PO.IM02.06 · 免疫学
新型 ASPORIN/CRABP2 轴驱动从腺癌到神经内分泌样前列腺癌的细胞状态可塑性
Novel ASPORIN/CRABP2 axis drives cell-state plasticity from adenocarcinoma to neuroendocrine-like prostate cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：由于神经内分泌（NE）样变体从腺癌中快速出现，前列腺癌（PCa）仍是一种高度致死性疾病。据预测，原发性 NEPC 将从 17% 的局限性 PCa 中出现，而治疗相关性 NEPC 将占晚期 PCa 的 20%。临床上，与 CRPC 患者（24%）相比，NEPC 患者常伴有内脏转移（62%，P<0.001），总生存不佳，仅 12-17 个月。因此，探索从腺癌到 NEPC 细胞状态转变背后的未知机制将有助于鉴定新靶点，以克服谱系可塑性并改善 PCa 患者生存。
方法：我们通过过表达 cMyc 并敲除 Pten（伴或不伴突变型 p53（R172H））建立了一种新型惰性及转移性 PCa 小鼠模型。对惰性和侵袭性小鼠以及 ASPORIN（ASPN）敲低（KD）进行全局转录谱分析，以鉴定差异基因表达、生物学及通路分析。ASPN 异位过表达（OE）和 KD 克隆使用 RT-PCR 和蛋白质印迹分析确认了 ASPN 的生物学功能及靶向蛋白/基因。ASPN 的体外功能在 Incucyte® 活体成像系统、集落生长实验和增殖实验中进行了分析。
结果：新型 Pten fl/fl；Hi-Myc；Trp53 R172H/+；Rosa-26；PB-Cre4+（PCTP Luc）小鼠在表型上类似于 PCa 向肺、肝、腹股沟淋巴结和肠道的内脏转移，与年龄匹配的惰性 Pten fl/fl；Hi-Myc；Rosa-26 Luc；PB-Cre4+（PCP Luc）相比生存不佳。对 PCTP 小鼠 PCa 腺癌组织的无偏 RNA-Seq 分析揭示了独特的细胞外基质（ECM）蛋白簇与肝和肺转移之间的显著关联。Asporin（ASPN）成为前 25 个基因中差异表达最显著的 ECM 蛋白。一致地，ASPN 敲低和过表达与 PCa 增殖和集落生长的细胞表型直接相关。在机制上，ASPN 的调控影响 pERK、CyclinD3、EMT 蛋白以及神经内分泌样表型，还影响细胞分化相关转录组，如细胞视黄酸结合蛋白 2（CRABP2），以及嗜铬粒蛋白 A（CHGA）、NeuroD1 和 INSM1。利用公开可用的 TCGA 数据库，我们观察到 ASPN 和 CRABP2 在 PCa 组织（N=497）中相对于正常组织（N=52）的显著过表达（P<0.001）。最后，ASPN 在永生化 RWPE-1 细胞中的异位过表达确认了侵袭性的体外表型。
结论：我们首次探索了 ASPN/CRABP2 及其他参与腔上皮向神经内分泌分化的信号通路的影响，并验证 CRABP2 为预防 NEPC 转分化的新靶点。我们的发现支持 ASPN 的新作用、机制及其作为 PCa 和其他内脏转移性疾病靶点的地位。
查看英文原文 English abstract
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.
利益披露 Disclosure
P. Seshacharyulu, None..
S. Lall, None..
S. Halder, None..
S. Muniyan, None..
Z. W. Alsafwani, None..
R. Chirravuri-Venkata, None..
M. P. Ponnusamy, None.
S. K. Batra,
Sanguine Diagnostics and Therapeutics, Inc. Other, SKB is a founding member of Sanguine Diagnostics and Therapeutics, Inc.