Prostate Androgen Research Articles

LncRNA PART1 Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating TFAP2C/DUSP5 Axis via miR-302a-3p.

Myocardial ischemia-reperfusion injury (MIRI) refers to the damage of cardiac function caused by restoration of blood flow perfusion in ischemic myocardium. However, long non-coding RNA prostate androgen regulated transcript 1 (PART1)'s role in MIRI remain unclear. Immunofluorescence detected LC3 expression. Intermolecular relationships were verified by dual luciferase reporter assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and transferase-mediated dUTP nick-end labeling (TUNEL) assays analyzed cell viability and apoptosis. The release of lactate dehydrogenase was tested via enzyme-linked immunosorbent assay (ELISA). Left anterior descending coronary artery surgery induced a MIRI mouse model. Infarct area was detected by 2,3,5-triphenyltetrazolium chloride staining. Hematoxylin and eosin staining examined myocardial injury. ELISA evaluated myocardial marker (creatine kinase MB) level. PART1 was decreased in hypoxia/reoxygenation (H/R) induced AC16 cells and MIRI mice. PART1 upregulation attenuated the increased levels of Bax, beclin-1 and the ratio of LC3II/I, and enhanced the decrease of Bcl-2 and p62 expression in H/R-treated cells. PART1 upregulation alleviated H/R-triggered autophagy and apoptosis via miR-302a-3p. Mechanically, PART1 targeted miR-302a-3p to upregulate transcription factor activating enhancer-binding protein 2C (TFAP2C). TFAP2C silencing reversed the protected effects of miR-302a-3p inhibitor on H/R treated AC16 cells. We further established TFAP2C combined to dual-specificity phosphatase 5 (DUSP5) promoter and activated DUSP5. TFAP2C upregulation suppressed H/R-stimulated autophagy and apoptosis through upregulating DUSP5. Overexpressed PART1 reduced myocardial infarction area and attenuated MIRI in mice. PART1 improved the autophagy and apoptosis in H/R-exposed AC16 cells through miR-302a-3p/TFAP2C/DUSP5 axis, which might provide novel targets for MIRI treatment.

Mining the ZINC database of natural products for specific, testosterone-like, OXER1 antagonists

OXER1, the receptor for the oxidized arachidonic acid metabolite 5-oxo-ETE has been reported to play a significant role in inflammatory responses, being responsible for leucocyte chemotactic responses. Recently, we have identified OXER1 (GPR170) as a membrane receptor for androgens in prostate and breast cancer cells. Testosterone action via OXER1 induces specific Ca2+ release from intracellular organelles, modifies polymerized actin distribution induces apoptosis and decreases cancer cell migration. These actions are antagonized by 5-oxo-ETE. In addition, 5-oxo-ETE through a Gαi protein decreases cAMP, an action antagonized by testosterone. In this work, we mined the ZINC15 database, using QSAR, for natural compounds able to signal through Gαi and Gβγ simultaneously, mimicking testosterone actions, as well as for specific Gβγ interactors, inhibiting 5-oxo-ETE tumor promoting actions. We were able to identify four druggable Gαβγ and seven Gβγ specific OXER1 interactors. We further confirmed by bio-informatic methods their binding to the 5-oxo-ETE/testosterone binding groove of the receptor, their ADME properties and their possible interaction with other receptor and/or enzyme targets. Two compounds, ZINC04017374 (Naphthofluorescein) and ZINC08589130 (Puertogaline A) were purchased, tested in vitro and confirmed their OXER1 Gβγ and Gαβγ activity, respectively. The methodology followed is useful for a better understanding of the mechanism by which OXER1 mediates its actions, it has the potential to provide structural insights, in order to design small molecular specific interactors and ultimately design new anti-inflammatory and anti-cancer agents. Finally, the methodology may also be useful for identifying specific agonists/antagonists of other GPCRs.

Regulation of Prostate Androgens by Megalin and 25-hydroxyvitamin D Status: Mechanism for High Prostate Androgens in African American Men.

These findings link vitamin D deficiency and the megalin protein to increased levels of prostate androgens, which may underpin the disparity in lethal prostate cancer in African America men.

