Inhibition Of Androgen Receptor Expression Research Articles

18β-Glycyrrhetinic acid synergizes with enzalutamide to counteract castration-resistant prostate cancer by inhibiting OATP2B1 uptake of dehydroepiandrosterone sulfate

Androgen dependence is a key feature of prostate cancer, and androgen deprivation is effective in treating prostate cancer. However, the disease often worsens and develops into castration-resistant prostate cancer after short-term control. The current study aimed to explore the mechanism of the synergistic action of 18β-glycyrrhetinic acid (18β-GA) and enzalutamide (ENZ) against prostate cancer. Our findings showed that 18β-GA significantly inhibited the expression of OATP2B1 and the transport of dehydroepiandrosterone sulfate (DHEAS) in LNCap and 22RV1 cells. It also downregulated the expression of androgen receptor (AR) to some extent. ENZ strongly inhibited AR expression, but it did not affect OATP2B1-mediated uptake of DHEAS. Compared to the effects of 18β-GA and ENZ alone, the combination of 18β-GA and ENZ significantly enhanced the inhibitory effects on AR, prostate-specific antigen (PSA) expression, tumor cell proliferation, and migration. The results obtained in castrated model mice matched the findings of in vitro experiments. 18β-GA significantly reduced the uptake of DHEAS mediated by OATP2B1 in mouse tumor tissues and cooperated with ENZ to further inhibit the expression of AR and PSA, combat the growth of tumor cells, and promote the apoptosis of tumor cells. In conclusion, 18β-GA considerably decreased the uptake of DHEAS and androgen production in cells by inhibiting the transport function of OATP2B1, while ENZ inhibited the nuclear translocation of AR and reduced the expression of AR. The combination of 18β-GA and ENZ can simultaneously inhibit androgen production and AR expression and exhibit a synergistic effect against castration and prostate cancer progression.

Abstract LB027: Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository

Abstract Second-generation androgen ablation therapies (ADT), enzalutamide, and abiraterone are initially effective on metastatic castration-resistant prostate cancer (mCRPC). However, nearly 30-40% of patients eventually become resistant to these treatments due to the presence of androgen-receptor variants (AR-V), which lack a ligand-binding domain. The grim reality that metastatic CRPC is virtually incurable for most patients poses a significant challenge for clinicians striving to enhance these individuals' quality of life and lifespan. Considering drug discovery efforts, we partnered with the National Cancer Institute's (NCI) Natural Product Repository, which possesses a diverse collection of natural product extracts derived from plant, marine, and microbial organisms. We developed high-throughput phenotypic screening assays to screen the crude natural product extract libraries on AR and AR-variants CRPC cell lines (C4-2B and 22Rv1). Several extracts inhibited the growth of both CRPC cell lines and were further confirmed for dose-dependent cell viability inhibition assays. Once we determined the IC50 dose, we performed a Western blot analysis that confirmed the inhibition of AR and AR-V7 expression in CRPC cells.In subsequent studies, we narrowed down by fractionating the most potent extracts and confirmed their ability to inhibit AR expression and signaling on CRPC cell lines. Currently, we are characterizing the potent compounds and their inhibitory activity on AR and AR-variants, in vitro and in vivo CRPC models. Many nutraceuticals have been shown to target AR signaling, synthesis, or degradation which effectively inhibited the growth of CRPC. Our collaborative efforts with NCI will permit us find N-terminal AR degrading molecules (AF-1), which are essential for the AR transcriptional activities of CRPC. In sum, discovery of ovel small molecules from our drug development program have the potential to translate these effective natural products into drugs with clinical benefits to treat CRPC. Citation Format: Neha Tyagi, Sepideh Hosseiniporgham, Ashish Tyagi, Balaji Chandrasekaran, Katherine Bonilla, Inna Kriger, James Saccchettini, Chendil Damodaran. Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB027.

