BRN4 Is a Novel Driver of Neuroendocrine Differentiation in Castration-Resistant Prostate Cancer and Is Selectively Released in Extracellular Vesicles with BRN2

Abstract

Purpose: Neuroendocrine prostate cancer (NEPC), an aggressive variant of castration-resistant prostate cancer (CRPC), often emerges after androgen receptor-targeted therapies such as enzalutamide or de novo, via trans-differentiation process of neuroendocrine differentiation. The mechanistic basis of neuroendocrine differentiation is poorly understood, contributing to lack of effective predictive biomarkers and late disease recognition. The purpose of this study was to examine the role of novel proneural Pit-Oct-Unc-domain transcription factors (TF) in NEPC and examine their potential as noninvasive predictive biomarkers.Experimental Design: Prostate cancer patient-derived xenograft models, clinical samples, and cellular neuroendocrine differentiation models were employed to determine the expression of TFs BRN1 and BRN4. BRN4 levels were modulated in prostate cancer cell lines followed by functional assays. Furthermore, extracellular vesicles (EV) were isolated from patient samples and cell culture models, characterized by nanoparticle tracking analyses, Western blotting, and realtime PCR.Results: We identify for the first time that: (i) BRN4 is amplified and overexpressed in NEPC clinical samples and that BRN4 overexpression drives neuroendocrine differentiation via its interplay with BRN2, a TF that was previously implicated in NEPC; (ii) BRN4 and BRN2 mRNA are actively released in prostate cancer EVs upon neuroendocrine differentiation induction; and (iii) enzalutamide treatment augments release of BRN4 and BRN2 in prostate cancer EVs, promoting neuroendocrine differentiation induction.Conclusions: Our study identifies a novel TF that drives NEPC and suggests that as adaptive mechanism to enzalutamide treatment, prostate cancer cells express and secrete BRN4 and BRN2 in EVs that drive oncogenic reprogramming of prostate cancer cells to NEPC. Importantly, EV-associated BRN4 and BRN2 are potential novel noninvasive biomarkers to predict neuroendocrine differentiation in CRPC.

Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells

“…The upregulation of αVβ3 in the donor cells (C4-2B in this study) may alter the cargo composition of the sEVs released by the cells and enable them to induce NED in the recipient cells. Previous examples of modulation of sEV cargo by a drug or a single molecule have been reported [ 35 – 37 ]. A recent publication [ 35 ] has also shown that enzalutamide treatment of LNCaP cells stably expressing AR induce changes in the sEV composition by increasing the levels of BRN2 and BRN4 mRNA.…”

Emerging role of tumor cell plasticity in modifying therapeutic response

“…546,547 In addition, a model of MUC1-driven lineage plasticity in PCa shows that MUC1 can upregulate the expression of BRN2 by recruitment of MYC and subsequent binding to the promoter region of BRN2, which further contributes to SOX2 expression. 548,549 This series of molecular regulations indicates that MUC1 may act as an upstream effector to regulate SOX2-induced lineage plasticity of neuroendocrine trans-differentiation in PCa. 548 Finally, by integrating systemic analyses of GEMM with patient clinical data, Zou et al 550 provided conclusive genetic evidence that drug-induced neuroendocrine trans-differentiation of PCSCs is one of the main reasons behind treatment failure.…”

“…551 Taken together, findings from different approaches, including clinical data, together with multiple in vitro and in vivo experimental models, strongly demonstrate that tumor cell plasticity-induced lineage switching enables PCa to escape from ARPI treatment. [543][544][545][548][549][550][551] Neuroendocrine trans-differentiation from NSCLC to SCLC. Similar observations of neuroendocrine trans-differentiation have been made for lung cancer, in which the transition from EGFR-mutant NSCLC to small cell lung cancer (SCLC) (Fig.…”

The epigenetic and transcriptional landscape of neuroendocrine prostate cancer

“…Notably, SOX2 is part of the 'reprogramming cocktail' of four pioneer transcription factors that together are capable of converting differentiated fibroblasts into induced pluripotent stem cells (Takahashi & Yamanaka 2016). More recently, another POU-domain transcription factor, BRN4, was found to interact with BRN2 and enhance its regulation of SOX2 (Bhagirath et al 2019). Importantly, BRN2 is directly repressed by AR, an example of the extensive, direct interplay that occurs between the key factors controlling cell identity in prostate cancer (Fig.…”