Androgen Receptor Knockdown Research Articles

Androgen receptor activation inhibits endothelial cell migration in vitro and angiogenesis in vivo

Our previous research revealed that androgen receptor (AR) activation reduces endothelial cell proliferation via non-genomic pathways. We hypothesized that AR activation might also affect endothelial cell migration, a critical step in angiogenesis. Our data demonstrates that treatment of human umbilical vein endothelial cells (HUVECs) with AR agonists, metribolone (R1881) or dihydrotestosterone (DHT), results in a dose-dependent reduction in migration, which can be reversed by AR antagonists or AR knockdown. Mechanistically, R1881 inhibits HUVEC migration by suppressing RhoA activity through the cSrc/FAK/paxillin pathway and promoting RhoA degradation via RhoA-p27 complex formation, ultimately resulting in RhoA ubiquitination. Transfection with constitutively active RhoA-V14 rescues the inhibitory effect of R1881 on HUVEC migration. Furthermore, R1881 elevates intracellular vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) levels but reduces VEGF secretion from HUVECs. This reduction is attributed to the formation of VEGF-CTGF complexes in the cytosol induced by R1881. Transfection with RhoA-V14 reduces CTGF levels and VEGF-CTGF complex formation, leading to enhanced VEGF secretion. Pre-treatment with WP631, a CTGF inhibitor, mitigates the R1881-induced reduction in VEGF secretion and HUVECs migration. In vivo assessments using zebrafish angiogenesis and mouse matrigel plug assays validate the anti-angiogenic effects of R1881. These findings provide insight into the molecular mechanisms through which AR activation modulates endothelial cell migration and angiogenesis.

Pleckstrin-2 Mediates the Activation of AKT in Prostate Cancer and Is Repressed by Androgen Receptor

PI3K-AKT and androgen receptor (AR) pathways are commonly activated in prostate cancers. Their reciprocal regulation makes it difficult to treat advanced prostate cancers. Here, we show that Pleckstrin-2 (PLEK2), a proto-oncoprotein involved in the activation and stabilization of AKT, connects these two pathways. We first provide genetic evidence that Plek2 deficiency largely reverted tumorigenesis in Pten prostate-specific knockout mice. Overexpression of PLEK2 promotes the proliferation and colony formation of prostate cancer cells in vitro. In addition, we found that PLEK2 is negatively regulated by AR. AR transcriptionally represses PLEK2 through binding to the PLEK2 promoter region. Overexpression of AR reduces PLEK2 expression, which inactivates AKT. Conversely, knockdown of AR in prostate cancer cells increases PLEK2 expression and activates the AKT pathway. This reciprocal inhibitory loop can be pharmacologically targeted using the PLEK2 inhibitor. We show that the PLEK2 inhibitor dose-dependently inhibits prostate cancer cell proliferation with the inactivation of AKT. Overall, our study uncovers the critical role of PLEK2 in prostate cancer proliferation and provides the rationale for targeting PLEK2 to treat prostate cancers.

Tanshinone analog inhibits castration-resistant prostate cancer cell growth by inhibiting glycolysis in an AR-dependent manner

Androgen receptor (AR) is one of the key targets for the treatment of Castration-resistant prostate cancer (CRPC). Current endocrine therapy can greatly improve CRPC patients. However, with the change of pathogenic mechanism, acquired resistance often leads to the failure of treatment. Studies have shown that tanshinone IIA (TS-IIA) and its derivatives have significant antitumor activity, and have certain AR targeting effects, but the mechanism is unknown. In this study, the TS-IIA analog TB3 was found to significantly inhibit the growth of CRPC in vitro and in vivo. Molecular docking, cellular thermal shift assay and CHX experiments confirmed that AR was the target of TB3 and promoted the degradation of AR. Furthermore, TB3 can significantly inhibit glycolysis metabolism by targeting the AR/PKM2 axis..The addition of pyruvic acid could significantly alleviate the inhibitory effect of TB3 on CRPC cells. Besides, the knockdown of AR or PKM2 also could reverse the effect of TB3 on CRPC cells. Taken together, our study suggests that TS-IIA derivative TB3 inhibits glycolysis to prevent the CRPC process by targeting the AR/PKM2 axis.