Abstract 2105: Targeting the “Achilles Heel” of androgen receptor activity in castration resistant prostate cancer

Abstract Background: Each year, millions of men are diagnosed with prostate cancer (CaP). The unchecked activation of the Androgen Receptor (AR) spurs CaP development and progression. Mutations or the presence of AR splice variants can add complexity to tumor ecology, leading to chemoresistance. This study aims to develop novel inhibitors targeting the “Achilles Heel” of AR activity: the N-terminal domain (NTD). This would circumvent the limitations of LBD (Ligand Binding Domain) therapies and offer a superior treatment option for patients. Though the AR-NTD is an intrinsically disordered protein, which complicates structure-based drug design, we have developed a unique small molecule inhibitor, ASR600, which specifically targets AR-NTD and promotes AR and AR- variants degradation by ubiquitination at previously unknown sites. Methods: Mass-spec analysis was performed to identify distinct ubiquitination sites in AR's NTD. To perform spatial transcriptome analysis, CytAssist Visium (10X genomics) was used. Furthermore, we used western blotting, immunofluorescence, immunoprecipitation, and PDX mice studies in CRPC mouse models to examine the impact of ASR600 on CRPC. Results: Four AR ubiquitination sites: K845, K847, and K913 (LBD) and K311 (NTD) are already identified. Interestingly, ASR600 continued to inhibit AR expression even with mutated ubiquitination sites. Mass spectrometry analysis of ASR-mediated mono-ubiquitinated N-terminal AR identified K16 as a unique ubiquitin acceptor. Altering this ubiquitination site rescues AR expression from ASR600-mediated degradation, emphasizing AR N-terminus as an ASR600 target. To discern the role of the K16 ubiquitination site in AR protein turnover, we evaluated AR protein stability in LNCaP cells that stably expressed ARWT or AR-K16R constructs. Our data indicates the pivotal importance of K16 for AR transcriptional activity. Invivo, ASR600, when used as a sole agent, exhibited inhibitory responses in PDX of clinically aggressive, AR-expressing tumors. Spatial gene expression analysis revealed marked expression differences in AR signature genes between control and ASR600-treated PDX tissues. Crucially, AR signaling, and related markers mainly aligned with epithelial markers rather than stromal ones. In contrast, the ASR600-treated PDX tumor showcased reduced AR signaling within epithelial cells. Conclusion: Targeting AR-NTD has received a lot of attention, but its innately disordered structure has hindered progress. ASR600 targets AR-NTD specifically by ubiquitinating the protein at a novel location, K16. Our in vivo, PDX studies, combined with the spatial analysis, lay a strong foundation for initiating IND-enabling TOX studies, followed by a phase-I clinical trial for ASR-600 in CRPC patients. Citation Format: Ashish Tyagi, Balaji Chandrasekaran, Arun K. Sharma, Chendil Damoadaran. Targeting the “Achilles Heel” of androgen receptor activity in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2105.

Androgen receptor is a determinant of melanoma targeted drug resistance

Melanoma provides a primary benchmark for targeted drug therapy. Most melanomas with BRAFV600 mutations regress in response to BRAF/MEK inhibitors (BRAFi/MEKi). However, nearly all relapse within the first two years, and there is a connection between BRAFi/MEKi-resistance and poor response to immune checkpoint therapy. We reported that androgen receptor (AR) activity is required for melanoma cell proliferation and tumorigenesis. We show here that AR expression is markedly increased in BRAFi-resistant melanoma cells, and in sensitive cells soon after BRAFi exposure. Increased AR expression is sufficient to render melanoma cells BRAFi-resistant, eliciting transcriptional changes of BRAFi-resistant subpopulations, including elevated EGFR and SERPINE1 expression, of likely clinical significance. Inhibition of AR expression or activity blunts changes in gene expression and suppresses proliferation and tumorigenesis of BRAFi-resistant melanoma cells, promoting clusters of CD8+ T cells infiltration and cancer cells killing. Our findings point to targeting AR as possible co-therapeutical approach in melanoma treatment.

