Targeting hyaluronan-mediated motility receptor (HMMR) enhances response to androgen receptor signalling inhibitors in prostate cancer.

BACKGROUND: Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs. METHODS: RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs). RESULTS: HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs. CONCLUSION: Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs.

A Boolean-based machine learning framework identifies predictive biomarkers of HSP90-targeted therapy response in prostate cancer.

Precision medicine has emerged as an important paradigm in oncology, driven by the significant heterogeneity of individual patients' tumour. A key prerequisite for effective implementation of precision oncology is the development of companion biomarkers that can predict response to anti-cancer therapies and guide patient selection for clinical trials and/or treatment. However, reliable predictive biomarkers are currently lacking for many anti-cancer therapies, hampering their clinical application. Here, we developed a novel machine learning-based framework to derive predictive multi-gene biomarker panels and associated expression signatures that accurately predict cancer drug sensitivity. We demonstrated the power of the approach by applying it to identify response biomarker panels for an Hsp90-based therapy in prostate cancer, using proteomic data profiled from prostate cancer patient-derived explants. Our approach employs a rational feature section strategy to maximise model performance, and innovatively utilizes Boolean algebra methods to derive specific expression signatures of the marker proteins. Given suitable data for model training, the approach is also applicable to other cancer drug agents in different tumour settings.

The role of RHAMM in cancer: Exposing novel therapeutic vulnerabilities.

Receptor for hyaluronic acid-mediated motility (RHAMM) is a cell surface receptor for hyaluronic acid that is critical for cell migration and a cell cycle protein involved in microtubule assembly and stability. These functions of RHAMM are required for cellular stress responses and cell cycle progression but are also exploited by tumor cells for malignant progression and metastasis. RHAMM is often overexpressed in tumors and is an independent adverse prognostic factor for a number of cancers such as breast and prostate. Interestingly, pharmacological or genetic inhibition of RHAMM in vitro and in vivo ablates tumor invasiveness and metastatic spread, implicating RHAMM as a potential therapeutic target to restrict tumor growth and improve patient survival. However, RHAMM's pro-tumor activity is dependent on its subcellular distribution, which complicates the design of RHAMM-directed therapies. An alternative approach is to identify downstream signaling pathways that mediate RHAMM-promoted tumor aggressiveness. Herein, we discuss the pro-tumoral roles of RHAMM and elucidate the corresponding regulators and signaling pathways mediating RHAMM downstream events, with a specific focus on strategies to target the RHAMM signaling network in cancer cells.

Harnessing the Heterogeneity of Prostate Cancer for Target Discovery Using Patient-Derived Explants.

Prostate cancer is a complex and heterogeneous disease, but a small number of cell lines have dominated basic prostate cancer research, representing a major obstacle in the field of drug and biomarker discovery. A growing lack of confidence in cell lines has seen a shift toward more sophisticated pre-clinical cancer models that incorporate patient-derived tumors as xenografts or explants, to more accurately reflect clinical disease. Not only do these models retain critical features of the original tumor, and account for the molecular diversity and cellular heterogeneity of prostate cancer, but they provide a unique opportunity to conduct research in matched tumor samples. The challenge that accompanies these complex tissue models is increased complexity of analysis. With over 10 years of experience working with patient-derived explants (PDEs) of prostate cancer, this study provides guidance on the PDE method, its limitations, and considerations for addressing the heterogeneity of prostate cancer PDEs that are based on statistical modeling. Using inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) as an example of a drug that induces robust proliferative response, we demonstrate how multi-omics analysis in prostate cancer PDEs is both feasible and essential for identification of key biological pathways, with significant potential for novel drug target and biomarker discovery.

Lipidomic Profiling of Clinical Prostate Cancer Reveals Targetable Alterations in Membrane Lipid Composition.

