Chaperone-mediated autophagy promotes PCa survival during ARPI through selective proteome remodeling.

The androgen receptor (AR) plays an important role in PCa metabolism, with androgen receptor pathway inhibition (ARPI) subjecting PCa cells to acute metabolic stress caused by reduced biosynthesis and energy production. Defining acute stress response mechanisms that alleviate ARPI stress and therefore mediate prostate cancer (PCa) treatment resistance will help improve therapeutic outcomes of patients treated with ARPI. We identified the up-regulation of chaperone-mediated autophagy (CMA) in response to acute ARPI stress, which persisted in castration-resistant PCa (CRPC); previously undefined in PCa. CMA is a selective protein degradation pathway and a key stress response mechanism up-regulated under several stress stimuli, including metabolic stress. Through selective protein degradation, CMA orchestrates the cellular stress response by regulating cellular pathways through selective proteome remodeling. Through broad-spectrum proteomic analysis, CMA coordinates metabolic reprogramming of PCa cells to sustain PCa growth and survival during ARPI; through the upregulation of mTORC1 signaling and pathways associated with PCa biosynthesis and energetics. This not only promoted PCa growth during ARPI, but also promoted the emergence of CRPC in-vivo. During CMA inhibition, PCa metabolism is compromised, leading to ATP depletion, resulting in a profound anti-proliferative effect on PCa cells, and is enhanced when combined with ARPI. Furthermore, CMA inhibition prevented in-vivo tumour formation, and also re-sensitized enzalutamide-resistant cell lines in-vitro. The profound anti-proliferative effect of CMA inhibition was attributed to cell cycle arrest mediated through p53 transcriptional repression of E2F target genes. In summary, CMA is an acute ARPI stress response mechanism, essential in alleviating ARPI induced metabolic stress, essential for ensuring PCa growth and survival. CMA plays a critical role in the development of ARPI resistance in PCa.

Regulation of AR mRNA translation in response to acute AR pathway inhibition.

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid-liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.

Androgen receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling.

Treatment-induced adaptive pathways converge to support androgen receptor (AR) reactivation and emergence of castration-resistant prostate cancer (PCa) after AR pathway inhibition (ARPI). We set out to explore poorly defined acute adaptive responses that orchestrate shifts in energy metabolism after ARPI and identified rapid changes in succinate dehydrogenase (SDH), a TCA cycle enzyme with well-known tumor suppressor activity. We show that AR directly regulates transcription of its catalytic subunits (SDHA, SDHB) via androgen response elements (AREs). ARPI acutely suppresses SDH activity, leading to accumulation of the oncometabolite, succinate. Succinate triggers calcium ions release from intracellular stores, which in turn phospho-activates the AR-cochaperone, Hsp27 via p-CaMKK2/p-AMPK/p-p38 axis to enhance AR protein stabilization and activity. Activation of this pathway was seen in tissue microarray analysis on prostatectomy tissues and patient-derived xenografts. This adaptive response is blocked by co-targeting AR with Hsp27 under both in vitro and in vivo studies, sensitizing PCa cells to ARPI treatments.

A polymeric paste-drug formulation for local treatment of upper tract urothelial carcinoma.

BACKGROUND: Intravesical instillation of chemo- or immunotherapy is commonly used in bladder cancer. Upper tract urothelial carcinoma (UTUC) shares similar pathological features, but current formulations are not suitable for direct instillation to the upper urinary tract. OBJECTIVE: To evaluate in vivo applicability, characteristics and toxicity of ST-UC, a mucoadhesive polymeric paste formulation of gemcitabine, for upper urinary tract instillation. MATERIAL AND METHODS: Three pigs received 10 ml of ST-UC (100 mg/ml gemcitabine) retrogradely into 1 renal pelvis for pharmacokinetic studies. Four days later, a second injection into the contralateral renal pelvis was followed by serial euthanasia of the pigs and nephroureterectomy after 1, 3, and 6 hours. Adverse effects were monitored. Urine, serum, and tissue gemcitabine concentrations were measured, along with histologic examination of the upper urinary tract. RESULTS: Retrograde instillation of ST-UC was well tolerated with mild, completely receding hydronephrosis. Urine gemcitabine concentrations were highest in the first 3-hour collection interval. Hundred percent of gemcitabine was recovered in the urine within 24 hours. Serum peak concentrations (cmax) of gemcitabine were low at 5.5 µg/ml compared to the 10 to 30 µg/ml levels observed after a single intravenous dose of 1,000 mg/m2 gemcitabine. The formulation was still traceable after one hour and gemcitabine tissue concentrations are supportive of this extended drug exposure. No major histopathological changes were observed. The main limitation of this study is the lack of antitumor activity data. CONCLUSION: This preclinical evaluation of ST-UC demonstrated feasible instillation in the renal pelvis, no significant safety concerns, and sustained release of gemcitabine.