Androgen increases klotho expression via the androgen receptor-mediated pathway to induce GCs apoptosis

BackgroundMany epidemiological studies have shown that anovulatory polycystic ovary syndrome (PCOS) is accompanied by hyperandrogenism. However, the exact mechanism of hyperandrogen-induced anovulation remains to be elucidated. In this study, we aimed to investigate the potential mechanism of anovulation in PCOS. To investigate the role of klotho as a key factor in the androgen receptor (AR)-mediated development of PCOS, we investigated the effects of testosterone on ovarian klotho expression in vivo and in vitro.ResultsTestosterone propionate (TP)-induced rats showed cycle irregularity, hyperandrogenism, polycystic ovarian changes, dyslipidemia. However, inhibition of AR expression could relieve PCOS traits. We also found that AR and klotho showed relatively high expression in PCOS rat ovarian tissue and in TP-induced granulosa cells (GCs), which was inhibited by the addition of flutamide. TP-induced GCs apoptosis was suppressed by AR antagonist, as well as silencing klotho expression in human GCs. Chromatin immunoprecipitation assay demonstrated that AR indirectly binds to the klotho promoter.ConclusionsOur results demonstrated TP mediates the expression of klotho via androgen receptor and klotho alterations could be a reason for ovarian dysfunction in PCOS.

A COP1-GATA2 axis suppresses AR signaling and prostate cancer

Androgen receptor (AR) signaling is crucial for driving prostate cancer (PCa), the most diagnosed and the second leading cause of death in male patients with cancer in the United States. Androgen deprivation therapy is initially effective in most instances of AR-positive advanced or metastatic PCa. However, patients inevitably develop lethal castration-resistant PCa (CRPC), which is also resistant to the next-generation AR signaling inhibitors. Most CRPCs maintain AR expression, and blocking AR signaling remains a main therapeutic approach. GATA2 is a pioneer transcription factor emerging as a key therapeutic target for PCa because it promotes AR expression and activation. While directly inhibiting GATA2 transcriptional activity remains challenging, enhancing GATA2 degradation is a plausible therapeutic strategy. How GATA2 protein stability is regulated in PCa remains unknown. Here, we show that constitutive photomorphogenesis protein 1 (COP1), an E3 ubiquitin ligase, drives GATA2 ubiquitination at K419/K424 for degradation. GATA2 lacks a conserved [D/E](x)xxVP[D/E] degron but uses alternate BR1/BR2 motifs to bind COP1. By promoting GATA2 degradation, COP1 inhibits AR expression and activation and represses PCa cell and xenograft growth and castration resistance. Accordingly, GATA2 overexpression or COP1 mutations that disrupt COP1-GATA2 binding block COP1 tumor-suppressing activities. We conclude that GATA2 is a major COP1 substrate in PCa and that COP1 promotion of GATA2 degradation is a direct mechanism for regulating AR expression and activation, PCa growth, and castration resistance.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.

Abstract 1527: Developing a multifaceted approach to target androgen receptor (AR) in castration resistant prostate cancer

Abstract Androgen receptor (AR) signaling is also regulated epigenetically by histone-modifying enzymes (histone deacetylases-HDACs) that result in the development and progression of prostate cancer (CaP). It has been established that the inhibition of HDACs suppresses CaP proliferation by inhibiting AR signaling via multiple mechanisms, including AR degradation. Hence, the goal of this study was to develop small molecules that employ multiple mechanisms to inhibit AR expression and promote its degradation, which would effectively control the emergence and treatment of castration-resistant prostate cancer (CRPC). Several rationally designed antiandrogen equipped HDAC inhibitors (HDACis) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo models. Results demonstrated that these compounds' target (HDAC and AR) binding properties translated into potent anticancer activity against AR+ CRPC cell lines and to a lesser extent in AR- CRPC cell lines. In addition, among these compounds, we found that SB-146, a nontoxic agent, not only inhibited the growth of AR+ CRPC cells at nanomolar IC50s without sparing toxicity to healthy prostate epithelial cells. Molecular analysis confirmed SB-146 downregulated the expressions of both AR and AR-spice variants (AR-SV) in CRPC cells. Further analyses revealed downregulation of AR downstream (PSA, HOXB13 and FOXA1) targets as well as AR-SV’s (NUP210 and SLC3A2) in CRPC cells. SB-146 was also able to overcome DHT-induced AR and AR-SV signaling in C4-2B and 22Rv1 cells. Assessment of SB-146’s therapeutic efficacy on in vivo models revealed that oral administration of this compound abrogated AR+ and AR-SV (C4-2B and 22Rv1) tumor growth in xenotransplanted nude mice models. Finally, immunohistochemical analysis confirmed SB-146’s target of both AR and AR-SVs on xenografted tumor tissues. Together these results confirm that SB-146 is a potent agent that inhibits AR signaling in CRPC cells. Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Subhasish Tapadar, Bocheng Wu, Oyelere Adegboyega, Chendil Damodaran. Developing a multifaceted approach to target androgen receptor (AR) in castration resistant 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 1527.

