microRNA Determinants of Neuroendocrine Differentiation in Prostate Cancer

Prostate cancer (PCa) is fueled by male hormones called androgens that act via binding to androgen receptors (ARs). Therefore, ablation of AR signaling by androgen deprivation therapy (ADT) is the goal of first-line therapy that results in cancer regression initially. However, 2 to 3 years after ADT, the disease develops into castration-resistant PCa (CRPC), which has limited therapeutic options and a poor overall survival rate of less than 3 years. The mainstay of therapy for patients with metastatic spread is the second line of hormonal therapy targeting AR, such as the androgen pathway inhibitors (APIs), enzalutamide and abiraterone, which improve survival initially. However, patients eventually develop resistance as tumors develop other bypass mechanisms such as restored AR signaling, AR bypass signaling, or complete AR independence. A significant number of tumors that become AR-independent progress into a more lethal variant called neuroendocrine (NE) PCa (NEPC).

NEPC is a very aggressive variant of advanced CRPC that manifests in ~30% of metastatic cases, either because of API treatment or even in treatment-naïve cases. This lethal form of PCa arises via a reversible trans-differentiation process, referred to as NE differentiation (NED), wherein PCa cells switch their lineage and convert into another cellular phenotype. After NED, PCa cells exhibit NE features that are characterized by expression of neuronal markers such as enolase 2, chromogranin A, and synaptophysin. Clinically, NEPC manifests as the presence of visceral metastatic disease, including metastasis to the liver, lung, central nervous system, or bone; low-serum PSA levels relative to disease burden; and limited response to AR signaling inhibitors. The prognosis of NEPC is very poor, and there is a lack of effective systemic therapies, as the molecular basis of how PCa cells undergo NED is poorly understood. A few recent genomic studies have been conducted to understand NEPC at a molecular level. Though these studies reported key NE-associated genomic alterations such as loss of the tumor suppressor genes, RB1 and TP53 and amplification of Aurora kinase A and N-Myc, the role of small non-coding RNAs in NEPC has not been systematically explored.

MicroRNAs are small non-coding RNAs that are important gene regulators and impact all fundamental cellular processes. MicroRNAs suppress gene expression post-transcriptionally by binding to the 3'- untranslated regions of cognate mRNA target genes. Considering the important regulatory role of microRNAs, this is a critical drawback in the field that contributes to a poor understanding of the molecular basis of NEPC. MicroRNAs behave as cell-dependent tumor suppressors or oncogenes, regulating tumorigenesis through a plethora of oncogenic mechanisms. Further, owing to their small size and resistance to endogenous RNase activity, microRNAs are stable biomarkers that can be readily detected in formalin-fixed tissues.

With a goal of understanding the molecular mechanisms underlying aggressive NEPC and resistance to APIs, in the present proposal, we will systematically investigate for the first time those microRNAs that are involved in inducing NED states in PCa. We will define the microRNA alterations associated with progression of advanced CRPC to an androgen-independent NE state by using clinical patient samples and cell culture models and performing small RNA sequencing. Further, we will analyze the significantly altered microRNAs by real-time PCR in a validation cohort of CRPC patients with/without NED and correlate with clinical data to identify microRNAs that can be predictive biomarkers to assess NED in advanced PCa. We propose that these microRNAs can also be used to guide clinical decision-making, as they can distinguish patients who will respond to treatment with APIs versus non-responders. We will study the role of two significantly altered microRNA clusters in NE PCa. We expect the proposed studies to be completed in 3 years.

The proposed research will benefit patients suffering from PCa that are at an advanced, metastatic stage and are not responsive to drugs that inhibit the androgen pathway. Currently, there are huge gaps in understanding how advanced, metastatic PCa switches to lethal NEPC upon androgen withdrawal, contributing to the lack of robust molecular markers for its diagnosis, prognosis, and therapy. This study will enhance our understanding of the molecular basis of this switch that will eventually form the basis of new ways to diagnose and treat this lethal form of PCa. Thus, the proposed study has high transformative and translational applicability as it will (1) lead to identification of novel anti-cancer targets for developing effective strategies for treatment of NEPC, (2) lead to development of effective molecular biomarkers for predicting the likelihood of development of NEPC in patients suffering from advanced CRPC, and (3) help identify patie .......