventing RNA Pol II-associated DNA damage. AR down-modulation or pharmacological inhibition suppresses melanomagenesis, with increased intratumoral infiltration of macrophages and, in an immune-competent mouse model, cytotoxic T cells. AR provides an attractive target for improved management of melanoma independent of patient sex.

Epigenetic modulation of AR gene expression in prostate cancer DU145 cells with the combination of sodium butyrate and 5'-Aza-2'-deoxycytidine.

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. Castration-resistant prostate cancer (CRPC) is a consequence of androgen deprivation therapy. Unchecked CRPC followed by metastasis is lethal. Some CRPCs show decreased AR gene expression due to epigenetic mechanisms such as DNA methylation and histone deacetylation. The aim of this study was to epigenetically modulate the methylated state of the AR gene leading to targeted demethylation and AR gene expression in androgen-independent human prostate cancer DU145 cell line, representing the CRPC model with very low or undetectable AR levels. The cell treatment was based on single and combined applications of two epigenetic inhibitors, sodium butyrate (NaB) as histone deacetylases inhibitor and 5'-Aza-2'-deoxycytidine (Aza-dC) as DNA methyltransferases inhibitor. We found that the Aza-dC in combination with NaB may help reduce the toxicity of higher NaB concentrations in cancer cells. In normal RWPE-1 cells and even stronger in cancer DU145 cells, the combined treatment induced both AR gene expression on the mRNA level and increased histone H4 acetylation in AR gene promoter. Also activation and maintenance of G2/M cell cycle arrest and better survival in normal RWPE-1 cells compared to cancer DU145 cells were observed after the treatments. These results imply the selective toxicity effect of both inhibitors used and their potentially more effective combined use in the epigenetic therapy of prostate cancer patients.

Androgen receptor expression predicts different clinical outcomes for breast cancer patients stratified by hormone receptor status.

In this study we sought to correlate androgen receptor (AR) expression with tumor progression and disease-free survival (DFS) in breast cancer patients. We investigated AR expression in 450 breast cancer patients. We found that breast cancers expressing the estrogen receptor (ER) are more likely to co-express AR compared to ER-negative cancers (56.0% versus 28.1%, P < 0.001). In addition, we found that AR expression is correlated with increased DFS in patients with luminal breast cancer (P < 0.001), and decreased DFS in TNBC (triple negative breast cancer, P = 0.014). In addition, patients with HR+ tumors (Hormone receptor positive tumors) expressing low levels of AR have the lowest DFS among all receptor combinations. We also propose a novel prognostic model using AR receptor status, BRCA1, and present data showing that our model is more predictive of disease free survival compared to the traditional TMN staging system.

Androgen receptor as a regulator of ZEB2 expression and its implications in epithelial-to-mesenchymal transition in prostate cancer.

Zinc finger E-box-binding protein 2 (ZEB2) is known to help mediate the epithelial-to-mesenchymal transition, and thereby it facilitates cancer metastasis. This study was initiated to explore whether ZEB2 expression differs in prostate cancer (PCa, n=7) and benign prostatic hyperplasia (BPH, n=7) tissues. In PCa tissues, the levels of both immunoreactive ZEB2 and androgen receptor (AR) were found to be significantly higher (P<0.05) when compared with BPH tissues. Co-regulation of AR and ZEB2 prompted us to investigate the role of androgenic stimuli in ZEB2 expression. ZEB2 expression was found to be significantly (P<0.05) upregulated after androgen stimulation and downregulated following AR silencing in LNCaP cells, an androgen-dependent PCa cell line. This finding suggested AR as a positive regulator of ZEB2 expression in androgen-dependent cells. Paradoxically, androgen-independent (AI) cell lines PC3 and DU145, known to possess low AR levels, showed significantly (P<0.05) higher expression of ZEB2 compared with LNCaP cells. Furthermore, forced expression of AR in PC3 (PC3-AR) and DU145 (DU-AR) cells led to reductions in ZEB2 expression, invasiveness, and migration. These cells also exhibited an increase in the levels of E-cadherin (a transcriptional target of ZEB2). Co-transfection of AR and ZEB2 cDNA constructs prevented the decline in invasiveness and migration to a significant extent. Additionally, ZEB2 downregulation was associated with an increase in miR200a/miR200b levels in PC3-AR cells and with a decrease in miR200a/miR200b levels in AR-silenced LNCaP cells. Thus, AR acts as a positive regulator of ZEB2 expression in androgen-dependent cells and as a negative regulator in AI PCa cells.

