Novel ASPORIN/CRABP2 axis drives cell-state plasticity from adenocarcinoma to neuroendocrine-like prostate cancer

Background : Prostate cancer (PCa) remains a highly lethal disease due to the rapid emergence of neuroendocrine (NE)-like variants from adenocarcinoma. It is predicted that de novo NEPC will emerge from 17% of localized PCa, while treatment-related NEPC will account for 20% of advanced PCa. Clinically, NEPC patients frequently harbor visceral metastasis (62%, P<0.001) compared with CRPC patients (24%), with poor overall survival of 12-17 months. Thus, exploring the unknown mechanism underlying cell-state transition from adeno to NEPC will help identify new targets to overcome lineage plasticity and improve PCa patient survival. Methods: We developed a novel indolent and metastatic PCa mouse model by overexpressing cMyc and knocking out Pten with/without Mutant p53 (R172H). Global transcriptional profiling was performed in indolent and aggressive mice and ASPORIN (ASPN) knockdown (KD) to identify differential gene expression, biological, and pathway analyses. ASPN ectopic overexpression (OE) and KD clones confirmed ASPN biological function and on-target proteins/genes using RT-PCR and western blot analyses. ASPN in vitro function was analyzed in the Incucyte® live imaging system, colony growth assay, and proliferation assays. Results: The novel P ten fl/fl ; Hi-Myc; T rp53 R172H/+ ; Rosa-26; PB- C re4 + (PCTP Luc ) mouse exhibits phenotypic resemblance to PCa visceral metastasis to the lung, liver, inguinal lymph node, and intestine with poor survival as compared with age-matched indolent P ten fl/fl ; Hi-Myc; Rosa-26 Luc ; P B-Cre4 + (PCP Luc ). Unbiased RNA-Seq analysis of PCa adenocarcinoma tissues from PCTP mice revealed a significant association between unique extracellular matrix (ECM) protein clusters and liver and lung metastasis. Asporin (ASPN) emerged as the top differentially expressed ECM protein among the top 25 genes. Consistently, ASPN knockdown and overexpression were directly associated with PCa cellular phenotypes of proliferation and colony growth. Mechanistically, ASPN modulation impacts pERK, CyclinD3, EMT proteins, and a neuroendocrine-like phenotype, as well as cell differentiation-related transcriptomes such as cellular retinoic acid-binding protein 2 (CRABP2), along with Chromogranin A (CHGA), NeuroD1, and INSM1. Using a publicly available TCGA database, we observed a significant overexpression of ASPN and CRABP2 in PCa tissues (N=497) relative to normal (N=52) (P<0.001). Finally, ASPN ectopic overexpression in immortalized RWPE-1 cells confirmed an aggressive in vitro phenotypes. Conclusion: For the first time, we explored the influence of ASPN/CRABP2 and other signaling involved in luminal epithelial to neuroendocrine differentiation and validated CRABP2 as a novel target to prevent NEPC transdifferentiation. Our findings support ASPN's new role, mechanism(s) and as a target for PCa and other visceral metastasis disease.