PAX6 promotes neuroendocrine phenotypes of prostate cancer via enhancing MET/STAT5A-mediated chromatin accessibility

Neuroendocrine prostate cancer (NEPC) is a highly aggressive form of prostate cancer characterized by the loss of androgen receptor (AR) signaling and the acquisition of neuroendocrine (NE) characteristics. However, the underlying molecular mechanism, especially related to the drivers or the determinants responsible for NE transdifferentiation, remains unclear. In this study, we compared gene expression profiling of NEPC and non-NEPC specimens and paid a particular attention to those transcriptional factors affecting neural differentiation during development. We identified that Paired box 6 (Pax6) expression was significantly elevated in NEPC and negatively regulated by AR signaling. While knock-down of Pax6 in NEPC cells inhibited NEPC phenotypes, over-expression of Pax6 in non-NEPC cells led to development of NEPC. Mechanistically, loss of AR resulted in an enhanced expression of Pax6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the c-Met/Stat5a signaling. By performing ATAC-seq, we found that high expression level of Pax6 elicited enhanced chromatin accessibility mainly through attenuation of H4K20me3 that usually causes chromatin silence in cancer cells. Taken together, these findings highlight PAX6 as a novel molecule to drive NEPC progression and suggest that it might serve as a potential target for the management of NEPC. To investigate the role of PAX6 in the differentiation of LNCaP cell line from androgen-sensitive to androgen-insensitive, we established an androgen-insensitive cell line similar to NEPC by treating LNCaP cells with enzalutamide for 30 days. Morever, we overexpressed PAX6 in LNCaP cells. Besides, we established DU145_shPAX6 cell lines to investigate whether PAX6 knockdown could repress the neuroendocrine transdifferentiation of NE-like cells. We then performed gene expression profiling analyzing data obtained from RNA-seq of LNCAP, LNCAP_ENZ, DU145-shCon and DU145_shPAX6 cells. We also performed ATAC seq to explore the chromatin accessibility in LNCaP_oePAX6, LNCaP_oeSTAT5A compared with LNCaP_con cells.

神经内分泌前列腺癌（Neuroendocrine prostate cancer, NEPC）是一类侵袭性极强的前列腺癌亚型，其特征为雄激素受体（androgen receptor, AR）信号通路缺失，并获得神经内分泌（neuroendocrine, NE）表型。然而，其潜在分子机制，尤其是与神经内分泌转分化相关的驱动因子或决定因素仍不明确。本研究对神经内分泌前列腺癌与非神经内分泌前列腺癌标本的基因表达谱进行了比较分析，并着重关注了发育过程中影响神经分化的转录因子。研究发现，配对盒6（Paired box 6, Pax6）在神经内分泌前列腺癌中表达显著升高，且其表达受AR信号通路负向调控。在神经内分泌前列腺癌细胞中敲低Pax6可抑制其神经内分泌表型，而在非神经内分泌前列腺癌细胞中过表达Pax6则可诱导其向神经内分泌前列腺癌表型转化。从机制上来说，AR表达缺失会导致Pax6表达上调，后者通过c-Met/Stat5a信号通路重编程前列腺癌细胞的谱系可塑性，使其获得神经内分泌表型。通过转座酶可及性测序（ATAC-seq）分析，本研究发现Pax6高表达主要通过减弱肿瘤细胞中通常介导染色质沉默的组蛋白H4赖氨酸20三甲基化（H4K20me3），从而增强染色质可及性。综上，本研究结果揭示Pax6是驱动神经内分泌前列腺癌进展的新型分子，提示其有望成为神经内分泌前列腺癌临床管理的潜在治疗靶点。为探究Pax6在雄激素敏感型LNCaP细胞系向雄激素非敏感型转化过程中的作用，本研究通过用恩扎卢胺（enzalutamide）持续处理LNCaP细胞30天，成功构建了类似神经内分泌前列腺癌的雄激素非敏感细胞模型。此外，本研究还在LNCaP细胞中过表达Pax6，并构建了DU145_shPAX6细胞系，以验证Pax6敲低是否能够抑制神经样细胞的神经内分泌转分化。本研究对LNCaP、LNCAP_ENZ、DU145-shCon及DU145_shPAX6细胞的RNA测序（RNA-seq）数据进行了基因表达谱分析；同时还通过ATAC-seq检测了LNCaP_oePAX6、LNCaP_oeSTAT5A与LNCaP_con细胞的染色质可及性水平。