Paternally Expressed Gene 10 (PEG10) Promotes Growth, Invasion, and Survival of Bladder Cancer.

Paternally expressed gene 10 (PEG10) has been associated with neuroendocrine muscle-invasive bladder cancer (MIBC), a subtype of the disease with the poorest survival. In this work, we further characterized the expression pattern of PEG10 in The Cancer Genome Atlas database of 412 patients with MIBC, and found that, compared with other subtypes, PEG10 mRNA level was enhanced in neuroendocrine-like MIBC and highly correlated with other neuroendocrine markers. PEG10 protein level also associated with neuroendocrine markers in a tissue microarray of 82 cases. In bladder cancer cell lines, PEG10 expression was induced in drug-resistant compared with parental cells, and knocking down of PEG10 resensitized cells to chemotherapy. Loss of PEG10 increased protein levels of cell-cycle regulators p21 and p27 and delayed G1-S-phase transition, while overexpression of PEG10 enhanced cancer cell proliferation. PEG10 silencing also lowered levels of SLUG and SNAIL, leading to reduced invasion and migration. In an orthotopic bladder cancer model, systemic treatment with PEG10 antisense oligonucleotide delayed progression of T24 xenografts. In summary, elevated expression of PEG10 in MIBC may contribute to the disease progression by promoting survival, proliferation, and metastasis. Targeting PEG10 is a novel potential therapeutic approach for a subset of bladder cancers.

Design and Characterization of Injectable Poly(Lactic-Co-Glycolic Acid) Pastes for Sustained and Local Drug Release.

PURPOSE: We describe the preparation of injectable polymeric paste (IPP) formulations for local and sustained release of drugs. Furthermore, we include the characterization and possible applications of such pastes. Particular attention is paid to characteristics relevant to the successful clinical formulation development, such as viscosity, injectability, degradation, drug release, sterilization, stability performance and pharmacokinetics. METHODS: Paste injectability was characterized using measured viscosity and the Hagen-Poiseuille equation to determine injection forces. Drug degradation, release and formulation stability experiments were performed in vitro and drug levels were quantified using HPLC-UV methods. Pharmacokinetic evaluation of sustained-release lidocaine IPPs used five groups of six rats receiving increasing doses subcutaneously. An anti-cancer formulation was evaluated in a subcutaneous tumor xenograft mouse model. RESULTS: The viscosity and injectability of IPPs could be controlled by changing the polymeric composition. IPPs demonstrated good long-term stability and tunable drug-release with low systemic exposure in vivo in rats. Preliminary data in a subcutaneous tumor model points to a sustained anticancer effect. CONCLUSIONS: These IPPs are tunable platforms for local and sustained delivery of drugs and have potential for further clinical development to treat a number of diseases.

Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models.

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

A polymeric paste-drug formulation for intratumoral treatment of prostate cancer.

OBJECTIVE: Focal therapy has emerged as a treatment option for low- to intermediate-risk localized prostate cancer (PCa) patients, to balance the risks for urinary and sexual morbidity of radical treatment with the psychological burden of active surveillance. In this context, we developed ST-4PC, an injectable, polymeric paste formulation containing docetaxel (dtx) and bicalutamide (bic) for image-guided focal therapy of PCa. The objective of this work was to evaluate the in vitro characteristics and in vivo toxicity and efficacy of ST-4PC. MATERIAL AND METHODS: In vitro drug release was evaluated using high-performance liquid chromatography. In vivo toxicity of blank- and drug-loaded ST-4PC was assessed in mice and rats. Tumor growth inhibition was evaluated in LNCaP subcutaneous (s.c.) and LNCaP-luc orthotopic xenograft models. Using the s.c. model, mice were monitored weekly for weight loss, tumor volume (TV) and serum PSA. For the orthotopic model, mice were additionally monitored for bioluminescence as measure of tumor growth. RESULTS: ST-4PC demonstrated a sustained and steady release of incorporated drugs with 50% dtx and 20% bic being released after 14 days. While no systemic toxicity was observed, dose-dependent local side effects from dtx developed in the s.c. but not in the orthotopic model, illustrating the limitations of s.c. models for evaluating local cytotoxic therapy. In the s.c. model, 0.1%/4% and 0.25%/4% dtx/bic ST-4PC paste significantly reduced PSA progression, but did not have a significant inhibitory effect on TV. ST-4PC loaded with 1%/4% dtx/bic significantly reduced TV, serum PSA, and bioluminescence in the orthotopic xenograft model. Compared with drugs dissolved in DMSO, ST-4PC significantly delayed tumor growth. CONCLUSION: Image-guided focal therapy using ST-4PC demonstrated promising inhibition of PSA progression and orthotopic tumor growth in vivo without significant toxicity, and warrants further clinical evaluation.