TGF-β controls stromal telomere length through epigenetic modifications.

The online version contains supplementary material available at 10.1007/s13205-022-03346-5.

Testosterone therapy in prostate cancer: is it still a controversy?

The benefits of testosterone therapy (TTh) in the hypogonadal male can be dramatic. Historically, TTh has been contraindicated in prostate cancer (PCa). Current evidence has redefined our understanding of the influence serum testosterone has on prostatic androgen activity. Increasing numbers of hypogonadal men with coexisting PCa emphasizes the importance of describing those who may safely receive TTh. This review aims to present literature that evaluates the efficacy and safety of TTh in men with coexisting PCa. Our study, a comprehensive review of published literature regarding TTh in men with a history of PCa, consisted of studies conducted from the 1940s to 2022. Our review discusses evidence in accordance with previous studies that TTh has a role in patients with localized PCa as it has not been reported to increase rates of recurrence or progression of PCa. The use of TTh in hypongonadal men with a localized PCa has been shown to have positive clinical outcomes without increasing the rate of disease progression or recurrence. Further research, in a randomized controlled setting, is warranted.

Effect of virgin coconut oil, lauric acid and myristic acid on serum and prostatic markers of benign prostatic hyperplasia

Purpose: To investigate the effect of virgin coconut oil, lauric acid and myristic acid on serum and prostatic androgens in testosterone-induced prostatic hyperplasia.Methods: Benign prostatic hyperplasia (BPH) was induced in the animals by repeated subcutaneous injection of testosterone propionate (5 mg/kg) at the inguinal region once a day for 28 days. Thereafter, BPH was treated for 56 days by oral administration of virgin coconut oil, lauric acid and myristic acid. Following the treatment period, the rats were sacrificed and blood samples were collected through cardiac puncture for biochemical analysis.Results: Virgin coconut oil, lauric acid and myristic acid led to a significant reduction (p < 0.05) in serum prostatic acid phosphatase (PAcP), prostate specific antigen (PSA) and dihydrotestosterone (DHT) levels, and also in prostatic DHT level.Conclusion: This study provides evidence that virgin coconut oil, lauric acid and myristic acid may be useful in the management of BPH because they exerted some anti-proliferative effects in the development and progression of BPH. Therefore, coconut may be a potential functional food for the management of BPH patients because it is rich in both lauric and myristic acid. However, further investigations, including clinical trials are required to buttress this.

Prostate Cancer

Prostate cancer is the most commonly diagnosed noncutaneous malignancy in men in the United States. This chapter discusses the epidemiology, pathogenesis, and diagnosis of prostate cancer, as well as risk factors, the use of digital rectal examination and prostate-specific antigen measurement for screening, and staging for the disease. Also reviewed are the natural history of untreated prostate cancer; the treatment of localized and advanced prostate cancer, including prostatectomy, radiation therapy, and androgen deprivation therapy; and the prevention of prostate cancer. Figures illustrate the incidence rates of prostate cancer by race, age-adjusted and/or age-specific cancer of the prostate, the risk of a diagnosis in 20 years (based on being cancer free at certain ages), the 5-year survival rate, and the overall survival in patients with early prostate cancer treated with observation or radical prostatectomy. Tables in this chapter review the clinical staging definitions and the combined-modality staging approach to prostate cancer. This chapter contains 6 figures, 8 tables and 104 references Keywords: Prostate cancer, digital rectal exam, PSA, prostatectomy, men's health, prostate cancer screening

Abstract 1781: Correlates of androgen deprivation and gut microbiome in mouse Pten-deficient prostate cancer

