Androgen Receptor mRNA Research Articles

Abstract 503: MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer and third deadliest cancer in American men, yet the only established risk factors are familial genetics, age and race. Men of African Ancestry (moAA) have higher PCa incidence rates when compared to Caucasian men in the United States, which are due to differences in genetic susceptibility variants. Moreover, high mortality rates of PCa are associated with castration-resistant prostate cancer (CRPC) due to maintenance of androgen receptor (AR) signaling in PCa cells following androgen ablation therapy. The 8q24 chromosomal locus is a highly susceptible PCa region that has frequent amplifications of the c-MYC oncogene and downstream PVT1 gene. PVT1 is a long non-protein coding gene that encodes six annotated microRNAs (miRNAs), including microRNA-1205 (miR-1205), yet the function of these miRNAs are poorly understood. To elucidate the role of miR-1205 in PCa, we examined miR-1205 mRNA expression in a cohort of normal (n=22), benign (n=42), and malignant (n=26) histologically confirmed prostatic tissues obtained from prostatectomy or transrectal biopsies of moAA men in Ibadan, Nigeria. One-way ANOVA analysis determined changes in the relative expression of miR-1205 between groups (F(2,87) = 1.153) and a Tukey post hoc test revealed decreased miR-1205 expression in benign (4.61 ± 7.5) and malignant tumors (3.39 ± 3.53) when compared to normal tissues (6.55 ± 9.5). These data suggest that miR-1205 may function as a miRNA tumor suppressor and is characteristic to moAA-associated PCa. To elucidate the molecular mechanism of miR-1205, we examined AR and c-MYC mRNA expression using androgen-dependent LNCaP and CRPC C4-2B and 22RV1 cells. We observed a two-fold decrease of miR-1205 expression and overexpression of AR and c-MYC in C4-2B and 22RV1 cells when compared to LNCaP, suggesting that miR-1205 may regulate AR and c-MYC signaling in CRPC. Next, we identified Fry-like (FRYL) as a putative target using a miSVR computer algorithm and subsequently performed whole transcriptome analysis on prostate tumors and adjacent normal tissue from fourteen PCa patients using the Galaxy web platform. FRYL and AR overexpression was observed in diseased patients suggesting that FRYL may function as an oncogene. Moreover, FRYL was overexpressed in C4-2B and 22RV1 cells when compared to LNCaP, further suggesting a role in CRPC development. C4-2B cells transfected with miR-1205 and the 3'UTR of FRYL in a luciferase expressing vector revealed a significant decrease in luciferase activity when compared to control cells, indicating direct binding of miR-1205 to the 3'UTR of FRYL. These observations strongly suggest that miR-1205 acts as a tumor suppressor that may regulate AR and c-MYC expression, and directly targets the 3'UTR of FRYL in PCa cells. Further understanding the role of miR-1205 regulation of FRYL, AR, and c-MYC signaling may provide novel insights into the molecular mechanisms of CRPC. Citation Format: Michelle K. Naidoo, Dibash K. Das, Adeodat Ilboudo, Akintunde Orunmuyi, Gabriel O. Ogun, S. A. Adebayo, E. O. Olapade-Olaopa, Olorunseun Ogunwobi. MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 503.

Abstract 5904: Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression

Abstract Purpose Prostate cancer (CaP) driven by androgen receptor (AR) can be targeted therapeutically by androgen deprivation therapy (ADT); however, in 10-20% of cases, ADT fails, allowing disease recurrence. Mechanisms for recurrence include dysregulation of AR at the gene, RNA, and protein level. Recently, AR negative cell growth has been implicated as a mechanism of resistance. This study aims to identify mechanisms of drug resistance in castration-resistant prostate cancer (CRPC) that can be exploited therapeutically to reduce disease recurrence. Experimental Procedures Using the BCaP model, a novel series of cell lines derived from a single human prostate epithelial cell, we can study the progression of CaP from benign to metastasis. Additionally, using the LNCaP-C4 series we can study progression to CRPC. To determine the RNA binding capacity of DDX3, we utilized 1) RIP-qPRC for identification of mRNA targets and 2) RNAscope for visual colocalization. Changes in RNA and protein expression were determined using qPCR, Western blot, and IF. Results DDX3 is an ATP-dependent RNA helicase that can aid translation of target mRNAs, or in stress conditions, form ribonucleoprotein granules that prevent translation. While DDX3 is implicated in several cancers, its role as a translational regulator in CaP remains unstudied. In BCaP and LNCaP-C4, DDX3 protein expression increases through progression, concurrent with localization to cytoplasmic puncta. RIP-qPCR identified AR as an mRNA target of DDX3 in the metastatic and CRPC cell lines. Because of the dual role of DDX3 in translational control, AR expression in these models was investigated; while AR mRNA expression increased through progression, AR protein expression decreased. Treatments in vitro with RK33, a commercially available inhibitor of DDX3, restored the protein expression of AR and downstream AR signaling (PSA, Nkx3.1). These data suggest DDX3 acts as a translational repressor of AR in metastasis and CRPC. To confirm that increased DDX3 expression is sufficient to reduce AR protein expression, we overexpressed DDX3 using two models: induced cellular stress and a DDX3 expression vector. Here, we saw DDX3 localized to puncta, and subsequent reduction in AR and PSA protein. Because DDX3 is sufficient to reduce AR protein expression in metastases and CRPC, co-treatment of DDX3 inhibitors with anti-androgen therapy may prevent AR negative cell growth underlying recurrence. in vitro co-treatments with RK33 and bicalutamide, an AR antagonist, show decreased proliferation compared to either treatment alone, suggesting increased efficacy of bicalutamide with DDX3 inhibition. Conclusions DDX3 as a repressor of AR translation could have clinical implications as a mechanism of resistance to ADT. Based on preliminary data, DDX3 could be contributing to the regulation of AR post-transcriptionally, and targeting DDX3 could reduce resistance and disease recurrence. Citation Format: Jordan E. Vellky, William Ricke. Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5904.