GABBR2 as a Downstream Effector of the Androgen Receptor Induces Cisplatin Resistance in Bladder Cancer.

The precise molecular mechanisms responsible for resistance to cisplatin-based chemotherapy in patients with bladder cancer remain elusive, while we have indicated that androgen receptor (AR) activity in urothelial cancer is associated with its sensitivity. Our DNA microarray analysis in control vs. AR-knockdown bladder cancer sublines suggested that the expression of a GABA B receptor GABBR2 and AR was correlated. The present study aimed to determine the functional role of GABBR2 in modulating cisplatin sensitivity in bladder cancer. AR knockdown and dihydrotestosterone treatment considerably reduced and induced, respectively, GABBR2 expression, and the effect of dihydrotestosterone was at least partially restored by an antiandrogen hydroxyflutamide. A chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of GABBR2 in bladder cancer cells. Meanwhile, GABBR2 expression was significantly elevated in a cisplatin-resistant bladder cancer subline, compared with control cells. In AR-positive bladder cancer cells, knockdown of GABBR2 or treatment with a selective GABA B receptor antagonist, CGP46381, considerably enhanced the cytotoxic activity of cisplatin. However, no additional effect of CGP46381 on cisplatin-induced growth suppression was seen in GABBR2-knockdown cells. Moreover, in the absence of cisplatin, CGP46381 treatment and GABBR2 knockdown showed no significant changes in cell proliferation or migration. These findings suggest that GABBR2 represents a key downstream effector of AR signaling in inducing resistance to cisplatin treatment. Accordingly, inhibition of GABBR2 has the potential of being a means of chemosensitization, especially in patients with AR/GABBR2-positive bladder cancer.

NR5A2/HSD3B1 pathway promotes cellular resistance to second-generation antiandrogen darolutamide.

This study investigated cellular mechanisms in steroidogenesis responsible for treatment resistance to the novel antiandrogen agent darolutamide in prostate cancer. HSD3B1 was overexpressed in darolutamide-resistant cells and induced by darolutamide treatment and AR knockdown. Inversely, HSD3B1 knockdown increased cellular sensitivity to darolutamide. Similarly, its upstream regulator NR5A2 was up-regulated in darolutamide-resistant cells and induced by darolutamide treatment and AR knockdown. Inversely, NR5A2 knockdown and NR5A2 inhibitor ML180 decreased expression of various steroidogenic enzymes including HSD3B1, leading to increased cellular sensitivity to darolutamide. The NR5A2/HSD3B1 pathway promoted cellular resistance to darolutamide and targeting NR5A2/HSD3B1 pathway is a promising therapeutic strategy to overcome darolutamide resistance.

Androgen receptor transcriptional activity is required for heregulin-1β–mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while invivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. Invitro treatment with the ErbB3 ligand heregulin-1β (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK/ELK1 signalling pathway.

Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.

Abstract B041: AR suppresses MHC Class I expression and T-cell response in prostate cancer