Dysregulated lipid metabolism is a prominent feature of prostate cancer that is driven by androgen receptor (AR) signaling. Here we used quantitative mass spectrometry to define the "lipidome" in prostate tumors with matched benign tissues (n = 21), independent unmatched tissues (n = 47), and primary prostate explants cultured with the clinical AR antagonist enzalutamide (n = 43). Significant differences in lipid composition were detected and spatially visualized in tumors compared with matched benign samples. Notably, tumors featured higher proportions of monounsaturated lipids overall and elongated fatty acid chains in phosphatidylinositol and phosphatidylserine lipids. Significant associations between lipid profile and malignancy were validated in unmatched samples, and phospholipid composition was characteristically altered in patient tissues that responded to AR inhibition. Importantly, targeting tumor-related lipid features via inhibition of acetyl-CoA carboxylase 1 significantly reduced cellular proliferation and induced apoptosis in tissue explants. This characterization of the prostate cancer lipidome in clinical tissues reveals enhanced fatty acid synthesis, elongation, and desaturation as tumor-defining features, with potential for therapeutic targeting. SIGNIFICANCE: This study identifies malignancy and treatment-associated changes in lipid composition of clinical prostate cancer tissues, suggesting that mediators of these lipidomic changes could be targeted using existing metabolic agents.

A feedback loop between the androgen receptor and 6-phosphogluoconate dehydrogenase (6PGD) drives prostate cancer growth.

Alterations to the androgen receptor (AR) signalling axis and cellular metabolism are hallmarks of prostate cancer. This study provides insight into both hallmarks by uncovering a novel link between AR and the pentose phosphate pathway (PPP). Specifically, we identify 6-phosphogluoconate dehydrogenase (6PGD) as an androgen-regulated gene that is upregulated in prostate cancer. AR increased the expression of 6PGD indirectly via activation of sterol regulatory element binding protein 1 (SREBP1). Accordingly, loss of 6PGD, AR or SREBP1 resulted in suppression of PPP activity as revealed by 1,2-13C2 glucose metabolic flux analysis. Knockdown of 6PGD also impaired growth and elicited death of prostate cancer cells, at least in part due to increased oxidative stress. We investigated the therapeutic potential of targeting 6PGD using two specific inhibitors, physcion and S3, and observed substantial anti-cancer activity in multiple models of prostate cancer, including aggressive, therapy-resistant models of castration-resistant disease as well as prospectively collected patient-derived tumour explants. Targeting of 6PGD was associated with two important tumour-suppressive mechanisms: first, increased activity of the AMP-activated protein kinase (AMPK), which repressed anabolic growth-promoting pathways regulated by acetyl-CoA carboxylase 1 (ACC1) and mammalian target of rapamycin complex 1 (mTORC1); and second, enhanced AR ubiquitylation, associated with a reduction in AR protein levels and activity. Supporting the biological relevance of positive feedback between AR and 6PGD, pharmacological co-targeting of both factors was more effective in suppressing the growth of prostate cancer cells than single-agent therapies. Collectively, this work provides new insight into the dysregulated metabolism of prostate cancer and provides impetus for further investigation of co-targeting AR and the PPP as a novel therapeutic strategy.

Aberrations in circulating ceramide levels are associated with poor clinical outcomes across localised and metastatic prostate cancer.

BACKGROUND: Dysregulated lipid metabolism is associated with more aggressive pathology and poorer prognosis in prostate cancer (PC). The primary aim of the study is to assess the relationship between the plasma lipidome and clinical outcomes in localised and metastatic PC. The secondary aim is to validate a prognostic circulating 3-lipid signature specific to metastatic castration-resistant PC (mCRPC). PATIENTS AND METHODS: Comprehensive lipidomic analysis was performed on pre-treatment plasma samples from men with localised PC (N = 389), metastatic hormone-sensitive PC (mHSPC)(N = 44), or mCRPC (validation cohort, N = 137). Clinical outcomes from our previously published mCRPC cohort (N = 159) that was used to derive the prognostic circulating 3-lipid signature, were updated. Associations between circulating lipids and clinical outcomes were examined by Cox regression and latent class analysis. RESULTS: Circulating lipid profiles featuring elevated levels of ceramide species were associated with metastatic relapse in localised PC (HR 5.80, 95% CI 3.04-11.1, P = 1 × 10-6), earlier testosterone suppression failure in mHSPC (HR 3.70, 95% CI 1.37-10.0, P = 0.01), and shorter overall survival in mCRPC (HR 2.54, 95% CI 1.73-3.72, P = 1 × 10-6). The prognostic significance of circulating lipid profiles in localised PC was independent of standard clinicopathological and metabolic factors (P < 0.0002). The 3-lipid signature was verified in the mCRPC validation cohort (HR 2.39, 95% CI 1.63-3.51, P = 1 × 10-5). CONCLUSIONS: Elevated circulating ceramide species are associated with poorer clinical outcomes across the natural history of PC. These clinically actionable lipid profiles could be therapeutically targeted in prospective clinical trials to potentially improve PC outcomes.