Abstract Androgen receptor (AR) signaling plays a key role in the progression of prostate cancer and androgen deprivation therapy (ADT) is one the standard of care for men with advanced prostate cancer. However, AR also exerts effects on androgen responsive tissues and accumulating data indicates that AR is implicated in influencing gut flora. Commensal bacteria are essential for maintaining a healthy immune system which plays a major role in cancer surveillance. Moreover, cancer is known to promote commensal dysbiosis which can further impair immune function. Here, we investigate associations between commensal fecal bacteria and androgen deprivation in an immunocompetent transgenic mouse model of Pten-deficient prostate cancer. 16S rRNA amplicon sequencing was used to comprehensive profile and compare the fecal microbiomes of bearing conditional Pten-knockout mice with those of mice 5 weeks after surgical castration. Tumor burden in surgically castrated mice was significantly lower compared to that of intact mice, 57.45%, P<0.001. With regards to fecal microbial composition, ADT via surgical castration enhanced alpha diversity (Shannon index, P=0.006). Moreover, microbial communities differed between castrated and intact mice (PERMANOVA, P=0.003, NMDS stress=0.042). Notably, an individual mouse with a strong response to ADT showed marked differences in fecal microbe composition compared to other mice in the cohort. Taxa associated with ADT included Dorea, Oscillospora, Sutturella, Dehalobacterium, Akkermansia muciniphila, and Flexispira while Prevoltella, Allobacalum, Ruminococcus flavefaciens, Candidatus Arthromitus and Bacteroides. Dehalobacterium, Coprococcus and Ruminococcus gnavus were more abundant in fecal samples from castrated, low tumor burden mouse. Overall, Bacteriodes and Parabacteriodes were positively correlated to tumor burden. Taxon enrichment analysis (TSEA) revealed functional enrichments of lipid translocation, carbohydrate transport, amino acid transport and proteoglycans in cancer in castrated mice, whereas, enrichment of low-density lipoprotein particle binding, extracellular matrix assembly, inflammatory response to antigenic stimulus and ADP metabolic process. Functional profiles inferred from metagenomic 16S rRNA data revealed enrichment of glyoxylate and dicarboxylate metabolism, carbon metabolism and geraniol metabolism in castrated mice. In intact mice, lipopolysaccharide biosynthesis, streptomycin biosynthesis and amino sugar and nucleotide sugar metabolism were enriched. Notably, lower tumor burden was associated with porphyrin and chlorophyll metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation. This study models ADT in a preclinical model of prostate cancer and provides a broad characterization of the gut microbiome and its association to androgen deprivation. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Eri Banno, Naomi Ando, Noriko Sako, Nobutaka Shimizu, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Correlates of androgen deprivation and gut microbiome in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1781.

LncRNA PART1 Promotes Proliferation and Migration, Is Associated with Cancer Stem Cells, and Alters the miRNA Landscape in Triple-Negative Breast Cancer.

Simple SummaryLong non-coding RNAs (lncRNAs) play an important role in cancer progression. Herein we provide new information regarding the role of prostate androgen regulated transcript 1 (PART1). We show that the lncRNA PART1 is enriched in triple-negative breast cancers and cancer stem cell populations. We demonstrate its role in cancer cell and tumor growth and provide evidence for its association with worse survival in a subset of breast cancer patients. Importantly, our genome-wide analyses have revealed novel insights into the function of this lncRNA, demonstrating how it changes the microRNA (miRNA) landscape leading to genome-wide mRNA expression regulation. Our study suggests that PART1 represents an attractive target for the treatment of triple-negative breast cancers.Triple-negative breast cancers (TNBCs) are aggressive, lack targeted therapies and are enriched in cancer stem cells (CSCs). Novel therapies which target CSCs within these tumors would likely lead to improved outcomes for TNBC patients. Long non-coding RNAs (lncRNAs) are potential therapeutic targets for TNBC and CSCs. We demonstrate that lncRNA prostate androgen regulated transcript 1 (PART1) is enriched in TNBCs and in Aldefluorhigh CSCs, and is associated with worse outcomes among basal-like breast cancer patients. Although PART1 is androgen inducible in breast cancer cells, analysis of patient tumors indicates its androgen regulation has minimal clinical impact. Knockdown of PART1 in TNBC cell lines and a patient-derived xenograft decreased cell proliferation, migration, tumor growth, and mammosphere formation potential. Transcriptome analyses revealed that the lncRNA affects expression of hundreds of genes (e.g., myosin-Va, MYO5A; zinc fingers and homeoboxes protein 2, ZHX2). MiRNA 4.0 GeneChip and TaqMan assays identified multiple miRNAs that are regulated by cytoplasmic PART1, including miR-190a-3p, miR-937-5p, miR-22-5p, miR-30b-3p, and miR-6870-5p. We confirmed the novel interaction between PART1 and miR-937-5p. In general, miRNAs altered by PART1 were less abundant than PART1, potentially leading to cell line-specific effects in terms miRNA-PART1 interactions and gene regulation. Together, the altered miRNA landscape induced by PART1 explains most of the protein-coding gene regulation changes (e.g., MYO5A) induced by PART1 in TNBC.