Abstract Immunotherapy is a treatment option that has had limited success with prostate cancer patients. The major histocompatibility complex (MHC) Class I plays a pivotal role in the adaptive immune response by presenting neo-antigens on the surface of cancer cells to CD8+ T-cells. Prostate cancer cells have markedly lower expression of MHC Class I genes compared to immune-responsive cancers. Loss of MHC Class I is also associated with more aggressive disease and immune evasion in prostate cancer. However, the molecular mechanisms that control MHC Class I downregulation in prostate cancer are still unknown. We hypothesize that increasing MHC Class I expression in prostate cancer cells will increase antigen presentation and improve the efficacy of immunotherapy agents. To investigate the mechanism of MHC Class I regulation in prostate cancer cells, we conducted a whole-genome CRISPRi flow cytometry screen. In this screen, C42B cells containing a non-catalytic Cas9 (dCas9) were infected with a lentivirus that contained a sgRNA library of 100,000 guides targeting 20,000 genes. Infected cells were selected and stained with a pan-Class I MHC antibody, sorted, and the highest and lowest 25-30% of MHC Class I expressing cells were collected for analysis. As expected, knockdown of MHC Class I genes such as B2M, HLA-A, and TAP1 decreased MHC Class I expression. Surprisingly, knockdown of AR and AR co-factors GRHL2 and FOXA1 resulted in a dramatic increase of MHC Class I surface expression. Additional AR inhibition experiments using enzalutamide, an AR degrader, or charcoal-stripped serum also showed increased MHC Class I expression over time. These in vitro results were substantiated with RNA expression analyses of patient biopsy samples taken before or after neoadjuvant enzalutamide treatment, which showed significantly increased MHC Class I expression post-treatment. Results from a small library CRISPR screen, showed that elimination of androgen response elements upstream of MHC Class I genes increased MHC expression, providing a mechanism by which AR regulates MHC Class I. Importantly, AR knockdown was shown to increase antigen-specific T-cell response in co-culture and transgenic mouse models. These observations were further validated in a mCRPC phase II clinical trial. Bulk RNA expression analyses comparing responders and non-responders to anti-PD1 therapy in enzalutamide treated patients, showed decreased expression of AR and increased expression of MHC Class I in patients who responded to immunotherapy. Overall, our data show that AR suppresses MHC Class I expression in prostate cancer, and that androgen-targeted therapies can increase antigen presentation and improve T-cell response. By understanding how AR regulates MHC Class I expression, we can identify new combination treatments utilizing AR signaling inhibition and immunotherapy that lead to a improved anti-cancer response. Citation Format: Lisa Chesner, Julie Graff, Fanny Polesso, Alexis Smith, Arian Lundberg, Martin Sjoestroem, Zheng Xia, Simon Linder, Andries Bergman, Alan Ashworth, David Quigley, Wilbert Zwart, Luke Gilbert, Amy Moran, Felix Feng. AR suppresses MHC Class I expression and T-cell response in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B041.

Targeting benign prostate hyperplasia treatments: AR/TGF-β/NOX4 inhibition by apocynin suppresses inflammation and proliferation

IntroductionApocynin (Apo), an NADPH oxidase (NOX) inhibitor, has been widely used to treat various inflammatory diseases. However, the therapeutic effects of Apo on benign prostatic hyperplasia (BPH), a multifactorial disease associated with chronic inflammation and hormone imbalance, remain unknown. ObjectivesThe link between androgen signaling, reactive oxygen species (ROS), and prostate cell proliferation may contribute to the pathogenesis of BPH; therefore, the aim of this study was to identify the specific signaling pathway involved and to demonstrate whether the anti-oxidant Apo plays a role in the prevention and treatment of BPH. MethodsIngenuity pathway analysis and si-RNA transfection were conducted to demonstrate the androgen receptor (AR) and NOX4 linkage in BPH. Pathological markers of BPH were measured by H&E staining, immunoblotting, ELISA, qRT-PCR, and immunofluorescence to examine the effect of Apo. Rats stimulated with testosterone and BPH-1 cells were used as BPH models. ResultsAR and NOX4 network-mediated oxidative stress was upregulated in the BPH model. Next, we examined the effects of Apo on oxidative stress and chronic prostatic inflammation in BPH mouse models. In a testosterone-induced BPH rat model, Apo alleviated pathological prostate enlargement and suppressed androgen/AR signaling. Apo suppressed the upregulation of proinflammatory markers and promoted the expression of anti-oxidant factors. Furthermore, Apo regulated the TGF-β/Glut9/activin pathway and macrophage programming. In BPH-1 cells, Apo suppressed AR-mediated proliferation and upregulation of TGFB and NOX4 expression by alleviating oxidative stress. Apo activated anti-oxidant and anti-inflammatory systems and regulated macrophage polarization in BPH-1 cells. AR knockdown partially abolished the beneficial effects of Apo in prostate cells, indicating AR-dependent effects of Apo. ConclusionIn contrast with existing BPH therapies, Apo may provide a new application for prostatic disease treatment, especially for BPH, by targeting the AR/TGF-β/NOX4 signaling pathway.