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.

Abstract 711: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple-negative breast cancer (TNBC) MDA-MB-453 cells is associated with a reduction in cellular proliferation and viability

Abstract Targeted therapy for triple-negative breast cancer (TNBC) remains challenging due to poor understanding of its molecular etiology. Previous studies have shown that androgen receptors (AR) are widely expressed in approximately one-third of TNBC, and are referred to as luminal androgen receptors (LAR). AR has recently emerged as a prognostic and treatment-predictive biomarker in breast cancer. However, the role of AR in TNBC remained elusive. Treatment with AR-antagonist have been shown to suppress AR activation and signaling in TNBC and results in a corresponding reduction in tumor cell proliferation and survival due to the initiation of apoptosis in these tumor cells. γ-Tocotrienol (γT3) is a natural isoform of vitamin E that displays potent anticancer activity. Results show that treatment with 0-7 μM γT3 induces a dose-responsive significant decrease, whereas treatment with 35 nM dihydrotestosterone (DHT) results in a significant increase of AR expression and corresponding increase in growth of MDA-MB-453 TNBC cells. Combined treatment of 7 μM γT3 with 35 nM DHT significantly inhibits DHT-induced proliferation and AR activation in MDA-MB-453 cells. Western blot analysis shows that combined treatment with γT3 and DHT induced a significant decrease in phosphorylated ERK1/2 (active form) expression, but had no effect on total ERK1/2 levels in MDA-MB-453 TNBC cells. Flow cytometry analysis shows that γT3 treatment was also found to induce G0/G1 cell cycle arrest and significantly increased the number of apoptotic and necrotic cells, as compared to MDA-MB-453 TNBC cells in the vehicle-treated control group. Western blot analysis shows that γT3 treatment also inhibited DHT-induced elevations in CDK2, CDK4, CDK6 and cyclin D1 expression (cell cycle progression biomarkers) and induces a significant increase in the expression of tumor suppressor protein, p53, in MDA-MB-453 TNBC cells. Similar treatment with 7 µM γT3 induces a corresponding significant increase in apoptosis biomarker expression of cleaved caspase-3 and Bax in MDA-MB-453 TNBC cells. In summary, results from these studies demonstrated that γT3 treatment inhibits AR expression, activation and signaling, as well as DHT-dependent cell proliferation and viability in TNBC. These finding also suggest that the LAR may be a potential therapeutic target for the treatment TNBC. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Tasmin A. Sultana, Nayef Aldabaan, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple-negative breast cancer (TNBC) MDA-MB-453 cells is associated with a reduction in cellular proliferation and viability [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 711.

LncRNA LINC00667 aggravates the progression of hepatocellular carcinoma by regulating androgen receptor expression as a miRNA-130a-3p sponge