Abstract 4373: AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells

Abstract The transcriptional coactivator YAP1 is a key nuclear effector of the Hippo pathway. STK4/MST1 and LATS1/2 are the core kinase components of the Hippo pathway, which restricts organ size and prevents tumorigenesis by attenuating cell proliferation and inducing cell death. Previously, we have reported that STK4/Hippo-YAP1 signaling could play a critical role in prostate cancer progression and therapeutic relapse. Here, we investigated the effects of androgen hormone signaling on YAP1 expression and post-transcriptional modifications in prostate cancer cells. We demonstrated that androgen exposure reduced Ser127 phosphorylation on YAP1 in a time-dependent manner in the castration-sensitive prostate cancer cell line, LNCaP, but without altering the levels of YAP1-Ser127 phosphorylation in the C4-2 cell line, a castration-resistant subline of LNCaP cells. As demonstrated by imaging and cell fractionation methods, androgen exposure promoted the nuclear accumulation of YAP1 in LNCaP; however, regardless of androgen exposure, YAP1 was accumulated in the nucleus of C4-2 cells. In addition, we demonstrated that androgen exposure reduced YAP1-Ser127 phosphorylation that was induced by okadaic acid, a potent activator of Ser/Thr phosphatases PP1 and PP2A. Also, we demonstrated that androgen exposure increased PP2A/B protein expression. Moreover, reduction of phospho-Ser127-YAP1 was correlated with increases in total YAP1 protein, which coincided with androgen receptor (AR) nuclear accumulation by androgen. Consistent with these observation, the genetic (siRNA) or the pharmacologic (enzalutamide) inhibition of AR signaling attenuated the expression of YAP1 protein. Furthermore, our analysis of the publicly available TCGA (The Cancer Genome Atlas) set indicated that YAP1 and AR mRNA expressions were positively correlated in prostate clinical samples. These observations suggest that YAP1 is a direct target of androgen hormone signaling, implicating that the AR-PP2A/B-YAP1 axis is a viable cancer drug target. Citation Format: Bekir Cinar, Carlos S. Moreno. AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4373.

Abstract 3946: Androgen receptor drives differential gene expression in KRAS-mediated non-small cell lung cancer

Abstract The Cancer Genome Atlas (TCGA) has identified alterations such as amplification, deletion, or mutation of androgen receptor (AR) in about 5% of human lung squamous cell carcinoma and adenocarcinoma. The expression of AR in human lung appears to play a crucial role in lung development and type II pneumocytes (PTII) maturation, but unlike in prostate cancer, the role of AR is not well known in non-small cell lung cancer (NSCLC). In our study, we demonstrated AR in NSCLC cell lines translocated into the nucleus over time when stimulated with synthetic androgen R1881, while addition of enzalutamide (MDV3100) or AR siRNA reduced AR nuclear localization. Using an NSCLC human tissue microarray also revealed that 10 out of 88 patients (11%) have positive AR immunohistochemical staining. Preliminary data from clonogenic assay indicated that enzalutamide might have radiosensitizing effect on certain NSCLC cells at 2 Gy, further suggesting the involvement of AR in lung cancer and its potential therapeutic value as a target. With the induction of R1881 for 30 minutes to 24 hours, certain NSCLC cells display decreased AR mRNA (0.5-0.8 fold), while others show modest increase (up to 1.5 fold), suggesting that AR regulation in these cells might be different due to their mutational landscapes. We then used predesigned 384 well panels from Bio-Rad to survey the effects of R1881, enzalutamide, and AR siRNA on mRNA expression levels of selected NSCLC cells. Distinct expression profiles were observed between cells that have wild-type and mutated KRAS. Taken together, these findings suggest that the AR signaling could be different based on KRAS mutational profiles of NSCLCs, and further work is required to reveal the underlying mechanism. Chromatin immunoprecipitation (ChIP) assay and RNA sequencing will be utilized to investigate AR interactions with androgen response elements and differential expressions in KRAS-driven NSCLC cells, respectively. Citation Format: Albert Roy Wang, Hope Beyer, Sean Brennan, Shannon Stiles, Dylan Wiese, Darya Buehler, Anwaar Saeed, Andrew M. Baschnagel, Gopal Iyer. Androgen receptor drives differential gene expression in KRAS-mediated non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3946.