PTENgenomic deletion predicts prostate cancer recurrence and is associated with low AR expression and transcriptional activity

BackgroundProstate cancer (PCa), a leading cause of cancer death in North American men, displays a broad range of clinical outcome from relatively indolent to lethal metastatic disease. Several genomic alterations have been identified in PCa which may serve as predictors of progression. PTEN, (10q23.3), is a negative regulator of the phosphatidylinositol 3-kinase (PIK3)/AKT survival pathway and a tumor suppressor frequently deleted in PCa. The androgen receptor (AR) signalling pathway is known to play an important role in PCa and its blockade constitutes a commonly used treatment modality. In this study, we assessed the deletion status of PTEN along with AR expression levels in 43 primary PCa specimens with clinical follow-up.MethodsFluorescence In Situ Hybridization (FISH) was done on formalin fixed paraffin embedded (FFPE) PCa samples to examine the deletion status of PTEN. AR expression levels were determined using immunohistochemistry (IHC).ResultsUsing FISH, we found 18 cases of PTEN deletion. Kaplan-Meier analysis showed an association with disease recurrence (P=0.03). Concurrently, IHC staining for AR found significantly lower levels of AR expression within those tumors deleted for PTEN (P<0.05). To validate these observations we interrogated a copy number alteration and gene expression profiling dataset of 64 PCa samples, 17 of which were PTEN deleted. We confirmed the predictive value of PTEN deletion in disease recurrence (P=0.03). PTEN deletion was also linked to diminished expression of PTEN (P<0.01) and AR (P=0.02). Furthermore, gene set enrichment analysis revealed a diminished expression of genes downstream of AR signalling in PTEN deleted tumors.ConclusionsAltogether, our data suggest that PTEN deleted tumors expressing low levels of AR may represent a worse prognostic subset of PCa establishing a challenge for therapeutic management.

Human α2β1HI CD133+VE Epithelial Prostate Stem Cells Express Low Levels of Active Androgen Receptor

Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α2β1 HI CD133+VE), transiently amplifying (α2β1 HI CD133–VE) and terminally differentiated (α2β1 LOW CD133–VE) cells. AR transcript and protein expression was confirmed in α2β1 HI CD133+VE and CD133–VE progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α2β1 HI CD133+VE stem cells and 68% (±12%) in α2β1 HI CD133–VE transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α2β1 HI CD133+VE stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133+VE cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis.

Abstract C56: The combined loss of SSeCKS/Gravin/AKAP12 and Rb in the prostate promotes spontaneous lymph node metastases with basal-luminal transitional phenotypes

Abstract Introduction: Previous data indicated that the loss of SSeCKS/Gravin/AKAP12 (“SSeCKS”) or Rb correlates with prostate cancer (CaP) progression and that their deletion results in increased prostatic hyperplasia (1,2). SSeCKS re-expression in MAT-LyLu prostate cancer cells has little effect on the growth potential of primary-site tumors and on the ability of metastatic cells to colonize distal sites, yet it suppresses the growth of macrometastases by suppressing tumor-encoded vascular endothelial growth factor expression and neovascularization of the metastatic niche(3). We demonstrated recently that SSeCKS-null (Akap12−/−) mouse embryo fibroblasts (KO-MEF) suffer from an Rb-dependent premature senescence, and that the hyperplastic prostates in KO mice exhibit markers of increased senescence (4). Experimental Procedures: In order to determine whether SSeCKS and Rb act synergistically to promote CaP progression, KO mice were crossed to mice with prostate-specific conditional Rb deletion (Pb-Cre;Rbfl/fl). Results: Compared to controls (WT, Akap12−/− or Pb-Cre;Rbfl/fl mice), 100% of Akap12−/−;Pb-Cre;Rbfl/fl male mice showed evidence of low grade prostatic intraepithelial neoplasia (LG-PIN), with a smaller percentage (22%) displaying high grade (HG) PIN by 10 months of age. These lesions were marked by increased levels of cytoplasmic E-cadherin (compared to the cell-cell junctional staining in normal or hyperplastic luminal prostate layers) in LGPIN and by the loss of E-cadherin expression in HG-PIN. Unexpectedly, Akap12−/−;Pb-Cre;Rbfl/fl males showed increased numbers (1.8- to 2.5-fold) of p63-positive basal cells in all four lobes compared with control mouse prostates. Importantly, whereas control mice showed neither primary-site prostate cancer nor lymph node (LN) metastasis, &amp;gt;/80% of the Akap12−/−;Pb-Cre;Rbfl/fl males displayed metastases in pelvic or inguinal LN. These metastases did not stain for E-cadherin, but were positive for both basal (5 and 14) and luminal (8) cytokeratins, and low levels of androgen receptor. Foci within these lesions were positive for the basal marker, p63, and for the mesenchymal marker, vimentin. The LN lesions contained deletions of their floxed Rb loci, as shown by nested PCR assays, corroborating that they derived from the transgenic prostate cells. Conclusions: The combined loss of SSeCKS and Rb leads to early CaP initiation marked by increased basal cell proliferation in all four lobes, as well as the marked increase in LN metastasis of tumor cells with a basal-luminal transitional phenotype. These data strongly suggest that SSeCKS suppresses parameters of CaP initiation and metastasis. Citation Format: Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, Barbara Foster, Irwin H. Gelman. The combined loss of SSeCKS/Gravin/AKAP12 and Rb in the prostate promotes spontaneous lymph node metastases with basal-luminal transitional phenotypes [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C56.