Emerging studies have found long noncoding RNAs, widely expressed in eukaryotes, crucial regulators in the progression of human cancers, including hepatocellular carcinoma (HCC). Although the long intergenic noncoding RNA 667 (LINC00667) can promote the progression of a variety of cancer types, the expression pattern, the role in cancer progression, and the molecular mechanism involved in HCC remain unclear. This study aims to investigate the function and mechanism of LINC00667 in HCC progression. The effects of LINC00667 silencing in cell proliferation, cell migration, and cell invasion, and androgen receptor (AR) expression were determined with loss-of-function phenotypic analysis in Huh-7 and HCCLM3 cells, and subsequently testified in vivo in tumor growth. We found that the expression of LINC00667 was upregulated in HCC tissues and cell lines. Upregulation of LINC00667 was significantly associated with the unfavorable prognosis of HCC in our study patients. On the other hand, low expression of LINC00667 significantly inhibited the cell proliferation, cell migration and cell invasion of HCC in vitro and tumor growth in vivo. This inhibitory effect could be counteracted by miR-130a-3p inhibitor. LINC00667 reduced the inhibition of AR expression by miR-130a-3p, which correlated with the progression of HCC. Our finding suggests LINC00667 is a molecular sponge in the miR-130s-3p/AR signal pathway in the progression of HCC, in which it relieves the repressive function of miR-130a-3p on the AR expression. This indicates LINC00667 functions as a tumor promotor in promoting HCC progression through targeting miR-130a-3p/AR axis, making a novel biomarker and potential therapeutic target for HCC.

MicroRNA‑149 inhibits cancer cell malignant phenotype by regulating Akt1 in C4‑2 CRPC cell line

Prostate cancer (PCa) is an androgen-dependent disease. Androgen receptor (AR) has a crucial role in the development and progression of PCa. Recently, several microRNAs (miRNAs/miRs) involved in AR regulation have been associated with castration-resistant prostate cancer (CRPC), the terminal stage of PCa. Nevertheless, the precise mechanism remains unclear. The present study aimed to identify a novel miR-149 regulatory network and potential therapeutic target for CRPC. It was found that ectopic expression of miR-149 mimic could inhibit AR expression, repress epithelial-mesenchymal transition, induce cell cycle arrest and apoptosis in CRPC cell line C4-2, whereas the miR-149 inhibitor exerted the opposite effects. Furthermore, it was also revealed that miR-149 could reduce the functional activity of the PI3K/Akt1 signaling pathway by targeting Akt1 protein, the key regulatory factor of the PI3K/Akt1 signaling pathway. Knockdown of Akt1 by short hairpin RNA increased apoptosis, reduced proliferation, and restrained migration and invasion in CRPC cells, with the effect of AR inhibition. In conclusion, these results revealed that miR-149 acts as a tumor suppressor in CRPC cell line C4-2 and restrains its progression through the AR signaling pathway by targeting Akt1. The miR-149/Akt1/AR regulatory pathway may represent a novel PCa therapeutic target.

Abstract 2572: 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 proliferation, migration and epithelial-to-mesenchymal transition (EMT)

Abstract Triple negative breast cancer (TNBC) is an aggressive invasive malignancy with the lowest 5-years survival rate. Gene expression profiling indicates that TNBC is a heterogeneous disease and at present, there is no targeted therapy available for treatment. Approximately one third of TNBC expressed androgen receptor (AR) and evaluation of AR positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. 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. Previous studies have shown that AR inhibition or AR knockdown significantly reduces baseline proliferation, anchorage-independent growth, migration and invasion and increase apoptosis in different TNBC cell lines. γ-Tocotrienol is a natural isoform of vitamin E that displays potent anticancer activity. Studies were conducted to determine if γ-tocotrienol effects on AR levels and activity play a role in mediating γ-tocotrienol induced inhibition of TNBC cell proliferation, migration and epithelial-to-mesenchymal transition (EMT). In vitro studies showed that treatment with 0-12 μM γ-tocotrienol induced a dose-responsive significant decrease in TNBC MDA-MB-231 and MDA-MB-453 cell proliferation and viability. Treatment with 35 nM dihydrotestosterone (DHT) resulted in an increase of AR expression and corresponding increase in the growth of both TNBC cell lines. Treatment of MDA-MB-231 and MDA MB-453 cells with γ-tocotrienol (5 μM and 7 μM, respectively), significantly inhibited DHT-induced proliferation of both TNBC cell lines. Western blot analysis showed that γ-tocotrienol treatment significantly reduced DHT-induced cytoplasmic and nuclear AR expression in MDA-MB-453 cells, but induced only a slight and insignificant reduction in AR expression in MDA-MB-231 cells. Immunocytochemistry studies confirmed that γ-tocotrienol treatment induced a decrease in cytoplasmic and nuclear AR expression in both TNBC cell lines, but this decrease was only found to be significant in MDA-MB-453 cells. Other studies showed that γ-tocotrienol treatment significantly reduced cancer cell migration and this finding was associated with an increased expression in cytokeratin 8 (an epithelial cell biomarker) and decrease in vimentin (a mesenchymal cell biomarker) in both TNBC cell lines, and is an indication of a reversal in EMT. In summary, these results demonstrated that γ-tocotrienol treatment inhibits AR expression as well as DHT-dependent cell proliferation, migration and EMT in TNBC. These findings also suggest that AR may be a potential therapeutic target for the treatment of both LAR and non-LAR TNBC subtypes. Citation Format: Tasmin A. Sultana, Nayef Aldabaan, 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 proliferation, migration and epithelial-to-mesenchymal transition (EMT) [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 2572.

