Androgen Receptor Degradation Enhancer ASC-J9 Research Articles

Targeting the androgen receptor (AR) with AR degradation enhancer ASC-J9® led to increase docetaxel sensitivity via suppressing the p21 expression

Chemotherapy with docetaxel remains the effective therapy to suppress castration resistant prostate cancer (CRPC) in some patients. However, most chemotherapy with docetaxel eventually fails with the development of docetaxel resistance after 18-weeks of treatment. Here we found docetaxel treatment might have an adverse effect of increasing the androgen receptor (AR) protein level in the CRPC cells, and combining docetaxel with anti-AR therapy using AR-shRNA or the AR degradation enhancer ASC-J9® may increase docetaxel sensitivity to better suppress the CRPC cell growth. Mechanism dissection found docetaxel might have the adverse effect of increasing the AR protein stability via suppressing the AR ubiquitination due to the increased AR phosphorylation. The consequence of such increased AR protein may then lead to increase p21 expression via transcriptional regulation. Preclinical studies with in vitro cells lines also demonstrated that targeting AR with ASC-J9® led to suppressing the AR-increased p21 expression to improve the docetaxel sensitivity in the CRPC cells that already developed docetaxel resistance. Together, these results suggest that a combined therapy of docetaxel and ASC-J9® is a novel therapy to better suppress CRPC in patients that already developed docetaxel resistance.

ASC-J9® suppresses prostate cancer cell invasion via altering the sumoylation-phosphorylation of STAT3

The androgen-deprivation therapy (ADT) to either reduce the androgen biosynthesis (for example, Abiraterone) or to prevent binding of androgen to the androgen receptor (AR), for example using Casodex or Enzalutamide, which may result in .decrease of the prostate cancer (PCa) cell growth, yet may also increase the PCa cell invasion. In contrast, the recently identified AR degradation enhancer ASC-J9® may function via degrading the AR protein to simultaneously suppress the PCa cell proliferation and invasion. The details of this unique mechanism, however, remain unclear. Here we found that ASC-J9® could suppress PCa cell invasion via inducing the sumoylation of STAT3, thereby inhibiting the STAT3 phosphorylation that led to suppress the EMT-SNAIL2 signals in both PCa DU145 and PC3 AR-negative cells. Mutation of lysine-679 on the sumoylation site of the STAT3 effectively blocked the ASC-J9®-suppressed PCa cell invasion in both in vitro cell lines and in vivo mouse models. These results suggest that in addition to degrading AR to suppress PCa cell proliferation, ASC-J9® can also function through an AR-independent mechanism via modulating the STAT3 sumoylation to alter the phospho-STAT3 status to suppress the PCa cell invasion. These dual functions of ASC-J9® to suppress PCa proliferation and invasion (via altering STAT3 sumoylation) may help us to develop a better anti-AR compound that may overcome the current antiandrogens' unwanted side-effect of increasing the metastasis to better suppress the castration-resistant PCa progression.

Androgen receptor promotes melanoma metastasis via altering the miRNA-539-3p/USP13/MITF/AXL signals.

Early studies demonstrated that male melanoma patients have worse survival than female patients, yet the detailed mechanisms for this gender difference remain unclear. We analyzed around 100 cases of human melanoma and found that androgen receptor (AR) positive melanoma patients have worse survival outcomes compared with AR-negative melanoma patients. Here we report that AR can have positive roles to increase melanoma cell invasion in multiple cell lines in vitro and a mouse model in vivo. Mechanism dissection suggest that AR increases melanoma cell invasion via modulating the MITF-AXL signals via altering the miRNA-539-3p/USP13 signaling to increase MITF protein degradation through a reduction of de-ubiquitination. Restoring MITF can reverse AR-enhanced melanoma cell invasion. Together, our results demonstrate that AR can promote melanoma metastasis via altering the miRNA-539-3p/USP13/MITF/AXL signal and targeting this newly identified signal with AR degradation enhancer ASC-J9 may help us to better suppress the melanoma metastasis.

Antiandrogen Therapy with Hydroxyflutamide or Androgen Receptor Degradation Enhancer ASC-J9 Enhances BCG Efficacy to Better Suppress Bladder Cancer Progression.