LncRNA PART1, manipulated by transcriptional factor FOXP2, suppresses proliferation and invasion in ESCC by regulating the miR‑18a‑5p/SOX6 signaling axis.

An increasing number of studies have demonstrated that long non-coding (lnc)RNAs are associated with tumor invasion, metastasis and the prognosis of patients with a variety of different tumors. However, the roles of lncRNA prostate androgen regulated transcript 1 (PART1) in esophageal squamous cell carcinoma (ESCC) remain unknown. In the present study, reverse transcription-quantitative PCR was performed to investigate the levels of PART1, SRY-box transcription factor 6 (SOX6) and miR-18a-5p in ESCC tissues and cells. The functions of PART1 in ESCC were demonstrated using Cell Counting Kit-8 and Matrigel assays. Promoter activity and dual-luciferase reporter assays, RNA immunoprecipitation and western blot analyses were also used to determine the potential mechanisms of PART1 in ESCC cell lines. It was found that PART1 and SOX6 were both downregulated in ESCC tissues and cells, and their low expression levels were associated with TNM stage, lymph node metastasis and poor prognosis in patients with ESCC. Forkhead box protein P2 (FOXP2) exhibited low expression level in ESCC tissues, and its expression was positively correlated with PART1 expression level in ESCC tissues. FOXP2 was found to bind to the promoter region of PART1 to regulate its expression in ESCC cells. Functionally, PART1 overexpression suppressed cell proliferation and invasion, whereas PART1 downregulation promoted cell proliferation and invasion in the ESCC cell lines. Mechanistically, PART1 functions as a competing endogenous (ce)RNA by sponging miR-18a-5p, resulting in the upregulation of the downstream target gene, SOX6, coupled with the inactivation of the β-catenin/c-myc signaling axis, to suppress ESCC cell proliferation and invasion. In conclusion, data from the present study unveil a potential ceRNA regulatory pathway, in which PART1 affects SOX6 expression level by sponging miR-18a-5p, to ultimately suppress ESCC development and progression.

LncRNA PART1 promotes lung squamous cell carcinoma progression via miR-185-5p/Six1 axis

Dysregulation of the long non-coding RNA prostate androgen regulated transcript 1 (lncRNA PART1) is involved in the tumorigenesis of various cancers. However, little is known about its function and molecular mechanism in the development of lung squamous cell carcinoma (LSCC). In this study, we examined the expression of PART1 in LSCC clinical tissue samples and cell lines, and gain- and loss-of-function experiments were performed to explore the function of PART1 in LSCC proliferation, invasion and migration. We found that PART1 was overexpressed in both LSCC tissues and cell lines. Functional studies revealed that PART1 knockdown significantly suppressed cell proliferation, invasion and migration but enhanced apoptosis in LSCC cells, whereas overexpression of PART1 showed the opposite results. Mechanistically, we identified that PART1 acted as a sponge of miR-185-5p, and sineoculis homeobox homolog 1 (Six1) was a direct downstream target of miR-185-5p. Moreover, restoration of miR-185-5p or silencing of Six1 partially abolished the oncogenic effect of PART1 in LSCC cells. Clinically, The areas under the receiver operating characteristic (ROC) curve of PART1, miR-185-5p, and Six1 were 0.7857, 0.7332, 0.8112, respectively. Notably, high PART1, low miR-185-5p, and high Six1 expressions were significantly associated with severe clinical parameters and were the independent risk factors for poor prognosis of LSCC patients. Thus, we concluded that the PART1/miR-185-5p/Six1 axis might serve as a novel biomarker for the diagnosis and treatment of LSCC.