Abstract PR02: β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor

Abstract Background: Androgen receptor (AR) has emerged as a new target for treating TNBC. AR is expressed in 10-43% of TNBCs. Although there are conflicting reports in the literature about the effect of AR status on TNBC prognosis, agents targeting AR signaling (enzalutamide) are already being evaluated in AR-positive TNBCs in early-stage clinical trials. However, no study so far has evaluated the association/correlation of AR status with ethnicity in TNBCs and downstream effects of AR loss in TNBCs. Given the association of AR loss with poor prognosis in breast cancer and that the African American (AA) with TNBC suffers aggressive disease course when compared to European American (EA) TNBCs, we hypothesized that AR loss might be an underlying cause of aggressive disease course in AR-negative TNBCs. Thus, in this project we aimed to study if loss or gain of AR in AA and EA TNBCs regulates the expression of β-catenin and leads to more aggressive disease course by activating downstream canonical Wnt-beta catenin signaling. Methods: We evaluated AR expression immunohistochemically in 424 formalin-fixed, paraffin-embedded samples from TNBC patients for whom complete clinicopathologic and overall survival (OS) data were available. Samples with <1% nuclear-stained cells were considered quadruple-negative. We also performed gene set enrichment analysis for the AR low and high group in AA and EA TNBCs using the publicly available TCGA gene dataset. Finally, we compared levels of 187 proteins with suspected involvement in breast tumorigenesis in AR-high vs AR-low TNBCs (using median AR expression as a cutpoint, as the IHC-derived AR status of these samples was unknown). Results: IHC staining of AR indicated that 79.5% of AA TNBCs (n=214) and 70% of EA TNBCs (n=210) were AR negative. Loss of AR was associated with poor overall survival in adjuvant-treated high Ki67 (>14%) (HR=1.72; p=0.095) AA TNBC (n=98) when compared to EA TNBCs (n=80). These data were validated by our in silico findings, which suggested that EA TNBCs (n=81) exhibited higher levels of AR mRNA compared to AA TNBCs (n=41) (p<0.05). Similarly, AR protein expression was higher in EA TNBCs (n=75) than AA TNBCs (n=32) (p<0.05). We further observed that β-catenin protein levels are higher in AA AR-low TNBCs compared with AA AR-high TNBCs (median AR expression used as cut point; p<0.05). This was strengthened by our observation in a cohort of 142 TNBCs wherein AAs with AR-negative TNBC showed a preponderance of cells with nuclear β-catenin staining via IHC compared with EA TNBCs that lack AR. Our GSEA analysis results indicated that Wnt/β-catenin signaling was the top-enriched gene ontology in the AR-low subgroup compared to AR-high subgroup of TNBCs. Furthermore, proteomic data revealed that β-catenin and Dvl3 expression was significantly upregulated in the AR-low subgroup when compared with AR-high group, suggesting that Wnt signaling is in an overdrive in the AR-low subgroup, especially in AA TNBCs. Conclusion: This study suggests that increased expression of β-catenin coupled with AR loss in AAs may underlie the ethnic disparity in outcomes among TNBC patients and strongly supports the prognostic role of AR and β-catenin in this breast cancer subtype. Citation Format: Karuna Mittal, Shristi Bhattarai, Sergey Klimov, Uma Krishnamurthi, Xiaoxian Li, Ceyda Sonmez Wetherilt, Mohammad A. Aleskandaran, Andrew A. Green, Emad A. Rakha, Ian O. Ellis, Guilherme Cantuaria, Guanhao Wei, Remus Mihai Osan, Meenakshi V. Gupta, Upender Manne, Padmashree C.G Rida, Ritu Aneja. β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR02.

Androgen receptor induces EPHA3 expression by interacting with transcription factor SP1.