Higher expression of androgen receptor is a significant predictor for better endocrine-responsiveness in estrogen receptor-positive breast cancers

The aim was to investigate the implications of androgen receptor (AR) expression levels on outcomes for estrogen receptor (ER)-positive tumors. Immunohistochemically AR levels were determined from tissue microarrays of 614 ER-positive patients who received adjuvant endocrine with or without chemotherapy between November 1999 and August 2005. Characteristics and survival were analyzed using a Chi-square test, Kaplan-Meier methods, and Cox's models. AR levels were categorized into 3 subgroups as follows: low, AR < 10%; intermediate, 10% ≤ AR < 50%; high, AR ≥ 50%. Low, intermediate, and high AR levels were observed in 29.0, 44.0, and 27.0% of patients, respectively. High AR was associated with smaller size, nodal uninvolvement, grade I/II tumor, higher progesterone receptor expression, and lower proliferation index. With a median follow-up of 70.9 months, the high AR subgroup showed better survival, and these associations were maintained in 119 patients who received endocrine therapy alone [hazard ratio (HR), 0.111; 95% CI, 0.013-0.961 for disease-free survival (DFS); HR, 0.135; 95% CI, 0.015-1.208 for overall survival (OS)]. No significant benefits from chemotherapy were demonstrated in the high AR subgroup; however, the benefit from chemotherapy was significant among 448 AR-intermediate or -low patients (HR, 2.679; 95% CI, 1.452-4.944 for DFS; HR, 3.371; 95% CI, 1.611-7.052 for OS). High AR is an independent prognostic factor and a significant predictor for better endocrine-responsiveness in ER-positive tumors. AR-low or -intermediate levels could give an additional indication for use of chemotherapy in ER-positive tumors.

Inhibition of the Acetyltransferases p300 and CBP Reveals a Targetable Function for p300 in the Survival and Invasion Pathways of Prostate Cancer Cell Lines

Inhibitors of histone deacetylases have been approved for clinical application in cancer treatment. On the other hand, histone acetyltransferase (HAT) inhibitors have been less extensively investigated for their potential use in cancer therapy. In prostate cancer, the HATs and coactivators p300 and CBP are upregulated and may induce transcription of androgen receptor (AR)-responsive genes, even in the absence or presence of low levels of AR. To discover a potential anticancer effect of p300/CBP inhibition, we used two different approaches: (i) downregulation of p300 and CBP by specific short interfering RNA (siRNA) and (ii) chemical inhibition of the acetyltransferase activity by a newly developed small molecule, C646. Knockdown of p300 by specific siRNA, but surprisingly not of CBP, led to an increase of caspase-dependent apoptosis involving both extrinsic and intrinsic cell death pathways in androgen-dependent and castration-resistant prostate cancer cells. Induction of apoptosis was mediated by several pathways including inhibition of AR function and decrease of the nuclear factor kappa B (NF-κB) subunit p65. Furthermore, cell invasion was decreased upon p300, but not CBP, depletion and was accompanied by lower matrix metalloproteinase (MMP)-2 and MMP-9 transcriptions. Thus, p300 and CBP have differential roles in the processes of survival and invasion of prostate cancer cells. Induction of apoptosis in prostate cancer cells was confirmed by the use of C646. This was substantiated by a decrease of AR function and downregulation of p65 impairing several NF-κB target genes. Taken together, these results suggest that p300 inhibition may be a promising approach for the development of new anticancer therapies.