ELOVL5 Is a Critical and Targetable Fatty Acid Elongase in Prostate Cancer.

The androgen receptor (AR) is the key oncogenic driver of prostate cancer, and despite implementation of novel AR targeting therapies, outcomes for metastatic disease remain dismal. There is an urgent need to better understand androgen-regulated cellular processes to more effectively target the AR dependence of prostate cancer cells through new therapeutic vulnerabilities. Transcriptomic studies have consistently identified lipid metabolism as a hallmark of enhanced AR signaling in prostate cancer, yet the relationship between AR and the lipidome remains undefined. Using mass spectrometry-based lipidomics, this study reveals increased fatty acyl chain length in phospholipids from prostate cancer cells and patient-derived explants as one of the most striking androgen-regulated changes to lipid metabolism. Potent and direct AR-mediated induction of ELOVL fatty acid elongase 5 (ELOVL5), an enzyme that catalyzes fatty acid elongation, was demonstrated in prostate cancer cells, xenografts, and clinical tumors. Assessment of mRNA and protein in large-scale data sets revealed ELOVL5 as the predominant ELOVL expressed and upregulated in prostate cancer compared with nonmalignant prostate. ELOVL5 depletion markedly altered mitochondrial morphology and function, leading to excess generation of reactive oxygen species and resulting in suppression of prostate cancer cell proliferation, 3D growth, and in vivo tumor growth and metastasis. Supplementation with the monounsaturated fatty acid cis-vaccenic acid, a direct product of ELOVL5 elongation, reversed the oxidative stress and associated cell proliferation and migration effects of ELOVL5 knockdown. Collectively, these results identify lipid elongation as a protumorigenic metabolic pathway in prostate cancer that is androgen-regulated, critical for metastasis, and targetable via ELOVL5. SIGNIFICANCE: This study identifies phospholipid elongation as a new metabolic target of androgen action that is critical for prostate tumor metastasis.

Synthesis and fluorine-18 radiolabeling of a phospholipid as a PET imaging agent for prostate cancer.

INTRODUCTION: Altered lipid metabolism and subsequent changes in cellular lipid composition have been observed in prostate cancer cells, are associated with poor clinical outcome, and are promising targets for metabolic therapies. This study reports for the first time on the synthesis of a phospholipid radiotracer based on the phospholipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (PC44:12) to allow tracking of polyunsaturated lipid tumor uptake via PET imaging. This tracer may aid in the development of strategies to modulate response to therapies targeting lipid metabolism in prostate cancer. METHODS: Lipidomics analysis of prostate tumor explants and LNCaP tumor cells were used to identify PC44:12 as a potential phospholipid candidate for radiotracer development. Synthesis of phosphocholine precursor and non-radioactive standard were optimised using click chemistry. The biodistribution of a fluorine-18 labeled analogue, N-{[4-(2-[18F]fluoroethyl)-2,3,4-triazol-1-yl]methyl}-1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine ([18F]2) was determined in LNCaP prostate tumor-bearing NOD SCID gamma mice by ex vivo biodistribution and PET imaging studies and compared to biodistribution of [18F]fluoromethylcholine. RESULTS: [18F]2 was produced with a decay-corrected yield of 17.8 ± 3.7% and an average radiochemical purity of 97.00 ± 0.89% (n = 6). Molar activity was 85.1 ± 3.45 GBq/µmol (2300 ± 93 mCi/µmol) and the total synthesis time was 2 h. Ex vivo biodistribution data demonstrated high liver uptake (41.1 ± 9.2%ID/g) and high splenic uptake (10.9 ± 9.1%ID/g) 50 min post-injection. Ex vivo biodistribution showed low absolute tumor uptake of [18F]2 (0.8 ± 0.3%ID/g). However, dynamic PET imaging demonstrated an increase over time of the relative tumor-to-muscle ratio with a peak of 2.8 ± 0.5 reached 1 h post-injection. In contrast, dynamic PET of [18F]fluoromethylcholine demonstrated no increase in tumor-to-muscle ratios due to an increase in both tumor and muscle over time. Absolute uptake of [18F]fluoromethylcholine was higher and peaked at 60 min post injection (2.25 ± 0.29%ID/g) compared to [18F]2 (1.44 ± 0.06%ID/g) during the 1 h dynamic scan period. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This study demonstrates the ability to radiolabel phospholipids and indicates the potential to monitor the in vivo distribution of phospholipids using fluorine-18 based PET.