Erythropoietin‑producing hepatocellular carcinoma cell surface type‑A receptor3 (EPHA3) has been found to promote the proliferation and survival of prostate cancer (PCa) cell lines and prostate tumor development in nude mice. However, the regulation of EPHA3 in PCa remains largely unknown. This study is aimed to investigate the association between EPHA3 expression and androgen receptor (AR) signaling and the potential mechanism. We determined mRNA and protein levels of EPHA3 and AR signaling‑related genes in the PCa cell line 22Rv1 by reverse transcription‑polymerase chain reaction (RT‑PCR) and western blotting, respectively. The EPHA3 mRNA and protein levels were both found to be elevated by dihydrotestosterone (DHT) hormone in a dose‑ and time‑dependent manner, as AR and prostate‑specific antigen (PSA) expression were increased. Similarly, EPHA3 protein levels were also increased in the PCa cell line LNCaP stimulated with DHT or mibolerone (Mib). Overexpression of pEGFP‑AR in 22Rv1 cells significantly increased the EphA3 level, while AR knockdown with small interfering RNA (siRNA) for AR (siAR) markedly decreased the expression of EPHA3. The key EPHA3 promoter region associated with AR regulation was evaluated by co‑transfection of various pGL3‑basic‑luciferase reporter plasmids, containing EPHA3 core promoter fragments differing in length, with the AR plasmid or siAR into 22Rv1 cells. AR overexpression in 22Rvl cells raised the EphA3 promoter transcription activity of pGL3‑EPHA3‑Luc (EPHA3‑Luc)‑789, and vice versa. Similarly, luciferase activity of EPHA3‑Luc‑317 was also clearly affected. However, truncated EPHA3‑Luc‑237 without the transcription factor specific protein 1 (SP1) binding sites or EPHA3‑Luc‑789ΔSP1 with modified SP1 binding sites clearly decreased EPHA3 promoter activity regardless of whether AR was overexpressed or blocked. Treatment of 22Rv1 cells with 10 and 100nM of the SP1 inhibitor mithramycinA for 24 and 48h significantly reduced EPHA3 mRNA and protein levels. Additionally, selective inhibition of SP1 with siRNA SP1 (siSP1) at various concentration from 25 to 75nM, reduced the EPHA3 protein level in PCa LNCaP cells, accordingly. Co‑immunoprecipitation (co‑IP) and chromatin IP (ChIP) assays were performed to determine whether AR forms a transcription factor complex with Sp1 that binds the EPHA3 core promoter region to sense androgen induction. The result suggests that the interaction of AR and SP1 contributes to regulate EPHA3 expression, and the SP1 binding sites (‑295~‑261) in the EPHA3 core promoter region is crucial to the regulation of EPHA3 expression in response to androgen hormone stimuli.

AR negative triple negative or “quadruple negative” breast cancers in African American women have an enriched basal and immune signature

There is increasing evidence that Androgen Receptor (AR) expression has prognostic usefulness in Triple negative breast cancer (TNBC), where tumors that lack AR expression are considered “Quadruple negative” Breast Cancers (“QNBC”). However, a comprehensive analysis of AR expression within all breast cancer subtypes or stratified by race has not been reported. We assessed AR mRNA expression in 925 tumors from The Cancer Genome Atlas (TCGA), and 136 tumors in 2 confirmation sets. AR protein expression was determined by immunohistochemistry in 197 tumors from a multi-institutional cohort, for a total of 1258 patients analyzed. Cox hazard ratios were used to determine correlations to PAM50 breast cancer subtypes, and TNBC subtypes. Overall, AR-negative patients are diagnosed at a younger age compared to AR-positive patients, with the average age of AA AR-negative patients being, 49. AA breast tumors express AR at lower rates compared to Whites, independent of ER and PR expression (p<0.0001). AR-negative patients have a (66.60; 95% CI, 32–146) odds ratio of being basal-like compared to other PAM50 subtypes, and this is associated with an increased time to progression and decreased overall survival. AA “QNBC” patients predominately demonstrated BL1, BL2 and IM subtypes, with differential expression of E2F1, NFKBIL2, CCL2, TGFB3, CEBPB, PDK1, IL12RB2, IL2RA, and SOS1 genes compared to white patients. Immune checkpoint inhibitors PD-1, PD-L1, and CTLA-4 were significantly upregulated in both overall “QNBC” and AA “QNBC” patients as well. Thus, AR could be used as a prognostic marker for breast cancer, particularly in AA “QNBC” patients.

Characterization of novel cell lines derived from a MYC-driven murine model of lethal metastatic adenocarcinoma of the prostate.

Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer. We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively. Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection. These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Androgen receptor expression in circulating tumor cells of metastatic breast cancer patients.

1087Background: The androgen receptor (AR) is of clinical relevance in metastatic breast cancer (mBC): AR has been associated with resistance to endocrine therapy and could be a potential target for therapy, especially in the triple negative (TN) subtype. A minimal-invasive way to determine AR expression is by characterization of circulating tumor cells (CTCs). We therefore assessed AR mRNA expression in CTCs (CTC-AR) from mBC patients representing different breast cancer subtypes in relation to outcome on endocrine therapy and 25 genes related to the ER and AR pathways. Furthermore, we assessed AR in matched primary tumors and CTC samples taken at advanced disease. Methods: AR and AR- and ER-related gene expression levels were measured in CellSearch-enriched CTCs from 133 mBC patients with ≥5 CTCs and in 48 matched formalin-fixed paraffin embedded primary tissues using quantitative reverse-transcriptase PCR. AR was considered positive if the expression was 1 standard deviation higher than the expression ...

Gene Expression Analysis of Immunomagnetically Enriched Circulating Tumor Cell Fraction in Castration-Resistant Prostate Cancer.

Molecular characterization of tumors could be a key to therapeutic decision-making with regards to targeted therapies in castration-resistant prostate cancer (CRPC). A convenient solution may be non-invasive liquid biopsy testing of circulating tumor cells (CTCs). For this reason, CTC-enriched samples obtained by immunomagnetic separation (AdnaTest®) were studied as a source material for high-throughput gene expression analysis using BioMark™. CTC-enriched samples from 41 CRPC patients previously determined to be CTC positive using the AdnaTest® were retrospectively re-analysed for androgen receptor (AR) messenger RNA (mRNA), using the updated AdnaTest®. Blood samples were drawn two times from each patient: at the time of CRPC diagnosis and after the third docetaxel cycle. A gene expression panel of 27 genes related to CRPC therapeutic decision-making, including AR full length (ARFL) and splice variant 7 (ARV7), was retrospectively analyzed on a BioMark™ platform in 29 of 41 patients. The AdnaTest® detected AR mRNA in three-quarters of CTC-positive samples taken at the time of CRPC diagnosis and after the third docetaxel cycle. AR detection was associated with a shorter disease-specific survival (45.0 vs. 20.4months) at the time of CRPC diagnosis. ARFL expression at the time of CRPC diagnosis, measured on the BioMark™ platform, was associated with a lower decrease of serum level of prostate-specific antigen (sPSA) (p = 0.029), i.e., worse therapy response. ARV7 was found in 38% of the ARFL--positive samples at both analyzed timepoints. Detection of AR expression by AdnaTest® in CTC-enriched samples may help predict patients' survival. These AdnaTest® CTC-enriched samples can be used in a high-throughput quantitative polymerase chain reaction (qPCR) analysis of gene expression, provided that the specificity of the assay for each individual gene is properly validated. The BioMark™ platform can be used for the simultaneous detection of ARFL and ARV7 and other genes in CTC-enriched samples from CRPC patients.

Soy Protein Supplementation Is Not Adipogenic Or Estrogenic In Young Men When Combined With Resistance Training

PURPOSE: Sex hormone physiology (e.g., estradiol, testosterone) may be affected by soy and/or whey protein consumption. Alterations in sex hormones due to resistance training (RT) and/or protein supplementation may explain meaningful variation in adipocyte and skeletal myocyte size alterations. Consequential molecular signaling in these cell types remain unclear. Therefore, we examined effects of RT and soy (SPC), whey (WPC), or placebo (PLA) supplementation in young men. METHODS: 47 healthy, young men were partitioned into PLA, SPC, or WPC groups and completed 12 weeks of RT. Body composition, serum hormones, androgen signaling markers in myocytes, and estrogen signaling markers in adipocytes were examined using DXA, ELISA, western blotting, PCR, and immunohistochemistry. RESULTS: Testosterone increased over time, but more so in subjects consuming WPC (p<0.05). Adipocyte mRNA expression of the estrogen receptor alpha increased (p<0.05), as did hormone sensitive lipase over time (p<0.05). Skeletal muscle androgen receptor mRNA expression increased while ornithine decarboxylase mRNA decreased over time (p<0.05). Alterations in body composition, adipocyte, and myocyte morphology were not significantly different between groups (p>0.05). Changes in 17β-estradiol and testosterone explained <3% of alterations in adipocyte and myocyte size. CONCLUSIONS: These data suggest primarily RT-mediated effects with little influence of protein type and hormonal changes.

The Expression of Androgen Receptor and Its Variants in Human Prostate Cancer Tissue according to Disease Status, and Its Prognostic Significance.

