Pax5在小细胞肺癌发生以及肺腺癌向小细胞肺癌转化中的功能和机制研究

Small cell lung cancer (SCLC) is the most malignant subtype of lung cancer. It is characterized by aggressive growth and early onset of metastasis. In addition, transformation to SCLC is found to be an acquired drug resistance mechanism in EGFR-mutant lung adenocarcinoma. Therefore, it is necessary to understand the biological mechanism of SCLC. By comparing the transcription profilling between H209 (SCLC) and A549 (lung adenocarcinoma cell line with low metastatic capability), we found that H209 sepifically expresses a series of lymphocyte restricted proteins including Pax5. Pax5 is a transcription factor and plays important role in determining B lymphocyte development. Ectopic expression of Pax5 in Lewis Lung Carcinoma cells （LLC1, which do not express Pax5）promotes lung metastasis and, in addition, angiogenesis. Furthermore, LLC1 expressing Pax5 can metastasize to kidney. The kidney metastases exhibit features typical for SCLC and positivity for TTF1, CgA and NSE by IHC. These preliminary data suggest that Pax5 may promote transition of lung adenocarcinoma to SCLC. Here, the objective of this proposal is to systematically focus on the role of Pax5 in lung cancer progression using multiple techniques including immunohistochemistry, single cell RT-PCR, Lectin perfusion, matrigel 3D culture and 3C. Specifically, we will investigate the connection between the expression of Pax5 in tumorigenesis of SCLC, the connection between Pax5 and SCLC transformation. We will explore the mechanism of Pax5 regulate gene transcription and and the environmental factors that contribute to the ectopic expression of Pax5. Hopefully we may gain significant insight into the change of regulatory network associated with tumorigenesis and metastasis in solid tumors.

肺腺癌转化成小细胞肺癌是肺腺癌继发性耐药的原因之一，小细胞肺癌发生及转化机制的研究已成为临床亟待解决的主要问题。利用临床不同亚型人肺癌样本，我们发现小细胞肺癌细胞表达生理状态下仅表达于脑和淋巴细胞的转录因子Pax5，将该蛋白在肺腺癌细胞中过表达并接种于小鼠皮下，2月后转移至肾脏的肺癌细胞表现出小细胞肺癌性状，提示Pax5很可能是致使小细胞肺癌发生或者转化中的关键因子。本项目将利用临床肿瘤样本，自发肺腺癌和小细胞肺癌小鼠模型，采用单细胞RT-PCR、CRISPR/Cas9等多种遗传学、分子生物学和细胞生物学技术手段，全面分析Pax5在小细胞肺癌发生和转化中的功能，并利用Hi-C、Chia-Pet等技术解析Pax5改变表皮细胞基因转录谱的分子机制，阐明致使Pax5在表皮细胞异位表达的细胞内外环境因素，研究结果将有助于理解小细胞肺癌发生和转化的分子机制,为肺癌临床诊疗和预后提供实验基础。

不同来源肿瘤会根据是否表达神经内分泌特征性蛋白分为神经内分泌型肿瘤和非神经内分泌型肿瘤，具有神经内分泌特征的肿瘤转移能力强、预后差。临床发现神经内分泌和非神经内分泌类型的转化是肿瘤治疗过程中耐药的主要原因，但是转化机制不清楚。利用单细胞转录组测序，我们发现在神经内分泌型肿瘤小细胞肺癌（SCLC）组织中存在缺失神经内分泌特征的肿瘤细胞，与经典的具有神经内分泌特征的SCLC细胞比较，这些细胞不表达PAX5。随后我们在原发SCLC的小鼠模型中敲除PAX5，发现肿瘤细胞丢失了神经内分泌特征，在原发肺腺癌小鼠模型中表达PAX5，小鼠则产生具有神经内分泌特征的小细胞肺癌。随后的机制研究发现，PAX5能够促进H3K9me3转移酶SUV39H1A在染色质的富集，从而导致染色质致密化，抑制了NOTCH，Hippo,TGFb等非神经内分泌肿瘤中激活的通路基因的表达，同时促进促血管生成因子如VEGFA的表达，生成了大量结构异常的血管，进一步促进神经内分泌表型的产生，抗血管生成药物贝伐单抗能够阻止肺腺癌向小细胞肺癌的转化，本项目发现了肿瘤细胞神经内分泌表型和非神经内分泌表型转化的关键蛋白之一，并提示在肺腺癌治疗中加入贝伐单抗会阻止向小细胞肺癌的转化，利于肺腺癌的治疗。