Glycolysis inhibition and apoptosis induction in human prostate cancer cells by FV-429-mediated regulation of AR-AKT-HK2 signaling network

Prostate cancer (PCa) depends on androgen receptor (AR) signaling to regulate cell metabolism, including glycolysis, and thereby promotes tumor growth. Glycolysis is overactive in PCa and associated with poor prognosis, but the therapeutic efficacy of glycolysis inhibitors has thus far been limited by their inability to induce cell death. FV-429, a flavonoid derivative of Wogonin, is a glycolysis inhibitor that has shown anti-cancer promise. In this study, we used FV-429 as an anti-PCa agent and investigated its mechanisms of action. In vitro, both the glycolytic ability and the viability of PCa cells were inhibited by FV-429. We found that FV-429 could induce mitochondrial dysfunction and apoptosis, with AKT-HK2 signaling pathway playing a key role. In addition, FV-429 had a pro-apoptotic effect on human prostate cancer cells that relied on the inhibition of AR expression and activity. In vivo, FV-429 exerted significant tumor-repressing activity with high safety in the xenograft model using LNCaP cells. In summary, we demonstrated that FV-429 induced glycolysis inhibition and apoptosis in human prostate cancer cells by downregulating the AR-AKT-HK2 signaling network, making FV-429 a promising candidate as one therapeutic agent for advanced PCa.

Evaluation of the effects of androgenic Chinese herbal medicines on androgen receptors and tumor growth in experimental prostate cancer models

Ethnopharmacological relevanceMany prostate cancer (PCa) patients in Mainland China and other Asian countries often use Chinese herbal medicines as an adjuvant treatment while receiving Western medicines. However, concerns have been raised about the potential herb-drug interaction when using herbal medicines containing phytoandrogens. Aim of the studyThis study aimed to investigate the effects of the selected 21 Chinese herbal medicines on the proliferation and tumor growth using the relevant in vitro and in vivo models of PCa. Materials and methodsAfter treatment of LNCaP and 22Rv1 cells with different concentrations of 70% ethanol extracts of the 21 selected herbal medicines for 48 h, the proliferative activity, the effects on androgen receptor (AR) and prostate specific antigen (PSA) were determined. The anti-tumor effects of the 21 herbs on PCa growth were also investigated on a subcutaneous mouse model of PCa. ResultsThe results showed that Epimedii Folium (EF) and Codonopsis Radix (CNR) could significantly increase the cell viability in LNCaP cells (p < 0.05 for both) and 22Rv1 cells (p < 0.05 for both), protein expressions of AR in LNCaP cells (p < 0.05 for both) and 22Rv1 cells (p < 0.05 for both), and PSA (p < 0.05 for both) in LNCaP cells. EF, CNR, and Cistanches Herba (CCH) markedly accentuated the tumor growth (p < 0.05 for three drugs) and AR expression (p < 0.05 for three herbs) in tumor tissues. On the other hand, treatment with Astragali Radix (AGR), Chuanxiong Rhizoma (CXR) and Bruceae Fructus (BF) significantly inhibited the cell viability in LNCaP cells (p < 0.05, p < 0.05 and p < 0.001, respectively) and in 22Rv1 cells (p < 0.05, p < 0.05 and p < 0.001, respectively), and the protein expression of AR in LNCaP cells (p < 0.05 for three herbs) and 22Rv1 cells (p < 0.05, p < 0.05 and p < 0.001, respectively), and the protein expression of PSA (p < 0.05 for three herbs) in LNCaP cells, as well as tumor growth (p < 0.05 for three herbs) and the AR expression (p < 0.05 for AGR and CXR, p < 0.001 for BF) in tumor tissues. ConclusionOur results revealed that AGR, CXR and BF suppressed the PCa development via inhibition of AR expression, while EF, CNR and CCH promoted the development and progression of PCa via enhancement of AR expression. The results strongly suggest that caution should be exercised when using androgenic Chinese herbal medicines in PCa patients.