Moving Towards Precision Urologic Oncology: Targeting Enzalutamide-resistant Prostate Cancer and Mutated Forms of the Androgen Receptor Using the Novel Inhibitor Darolutamide (ODM-201).

Darolutamide (ODM-201) is a novel androgen receptor (AR) antagonist with a chemical structure distinctly different from currently approved AR antagonists that targets both wild-type and mutated ligand binding domain variants to inhibit AR nuclear translocation. Here, we evaluate the activity of darolutamide in enzalutamide-resistant castration resistant prostate cancer (CRPC) as well as in AR mutants detected in patients after treatment with enzalutamide, abiraterone, or bicalutamide. Darolutamide significantly inhibited cell growth and AR transcriptional activity in enzalutamide-resistant MR49F cells in vitro, and led to decreased tumor volume and serum prostate-specific antigen levels in vivo, prolonging survival in mice bearing enzalutamide-resistant MR49F xenografts. Moreover, darolutamide inhibited the transcriptional activity of AR mutants identified in the plasma of CRPC patients progressing on traditional therapies. In particular, darolutamide significantly inhibited the transcriptional activity of the F877L, H875Y/T878A, F877L/T878A, and the previously unreported T878G AR mutants, that transform enzalutamide into a partial agonist. In silico cheminformatics computer modeling provided atomic level insights confirming darolutamide antagonist effect in F877L and T878G AR mutants. In conclusion, our results provide a rationale for further clinical evaluation of darolutamide in enzalutamide-resistant CRPC, in particular in combination with circulating tumor DNA assays that detect AR mutants sensitive to darolutamide, in a precision oncology setting. PATIENT SUMMARY: In this study we evaluated the novel drug darolutamide in preclinical models of prostate cancer. We found that darolutamide delays growth of enzalutamide-resistant prostate cancer, in particular in cells with mutated forms of the androgen receptor after previous treatment. Our data supports further evaluation of darolutamide in clinical trials.

Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis.

Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors.

A Novel Triazole Nucleoside Suppresses Prostate Cancer Cell Growth by Inhibiting Heat Shock Factor 1 and Androgen Receptor.

A novel triazole nucleoside analogue was discovered to exhibit potent anticancer activity in prostate cancer cells via down-regulating heat shock factor 1 (HSF1) and related heat shock proteins, along with the consequential inhibition of androgen receptor (AR) expression and transactivation, arresting the cell cycle in AR-governed phase. This triazole nucleoside therefore constitutes a novel structural paradigm and potential drug candidate for prostate cancer through inhibition of HSF1 and AR.

siRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer.

PURPOSE: Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model. EXPERIMENTAL DESIGN: Gene silencing of a luciferase reporter gene in the PC-3M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models. RESULTS: LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. LNP CLU-siRNA potently suppressed CLU and AR ASO-induced CLU and AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared with AR-ASO monotherapy. In subcutaneous ENZ-R LNCaP xenografts, combinatory treatment of LNP CLU-siRNA plus AR-ASO significantly suppressed tumor growth and serum PSA levels compared with LNP LUC-siRNA (control) and AR-ASO. CONCLUSIONS: LNP siRNA can silence target genes in vivo and enable inhibition of traditionally non-druggable genes like CLU and other promising cotargeting approaches in ENZ-R CRPC therapeutics.

Inhibition of the HER2-YB1-AR axis with Lapatinib synergistically enhances Enzalutamide anti-tumor efficacy in castration resistant prostate cancer.

Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

Hsp27 Inhibition with OGX-427 Sensitizes Non-Small Cell Lung Cancer Cells to Erlotinib and Chemotherapy.