PurposeTo evaluate changes in the expression of androgen receptor (AR) and its variants (ARVs) in human prostate cancer (PCa) tissues according to disease status, and its prognostic significance following radical prostatectomy (RP).Materials and MethodsA total of 282 PCa cases were evaluated, which included 252 localized PCa, 8 metastatic castration resistant prostate cancer (CRPC), and 22 benign prostatic hyperplasia (BPH) cases. Samples were collected from patients who underwent RP or transurethral resection and were stored in ethically approved tissue banks. Quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry were performed for AR and ARVs. Each tissue was confirmed as cancerous (greater than 80%) using hematoxylin and eosin staining. AR and ARVs expression was compared according to disease status. The biochemical recurrence free survival (BCRFS) rates in men with localized PCa was analyzed according to AR and ARV7 expression using the Kaplan-Meier curve.ResultsOnly 58 of the 252 localized PCa were included in the analysis because of insufficient cancer tissue. AR and ARV7 mRNA expression was higher in the CRPC tissue than in the localized PCa tissue (p=0.025, p=0.002, respectively). In localized PCa tissue, high AR mRNA and protein level was associated with a low BCRFS rate (log-ranked, p=0.019, p<0.001, respectively).ConclusionsOverall AR and ARV7 mRNA expression levels were increased in CRPC tissues compared to localized PCa and BPH tissues. High AR protein and mRNA expression in the tumor tissue may be considered a predictive factor of BCRFS following RP.

Prolactin Induced Protein (PIP) is a potential biomarker for early stage and malignant breast cancer

ObjectivesBreast cancer (BC) is the second leading cause of cancer-related mortality in women. Bioinformatic analysis and expression screening showed that Prolactin Induced Protein (PIP) was differentially expressed in BC. The objective of this investigation was to characterize the expression pattern of PIP, an aspartyl proteinase, in malignant and non-malignant breast tissues. Materials and MethodsReal time quantitative PCR was employed to analyze PIP and androgen receptor (AR) mRNA levels in BC cell lines and 190 normal tissues and tumor samples. The tumor specimens were categorized based on TNM classification, anatomic stage, histologic grade and molecular subtype and expression pattern evaluated. To detect protein levels, immunohistochemistry followed by semi quantitative scoring was employed in the examination of 517 normal, benign, and invasive BC tissues. ResultsWe observed substantial downregulation of PIP transcription in cancer samples compared to normal breast tissue. mRNA levels were significantly downregulated (93 fold, P < 0.005) in advanced grades compared to lower grades. Transcript levels were also significantly lower (22 fold, P < 0.05) in triple negative tumors compared to hormone receptor positive tumors. Significant downregulation was observed in early stage samples of triple negative and hormone receptor positive tumors. Though PIP protein showed a wide range of expression levels in BC, early stage samples showed significant downregulation. ConclusionsPIP mRNA is significantly downregulated in early stage BC compared to normal breast tissue. Consequently, low PIP mRNA expression in BC tissues could potentially be used as a tissue based biomarker to assist pathologists in confirmation of early stage BC diagnosis.

Digital Circulating Tumor Cell Analyses for Prostate Cancer Precision Oncology.

<b/> In this issue of Cancer Discovery, Miyamoto and colleagues adapted their microfluidic CTC-iChip isolation platform with a digital RNA-PCR readout for eight prostate-specific transcripts and two assays for the androgen receptor mRNA splice variant ARV7 and the TMPRSS2-ERG translocation transcript. In patients with metastatic castrate-resistant prostate cancer at initiating abiraterone therapy in a first-line setting, the resulting RNA-based digital circulating tumor cell signatures identified patients with a shorter overall survival, and in patients with clinically localized disease, the signatures identified those with seminal vesicle invasion and pelvic lymph node involvement. Cancer Discov; 8(3); 269-71. ©2018 AACR.See related article by Miyamoto et al., p. 288.

Expression of Progesterone and Androgen Receptors in the Breast of Premenopausal Women, Considering Menstrual Phase.

Progesterone and androgens are important for normal development and tumorigenesis of the breast. Breast tissue samples from 49 premenopausal women were obtained. The progesterone receptors (PRA, PRB, PGRMC1 and PGRMC2) and the androgen receptor (AR) were determined in malignant and benign breast tumors and control tissues. The PRB and AR mRNA levels were highest in tumors. PGRMC1 and PGRMC2 mRNA levels were higher in malignant tumors compared to their paired normal tissues. PRA protein showed most immunostaining in benign tumors. PRB immunostaining varied according to menstrual phase. AR immunostaining was highest in the glands of malignant tumors. Progesterone and androgen receptors are differently regulated in tumors compared to normal breast tissues. A malignant breast tumor could appear PR-negative if collected in the luteal phase, but positive in the follicular phase. This finding may have clinical implications.