The JNK inhibitor AS602801 Synergizes with Enzalutamide to Kill Prostate Cancer Cells In Vitro and In Vivo and Inhibit Androgen Receptor Expression

In our previous study, we observed that androgen deprivation therapy (ADT) may induce a compensatory increase in MAPK or JNK signaling. Here, we tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with the AR inhibitor enzalutamide (ENZ) in androgen-sensitive LNCaP cells and androgen-resistant C4-2 and 22Rv1 cells. Enzalutamide combined with AS602801 synergistically killed LNCaP, C4-2, and 22Rv1 cells, and decreased migration and invasion of LNCaP and C4-2 cells. We studied the combination of enzalutamide with AS602801 in vivo using luciferase labeled LNCaP xenografts, and observed that combination of ENZ with AS602801 significantly suppressed tumor growth compared with either drug alone. Importantly, combination therapy resulted in dramatic loss of AR mRNA and protein. Surprisingly, mechanistic studies and Nanostring data suggest that AS602801 likely activates JNK signaling to induce apoptosis. Since AS602801 had sufficient safety and toxicity profile to advance from Phase I to Phase II in clinical trials, repurposing of this compound may represent an opportunity for rapid translation for clinical therapy of CRPC patients.

Pharmacological inhibition of androgen receptor expression induces cell death in prostate cancer cells.

The androgen receptor (AR) plays an important role in the pathogenesis and development of prostate cancer (PCa). Mostly, PCa progresses to androgen-independent PCa, which has activated AR signaling from androgen-dependent PCa. Thus, inhibition of AR signaling may be an important therapeutic target in androgen-dependent and castration-resistant PCa. In this study, we determined the anticancer effect of a newly found natural compound, sakurasosaponin (S-saponin), using androgen-dependent and castration-resistant PCa cell lines. S-saponin induces mitochondrial-mediated cell death in both androgen-dependent (LNCaP) and castration-resistant (22Rv1 and C4-2) PCa cells, via AR expression. S-saponin treatment induces a decrease in AR expression in a time- and dose-dependent manner and a potent decrease in the expression of its target genes, including prostate-specific antigen (PSA), transmembrane protease, serin 2 (TMPRSS2), and NK3 homeobox 1 (NKX3.1). Furthermore, S-saponin treatment decreases B-cell lymphoma-extra large (Bcl-xL) and mitochondrial membrane potential, thereby increasing the release of cytochrome c into the cytosol. Moreover, Bcl-xL inhibition and subsequent mitochondria-mediated cell death caused by S-saponin were reversed by Bcl-xL or AR overexpression. Interestingly, S-saponin-mediated cell death was significantly reduced by a reactive oxygen species (ROS) scavenger, N-acetylcystein. Animal xenograft experiments showed that S-saponin treatment significantly reduced tumor growth of AR-positive 22Rv1 xenografts but not AR-negative PC-3 xenografts. Taken together, for the first time, our results revealed that S-saponin induces mitochondrial-mediated cell death in androgen-dependent and castration-resistant cells through regulation of AR mechanisms, including downregulation of Bcl-xL expression and induction of ROS stress by decreasing mitochondrial membrane potential.