Fatty Acid Oxidation Is an Adaptive Survival Pathway Induced in Prostate Tumors by HSP90 Inhibition.

HSP90 is a molecular chaperone required for stabilization and activation of hundreds of client proteins, including many known oncoproteins. AUY922 (luminespib), a new-generation HSP90 inhibitor, exhibits potent preclinical efficacy against several cancer types including prostate cancer. However, clinical use of HSP90 inhibitors for prostate cancer has been limited by toxicity and treatment resistance. Here, we aimed to design an effective combinatorial therapeutic regimen that utilizes subtoxic doses of AUY922, by identifying potential survival pathways induced by AUY922 in clinical prostate tumors. We conducted a proteomic analysis of 30 patient-derived explants (PDE) cultured in the absence and presence of AUY922, using quantitative mass spectrometry. AUY922 significantly increased the abundance of proteins involved in oxidative phosphorylation and fatty acid metabolism in the PDEs. Consistent with these findings, AUY922-treated prostate cancer cell lines exhibited increased mitochondrial mass and activated fatty acid metabolism processes. We hypothesized that activation of fatty acid oxidation is a potential adaptive response to AUY922 treatment and that cotargeting this process will sensitize prostate cancer cells to HSP90 inhibition. Combination treatment of AUY922 with a clinical inhibitor of fatty acid oxidation, perhexiline, synergistically decreased viability of several prostate cancer cell lines, and had significant efficacy in PDEs. The novel drug combination treatment induced cell-cycle arrest and apoptosis, and attenuated the heat shock response, a known mediator of HSP90 treatment resistance. This combination warrants further preclinical and clinical investigation as a novel strategy to overcome resistance to HSP90 inhibition. IMPLICATIONS: Metabolic pathways induced in tumor cells by therapeutic agents may be critical, but targetable, mediators of treatment resistance.

Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis.

Fatty acid ß-oxidation (FAO) is the main bioenergetic pathway in human prostate cancer (PCa) and a promising novel therapeutic vulnerability. Here we demonstrate therapeutic efficacy of targeting FAO in clinical prostate tumors cultured ex vivo, and identify DECR1, encoding the rate-limiting enzyme for oxidation of polyunsaturated fatty acids (PUFAs), as robustly overexpressed in PCa tissues and associated with shorter relapse-free survival. DECR1 is a negatively-regulated androgen receptor (AR) target gene and, therefore, may promote PCa cell survival and resistance to AR targeting therapeutics. DECR1 knockdown selectively inhibited ß-oxidation of PUFAs, inhibited proliferation and migration of PCa cells, including treatment resistant lines, and suppressed tumor cell proliferation and metastasis in mouse xenograft models. Mechanistically, targeting of DECR1 caused cellular accumulation of PUFAs, enhanced mitochondrial oxidative stress and lipid peroxidation, and induced ferroptosis. These findings implicate PUFA oxidation via DECR1 as an unexplored facet of FAO that promotes survival of PCa cells.