Recent studies suggest that the androgen receptor (AR) might play important roles in influencing bladder cancer progression, yet its clinical application remains unclear. Here, we developed a new combined therapy with Bacillus Calmette-Guérin (BCG) and the AR degradation enhancer ASC-J9 or antiandrogen hydroxyflutamide (HF) to better suppress bladder cancer progression. Mechanism dissection revealed that ASC-J9 treatment enhanced BCG efficacy to suppress bladder cancer cell proliferation via increasing the recruitment of monocytes/macrophages that involved the promotion of BCG attachment/internalization to the bladder cancer cells through increased integrin-α5β1 expression and IL6 release. Such consequences might then enhance BCG-induced bladder cancer cell death via increased TNFα release. Interestingly, we also found that ASC-J9 treatment could directly promote BCG-induced HMGB1 release to enhance the BCG cytotoxic effects for suppression of bladder cancer cell growth. In vivo approaches also concluded that ASC-J9 could enhance the efficacy of BCG to better suppress bladder cancer progression in BBN-induced bladder cancer mouse models. Together, these results suggest that the newly developed therapy combining BCG plus ASC-J9 may become a novel therapy to better suppress bladder cancer progress.

Androgen receptor promotes abdominal aortic aneurysm development via modulating inflammatory interleukin-1α and transforming growth factor-β1 expression.

Sex difference is a risk factor for abdominal aortic aneurysm (AAA) formation yet the reason for male predominance remains unclear. Androgen and the androgen receptor (AR) influence the male sex difference, indicating that AR signaling may affect AAA development. Using angiotensin II–induced AAA in apolipoprotein E null mouse models (82.4% AAA incidence), we found that mice lacking AR failed to develop AAA and aorta had dramatically reduced macrophages infiltration and intact elastic fibers. These findings suggested that AR expression in endothelial cells, macrophages, or smooth muscle cells might play a role in AAA development. Selective knockout of AR in each of these cell types further demonstrated that mice lacking AR in macrophages (20% AAA incidence) or smooth muscle cells (12.5% AAA incidence) but not in endothelial cells (71.4% AAA incidence) had suppressed AAA development. Mechanism dissection showed that AR functioned through modulation of interleukin-1α (IL-1α) and transforming growth factor-β1 signals and by targeting AR with the AR degradation enhancer ASC-J9 led to significant suppression of AAA development. These results demonstrate the underlying mechanism by which AR influences AAA development is through IL-1α and transforming growth factor-β1, and provides a potential new therapy to suppress/prevent AAA by targeting AR with ASC-J9.

AB130. ASC-J9 suppresses renal cell carcinoma progression by targeting an androgen receptor-dependent HIF2α/VEGF signaling pathway.

Males have a higher incidence of renal cell carcinoma (RCC) than females, but the reason for this gender difference is unknown. Addressing this question, we report the discovery of an androgen receptor (AR)-induced HIF2α/VEGF signal that drives RCC progression. AR attenuation or augmentation in RCC cells altered their proliferation, migration, and invasion in multiple models in vitro and in vivo. Mechanistic investigations revealed that AR targeting inhibited RCC cell migration and invasion by modulating HIF2α/VEGF signals at the level of mRNA and protein expression. Interrupting HIF2α/VEGF signals with inhibitors of either HIF2α or VEGF was sufficient to suppress RCC progression. Similarly, the specific AR degradation enhancer ASC-J9 was sufficient to suppress AR-induced HIF2α/VEGF signaling and RCC progression in multiple models in vitro and in vivo. Taken together, our results revealed a novel role for AR in RCC initiation and progression with implications for novel therapeutic strategies.

ASC-J9 suppresses renal cell carcinoma progression by targeting an androgen receptor-dependent HIF2α/VEGF signaling pathway.

Males have a higher incidence of renal cell carcinoma (RCC) than females, but the reason for this gender difference is unknown. Addressing this question, we report the discovery of an androgen receptor (AR)-induced HIF2α/VEGF signal that drives RCC progression. AR attenuation or augmentation in RCC cells altered their proliferation, migration, and invasion in multiple models in vitro and in vivo. Mechanistic investigations revealed that AR targeting inhibited RCC cell migration and invasion by modulating HIF2α/VEGF signals at the level of mRNA and protein expression. Interrupting HIF2α/VEGF signals with inhibitors of either HIF2α or VEGF was sufficient to suppress RCC progression. Similarly, the specific AR degradation enhancer ASC-J9 was sufficient to suppress AR-induced HIF2α/VEGF signaling and RCC progression in multiple models in vitro and in vivo. Taken together, our results revealed a novel role for AR in RCC initiation and progression with implications for novel therapeutic strategies.