Targeting Androgen Regulation of TMPRSS2 and ACE2 as a Therapeutic Strategy to Combat COVID-19

The COVID-19 pandemic is expected to have an adverse effect on the progression of multiple cancers, including prostate cancer, due to the ensuing cytokine storm and associated oncogenic signaling. Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and co-receptor TMPRSS2 by androgen in mouse tissues and human prostate and lung cell lines. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. In an overexpression model, and the prostate and lung cells, Camostat – a TMPRSS2 inhibitor, blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction. Thus providing evidence for the first time a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike protein, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens such as enzalutamide/AR-PROTAC, or Camostat treatment attenuated the SARS-CoV-2 S-mediated entry in lung and prostate cells. Together, our preclinical data provide a strong rationale for clinical evaluations of the TMPRSS2 inhibitors, androgen-deprivation therapy and androgen receptor antagonists alone or in combination with anti-viral drugs as early as clinically possible to prevent inflammation driven COVID-19 progression.Funding: Research in I.A.A laboratory is supported by NIH (1-R01 CA249210-0), Department of Defense Idea Development Award (W81XWH-17-0404), and Sarcoma Foundation of America Research Award to I.A.A.Ethical Approval: In vivo mouse studies were performed following protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and in compliance with all regulatory standards. C57BL/6J mice were procured from Jackson Laboratory (000664) and breed in house.Declaration of Interest: None to declare

113: Hematologic Changes Associated with Prostate Radiotherapy and Androgen Deprivation Therapy

113: Hematologic Changes Associated with Prostate Radiotherapy and Androgen Deprivation Therapy

Efficacy and safety of apalutamide in Japanese patients with nonmetastatic castration-resistant prostate cancer: a subgroup analysis of a randomized, double-blind, placebo-controlled, Phase-3 study

BackgroundIn the global Phase-3 Selective Prostate Androgen Receptor Targeting with ARN-509 study, apalutamide plus ongoing androgen deprivation therapy (ADT) significantly increased metastasis-free survival (MFS) and improved other clinical outcomes in men with nonmetastatic castration-resistant prostate cancer (nm-CRPC) who were at high risk of developing metastases. In this subpopulation analysis of Selective Prostate Androgen Receptor Targeting with ARN-509 study, the efficacy and safety of apalutamide plus ADT were evaluated in Japanese patients with nm-CRPC.MethodsThe primary efficacy end point was MFS. Secondary efficacy end points were time to metastasis, progression-free survival, symptomatic progression, initiation of cytotoxic chemotherapy, and overall survival. Safety and pharmacokinetic parameters were also assessed.ResultsFifty-five Japanese patients with ongoing ADT were randomized (apalutamide: n = 34, placebo: n = 21). Median treatment duration was 5.7 months in the apalutamide group and 11.0 months in the placebo group. Median MFS was not reached in the apalutamide group (95% confidence interval: 10.97, not estimable) and was 18.23 months (95% confidence interval: 11.04, 18.50) in the placebo group. Secondary end points were improved in the apalutamide group. The safety profile of apalutamide with ADT was comparable with the global population, and no new safety signals were identified in this Japanese subpopulation. Although, apalutamide exposure tended to be higher in the Japanese subpopulation compared with the non-Japanese population, this was likely to be explained by body weight and considered not clinically meaningful.ConclusionIn the Japanese subpopulation, treatment with apalutamide with ADT resulted in favorable efficacy outcomes with comparable benefit-risk profile to the global population with nm-CRPC who are at high-risk of developing metastases.

Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis.