Abstract P4-08-01: Targeting CDK7 enhances the antitumor efficacy of enzalutamide in androgen receptor-positive triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Among TNBC subtypes, the luminal androgen receptor (LAR) subtype expresses high levels of androgen receptor (AR), tends to be less proliferative, and generally responds poorly to neoadjuvant therapy. Previous studies have shown that AR inhibition suppressed proliferation of AR+ or LAR subtype TNBC cells and tumor growth in xenograft models. Thus, AR is a promising therapeutic target for this TNBC subtype. Here, we identified kinase targets for enhancing the antitumor efficacy of the AR inhibitor enzalutamide in preclinical models and investigated the underlying molecular mechanisms. Methods: We performed a nonbiased high-throughput kinome siRNA screening (targeting 709 genes) to identify a synergistic partner for enhancing the antitumor efficacy of the AR inhibitor enzalutamide in AR+ LAR TNBC. The growth inhibitory effects of enzalutamide alone or combined with kinase inhibitors were determined using the sulforhodamine B staining assay and clonogenic assay. The effect of treatments on the expression of target proteins of interest was examined by Western blot analysis. Results: Enzalutamide was not effective as a single agent to inhibit the proliferation of AR+ TNBC cells (IC50 >15 µM in MDA-MB-453, CAL-51, CAL-148, MFM223, HCC2185, SUM149, SUM159, and SUM185 cells). Nonbiased high-throughput kinome siRNA screening identified PI3K/AKT/mTOR, cell cycle, and JNK pathways as potential canonical targets for combination with enzalutamide to enhance its antitumor efficacy in AR+ LAR TNBC. Among these pathways, inhibition of cell cycle progression using the CDK7 inhibitor UD-017 showed the most synergistic anti-proliferation effect with enzalutamide in AR+ LAR MDA-MB-453 (50.61% reduction in proliferation compared with enzalutamide alone, P < 0.001; and 48.98% reduction in proliferation compared with UD-017 alone, P < 0.001) and SUM185 (40.41% reduction in proliferation compared with enzalutamide alone, P < 0.05; and 42.76% reduction in proliferation compared with UD-017 alone, P < 0.05) TNBC cells. Furthermore, CDK7 knockdown using siRNA significantly enhanced the sensitivity of MDA-MB-453 (60.98%, P < 0.01) and SUM185 (30.99%, P < 0.01) cells to enzalutamide, and AR knockdown significantly enhanced the sensitivity of MDA-MB-453 (32.64%, P < 0.01) and SUM185 (43.62%, P < 0.01) cells to UD-017, in both cases with a sensitivity similar to that of enzalutamide plus UD-017. This result suggests that the synergy of enzalutamide and UD-017 results from specific targeting of AR and CDK7. Downstream target analysis revealed that the combination of enzalutamide and UD-017 dramatically reduced the expression of c-MYC. c-MYC knockdown using siRNA dramatically suppressed colony formation in both MDA-MB-453 and SUM185 cells at a degree similar to that of enzalutamide plus UD-017, whereas c-MYC overexpression reversed the synergistic effect of the combination treatment. This result suggests that UD-017 synergizes with enzalutamide through inhibition of c-MYC–mediated tumorigenesis. Conclusion: Our results suggest that enzalutamide synergizes with UD-017 by inhibiting c-MYC–mediated oncogenic activity. These in vitro data warrant future in vivo studies of the antitumor synergy of enzalutamide plus UD-017 in AR+ LAR TNBC models. Citation Format: Xuemei Xie, Marwa Manai, Jon A. Fuson, Troy Pearson, Dileep R. Rampa, Debu Tripathy, Jangsoon Lee, Naoto T. Ueno. Targeting CDK7 enhances the antitumor efficacy of enzalutamide in androgen receptor-positive triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-01.

Inhibition of androgen receptor enhanced the anticancer effects of everolimus through targeting glucose transporter 12.