Non-small cell lung cancer (NSCLC) is the most frequent cause of death from cancer worldwide. Despite the availability of active chemotherapy regimens and EGFR tyrosine kinase inhibitors, all advanced patients develop recurrent disease after first-line therapy. Although Hsp27 is a stress-induced chaperone that promotes acquired resistance in several cancers, its relationship to treatment resistance in NSCLC has not been defined. Understanding adaptive responses of acquired resistance will help guide new strategies to control NSCLC. Hsp27 levels were evaluated in an HCC827 erlotinib-resistant-derived cell line (HCC-827Resistant), and sensitivity to erlotinib was examined in Hsp27-overexpressing A549 cells. The role of Hsp27 in both erlotinib and cytotoxic treatment resistance was evaluated in HCC-827 and A549 NSCLC cells using the Hsp27 antisense drug OGX-427. The effect of OGX-427 in combination with erlotinib was also assessed in mice bearing A549 xenografts. Hsp27 is induced by erlotinib and protects NSCLC cells from treatment-induced apoptosis, whereas OGX-427 sensitizes NSCLC cells to erlotinib. Interestingly, increased resistance to erlotinib was observed when Hsp27 was increased either in HCC827 erlotinib-resistant or overexpressing A549 cells. Combining OGX-427 with erlotinib significantly enhanced antitumor effects in vitro and delayed A549 xenograft growth in vivo. OGX-427 also significantly enhanced the activity of cytotoxic drugs used for NSCLC. These data indicate that treatment-induced Hsp27 contributes to the development of resistance, and provides preclinical proof-of-principle that inhibition of stress adaptive pathways mediated by Hsp27 enhances the activity of erlotinib and chemotherapeutics.

Generation 2.5 antisense oligonucleotides targeting the androgen receptor and its splice variants suppress enzalutamide-resistant prostate cancer cell growth.

PURPOSE: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (ARFL) or variants (AR-Vs) in disease progression. EXPERIMENTAL DESIGN: To define functional roles of ARFL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown ARFL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts. RESULTS: ENZ-R-LNCaP cells express high levels of both ARFL and AR-V7 compared with CRPC-LNCaP; in particular, ARFL levels were approximately 12-fold higher than AR-V7. Both ARFL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of ARFL alone, or ARFL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both ARFL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of ARFL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts. CONCLUSIONS: These data identify the AR as an important driver of ENZ resistance, and while the contributions of ARFL and AR-Vs can vary across cell systems, ARFL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both ARFL and AR-Vs is a rational approach for AR-dependent CRPC.

Clusterin facilitates stress-induced lipidation of LC3 and autophagosome biogenesis to enhance cancer cell survival.

We define stress-induced adaptive survival pathways linking autophagy with the molecular chaperone clusterin (CLU) that function to promote anticancer treatment resistance. During treatment stress, CLU co-localizes with LC3 via an LIR-binding sequence within autophagosome membranes, functioning to facilitate LC3-Atg3 heterocomplex stability and LC3 lipidation, and thereby enhance autophagosome biogenesis and autophagy activation. Stress-induced autophagy is attenuated with CLU silencing in CLU(-/-) mice and human prostate cancer cells. CLU-enhanced cell survival occurs via autophagy-dependent pathways, and is reduced following autophagy inhibition. Combining CLU inhibition with anticancer treatments attenuates autophagy activation, increases apoptosis and reduces prostate cancer growth. This study defines a novel adaptor protein function for CLU under stress conditions, and highlights how co-targeting CLU and autophagy can amplify proteotoxic stress to delay cancer progression.

Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth.

Clusterin (CLU) is cytoprotective molecular chaperone that is highly expressed in castrate-resistant prostate cancer (CRPC). CRPC is also characterized by increased insulin-like growth factor (IGF)-I responsiveness which induces prostate cancer survival and CLU expression. However, how IGF-I induces CLU expression and whether CLU is required for IGF-mediated growth signaling remain unknown. Here we show that IGF-I induced CLU via STAT3-Twist1 signaling pathway. In response to IGF-I, STAT3 was phosphorylated, translocated to the nucleus and bound to the Twist1 promoter to activate Twist1 transcription. In turn, Twist1 bound to E-boxes on the CLU promoter and activated CLU transcription. Inversely, we demonstrated that knocking down Twist1 abrogated IGF-I induced CLU expression, indicating that Twist1 mediated IGF-I-induced CLU expression. When PTEN knockout mice were crossed with lit/lit mice, the resultant IGF-I deficiency suppressed Twist1 as well as CLU gene expression in mouse prostate glands. Moreover, both Twist1 and CLU knockdown suppressed prostate cancer growth accelerated by IGF-I, suggesting the relevance of this signaling not only in an in vitro, but also in an in vivo. Collectively, this study indicates that IGF-I induces CLU expression through sequential activation of STAT3 and Twist1, and suggests that this signaling cascade plays a critical role in prostate cancer pathogenesis.

Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo.

The progression to castration-resistant prostate cancer (CRPC) correlates with gain-of-function of the androgen receptor (AR) and activation of AKT. However, as single agents, AR or AKT inhibitors result in a reciprocal feedback loop. Therefore, we hypothesized that combination of an AKT inhibitor with an antiandrogen might result in a more profound, long-lasting remission of CRPC. Here, we report that the AKT inhibitor AZD5363 potently inhibits proliferation and induces apoptosis in prostate cancer cell lines expressing the AR and has anticancer activity in vivo in androgen-sensitive and castration-resistant phases of the LNCaP xenograft model. However, we found that the effect of castration-resistant tumor growth inhibition and prostate-specific antigen (PSA) stabilization is transient and resistance occurs with increasing PSA after approximately 30 days of treatment. Mechanistically, we found that single agent AZD5363 induces increase of AR binding to androgen response element, AR transcriptional activity, and AR-dependent genes such as PSA and NKX3.1 expression. These effects were overcome by the combination of AZD5363 with the antiandrogen bicalutamide, resulting in synergistic inhibition of cell proliferation and induction of apoptosis in vitro, and prolongation of tumor growth inhibition and PSA stabilization in CRPC in vivo. This study provides a preclinical proof-of-concept that combination of an AKT inhibitor with antiandrogen results in prolonged disease stabilization in a model of CRPC.

Cotargeting Androgen Receptor and Clusterin Delays Castrate-Resistant Prostate Cancer Progression by Inhibiting Adaptive Stress Response and AR Stability.

Although androgen receptor (AR) pathway inhibitors prolong survival in castrate-resistant prostate cancer (CRPC), resistance rapidly develops and is often associated with increased stress-activated molecular chaperones like clusterin (CLU) and continued AR signaling. Because adaptive pathways activated by treatment facilitate development of acquired resistance, cotargeting the stress response, activated by AR inhibition and mediated through CLU, may create conditional lethality and improve outcomes. Here, we report that CLU is induced by AR antagonism and silencing using MDV3100 and antisense, respectively, to become highly expressed in castrate- and MDV3100-resistant tumors and cell lines. CLU, as well as AKT and mitogen-activated protein kinase (MAPK) signalosomes, increase in response to MDV3100-induced stress. Mechanistically, this stress response is coordinated by a feed-forward loop involving p90rsk (RPS6KA)-mediated phosphoactivation of YB-1 with subsequent induction of CLU. CLU inhibition repressed MDV3100-induced activation of AKT and MAPK pathways. In addition, when combined with MDV3100, CLU knockdown accelerated AR degradation and repressed AR transcriptional activity through mechanisms involving decreased YB-1-regulated expression of the AR cochaperone, FKBP52. Cotargeting the AR (with MDV3100) and CLU (with OGX-011) synergistically enhanced apoptotic rates over that seen with MDV3100 or OGX-011 monotherapy and delayed CRPC LNCaP tumor and prostate-specific antigen (PSA) progression in vivo. These data indicate that cotargeting adaptive stress pathways activated by AR pathway inhibitors, and mediated through CLU, creates conditional lethality and provides mechanistic and preclinical proof-of-principle to guide biologically rational combinatorial clinical trial design.

Hsp27 regulates epithelial mesenchymal transition, metastasis, and circulating tumor cells in prostate cancer.

Defining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how to decrease morbidity and mortality in this disease. An important determinant of metastasis is epithelial-to-mesenchymal transition (EMT), and the mechanisms that control the process of EMT in cancer cells are still emerging. Here, we report that the molecular chaperone Hsp27 (HSPB1) drives EMT in prostate cancer, whereas its attenuation reverses EMT and decreases cell migration, invasion, and matrix metalloproteinase activity. Mechanistically, silencing Hsp27 decreased IL-6-dependent STAT3 phosphorylation, nuclear translocation, and STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6-mediated EMT via modulation of STAT3/Twist signaling. We observed a correlation between Hsp27 and Twist in patients with prostate cancer, with Hsp27 and Twist expression each elevated in high-grade prostate cancer tumors. Hsp27 inhibition by OGX-427, an antisense therapy currently in phase II trials, reduced tumor metastasis in a murine model of prostate cancer. More importantly, OGX-427 treatment decreased the number of circulating tumor cells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial. Overall, this study defines Hsp27 as a critical regulator of IL-6-dependent and IL-6-independent EMT, validating this chaperone as a therapeutic target to treat metastatic prostate cancer.