V-ATPase-dependent repression of androgen receptor in prostate cancer cells.

Prostate Cancer (PCa) is the most commonly diagnosed cancer and the third leading cause of death for men in the United States. Suppression of androgen receptor (AR) expression is a desirable mechanism to manage PCa. Our studies showed that AR expression was reduced in LAPC4 and LNCaP PCa cell lines treated with nanomolar concentrations of the V-ATPase inhibitor concanamycin A (CCA). This treatment decreased PSA mRNA levels, indicative of reduced AR activity. V-ATPase-dependent repression of AR expression was linked to defective endo-lysosomal pH regulation and reduced AR expression at the transcriptional level. CCA treatment increased the protein level and nuclear localization of the alpha subunit of the transcription factor HIF-1 (HIF-1α) in PCa cells via decreased hydroxylation and degradation of HIF-1α. The addition of iron (III) citrate restored HIF-1α hydroxylation and decreased total HIF-1α levels in PCa cells treated with CCA. Moreover, iron treatment partially rescued CCA-mediated AR repression. Dimethyloxalylglycine (DMOG), which prevents HIF-1α degradation independently of V-ATPase, also decreased AR levels, supporting our hypothesis that HIF-1α serves as a downstream mediator in the V-ATPase-AR axis. We propose a new V-ATPase-dependent mechanism to inhibit androgen receptor expression in prostate cancer cells involving defective endosomal trafficking of iron and the inhibition of HIF-1 α-subunit turnover.

Abstract 1589: Zinc inhibits androgen receptor expression to inhibit prostate cancer cell growth

Abstract Background: Prostate gland contains high level of intracellular zinc which is dramatically diminished during cancer development. Due to the obscure role of zinc in this process, therapeutic application using zinc and its supplement is very limited. This study aims to clarify the role(s) of zinc and its intervening mechanism. Material and methods: Treated by zinc chloride (15-150 µM), several prostate cancer cell lines were applied to confocal microscopy for intracellular trafficking of exogenous zinc, in vitro proliferation assays for their growth, prostate specific antigen (PSA)-based reporter-mediated transactivation, and Western blot for detection of androgen receptor (AR), PSA and ubiquitination. Further in vivo studies were performed to demonstrate the effect of zinc (10-20 mg/kg) on xenograft cancer growth using syngeneic animals followed by tumor analyses. Results: Zinc chloride suppressed androgen-dependent proliferation of human prostate cancer cells and accordingly zinc chloride dramatically inhibited androgen-mediated transactivation and several androgen target protein expressions, including PSA and p21. Further investigation showed that addition of zinc chloride strikingly downregulated AR protein levels after 4 hours up to 24 hours in both human LNCaP and murine TRAMP C2 prostate cancer cell lines. AR downregulation resulted from facilitated protein degradation instead of transcriptional control. Further in vivo study was carried out using syngeneic mice bearing C2 subcutaneous tumors. Peritoneal injection of zinc chloride significantly reduced tumor size. Analysis of these tumors revealed that there were reduced expression of AR and increased cell death. Conclusions: Zinc has been shown to inhibit incumbent oncogenic NF-κB pathway. These results also suggest that intracellular zinc inhibits cell growth via downregulation of AR to inhibit growth of prostate cancer. Considering that AR functions as a major effector in prostate cancer development and progression into castration resistant prostate cancer, loss of zinc may be a critical step for this devastating disease and further studies can be performed to develop zinc-based cancer therapeutics. Citation Format: Phuong Kim To, Young-Suk Cho, Se-Young Kwon, Taek Won Kang, Kyung Keun Kim, Chaeyong Jung. Zinc inhibits androgen receptor expression to inhibit prostate cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2017-1589

C-Met/miR-130b axis as novel mechanism and biomarker for castration resistance state acquisition.

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients' samples and by in silico analysis on an independent patient cohort from Taylor's platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients' risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.