Combined treatment with melatonin and insulin improves glycemic control, white adipose tissue metabolism and reproductive axis of diabetic male rats

AimsMelatonin treatment has been reported to be capable of ameliorating metabolic diabetes-related abnormalities but also to cause hypogonadism in rats. We investigated whether the combined treatment with melatonin and insulin can improve insulin resistance and other metabolic disorders in rats with streptozotocin-induced diabetes during neonatal period and the repercussion of this treatment on the hypothalamic-pituitary-gonadal axis. Main methodsAt the fourth week of age, diabetic animals started an 8-wk treatment with only melatonin (0.2 mg/kg body weight) added to drinking water at night or associated with insulin (NHP, 1.5 U/100 g/day) or only insulin. Animals were then euthanized, and the subcutaneous (SC), epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Hypothalamus was collected for gene expression and blood samples were collected for biochemical assays. Key findingsThe treatment with melatonin plus insulin (MI) was capable of maintaining glycemic control. In epididymal (EP) and subcutaneous (SC) adipocytes, the melatonin plus insulin (MI) treatment group recovered the insulin responsiveness. In the hypothalamus, melatonin treatment alone promoted a significant reduction in kisspeptin-1, neurokinin B and androgen receptor mRNA levels, in relation to control group. SignificanceCombined treatment with melatonin and insulin promoted a better glycemic control, improving insulin sensitivity in white adipose tissue (WAT). Indeed, melatonin treatment reduced hypothalamic genes related to reproductive function.

Association of androgen receptor (AR) copy number gain with ARV7 expression and response to chemotherapy.

180 Background: Emerging data suggests that ARV7 mRNA (ARV7+) expression and AR copy number gain/amplification (AR+) are associated with resistance to androgen deprivation therapy. Most studies thus far have evaluated either AR copy number or ARV7 mRNA, but not both markers. It is likely that both markers are indicators of genomic changes that are seen in the prostate cancer cells in response to tumour evolution or treatment. Here we developed a digital droplet PCR (ddPCR) based assay to evaluate plasma ARV7 mRNA expression in the same samples used for AR copy number analysis in castrate resistant prostate cancer (CRPC). This assay was use to evaluate the role of AR+ and ARV7+ in CRPC using samples collected from multiple centers and correlated with outcome. Methods: We collected plasma samples from patients with CRPC treated with chemotherapy (C) or next generation androgen targeted agents (NAR) to evaluate circulating tumour DNA (ctDNA) for AR and plasma mRNA for ARV7. Some patients had sequential samples and we correlated PSA response (reduction PSA≥50 confirmed 3wks later (RR)) with AR and ARV7 status and clinical features. Results: A total of 52 samples were included from 26 patients, with matched samples from some patients at different time points. Of the CRPC patients, AR+ was seen in 70% (18/26), AR- in 30% (8/26), ARV7+ in 67% (12/18) and ARV7- in 33% (6/18). 75% (9/12) patients with AR+ also had ARV7+. In matched samples: 3/7 had change in AR status, 2/7 had change in ARV7 status, including 1 patient where both changed; and interestingly there was a group with no AR or ARV7 detected. Number of patients expressing both markers AR+/ARV7+ in 50% (9/18), AR+/ARV7- in 17% (3/18), AR-/ARV7+ 17% (3/18) and AR-/ARV7- in 11% (2/18). RR to C or NAR was 63% (12/19) for AR+ and 75% (9/12) for ARV7+ patients. In AR+ patients, response to NAR was 58% (12/16) but response to chemotherapy was 100% (5/5). Conclusions: AR gain/amplification in CRPC was associated with ARV7+ expression and leads to lower response to NAR compared to C. This approach can be used to undertake integrated DNA and mRNA analysis, has wide-scale utility and may be used for analysis of a larger number of circulating biomarkers.