Correlation of sex hormone and androgen receptor with diabetes mellitus in elderly men

Aim. To investigate sex hormone and androgen receptor (AR) levels and to evaluate their relationship with diabetes mellitus (DM) in senile men.Methods. The cross-sectional study included 492 elderly men comprising 104 healthy subjects (mean age 71.4 ± 5.2 years), 259 subjects without DM (71.5 ± 5.0 years) and 129 DM patients (73.0 ± 6.3 years). Plasma concentrations of total testosterone (TT), free testosterone (FT), dehydroepiandrosterone sulphate, sex hormone-binding globulin (SHBG), estradiol (E2), luteinising hormone) and follicle-stimulating hormone (FSH) were determined. AR-positive cells were measured by flow cytometry.Results. TT concentrations were significantly lower in the DM group (13.8 ± 4.7 nmol/l) than in the healthy (17.1 ± 6.1 nmol/l) and non-diabetes groups (15.8 ± 6.0 nmol/l; all P < 0.01). FT, SHBG, AR-positive proportion (AR%) and AR fluorescence intensity showed a decreasing trend among the healthy, non-DM and DM groups, but the differences were not significant. TT, E2, E2/testosterone and SHBG were negatively correlated with blood glucose. SHBG was positively correlated and TT and AR% were negatively correlated with the course of DM. Logistic multiple regression analysis revealed that age, waist/hip ratio, FSH, SHBG and AR% are potential risk factors for DM.Conclusions. Low levels of TT, SHBG and AR may be potential risk factors for DM in elderly men.

Abstract 1622: Inhibition of the acetyltransferase p300 as a novel pro-apoptotic and anti-invasion approach for treatment of prostate cancer

Abstract Inhibitors of histone deacetylases have been approved for clinical application in cancer treatment. On the other hand, histone acetyltransferase inhibitors have not been investigated for their potential use in cancer therapy. In prostate cancer the histone acetyltransferases and coactivators p300 and CBP are upregulated and induce transcription of androgen-receptor responsive genes, even in the absence or in the presence of low levels of androgen receptor. To discover a potential anti-cancer effect of p300/CBP inhibition we used two different approaches: i) downregulation of p300 and CBP by specific siRNA and ii) chemical inhibition of the acetyltransferase activity by a newly developed small molecule. Knockdown of p300 by specific siRNA, but surprisingly not of CBP led to an increase of caspase-dependent apoptosis involving both extrinsic and intrinsic cell death pathways in androgen-dependent and castration-resistant prostate cancer cells. Induction of apoptosis was mediated by several pathways including inhibition of androgen-receptor responsive genes and decrease of expression levels of the NF-κB subunit p65 due to a higher turnover of the protein. Furthermore, cell invasion was decreased upon p300, but not CBP depletion and was accompanied by a lower MMP-9 transcription. Thus, p300 and CBP have differential roles in the processes of survival and invasion of prostate cancer cells. Induction of apoptosis in prostate cancer cells was confirmed by the use of the cell permeable small molecule inhibitor of p300/CBP′s acetyltransferase domain. Taken together, those results suggest that p300 inhibition is a promising approach for the development of new anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1622. doi:10.1158/1538-7445.AM2011-1622

The androgen receptor drives the sex-specific expression of vascular cell adhesion molecule-1 in endothelial cells but not lipid metabolism genes in monocyte-derived macrophages.

Anecdotal evidence suggests that male sex hormones are proatherogenic. We hypothesized that the male sex hormone receptor, the androgen receptor (AR), acts as a molecular switch in sex-specific inflammatory signaling in vascular cells. AR expression in human umbilical vein endothelial cells (HUVECs), human monocyte-derived macrophages (MDMs) or HeLa cells was modulated by transfection with AR siRNA or human AR cDNA expression vector. Activity and expression levels were measured by luciferase reporter assays, Western blotting or real-time PCR analysis. AR knockdown reduced expression of vascular cell adhesion molecule-1 (VCAM-1) in genetically male HUVECs. Conversely, AR upregulation in genetically female HUVECs induced VCAM-1 expression and increased dihydrotestosterone-stimulated monocyte adhesion. Co-transfection of an AR expression vector with VCAM-1 or NF-κB-reporter vectors into phenotypically female, AR-negative HeLa cells confirmed AR regulation of VCAM-1 expression as well as AR activation of NF-κB. AR upregulation was not sufficient to increase ICAM-1 levels in female HUVECs or lipoprotein metabolism gene expression in female MDMs, despite AR knockdown limiting expression in their male counterparts. AR acts as a molecular switch to induce VCAM-1 expression. Low AR levels in female HUVECs limit NF-κB/VCAM-1 induction and monocyte adhesion and could contribute to the gender bias in cardiovascular disease. Unidentified factors in female cells limit induction of other proatherogenic genes not primarily regulated by NF-κB.

