牙龈卟啉单胞菌通过调控TGFβ/Smad和Hippo信号通路促进食管鳞癌演进

Porphyromonas gingivalis (Pg)-induced microbiota dysbiosis in mouth/esophagus increases the risk of esophageal squamous cell carcinoma (ESCC) and correlates with shorter survivals in patients with ESCC. This research proposal is presented as follows: The malignant cell phenotypes in vitro and in vivo such as proliferation, migration an invasion are examined after Pg infection of ESCC cells. Furthermore, epithelial-mesenchymal transition (EMT)- and stemness-related markers and phenotypes are evaluated as well. To dissect the molecular mechanisms underlying Pg-induced aggressiveness, Pg-associated differentially expressed genes/proteins are identified and then key molecules, and biological pathways involved in Pg-induced progression are identified based on the composite indexes topological features of protein-protein interaction network and enrichment analysis of function (GO) and pathways (KEGG and Reactome pathways), respectively. Subsequently, two key molecules identified included TGFβ and RAC1. As such, phosphorylation of Smad2/3 and dephosphorylation of YAP/TAZ reflecting activities of TGFβ and Hippo pathways are determined. The target genes downstream of these two pathways are measured as well after Pg infection of ESCC cells and xenograted tumors. Seeking to determine the roles of GFβ and Hippo pathways, inhibitors, overexpression (wild-types or mutant-types) or knockdown of key molecules are employed to block the Pg-induced aggressiveness Using animal models of subcutaneous inoculation, lung metastasis via tail vein and footpad lymphnode metastasis of ESCC cells treated with Pg or different controls, To further evaluate the clinical relevance of Pg-induced signature markers in ESCC, three different cohorts including ESCC tissue array (264 ESCCs, cohort 1), 100 ESCC with follow-up data (cohort 2), and TCGA (95 ESCCs) and GEO (179 ESCCs, cohort 3) with follow-up data, are utilized to establish molecular classifier of ESCC associated with Pg infection. Our research results will shed more lights on molecular mechanisms of esophageal carcinogenesis and identify potential therapeutic molecular targets associated with Pg-induced aggressiveness in ESCC.

牙龈卟啉单胞菌（Pg）诱发的口腔/食管微生态紊乱增加罹患食管鳞癌风险并与生存期缩短显著相关。本项目拟用Pg感染增加食管鳞癌细胞侵袭转移等恶性表型，明确Pg诱导癌细胞EMT和干性表型及相关分子变化；鉴定Pg相关差异表达基因/蛋白谱，构建分子互作网络，拓扑结构特征综合评分确定关键分子TGFβ和RAC1，生物学通路富集法确定参与食管鳞癌恶性演进的关键通路；检测pSmad2/3和pYAP/TAZ变化、胞核分布及其下游靶基因变化，细胞或裸鼠皮下成瘤、尾静脉肺转移、爪垫淋巴结转移等动物模型中，关键分子抑制剂、野生或实变体过表达或沉默阻断Pg诱导的食管鳞癌侵袭转移，明确Pg激活TGFβ/Smad和抑制Hippo通路协同作用；组织芯片明晰Pg与关键分子相关性及这些分子表达异常的临床意义，建立基于公共数据库的Pg特征性分子预后分子模型。本研究可阐释Pg促进食管鳞癌恶性演进分子机制，并鉴定潜在分子干预靶点。

食管鳞癌是中国食管癌的主要组织学类型，约占所有食管癌的90%以上。口腔卫生不良、牙齿缺失、牙周炎和胃肠道肿瘤密切相关。牙龈卟啉单胞菌（Pg）是牙周疾病的关键致病菌，食管鳞癌组织中Pg丰度与肿瘤分化程度、淋巴结转移、临床分期及患者生存期缩短呈显著正相关。本项目应用Pg感染的细胞模型和裸鼠皮下荷瘤、尾静脉肺转移模型，证实了Pg感染促进食管鳞癌细胞生长、侵袭和转移；Pg感染的食管鳞癌细胞和组织中TLR4/MYD88介导了GARP表达上调和TGFβ激活，通过TGFβ/Smad2/3经典信号通路，替硝唑和TGFβ受体抑制剂能够阻断Pg的肿瘤促进作用；Pg感染ESCC细胞后Transgelin-2蛋白表达增加，应力纤维减少，丝状伪足和黏着斑增多，这些细胞骨架的变化均被Transgelin-2沉默逆转，并且Transgelin-2沉默后明显阻断了Pg对食管鳞癌细胞增殖、迁移和侵袭的促进作用；Pg诱导的E-cadherin表达降低和Vimentin表达增加亦被Transgelin-2沉默逆转；fimA基因缺失型Pg（fimA-/-Pg）侵入食管鳞癌细胞内细菌数目减少，pSmad2/3激活降低，促进刺激癌细胞生长、迁移和侵袭能力降低。本项目结果表明Pg通过TLR4/MYD88介导的TGFβ/Smad2/3经典信号通路激活，进一步通过Transgelin-2调控应力纤维相关的细胞骨架改变，促进食管鳞癌发生上皮间质转化和干性富集，促进ESCC进展；FimA作为Pg毒力因子菌毛的主要组分之一，介导了Pg促进食管鳞癌恶性进展，FimA是Pg相关疾病预防和诊治的潜在靶点分子。本项目阐明了Pg促进食管鳞癌恶性演进的分子机制，明确了FimA是Pg侵入细胞的关键毒力因子，清除Pg和阻断TGFβ/Smad2/3信号通路能够抑制Pg对食管鳞癌演进的促进作用，为Pg感染的食管鳞癌的精准治疗奠定了理论基础。