BackgroundHepatocellular carcinoma (HCC) is the most common primary hepatic malignancy worldwide. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been identified as effective markers for the detection of multiple cancers. This study aimed to illuminate the mechanism of prostate androgen regulated transcript 1 (PART1) in HCC.Materials and MethodsThe levels of PART1, miR-149-5p and mitogen-activated protein kinase 1 (MAP2K1) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay, and cell migration and invasion were evaluated by transwell assay. Dual-luciferase reporter assay was carried out to examine the relationship among PART1, miR-149-5p and MAP2K1. Western blot assay was conducted to measure the protein expression of MAP2K1.ResultsPART1 and MAP2K1 expression were greatly increased and miR-149-5p level was decreased in HCC tissues. Functional analysis revealed that the si-PART1 inhibited proliferation, migration and invasion of HCC cells. PART1 directly bound to miR-149-5p and miR-149-5p level was down-regulated by PART1. Moreover, restoration experiment demonstrated that the effect of PART1 knockdown on HCC cell progression could be partially rescued by miR-149-5p depletion. MiR-149-5p was predicted to target MAP2K1 and MAP2K1 expression was negatively modulated by miR-149-5p. Also, MAP2K1 rescued the inhibitory effects of miR-149-5p overexpression on proliferation, migration and invasion in HCC cells. Besides, the inhibition of miR-149-5p weakened the impact on MAP2K1 expression mediated by PART1 repression.ConclusionPART1 promoted proliferation, migration and invasion of HCC cells by regulating miR-149-5p/MAP2K1 axis.

Potential Novel Role of UDP Glucuronosyltransferases 2B11 and 2B28 in Crosstalk Between Androgen and Lipid Signalling

Androgens play critical roles in both prostate and breast cancer progression. Recent work has revealed extensive crosstalk between androgen and lipid signalling, including the ability of the androgen receptor to control lipid synthesis and uptake to fuel prostate cancer growth [1, 2]. Members of the UDP‐glucuronosyltransferase (UGT) enzyme family can metabolically inactivate multiple lipophilic small molecules including steroids such as androgens, which modulates both prostate and breast cancer progression [3, 4]. Moreover, transcriptional induction of UGTs by steroid‐liganded nuclear receptors (e.g. AR and ER) can create a feedback loop to control steroid signalling [3].The UGT2B11 and UGT2B28 enzymes are dramatically induced by androgens in prostate and breast cancer cells (up to 200‐fold); however, unlike some other UGTs they have little or no activity towards androgens and hence their functions have been enigmatic. We now have evidence that UGT2B11 and UGT2B28 function via two novel mechanisms that together indirectly drive cancer cell growth downstream of androgen signals. First, UGT2B11 and UGT2B28 control the activity of the SREBP lipid‐sensing and signalling pathway leading to increased expression of target genes involved in lipid synthesis/metabolism. They also appear to influence feedback regulation of nuclear SREBP via proteolytic turnover. Second, UGT2B11 and UGT2B28 heterodimerize with and inhibit known androgen‐metabolizing UGT enzymes, thus preventing androgen inactivation and clearance. These findings suggest a new mechanism for crosstalk between androgen and lipid signalling pathways in breast and prostate cancer cells, and prompt further investigation of UGTs as therapeutic targets.Support or Funding InformationNational Health and Medical Research Council of Australia, Flinders Foundation

Abstract P3-03-11: Notch inhibition promotes triple negative breast cancer growth