Sex Hormones and Androgen Receptor: Risk Factors of Coronary Heart Disease in Elderly Men

To investigate the variation of sex hormone and its receptor level in elderly male patients with coronary heart disease (CHD) and to evaluate the correlations between CHD and sex hormone as well as sex hormone receptor. Altogether 139 male CHD patients (CHD group) aged 60-92 years and 400 healthy men (control group) aged 60-90 years were included in this cross sectional study. The plasma concentrations of dehydroepiandrosterone sulfate (DHEAS), total testosterone (TT), free testosterone (FT), estradiol (E2), sex hormone binding globulin (SHBG), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured. The androgen receptor (AR) was tested by flow cytometry. Correlations between CHD and levels of sex hormones and AR were analyzed. Compared with the control group, the levels of DHEAS, TT, FT, SHBG, and the fluorescence intensity of AR in the CHD group significantly reduced (P < 0.05), while the levels of FSH and E2 significantly increased (P < 0.01). Age was negatively correlated with TT (r = -0.28, P = 0.00) and FT (r = -0.17, P = 0.01), while it was positively correlated with SHBG (r = 0.14, P = 0.04) and E2 (r = 0.33, P = 0.00). AR fluorescence intensity was negatively correlated with systolic blood pressure (r = -0.12, P = 0.01). Binary logistic regression analysis showed that TT, SHBG, and AR were all negatively correlated with CHD (P < 0.05). Elderly male patients with CHD are found to have low levels of DHEAS, TT, FT, SHBG, and AR, while high concentrations of E2 and FSH. Low levels of TT and SHBG may be the potential risk factors of CHD in elderly men.

P300 Regulates Androgen Receptor–Independent Expression of Prostate-Specific Antigen in Prostate Cancer Cells Treated Chronically with Interleukin-6

Prostate cancer is the most frequent non-skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor-dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6-treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.

Sea bass ( Dicentrarchus labrax) androgen receptor: cDNA cloning, tissue-specific expression, and mRNA levels during early development and sex differentiation

Androgens play key roles in vertebrate sex differentiation, gonadal maturation and reproductive behaviour and their actions are generally mediated through specific nuclear receptors. The present study describes the isolation, sequencing and characterization of the cDNA encoding the androgen receptor (AR) in the European sea bass. AR was cloned from a sea bass testis cDNA library and encoded a predicted protein of 767 residues, with a calculated molecular weight of 86.4 kDa and a theoretical p I of 6.34. Several domains present in all cloned ARs were identified. The domains corresponded to an amino-terminal hypervariable transcriptional activation domain (TAD), a central highly conserved DNA-binding domain (DBD), and a carboxy-terminal ligand-binding domain (LBD). Percentages of homology-similarity among these functional domains in teleost fish ranged between 9 and 75% for the TAD, 73 and 98% for the DBD, and 78 and 96% for the LBD when compared to those of the sea bass. Tissue-specific expression showed that AR was preferentially expressed in testis, ovaries, and brain. Some other tissues such as the head kidney, liver and spleen also showed AR expression although at very low levels. A semiquantitative PCR was developed to study the expression of AR mRNA during the period of development encompassed between 50 and 300 DPH in sea bass gonads. An experimental design, involving repeated size gradings, based on the fact that sea bass females are larger than males already at sex differentiation, was set to obtain a group consisting of the largest fish (female-dominant) and a group consisting of the smallest fish (male-dominant). The results showed very low mRNA expression levels of AR in the gonads during early development. Differences in AR expression between groups were first encountered at 150 DPH and became especially marked at 250 DPH with much higher levels in the male-dominant group. These sex-related differences in expression profiles between males and females by the time of sex differentiation, suggest an important role for AR controlling this process in the sea bass.