Androgen receptor enhances kidney stone-CaOx crystal formation via modulation of oxalate biosynthesis & oxidative stress.

Males develop kidney stones far more frequently than females with a ratio of 2–3:1, suggesting that androgen receptor (AR) signaling might play a key role in the development of nephrolithiasis. Using the cre-loxP system to selectively knock out AR in glyoxylate-induced calcium oxalate (CaOx) crystal mouse models, we found that the mice lacking hepatic AR had less oxalate biosynthesis, which might lead to lower CaOx crystal formation, and that the mice lacking kidney proximal or distal epithelial AR also had lower CaOx crystal formation. We found that AR could directly up-regulate hepatic glycolate oxidase and kidney epithelial NADPH oxidase subunit p22-PHOX at the transcriptional level. This up-regulation might then increase oxalate biosynthesis and oxidative stress that resulted in induction of kidney tubular injury. Targeting AR with the AR degradation enhancer ASC-J9 led to suppression of CaOx crystal formation via modulation of oxalate biosynthesis and oxidative stress in both in vitro and in vivo studies. Taken together, these results established the roles of AR in CaOx crystal formation.

Determination of androgen receptor degradation enhancer ASC-J9® in mouse sera and organs with liquid chromatography tandem mass spectrometry

A novel androgen receptor (AR) degradation enhancer ASC-J9® has displayed beneficial effects during the in vitro and in vivo studies for treatment of prostate cancer, liver cancer, bladder cancer and spinal and bulbar muscular atrophy (SBMA). It works mainly via the degradation of AR with minimal side effects on the tested mice. Here we developed a fast, robust and more sensitive method for the quantification of ASC-J9® in 100μL of mouse serum by using liquid chromatography tandem mass spectrometry (LC–MS/MS). The limit of quantification (LOQ) was found to be 5nM for ASCJ9®. This method was successfully applied to investigate the pharmacokinetics of ASC-J9® in mice serum samples and also the distribution of the drug in various mice organs after single dose injection with results showing that ASC-J9® could be quickly absorbed in vivo and had a relatively slow elimination half-life of 5.45h. The ASC-J9® also exhibited a higher tendency to accumulate in organs such as liver, testes and prostate.

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

New Therapeutic Approach to Suppress Castration-Resistant Prostate Cancer Using ASC-J9 via Targeting Androgen Receptor in Selective Prostate Cells

Using androgen receptor (AR) knockout mice to determine AR functions in selective prostate cancer (PCa) cells, we determined that AR might play differential roles in various cell types, either to promote or suppress PCa development/progression. These observations partially explain the failure of current androgen deprivation therapy (ADT) to reduce/prevent androgen binding to AR in every cell. Herein, we identified the AR degradation enhancer ASC-J9, which selectively degrades AR protein via interruption of the AR-AR selective coregulator interaction. Such selective interruption could, therefore, suppress AR-mediated PCa growth in the androgen-sensitive stage before ADT and in the castration-resistant stage after ADT. Mechanistic dissection suggested that ASC-J9 could activate the proteasome-dependent pathway to promote AR degradation through the enhanced association of AR-Mdm2 complex. The consequences of ASC-J9-promoted AR degradation included reduced androgen binding to AR, AR N-C terminal interaction, and AR nuclear translocation. Such inhibitory regulation could then result in suppression of AR transactivation and AR-mediated cell growth in eight different mouse models, including intact or castrated nude mice xenografted with androgen-sensitive LNCaP cells orandrogen-insensitive C81 cells and castrated nude mice xenografted with castration-resistant C4-2 and CWR22Rv1 cells, and TRAMP and Pten(+/-) mice. These results demonstrate that ASC-J9 could serve as an AR degradation enhancer that effectively suppresses PCa development/progression in the androgen-sensitive and castration-resistant stages.