Abstract Notch1 and Transforming Growth Factor-beta (TGF-β) signaling pathways in cancer have a paradoxical roles as they were shown to have both tumor suppressive and tumor promoting activities. The interaction between these two signaling pathways in cancer progression was addressed in a systematic way. In these studies, we utilized primary human mammary epithelial cells (HMEC) and various triple negative breast cancer (TNBC) cells and examined the molecular cross-talk between TGF-β and Notch signaling pathways using biochemical, molecular and cell biology techniques. First, we noticed lower expression of Notch1 in most of the TNBC cells compared to normal cells. Second, most of these TNBC cells overexpressed TMEPAI (transmembrane prostate androgen induced) protein, which was shown to switch TGF-β from a tumor suppressor to a tumor promoter role in breast cancer. Surprisingly, treatment of TNBC cells with Notch inhibitors increased their growth rate both in the absence or presence of TGF-β. Moreover, Notch inhibition relieved the growth suppression by TGF-β in normal cells. Both normal and TNBC cells showed increased growth in the presence of Notch inhibitors with decreased TGF-β signaling activity. Normal cells that express very low levels of TMEPAI both at mRNA and protein levels, overexpressed TMEPAI protein in the presence of various Notch inhibitors, suggesting that Notch negatively regulates TMEPAI expression. Correspondingly, knockdown of TMEPAI in TNBC cell lines showed increased expression of Notch1 and Jagged 1 suggesting a negative regulation of Notch1 by TMEPAI. Consistent with these observations, TMEPAI, a known binding partner of E3 ligases, causes increased turnover of Notch1 protein. In fact, over expression of active Notch1 in MDA-MB-231 cells inhibited tumor cell growth both in vitro and in vivo. Our results clearly suggest that a crosstalk exists between TGF-β and Notch1 signaling through TMEPAI. Therefore, our studies clearly indicate that use of Notch inhibitors is contraindicated in triple negative breast cancer patients. Citation Format: Pothana Saikumar, Prajjal K Singha, Manjeri A Venkatachalam. Notch inhibition promotes triple negative breast cancer growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-11.

Abstract 1019: P300/CBP inhibitor CCS1477 targets 22Rv1 prostate tumor AR and c-myc gene expression in vivo

Abstract Background: Histone acetyl transferases E1A binding protein (p300) and CREB binding protein (CBP) are known co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-myc and androgen receptor (AR). A large proportion of AR regulated gene expression has been shown to be dependent on p300 either through direct regulation of AR interaction with promoters of AR regulated genes or subsequent histone modification events. Both p300 and CBP are highly expressed in advanced prostate cancer and androgen deprivation leads to upregulation of both proteins. CCS1477 is a potent, selective inhibitor of the bromodomain in CBP/p300 that has been shown to inhibit prostate tumor cell proliferation in vitro and tumor growth in vivo. Methods: 22Rv1 prostate tumor cells that express both AR and AR variants were transplanted in nude mice. Established tumors were treated with CCS1477, 20mg per kg, once daily p.o. for 28 days. CCS1477 treatment resulted in virtually complete inhibition of tumor growth that was maintained up to 24 days after cessation of treatment by which time tumor recurrence was evident. Tumors were excised from vehicle and CCS1477 treated animals at day 7, day 28, and day 52; mRNA was isolated and gene expression analysis was carried using Affymetrix Clarion D microarrays. Differential gene expression based on fold change (FC) >1.5 and FDR-adjusted p-value <0.05 identified a number of genes with significant FC in CCS1477 vs vehicle treated control tumors. Results: Although ~1.5 fold downregulation of AR was maintained from day 7 to day 52, downregulation of AR target genes ETS2, TMPRSS2 and NKX3.1 recovered after treatment cessation. Similarly, expression of c-myc was significantly reduced at day 7 (-2.7 FC) and recovered by day 28. Of note, among the top downregulated genes were CIART and BHLHE40 circadian clock regulated genes that provide negative feedback loops that in conjunction with downregulation of AR and c-myc would disrupt circadian gene regulation. VEGFA mRNA that was downregulated >1.5 fold at all time points, together with c-myc and p300 are all under circadian regulation. Expression of the histone demethylase KDM3A was reduced >1.5 fold at all time points. KDM3A is known to function as an AR coactivator of key AR target genes including NKX3.1 and c-myc. Conclusions: Gene expression analysis of CCS1477 treated prostate tumors suggests an underlying mechanism involving the inhibition of key drivers of prostate cancer progression including AR and c-myc and a network of interacting pathways. Citation Format: Paul Elvin, Neil Pegg, Simone Daminelli, Izabela Eden, Barbara Young, Amy Prosser, Jenny Worthington, Nigel Brooks. P300/CBP inhibitor CCS1477 targets 22Rv1 prostate tumor AR and c-myc gene expression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1019.