Everolimus was designed as a mammalian target of rapamycin (mTOR) inhibitor. It has been proven as a targeted drug for gastric cancer (GC) therapy. However, long-term treatment with everolimus may cause severe side effects for recipients. Decreasing the dosage and attenuating the associated risks are feasible to promote clinical translation of everolimus. This study aimed to identify the underlying mechanisms of responses to everolimus and develop novel regimens for GC treatment. Our findings proved that there was a significant dose-dependent relationship of everolimus-induced GC cell apoptosis and glycolysis inhibition. Then, we found that a member of glucose transporter (GLUT12) family, GLUT12, was actively upregulated to counteract the anticancer effects of everolimus. GLUT12 might be overexpressed in GC. High expression of GLUT12 might be correlated with tumor progression and short survival time of GC patients. Bioinformatic analysis suggested that GLUT12 might be involved in regulating cancer development and metabolism. The experiments proved that GLUT12 significantly promoted GC growth, glycolysis and impaired the anticancer effects of everolimus. Androgen receptor (AR) is a classical oncogenic factor in many types of cancer. Everolimus elevated GLUT12 expression in an AR-dependent manner. Inhibition of AR activity abrogated the promotive effects on GLUT12 expression. Both in-vitro and in-vivo experiments demonstrated that GLUT12 knockdown augmented anticancer effects of everolimus. Enzalutamide, an AR inhibitor, or AR knockdown was comparable to GLUT12 suppression. This study identified the role of the AR/GLUT12 pathway in the development of poor responses to everolimus. Interference with AR/GLUT12 pathway may serve as a promising approach to promoting the translational application of everolimus in GC therapy.

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

Bioengineered polyester nanoparticles for the synergistic treatment of androgenic alopecia via the suppression of 5α-reductase and knockdown of androgen receptor.

Androgenic alopecia (AGA) is a common disease that negatively affects patients' physical and mental health. AGA can be treated with drugs that improve the perifollicular microenvironment, such as 5α-reductase inhibitors (e.g., dutasteride [DUT]), androgen receptor blockers, and minoxidil. However, the efficacy of these treatments is limited. Therefore, this study aimed to show that nanoparticles are effective as stable carriers with high curative benefits and little adverse effects. The in vitro study showed that PLGA-DUT/siAR@DPCM NPs could deliver both DUT and siAR to dermal papilla cells. They could successfully suppress 5α-reductase and knock down androgen receptor, respectively, and thereby promote cell proliferation. In the in vivo study, PLGA-DUT/siAR@DPCM NPs showed a significant therapeutic effect in an AGA mouse model. They successfully penetrated the stratum corneum and showed a clear targeting effect on hair follicles and surrounding tissues. PLGA-DUT/siAR@DPCM NPs could enable the targeted delivery of DUT and siAR through percutaneous penetration, enhancing phagocytosis and decreasing adverse effects. Thus, they have great potential in the clinical treatment of AGA.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

Abstract 3050: Equol as a radiosensitizer and therapeutic agent in prostate cancer

