Supporting Figures S5-S8 from Novel Selective Agents for the Degradation of Androgen Receptor Variants to Treat Castration-Resistant Prostate Cancer
Suriyan PonnusamyChristopher C. CossThirumagal ThiyagarajanKate WattsDong‐Jin HwangYali HeLuke A. SelthIain J. McEwanC. B. DukeJayaprakash PagadalaGeetika SinghRobert WakeChristopher LedbetterWayne D. TilleyTudor MoldoveanuJames T. DaltonDuane D. MillerRamesh Narayanan
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<p>S5: UT-155 prevents nuclear localization of AR and AR v567es. S6: UT-155 binds to AR AF-1 domain. S7: Results from microarray with SARDs.S8: UT-155 inhibits transactivation of AD1 and D567es AR transactivation. Metabolism properties of SARDs.</p>Keywords:
Androgen Receptor Antagonists
<div>Abstract<p>Systemic prostate cancer therapy requires androgen ablation, which inhibits the production or action of androgens. Prostate cancer ultimately relapses during androgen ablation, and an androgen depletion-independent (ADI) phenotype emerges. Aberrant androgen receptor (AR) activation underlies therapy resistance at this stage of the disease, and mounting evidence implicates the large and highly disordered AR NH<sub>2</sub>-terminal domain (NTD) as a key mediator of this activity. In this study, we investigated the role of the NTD transactivation unit 5 (TAU5) domain in mediating AR transcriptional activity in cell-based models of prostate cancer progression. AR replacement and Gal4-based promoter tethering experiments revealed that AR TAU5 had a dichotomous function, inhibiting ligand-dependent AR activity in androgen-dependent prostate cancer cells, while enhancing ligand-independent AR activity in ADI prostate cancer cells. Molecular dissection of TAU5 showed that a WxxLF motif was fully responsible for its ligand-independent activity. Mechanistically, WxxLF did not rely on an interaction with the AR ligand-binding domain to mediate ligand-independent AR activity. Rather, WxxLF functioned as an autonomous transactivation domain. These data show that ligand-dependent and ligand-independent AR activation rely on fundamentally distinct mechanisms, and define WxxLF as the major transactivation motif within the AR TAU5 domain. [Cancer Res 2007;67(20):10067–77]</p></div>
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Abstract Systemic prostate cancer therapy requires androgen ablation, which inhibits the production or action of androgens. Prostate cancer ultimately relapses during androgen ablation, and an androgen depletion-independent (ADI) phenotype emerges. Aberrant androgen receptor (AR) activation underlies therapy resistance at this stage of the disease, and mounting evidence implicates the large and highly disordered AR NH2-terminal domain (NTD) as a key mediator of this activity. In this study, we investigated the role of the NTD transactivation unit 5 (TAU5) domain in mediating AR transcriptional activity in cell-based models of prostate cancer progression. AR replacement and Gal4-based promoter tethering experiments revealed that AR TAU5 had a dichotomous function, inhibiting ligand-dependent AR activity in androgen-dependent prostate cancer cells, while enhancing ligand-independent AR activity in ADI prostate cancer cells. Molecular dissection of TAU5 showed that a WxxLF motif was fully responsible for its ligand-independent activity. Mechanistically, WxxLF did not rely on an interaction with the AR ligand-binding domain to mediate ligand-independent AR activity. Rather, WxxLF functioned as an autonomous transactivation domain. These data show that ligand-dependent and ligand-independent AR activation rely on fundamentally distinct mechanisms, and define WxxLF as the major transactivation motif within the AR TAU5 domain. [Cancer Res 2007;67(20):10067–77]
Androgen Receptor Antagonists
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Both androgen receptor (AR) and orphan receptor TR2 (TR2) belong to the steroid nuclear receptor superfamily and are expressed in prostate cancer tissue and cell lines. AR has been known to be involved in prostate proliferation and prostate cancer progression. AR binds to androgen response elements and regulates target gene expression via a mechanism involving coregulators. However, the function of TR2 in prostate and prostate cancer and the relationship between TR2 and AR in the prostate cancer is unclear.Transient transfection and CAT reporter gene assays were employed to assess AR-mediated transactivation. The expression level of prostate specific antigen (PSA) was measured by Northern blot analysis. The interaction between AR and TR2 was assessed by glutathione-S-transferase (GST) pull-down assay and mammalian two-hybrid system assay.Orphan nuclear receptor TR2 suppressed androgen-mediated transactivation in prostate cancer PC-3 cells, and over-expression of TR2 suppressed PSA expression. The suppression of AR mediated transactivation by TR2 is not due to competition for the limited coregulator availability by these two receptors, but possibly through the interaction between TR2 and AR nuclear receptors.TR2 may function as a negative modulator to suppress AR function in prostate cancer. Further studies on how to control TR2 function may result in the ability to modulate AR function in prostate cancer.