Pharmacodynamics effects of CDK4/6 inhibitor LEE011 (ribociclib) in high-risk, localised prostate cancer: a study protocol for a randomised controlled phase II trial (LEEP study: LEE011 in high-risk, localised Prostate cancer).

INTRODUCTION: Despite the development of new therapies for advanced prostate cancer, it remains the most common cause of cancer and the second leading cause of cancer death in men. It is critical to develop novel agents for the treatment of prostate cancer, particularly those that target aspects of androgen receptor (AR) signalling or prostate biology other than inhibition of androgen synthesis or AR binding. Neoadjuvant pharmacodynamic studies allow for a rational approach to the decisions regarding which targeted therapies should progress to phase II/III trials. CDK4/6 inhibitors have evidence of efficacy in breast cancer, and have been shown to have activity in preclinical models of hormone sensitive and castrate resistant prostate cancer. The LEEP trial aims to assess the pharmacodynamic effects of LEE011 (ribociclib), an orally bioavailable and highly selective CDK4/6 inhibitor, in men undergoing radical prostatectomy for high-risk, localised prostate cancer. METHODS AND ANALYSIS: The multicentre randomised, controlled 4:1 two-arm, phase II, open label pharmacodynamic study will recruit 47 men with high risk, localised prostate cancer who are planned to undergo radical prostatectomy. Participants who are randomised to receive the study treatment will be treated with LEE011 400 mg daily for 21 days for one cycle. The primary endpoint is the frequency of a 50% reduction in Ki-67 proliferation index from the pretreatment prostate biopsy compared to that present in prostate cancer tissue from radical prostatectomy. Secondary and tertiary endpoints include pharmacodynamic assessment of CDK4/6 cell cycle progression via E2F levels, apoptotic cell death by cleaved caspase-3, changes in serum and tumour levels of Prostate Specific Antigen (PSA), pathological regression, safety via incidence of adverse events and exploratory biomarker analysis. ETHICS AND DISSEMINATION: The protocol was approved by a central ethics review committee (St Vincent's Hospital HREC) for all participating sites (HREC/17/SVH/294). Results will be disseminated in peer-reviewed journals and at scientific conferences. DRUG SUPPLY: Novartis. PROTOCOL VERSION: 2.0, 30 May 2019 TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry (ACTRN12618000354280).

Evaluation of Small Molecule Drug Uptake in Patient-Derived Prostate Cancer Explants by Mass Spectrometry.

Patient-derived explant (PDE) culture of solid tumors is increasingly being applied to preclinical evaluation of novel therapeutics and for biomarker discovery. In this technique, treatments are added to culture medium and penetrate the tissue via a gelatin sponge scaffold. However, the penetration profile and final concentrations of small molecule drugs achieved have not been determined to date. Here, we determined the extent of absorption of the clinical androgen receptor antagonist, enzalutamide, into prostate PDEs, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser/desorption ionisation (MALDI) mass spectrometry imaging (MSI). In a cohort of 11 PDE tissues from eight individual patients, LC-MS/MS quantification of PDE homogenates confirmed enzalutamide (10 µM) uptake by all PDEs, which reached maximal average tissue concentration of 0.24-0.50 ng/µg protein after 48 h culture. Time dependent uptake of enzalutamide (50 µM) in PDEs was visualized using MALDI MSI over 24-48 h, with complete penetration throughout tissues evident by 6 h of culture. Drug signal intensity was not homogeneous throughout the tissues but had areas of markedly high signal that corresponded to drug target (androgen receptor)-rich epithelial regions of tissue. In conclusion, application of MS-based drug quantification and visualization in PDEs, and potentially other 3-dimensional model systems, can provide a more robust basis for experimental study design and interpretation of pharmacodynamic data.

Extracellular Fatty Acids Are the Major Contributor to Lipid Synthesis in Prostate Cancer.

Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); however, little is known about how extracellular FAs, such as those in the circulation, may support prostate cancer progression. Here, we show that increasing FA availability increased intracellular triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). Extracellular FAs are the major source (∼83%) of carbons to the total lipid pool in all cell lines, compared with glucose (∼13%) and glutamine (∼4%), and FA oxidation rates are greater in prostate cancer cells compared with PNT1 cells, which preferentially partitioned extracellular FAs into triacylglycerols. Because of the higher rates of FA oxidation in C4-2B cells, cells remained viable when challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate induced apoptosis, which was prevented by pretreatment of PC-3 cells with FAs, and this protective effect required DGAT-1-mediated triacylglycerol synthesis. These outcomes highlight for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential influence that obesity-associated dyslipidemia or host circulating has on prostate cancer progression. IMPLICATIONS: Extracellular-derived FAs are primary building blocks for complex lipids and heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior.

Patient-derived Models Reveal Impact of the Tumor Microenvironment on Therapeutic Response.

BACKGROUND: Androgen deprivation therapy is a first-line treatment for disseminated prostate cancer (PCa). However, virtually all tumors become resistant and recur as castration-resistant PCa, which has no durable cure. One major hurdle in the development of more effective therapies is the lack of preclinical models that adequately recapitulate the heterogeneity of PCa, significantly hindering the ability to accurately predict therapeutic response. OBJECTIVE: To leverage the ex vivo culture method termed patient-derived explant (PDE) to examine the impact of PCa therapeutics on a patient-by-patient basis. DESIGN SETTING AND PARTICIPANTS: Fresh PCa tissue from patients who underwent radical prostatectomy was cultured as PDEs to examine therapeutic response. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The impact of genomic and chemical perturbations in PDEs was assessed using various parameters (eg, AR levels, Ki67 staining, and desmoplastic indices). RESULTS AND LIMITATIONS: PDE maintained the integrity of the native tumor microenvironment (TME), tumor tissue morphology, viability, and endogenous hormone signaling. Tumor cells in this model system exhibited de novo proliferative capacity. Examination of the native TME in the PDE revealed a first-in-field insight into patient-specific desmoplastic stromal indices and predicted responsiveness to AR-directed therapeutics. CONCLUSIONS: The PDE model allows for a comprehensive evaluation of individual tumors in their native TME to ultimately develop more effective therapeutic regimens tailored to individuals. Discernment of novel stromal markers may provide a basis for applying precision medicine in treating advanced PCa, which would have a transformative effect on patient outcomes. PATIENT SUMMARY: In this study, an innovative model system was used to more effectively mimic human disease. The patient-derived explant (PDE) system can be used to predict therapeutic response and identify novel targets in advanced disease. Thus, the PDE will be an asset for the development of novel metrics for the implementation of precision medicine in prostate cancer.The patient-derived explant (PDE) model allows for a comprehensive evaluation of individual human tumors in their native tumor microenvironment (TME). TME analysis revealed first-in-field insight into predicted tumor responsiveness to AR-directed therapeutics through evaluation of patient-specific desmoplastic stromal indices.

A patient-derived explant (PDE) model of hormone-dependent cancer.

Breast and prostate cancer research to date has largely been predicated on the use of cell lines in vitro or in vivo. These limitations have led to the development of more clinically relevant models, such as organoids or murine xenografts that utilize patient-derived material; however, issues related to low take rate, long duration of establishment, and the associated costs constrain use of these models. This study demonstrates that ex vivo culture of freshly resected breast and prostate tumor specimens obtained from surgery, termed patient-derived explants (PDEs), provides a high-throughput and cost-effective model that retains the native tissue architecture, microenvironment, cell viability, and key oncogenic drivers. The PDE model provides a unique approach for direct evaluation of drug responses on an individual patient's tumor, which is amenable to analysis using contemporary genomic technologies. The ability to rapidly evaluate drug efficacy in patient-derived material has high potential to facilitate implementation of personalized medicine approaches.

Identification of Novel Response and Predictive Biomarkers to Hsp90 Inhibitors Through Proteomic Profiling of Patient-derived Prostate Tumor Explants.