Abstract The gastrointestinal microbiome and the metabolites it can produce have been linked to response to various cancer treatments, including in prostate cancer. Equol is a microbial metabolite that is exclusively produced by the conversion of soy isoflavones in the diet by specific intestinal bacteria. Interestingly, equol has previously been shown to exhibit anti-androgen effects on prostate cells and likewise may act as a radiosensitizer. We analyzed the microbial composition of fecal samples collected from men receiving stereotactic ablative radiation (SABR) versus observation to treat oligometastatic prostate cancer as part of the ORIOLE trial and found that patients harboring equol-producing bacteria had a lower risk of disease progression. We therefore aimed to study the effect of this compound on prostate cancer cell lines in vitro. MTT assay was used to assess the effects of equol treatment on cell viability in androgen receptor (AR) positive LNCaP cells and AR null PC3 cells over time. We assessed the ability of equol to reduce AR protein levels and/or subcellular localization in LNCaP cells using immunohistochemistry. Finally, western blotting was used to assess the effects of equol treatment on prostate specific antigen (PSA) levels to assess effects on the AR signaling pathway. Cell viability was significantly decreased over time in equol treated cells, but only in prostate cancer cell lines that were AR positive. The effects of equol on LNCaP viability was abrogated in an AR knockdown LNCaP cell line. Interestingly, AR protein levels were found to be markedly decreased upon equol treatment of LNCaP cells. We likewise observed a decrease in PSA protein levels in AR positive cells after equol treatment. Our results support the notion that equol can inhibit prostate cancer growth by inhibition of the androgen receptor. We are currently investigating whether combining equol exposure with radiation treatment produces a synergistic effect on DNA damage and reduction of prostate cancer cell viability and survival in vitro. Our future studies will investigate the effects of equol and equol-producing bacteria on tumor growth in in vivo models. Citation Format: Carli B. Jones, Jonathan B. Coulter, Janielle P. Maynard, Luke A. Mummert, Ryan M. Phillips, Phuoc T. Tran, Karen S. Sfanos. Equol as a radiosensitizer and therapeutic agent in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3050.

Abstract 967: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT)

Abstract Triple-negative breast cancers (TNBC) are a subset of breast cancer that is highly aggressive and has a poor prognosis. The TNBC subtype has no effective molecular targeted therapies. Gene expression studies have identified a subset of TNBC that is enriched with androgen receptors (AR) and androgen receptor signaling. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Interference with androgen signaling in TNBC with AR-inhibiting drugs have shown a reduction in epithelial-to-mesenchymal transition (EMT), which is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal-like stem cells. Approximately one-third of TNBC expressed AR, and evaluation of AR-positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. Previous studies have shown that AR inhibition or AR knockdown significantly reduces migration and invasion and EMT in different TNBC cell lines. γ-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer effects and have little or no effect on normal cell viability. Studies show that treatment with 0-7 µM γ-tocotrienol reduced AR levels, proliferation, migration and invasion in a dose-responsive manner in TNBC MDA-MB-231 and MDA MB-453 cell lines. Results also show that treatment with 5 µM (MDA-MB-231) and 7 µM (MDA-MB-453) γ-tocotrienol induced a reversal in EMT cell biomarkers, as well as reversal in EMT morphological and behavioral characteristics in these TNBC cell lines. Western blot analysis show that similar treatment with γ-tocotrienol significantly decreased DHT-induced N-cadherin (mesenchymal cell biomarker) and increased expression in cytokeratin 18 and E-cadherin (epithelial cell biomarkers) in these cells. AR signaling is known to play an important role in stimulating DHT-induced EMT by stimulating the STAT3/Snail signaling pathway in MDA-MB231. However, results in the present study shows that γ-tocotrienol treatment significantly inhibited activation of STAT3 and Snail proteins. Additional immunocytochemistry experiments showed that γ-tocotrienol treatment significantly inhibited DHT-induced cytoskeleton changes in TNBC MDA-MB-231 and MDA MB-453 cell lines. In conclusion, these results demonstrated that γ-tocotrienol treatment inhibits AR expression and DHT-dependent EMT and cytoskeleton changes in TNBC cells. These findings suggest that AR may be a potential therapeutic target for treating both LAR and non-LAR TNBC subtypes. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Nayef Aldabaan, Tasmin A. Sultana, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 967.

Curcumin Boosts Natural Killer Cell-Based Immunotherapy in Impeding Progression of Hepatocellular Carcinoma Through Androgen Receptor/UL16 Binding Protein-2 Signal