Abstract 923: FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells

Abstract Androgen receptor (AR) plays a critical function in driving prostate cancer (PCa) development and androgen deprivation therapy (ADT) is standard of care in treating PCa patients. Although patients generally response well, the cancer eventually relapse and progress to the castration-resistant stage of PCa (called CRPC), which can be further treated with more aggressive ADT. However, the tumors still develop resistance within a year and restored AR activity is found in the majority of relapsed tumors, indicating a pressing need for developing novel AR-targeted therapies. In PCa cells, AR activity is dependent on the chromatin binding of the pioneer factor, FOXA1. In particular, FOXA1 recognizes the H3K4me1,2 marked enhancers and its binding opens the chromatin for subsequent AR recruitment. Although the role of FOXA1 on AR signaling has been intensively studied, the mechanisms regulating FOXA1 binding to chromatin remain elusive. LSD1/KDM1A was initially identified as a transcription repressor through demethylation of H3K4me1,2. In contrast to its well-established corepressor function, LSD1 has been found to coactivate several transcription factors including AR. This AR coactivator function has been attributed to the phosphorylation of histone 3 on threonine 6 and threonine 11, which may switch LSD1 substrate specificity from H3K4me1,2 to the repressive mark H3K9me1,2. However, we have recently reported that the H3K4 demethylase activity of LSD1 persists at AR-regulated enhancer sites, including sites marked by H3T6ph, arguing against the coactivator function of LSD1 being generally controlled by this mechanism. Importantly, we have shown in a recent study that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 silencing impaired FOXA1 binding at AR-regulated enhancers, suggesting a role of LSD1 in regulating enhancer availability to AR. In the current study, we found that LSD1 inhibition globally disrupts FOXA1 chromatin binding prior to androgen stimulation and thus impairs further AR recruitment, resulting in the global inhibition of AR transcriptome. Using immunoprecipitation and mass-spectrometry assays, we discovered that LSD1 maintains FOXA1 binding by demethylating lysine K270, adjacent to its DNA binding domain. We further found that the expression of a methylation-deficient FOXA1 mutant (K270R) stabilizes AR chromatin binding and allows PCa cells resistant to enzalutamide treatment. Using a clinical tested LSD1 inhibitor, we have shown in vivothat LSD1 inhibition dramatically decreases PCa growth alone and in synergy with enzalutamide in AR/AR variants-positive CRPC models. These findings provide fundamental new insights into FOXA1, LSD1, and mechanisms that regulate the activity of AR in PCa cells. As LSD1 inhibitors are moving into the clinic, these new insights into LSD1 function strongly support LSD1 as a therapeutic target in CRPC. Citation Format: Shuai Gao, Sujun Chen, Dong Han, Wanting Han, Jude N. Owiredu, Steven P. Balk, Housheng H. He, Changmeng Cai. FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 923.

A pivotal role of androgen signaling in Notch-responsive cells in prostate development, maturation, and regeneration

Androgen signaling is essential for prostate development, morphogenesis, and regeneration. Emerging evidence also indicates a regulatory role of Notch signaling in prostate development, differentiation, and growth. However, the collaborative regulatory mechanisms of androgen and Notch signaling during prostate development, growth, and regeneration are largely unknown. Hairy and Enhancer of Split 1 (Hes1) is a transcriptional regulator of Notch signaling pathways, and its expression is responsive to Notch signaling. Hes1-expressing cells have been shown to possess the regenerative capability to repopulate a variety of adult tissues. In this study, we developed new mouse models to directly assess the role of the androgen receptor in prostatic Hes1-expressing cells. Selective deletion of AR expression in embryonic Hes1-expressing cells impeded early prostate development both in vivo and in tissue xenograft experiments. Prepubescent deletion of AR expression in Hes1-expressing cells resulted in prostate glands containing abnormalities in cell morphology and gland architecture. A population of castration-resistant Hes1-expressing cells was revealed in the adult prostate, with the ability to repopulate prostate epithelium following androgen supplementation. Deletion of AR in Hes1-expressing cells diminishes their regenerative ability. These lines of evidence demonstrate a critical role for the AR in Notch-responsive cells during the course of prostate development, morphogenesis, and regeneration, and implicate a mechanism underlying interaction between the androgen and Notch signaling pathways in the mouse prostate.