Inhibition of the heat shock protein 90 (Hsp90) chaperone is a promising therapeutic strategy to target expression of the androgen receptor (AR) and other oncogenic drivers in prostate cancer cells. However, identification of clinically-relevant responses and predictive biomarkers is essential to maximize efficacy and treatment personalization. Here, we combined mass spectrometry (MS)-based proteomic analyses with a unique patient-derived explant (PDE) model that retains the complex microenvironment of primary prostate tumors. Independent discovery and validation cohorts of PDEs (n = 16 and 30, respectively) were cultured in the absence or presence of Hsp90 inhibitors AUY922 or 17-AAG. PDEs were analyzed by LC-MS/MS with a hyper-reaction monitoring data independent acquisition (HRM-DIA) workflow, and differentially expressed proteins identified using repeated measure analysis of variance (ANOVA; raw p value <0.01). Using gene set enrichment, we found striking conservation of the most significantly AUY922-altered gene pathways between the discovery and validation cohorts, indicating that our experimental and analysis workflows were robust. Eight proteins were selectively altered across both cohorts by the most potent inhibitor, AUY922, including TIMP1, SERPINA3 and CYP51A (adjusted p < 0.01). The AUY922-mediated decrease in secretory TIMP1 was validated by ELISA of the PDE culture medium. We next exploited the heterogeneous response of PDEs to 17-AAG in order to detect predictive biomarkers of response and identified PCBP3 as a marker with increased expression in PDEs that had no response or increased in proliferation. Also, 17-AAG treatment led to increased expression of DNAJA1 in PDEs that exhibited a cytostatic response, revealing potential drug resistance mechanisms. This selective regulation of DNAJA1 was validated by Western blot analysis. Our study establishes "proof-of-principle" that proteomic profiling of drug-treated PDEs represents an effective and clinically-relevant strategy for identification of biomarkers that associate with certain tumor-specific responses.

New Opportunities for Targeting the Androgen Receptor in Prostate Cancer.

Recent genomic analyses of metastatic prostate cancer have provided important insight into adaptive changes in androgen receptor (AR) signaling that underpin resistance to androgen deprivation therapies. Novel strategies are required to circumvent these AR-mediated resistance mechanisms and thereby improve prostate cancer survival. In this review, we present a summary of AR structure and function and discuss mechanisms of AR-mediated therapy resistance that represent important areas of focus for the development of new therapies.

Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion.

The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.

Effect of FAK inhibitor VS-6063 (defactinib) on docetaxel efficacy in prostate cancer.

BACKGROUND: Docetaxel, the standard chemotherapy for metastatic castration-resistant prostate cancer (CRPC) also enhances the survival of patients with metastatic castration-sensitive prostate cancer (CSPC) when combined with androgen-deprivation therapy. Focal Adhesion Kinase (FAK) activation is a mediator of docetaxel resistance in prostate cancer cells. The aim of this study was to investigate the effect of the second generation FAK inhibitor VS-6063 on docetaxel efficacy in pre-clinical CRPC and CSPC models. METHODS: Docetaxel-resistant CRPC cells, mice with PC3 xenografts, and ex vivo cultures of patient-derived primary prostate tumors were treated with VS-6063 and/or docetaxel, or vehicle control. Cell counting, immunoblotting, and immunohistochemistry techniques were used to evaluate the treatment effects. RESULTS: Docetaxel and VS-6063 co-treatment caused a greater decrease in the viability of docetaxel-resistant CRPC cells, and a greater inhibition in PC3 xenograft growth compared to either monotherapy. FAK expression in human primary prostate cancer was positively associated with advanced tumor stage. Patient-derived prostate tumor explants cultured with both docetaxel and VS-6063 displayed a higher percentage of apoptosis in cancer cells, than monotherapy treatment. CONCLUSIONS: Our findings suggest that co-administration of the FAK inhibitor, VS-6063, with docetaxel represents a potential therapeutic strategy to overcome docetaxel resistance in prostate cancer.