Hepatocellular carcinoma (HCC) is hardly curable with present treatment modalities, especially for those in advanced disease stage. Several chronic hepatic disorders, including alcoholic liver disease, cirrhotic and viral hepatitis, can ultimately lead to occurrence of HCC. This study investigated the effects of curcumin on natural killer (NK) cell-based immunotherapies being applied to restrain the progress of HCC, and disclose valid message for better clinical application of curcumin. After receiving curcumin treatment, the HCC cells were co-cultured with NK cells to study cytotoxicity of NK cells. After thein vitrocytotoxicity assay, we determined the function of curcumin in modulating the cytotoxicity of NK cells. Additionally, the mechanism of curcumin on HCC cells was investigated by utilizing the following techniques: lentiviral infection/transfection, qPCR and Western-blot, thereby exploring the genetic alterations of differently treated HCC cells. HCC cell lines (SK-Hep1 and SUN423) were studied, and findings confirmed that curcumin significantly strengthened the cytotoxic NK cells to kill HCC cells. This effect was mainly achieved through regulating androgen receptor (AR)/UL162 signal, which led to efficacy enhancement of NK cell-based immunotherapy. Moreover, secretion of interferon-γwhich is an immunoregulator executing an anti-tumor effect was also elevated. In addition, curcumin strengthened the expression of ULBP2 in SK-Hep1 and SUN423 cells, while relatively weakening the expression of androgen receptor. The findings from this study can provide a theoretical base for further research and development of curcumin as a beneficial suppressor of HCC progression. We found that the cytotoxicity of NK cells towards HCC cells was gradually elevated as the HCC cells were treated with increased concentration of curcumin. Meanwhile, the secretion of interferon-γ, which was an immunoregulator exerting an anti-tumor effect, was also gradually elevated. In addition, the expression levels of ULBP2 in SK-Hep1 and SUN423 cells were continuously enhanced with increased concentration of curcumin in the pre-treatment of HCC cells, while the expression of androgen receptor was relatively weakened. Similar results were also obtained after the overexpression or knockdown of androgen receptor (AR) gene in SK-Hep1 and SUN423 cells. Therefore, this study found that curcumin can boost the NK cell-based immunotherapy in impeding progression of HCC through the androgen receptor/ULBP2 signal.

Ratio of the expression levels of androgen receptor splice variant 7 to androgen receptor in castration refractory prostate cancer.

In clinical samples, the expression of androgen receptor (AR) and of AR splice variant 7 (AR-V7) is higher in castration-resistant prostate cancer (CRPC) compared with that in hormone-sensitive prostate cancer (PCa). However, there are only a few reports on the ratio of the expression levels of AR-V7 to AR (AR-V7/AR) in prostate tissue. The present study evaluated AR-V7/AR expression in various types of human prostate tissues and CRPC cells. Pretreatment prostate tissue samples from patients with benign prostatic hyperplasia (BPH; n=18), Gleason score 7 (n=17), and Gleason score 8–10 (n=26) were collected at the time of prostate biopsy, and tissue samples from CRPC patients (n=10) were collected at the time of transurethral resection of the prostate. Furthermore, androgen-independent LNCaP cells were established. The mRNA expression levels of AR and AR-V7, cell proliferation and prostate-specific antigen (PSA) production were evaluated by reverse transcription quantitative PCR, MTS assay and chemiluminescent enzyme immunoassay, respectively. There was a significant difference in AR-V7/AR expression ratios between the CRPC group and the BPH and pre-treatment PCa groups (CRPC, 7%; BPH and pre-treatment PCa, 1%). Subsequently, we compared the AR and AR-V7 expression levels in CRPC samples with those in the pretreatment prostate tissues from the same patients. The results demonstrated that the AR-V7/AR ratio increased from 3 to 9% after CRPC onset. Furthermore, in vitro experiment demonstrated that AR-V7 expression in LNCaP cells was increased after transforming into CRPC cells. The AR-V7/AR ratio also increased from 0.05 to 0.3%. In addition, small interfering (si)-RNA-mediated knockdown of AR inhibited the proliferation of and PSA production from androgen-independent LNCaP cells; however, AR-V7 knockdown had no effect. Conversely, siRNA-mediated knockdown of both AR and AR-V7 inhibited the proliferation of VCAP cells. In summary, the findings from the present study demonstrated that AR-V7 expression and AR-V7/AR ratio were increased after the onset of CRPC, which had a limited role in CRPC cell proliferation. Further investigation is required to clarify the roles of AR other splice variants and AR-V7 in CRPC.