Targeting Androgen Receptor Leads to Suppression of Prostate Cancer via Induction of Autophagy

Androgen receptor has a critical role in prostate cancer development and progression. Cell death via autophagy may also contribute to prostate cancer progression. We determined the role and regulatory effects of androgen receptor on the autophagy process of prostate cancer cells. Using a series of morphological approaches, such as transmission electron microscopy, monodansylcadaverine (Sigma®) and GFP-LC3 fluorescence microscopy assay, and Western blot we monitored the autophagic process in 3 pairs of prostate cancer cell lines to study the relationship to androgen receptor signals. Androgen receptor knockdown in androgen receptor positive cells, such as LNCaP or CWRrv1 human prostate cancer cells, led to increased autophagy. Adding functional androgen receptor to androgen receptor negative cells, such as PC3 human prostate cancer cells, resulted in decreased autophagy. This suggests that androgen receptor could have a negative role in regulating autophagy. Mechanism dissection indicated that androgen receptor might repress autophagy via modulation of p62 expression. A therapeutic approach of targeting androgen receptor to increase autophagy using the androgen receptor degradation enhancer ASC-J9® suppressed prostate cancer growth. Findings provide evidence that the androgen receptor might promote prostate cancer cell growth via autophagy down-regulation. Targeting the androgen receptor via ASC-J9 might lead to tumor suppression via the induction of autophagy. This may represent a new, potential therapeutic approach and mechanism for prostate cancer.

New therapy targeting differential androgen receptor signaling in prostate cancer stem/progenitor vs. non-stem/progenitor cells

The androgen deprivation therapy (ADT) to systematically suppress/reduce androgens binding to the androgen receptor (AR) has been the standard therapy for prostate cancer (PCa); yet, most of ADT eventually fails leading to the recurrence of castration resistant PCa. Here, we found that the PCa patients who received ADT had increased PCa stem/progenitor cell population. The addition of the anti-androgen, Casodex, or AR-siRNA in various PCa cells led to increased stem/progenitor cells, whereas, in contrast, the addition of functional AR led to decreased stem/progenitor cell population but increased non-stem/progenitor cell population, suggesting that AR functions differentially in PCa stem/progenitor vs. non-stem/progenitor cells. Therefore, the current ADT might result in an undesired expansion of PCa stem/progenitor cell population, which explains why this therapy fails. Using various human PCa cell lines and three different mouse models, we concluded that targeting PCa non-stem/progenitor cells with AR degradation enhancer ASC-J9 and targeting PCa stem/progenitor cells with 5-azathioprine and γ-tocotrienol resulted in a significant suppression of the tumors at the castration resistant stage. This suggests that a combinational therapy that simultaneously targets both stem/progenitor and non-stem/progenitor cells will lead to better therapeutic efficacy and may become a new therapy to battle the PCa before and after castration resistant stages.

ASC-J9 Suppresses Castration-Resistant Prostate Cancer Growth through Degradation of Full-length and Splice Variant Androgen Receptors

Early studies suggested androgen receptor (AR) splice variants might contribute to the progression of prostate cancer (PCa) into castration resistance. However, the therapeutic strategy to target these AR splice variants still remains unresolved. Through tissue survey of tumors from the same patients before and after castration resistance, we found that the expression of AR3, a major AR splice variant that lacks the AR ligand-binding domain, was substantially increased after castration resistance development. The currently used antiandrogen, Casodex, showed little growth suppression in CWR22Rv1 cells. Importantly, we found that AR degradation enhancer ASC-J9 could degrade both full-length (fAR) and AR3 in CWR22Rv1 cells as well as in C4-2 and C81 cells with addition of AR3. The consequences of such degradation of both fAR and AR3 might then result in the inhibition of AR transcriptional activity and cell growth in vitro. More importantly, suppression of AR3 specifically by short-hairpin AR3 or degradation of AR3 by ASC-J9 resulted in suppression of AR transcriptional activity and cell growth in CWR22Rv1-fARKD (fAR knockdown) cells in which DHT failed to induce, suggesting the importance of targeting AR3. Finally, we demonstrated the in vivo therapeutic effects of ASC-J9 by showing the inhibition of PCa growth using the xenografted model of CWR22Rv1 cells orthotopically implanted into castrated nude mice with undetectable serum testosterone. These results suggested that targeting both fAR- and AR3-mediated PCa growth by ASC-J9 may represent the novel therapeutic approach to suppress castration-resistant PCa. Successful clinical trials targeting both fAR and AR3 may help us to battle castration-resistant PCa in the future.