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Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.
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All current clinically approved androgen deprivation therapies for prostate cancer target the C-terminal ligand-binding domain of the androgen receptor (AR). However, the main transactivation function of the receptor is localized at the AR N-terminal domain (NTD). Targeting the AR NTD directly is a challenge because of its intrinsically disordered structure and the lack of pockets for drugs to bind. Here, we have taken an alternative approach using the cochaperone BAG1L, which interacts with the NTD, to develop a novel AR inhibitor. We describe the identification of 2-(4-fluorophenyl)-5-(trifluoromethyl)-1,3-benzothiazole (A4B17), a small molecule that inhibits BAG1L–AR NTD interaction, attenuates BAG1L-mediated AR NTD activity, downregulates AR target gene expression, and inhibits proliferation of AR-positive prostate cancer cells. This compound represents a prototype of AR antagonists that could be key in the development of future prostate cancer therapeutics.
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<div>Abstract<p>Systemic prostate cancer therapy requires androgen ablation, which inhibits the production or action of androgens. Prostate cancer ultimately relapses during androgen ablation, and an androgen depletion-independent (ADI) phenotype emerges. Aberrant androgen receptor (AR) activation underlies therapy resistance at this stage of the disease, and mounting evidence implicates the large and highly disordered AR NH<sub>2</sub>-terminal domain (NTD) as a key mediator of this activity. In this study, we investigated the role of the NTD transactivation unit 5 (TAU5) domain in mediating AR transcriptional activity in cell-based models of prostate cancer progression. AR replacement and Gal4-based promoter tethering experiments revealed that AR TAU5 had a dichotomous function, inhibiting ligand-dependent AR activity in androgen-dependent prostate cancer cells, while enhancing ligand-independent AR activity in ADI prostate cancer cells. Molecular dissection of TAU5 showed that a WxxLF motif was fully responsible for its ligand-independent activity. Mechanistically, WxxLF did not rely on an interaction with the AR ligand-binding domain to mediate ligand-independent AR activity. Rather, WxxLF functioned as an autonomous transactivation domain. These data show that ligand-dependent and ligand-independent AR activation rely on fundamentally distinct mechanisms, and define WxxLF as the major transactivation motif within the AR TAU5 domain. [Cancer Res 2007;67(20):10067–77]</p></div>
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Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to
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Prostate cancer (PCa) is the leading cause of cancer in men in the USA, with more than 160,000 new cases each year. PCa patients receiving standard of care androgen deprivation therapy (ADT) eventually acquire what’s known as castration resistant prostate cancer (CRPC), and 20% of all PCa cases progress to this metastatic and incurable form of the disease. PCa is the second leading cause of cancer-related death in American men. Current FDA approved drugs for CRPC only provide a 2-5-month survival benefit due to the emergence of resistance to these therapies. Resistance involves continued androgen receptor (AR) signaling despite castrate serum levels of androgens. CRPC cells express elevated levels of both full-length AR (AR-FL), and constitutively active AR splice variants which lack the ligand binding domain (LBD) and display altered AR coactivator interactions, both of which contribute to persistent AR signaling. Overexpression of steroid-receptor coactivators are implicated in the progression of CRPC and amplify AR-mediated transcription by binding to either the activation function 2 (AF-2) surface formed by the AR-LBD, or the activation function 1 (AF-1) surface located in the amino-terminal domain of the receptor. Coactivator binding interactions enhance the recruitment of the basal transcriptional machinery and activate the transcription of AR target genes. Our lab has designed a screening paradigm to identify compounds with novel mechanisms of action to prevent or delay resistance. The screening paradigm uses a primary high content screening assay (HCS) designed to identify compounds that inhibit or disrupt protein-protein interactions (PPI’s) between AR and one of its coactivators, transcriptional intermediary factor 2 (TIF2), together with panels of counter screens and characterization assays to prioritize hit compounds that inhibit or disrupt AF-2 and/or AF-1 transactivation. The ideal hit compound would prevent the transcription of both AR-FL and AR-V7 thereby providing a dual approach to preventing CRPC progression.
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