Androgen Receptor Signaling Induces Cisplatin Resistance via Down-Regulating GULP1 Expression in Bladder Cancer.

The underlying molecular mechanisms of resistance to cisplatin-based systemic chemotherapy in bladder cancer patients remain to be elucidated, while the link between androgen receptor (AR) activity and chemosensitivity in urothelial cancer has been implicated. Our DNA microarray analysis in control vs. AR knockdown bladder cancer lines identified GULP1 as a potential target of AR signaling. We herein determined the relationship between AR activity and GULP1 expression in bladder cancer cells and then assessed the functional role of GULP1 in cisplatin sensitivity. Androgen treatment in AR-positive cells or AR overexpression in AR-negative cells considerably reduced the levels of GULP1 expression. Chromatin immunoprecipitation further showed direct interaction of AR with the promoter region of GULP1. Meanwhile, GULP1 knockdown sublines were significantly more resistant to cisplatin treatment compared with respective controls. GULP1 knockdown also resulted in a significant decrease in apoptosis, as well as a significant increase in G2/M phases, when treated with cisplatin. In addition, GULP1 was immunoreactive in 74% of muscle-invasive bladder cancers from patients who had subsequently undergone neoadjuvant chemotherapy, including 53% of responders showing moderate (2+)/strong (3+) expression vs. 23% of non-responders showing 2+/3+ expression (P = 0.044). These findings indicate that GULP1 represents a key downstream effector of AR signaling in enhancing sensitivity to cisplatin treatment.

Androgen receptor regulates the proliferation of myoblasts under appropriate or excessive stretch through IGF-1 receptor mediated p38 and ERK1/2 pathways

BackgroundAndrogen receptor (AR) exerts important roles in exercise-induced alterations of muscle mass, in which the proliferation and differentiation of satellite cells or myoblasts are crucial. Our previous study in C2C12 myoblasts demonstrated that 15% (mimic appropriate exercise) and 20% (mimic excessive exercise) stretches promoted and inhibited the proliferation respectively; and AR played a crucial role in 15% stretch-induced pro-proliferation through IGF-1-modulated PI3K/Akt, p38 and ERK1/2 pathways, but AR’s role in stretches-modulated proliferation of general myoblasts, especially 20% stretch, remains unclear, and the mechanisms need to be further clarified.MethodsFirstly, the discrepancy in proliferation and the above indicators between L6 (without AR) and C2C12 (with AR) myoblasts were compared under 15% or 20% stretch. Then the influences of transfection AR or exogenous IGF-1 treatment on proliferation and these indicators were detected in stretched L6 myoblasts.Results(1) Under un-stretched state, the proliferation of L6 was slower than C2C12 cells. Furthermore, AR knockdown in C2C12 myoblasts repressed, while AR overexpression in L6 myoblasts promoted the proliferation. (2) 15% stretch-induced increases in the proliferation and activities of p38 and ERK1/2 were lower in L6 than C2C12 cells; AR overexpression enhanced the proliferation of 15% stretched L6 cells accompanied with the increases of p38 and ERK1/2 activities. (3) 20% stretch-induced anti-proliferation and inhibition of p38 activity were severer in L6 than C2C12 myoblasts; AR overexpression reversed the anti-proliferation of 20% stretch and enhanced p38 activity in L6 myoblasts. (4) In stretched L6 myoblasts, AR overexpression increased IGF-1R level despite no detectable IGF-1; and recombinant IGF-1 increased the proliferation, the level of IGF-1R, and the activities of p38 and ERK1/2 in 15% stretched L6 myoblasts.ConclusionsThe study demonstrated AR's crucial roles in stretches-regulated proliferation of myoblasts, and increased AR fulfilled 15% stretch's pro-proliferation via activating IGF-1R- p38 and ERK1/2 pathways while decreased AR achieved 20% stretch's anti-proliferation via inhibiting IGF-1R- p38 pathway, which is useful to understand in depth the role and mechanisms of AR in appropriate exercise increasing while excessive exercise decreasing muscle mass.