Integration of class 1 HDAC inhibitors into current treatments for prostate cancer
Introduction: Prostate Cancer (CaP) is the most common non-cutaneous malignancy in men in the Western world and therefore a significant health burden worldwide. First-line standard-of-care therapies for organ confined, locally advanced prostate cancer (LAPC) are surgery and radiotherapy. Although highly effective, relapse to recurrent disease occurs in 20-50% of men and resistance to subsequent hormonal therapies results in progression to incurable castrate resistant prostate cancer (CRPC). Androgen signalling inhibitors (ASI), including Enzalutamide (ENZ) and Abiraterone (ABI), are effective treatments for metastatic CRPC however, resistance develops within 24-months of initial exposure in the majority of patients. Epigenetic silencing of cancer-related genes has been shown to be highly influential in CaP disease development and progression and epigenetic regulators, including histone deacetylases (HDAC), are commonly mutated and highly expressed in cancer, representing attractive clinical targets. Although five HDAC inhibitors (HDACi) have been FDA approved for myelodysplastic syndromes, evaluation of pan-HDACi in CaP clinical trials as single agents have been ineffective to date largely due to treatment-related toxicity and efficacy as monotherapies. To be able to incorporate HDACi into the treatment of CaP, the reduction of toxicity must be addressed including the use of a more specific targeted approach such as class specific HDACi. Class 1 HDAC isozymes are highly expressed in CaP and the Class 1 specific HDACi Entinostat (ENT) has been awarded FDA designation as a breakthrough therapy for aromatase inhibitor (AI)-resistant breast cancer in combination with hormone therapy demonstrating its potential to be used for treatment of solid tumours including CaP. In addition, the identification of an appropriate combination partner with HDACi and optimal scheduling of treatment will be beneficial in improving response to treatment and lowering the required dose, thereby improving dose-related toxicity. Hypothesis: Therefore, we hypothesise that Class 1 HDAC inhibition by ENT is sufficient to induce acetylation-based epigenomic alterations resulting in anti-tumour effects to synergise with current standards-of-care therapies, RT and ASI, and remain effective after castrate resistance, demonstrating its potential as a novel treatment for CaP. Methods: CaP cell lines were treated with ENT as a single agent and in combination with RT, ENZ and ABI and analysed for anti-tumour response in vitro, using cell viability, survival, and cell cycle analysis as well as transcriptomic and proteomic analysis by Western blot (WB), immunofluorescence (IF), qRT-PCR and RNAseq. In addition, two isogenic cell lines responsive to ENZ (PAR) and with acquired Enzalutamide-resistance (EnzR) were studied in vitro using similar methods. In vivo xenograft studies were carried out using PC3 cells showing synergistic response to ENT and ionising radiation (IR) and CWR-R1 PAR and EnzR cell lines for the effect of ENT as a novel treatment for enzalutamide resistant CRPC. Results: A combination of ENT and IR or ASI, resulted in an enhanced reduction in cell proliferation and survival providing evidence of the role of ENT in sensitising CaP cells to these therapies. An in vivo PC3 xenograft study demonstrated the radiosensitising effect of ENT in halting tumour growth and that this combination was well tolerated in a murine model supporting in vitro characterisation of this response in PC3 and C4-2B cells. In addition, molecular analyses through WB, IF, qRT-PCR and RNAseq showed a decrease in AR expression and downstream signalling in C4-2B cells treated with ENT, which was further suppressed in response to the combination treatment with ENZ. HDAC3 was suggested to be the most influential HDAC target in bringing about this response, as shown by siRNA mediated knockdown of individual HDAC isozymes. In vitro analysis of matched PAR and EnzR cell lines revealed EnzR cells to be more sensitive to ENT treatment than matched PAR cells by growth inhibition, survival and apoptotic cell death, and in CWR-R1 PAR and EnzR xenograft models, ENT effectively suppressed tumour growth and was well tolerated as measured by no significant alterations in body weight at study endpoint. Conclusions: Pan-HDACi, as single agents, have shown limited success in clinical trials for solid tumours including CaP primarily due to treatment related toxicity. However, we demonstrate the potential for the use of the more specific class-1 HDACi ENT in combination with current standard-of-care therapies radiotherapy and androgen signalling inhibitors in the treatment of CaP, including tumours with acquired ENZ resistance as a novel treatment for advanced refractory disease. ENT is an optimal candidate for clinical translation due to its pharmacokinetic and known toxicity profiles in patients and proven clinical efficacy for the treatment of AI-resistant breast cancer. This provides the basis for rapid translation into the clinical landscape of CaP, especially for patients with no current treatment options to better control disease and improve quality of life.
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