AR-V7 Androgen Receptor Variant as a Predictor of Response to Androgen-receptor Targeting Agents Used to Treat Castration-refractory Metastatic Prostate Cancer

Several systemic treatment options are currently available for patients with metastatic castration-refractory prostate cancer (mCRPC), including the androgen-receptor targeting agents (ARTA) enzalutamide and abiraterone, the taxanes docetaxel and cabazitaxel, and the radioisotope drug 223-radium dichloride. In some patients with mCRCP, alternative splicing of androgen receptor (AR) mRNA occurs, resulting in the formation of a truncated AR lacking the androgen-binding domain. These receptors activate downstream signalling pathways even without the ligand. Recent studies show that the presence of the AR-V7 (ARV - AR variants) splicing variant is associated with resistance to ARTA. Bec>ause the presence of AR-V7 does not affect the efficacy of other systemic therapies used in mCRCPs, particularly taxanes, AR-V7 is a candidate predictive biomarker for the individualisation of mCRCP treatment. Two types of assays based on mRNA or abnormal protein detection are used to detect AR-V7 in circulating tumour cells. To describe the current status of AR-V7 testing in mCRPC and possible applications of this method for predicting outcomes of ARTA therapy. The percentage of CTC AR-V7+ in ARTA-naive men is relatively low at baseline, but in patients pretreated with ARTA, the prevalence of AR-V7 increases to 19-34%. Given the relatively high expected prevalence, AR-V7 testing may be economically feasible in this population. The proportion of AR-V7+ patients responding to ARTA retreatment appears to be very low, at only 4.8%. AR-V7 testing could thus be useful if an ARTA switch is considered in a patient progressing onto an ARTA drug. Both protein-based tests and mRNA-based tests are currently undergoing clinical validation in prospective studies, with results expected within a year.Key words: prostate cancer - abiraterone - enzalutamide - alternative splicing - drug resistanceSubmitted: 30. 8. 2017Accepted: 5. 11. 2017 doc. MUDr. Tomáš Büchler, Ph.D. received honorary lectures and publications from Astellas and Janssen and a travel grant from Janssen. Supported by Ministry of Health, Czech Republic - conceptual development of research organization Thomayer Hospital - TN 0064190. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Differential research of inflammatory and related mediators in BPH, histological prostatitis and PCa.

Prostate cancer (PCa) is one of the most common male malignancies in the world. It was aimed to investigate differential expression of inflammatory and related factors in benign prostatic hyperplasia (BPH), prostate cancer (PCa), histological prostatitis (HP) and explore the role of Inducible nitric oxide synthase (iNOS), (VEGF) Vascular endothelial growth factor, androgen receptor (AR) and IL-2, IL-8 and TNF-α in the occurrence and development of prostate cancer. RT-PCR was used to detect the mRNA expression level of iNOS, VEGF, AR and IL-2, IL-8 and TNF-α in BPH, PCa and BPH+HP. Western blotting and immunohistochemical staining were used to detect the protein levels of various proteins in three diseases. The results showed the mRNA and protein levels of iNOS, VEGF and IL-2, IL-8 and TNF-α were significantly increased in PCa and BPH+HP groups compared with BPH group (p<.05), while the AR was significantly lower than those in PCa and BPH+HP groups (p<.05). There was no significant difference in the mRNA and protein levels of iNOS, VEGF, AR and IL-2, IL-8 and TNF-α between PCa and BPH+HP groups (p>.05). iNOS, VEGF, AR and IL-2, IL-8 and TNF-α are involved in the malignant transformation of prostate tissue and play an important role in the development and progression of Prostate cancer (PCa).

Diminished androgen and estrogen receptors and aromatase levels in hypogonadal diabetic men: reversal with testosterone.

One-third of males with type 2 diabetes (T2DM) have hypogonadism, characterized by low total and free testosterone concentrations. We hypothesized that this condition is associated with a compensatory increase in the expression of androgen receptors (AR) and that testosterone replacement reverses these changes. We also measured estrogen receptor and aromatase expression. This is a randomized double-blind placebo-controlled trial. Thirty-two hypogonadal and 32 eugonadal men with T2DM were recruited. Hypogonadal men were randomized to receive intramuscular testosterone or saline every 2 weeks for 22 weeks. We measured AR, ERα and aromatase expression in peripheral blood mononuclear cells (MNC), adipose tissue and skeletal muscle in hypogonadal and eugonadal males with T2DM at baseline and after 22 weeks of treatment in those with hypogonadism. The mRNA expression of AR, ERα (ESR1) and aromatase in adipose tissue from hypogonadal men was significantly lower as compared to eugonadal men, and it increased significantly to levels comparable to those in eugonadal patients with T2DM following testosterone treatment. AR mRNA expression was also significantly lower in MNC from hypogonadal patients compared to eugonadal T2DM patients. Testosterone administration in hypogonadal patients also restored AR mRNA and nuclear extract protein levels from MNC to that in eugonadal patients. In the skeletal muscle, AR mRNA and protein expression are lower in men with hypogonadism. Testosterone treatment restored AR expression levels to that comparable to levels in eugonadal men. We conclude that, contrary to our hypothesis, the expression of AR, ERα and aromatase is significantly diminished in hypogonadal men as compared to eugonadal men with type 2 diabetes. Following testosterone replacement, there is a reversal of these deficits.