Chen Yunru, Li Qing, Tu Kangsheng, Wang Yuanguo, Wang Xianghu, Liu Dandan, Chen Chen, Liu Donglian, Yang Rendong, Qiu Wei, Kang Ningling
Tumor Microenvironment and Metastasis Hormel Institute University of Minnesota Austin MN.
Present address: First Affiliated Hospital of Xi'an Jiaotong University Xi'an Shanxi P. R. China.
Hepatol Commun. 2019 Dec 20;4(2):268-283. doi: 10.1002/hep4.1452. eCollection 2020 Feb.
Transforming growth factor β (TGFβ) induces hepatic stellate cell (HSC) differentiation into tumor-promoting myofibroblast, although underlying mechanism remains incompletely understood. Focal adhesion kinase (FAK) is activated in response to TGFβ stimulation, so it transmits TGFβ stimulus to extracellular signal-regulated kinase and P38 mitogen-activated protein kinase signaling. However, it is unknown whether FAK can, in return, modulate TGFβ receptors. In this study, we tested whether FAK phosphorylated TGFβ receptor 2 (TGFβR2) and regulated TGFβR2 intracellular trafficking in HSCs. The FAKY397F mutant and PF-573,228 were used to inhibit the kinase activity of FAK, the TGFβR2 protein level was quantitated by immunoblotting, and HSC differentiation into myofibroblast was assessed by expression of HSC activation markers, alpha-smooth muscle actin, fibronectin, or connective tissue growth factor. We found that targeting FAK kinase activity suppressed the TGFβR2 protein level, TGFβ1-induced mothers against decapentaplegic homolog phosphorylation, and myofibroblastic activation of HSCs. At the molecular and cellular level, active FAK (phosphorylated FAK at tyrosine 397) bound to TGFβR2 and kept TGFβR2 at the peripheral plasma membrane of HSCs, and it induced TGFβR2 phosphorylation at tyrosine 336. In contrast, targeting FAK or mutating Y336 to F on TGFβR2 led to lysosomal sorting and degradation of TGFβR2. Using RNA sequencing, we identified that the transcripts of 764 TGFβ target genes were influenced by FAK inhibition, and that through FAK, TGFβ1 stimulated HSCs to produce a panel of tumor-promoting factors, including extracellular matrix remodeling proteins, growth factors and cytokines, and immune checkpoint molecule PD-L1. Functionally, targeting FAK inhibited tumor-promoting effects of HSCs and in a tumor implantation mouse model. FAK targets TGFβR2 to the plasma membrane and protects TGFβR2 from lysosome-mediated degradation, thereby promoting TGFβ-mediated HSC activation. FAK is a target for suppressing HSC activation and the hepatic tumor microenvironment.
转化生长因子β（TGFβ）可诱导肝星状细胞（HSC）分化为促肿瘤肌成纤维细胞，但其潜在机制仍未完全阐明。粘着斑激酶（FAK）在TGFβ刺激下被激活，进而将TGFβ信号传递至细胞外信号调节激酶和P38丝裂原活化蛋白激酶信号通路。然而，FAK是否能反过来调节TGFβ受体尚不清楚。在本研究中，我们检测了FAK是否磷酸化TGFβ受体2（TGFβR2）并调节其在HSCs中的细胞内转运。使用FAKY397F突变体和PF-573,228抑制FAK的激酶活性，通过免疫印迹法定量TGFβR2蛋白水平，并通过HSC激活标志物α-平滑肌肌动蛋白、纤连蛋白或结缔组织生长因子的表达评估HSCs向肌成纤维细胞的分化。我们发现，靶向FAK激酶活性可抑制TGFβR2蛋白水平、TGFβ1诱导的母亲对五体不全同源物的磷酸化以及HSCs的肌成纤维细胞活化。在分子和细胞水平上，活性FAK（酪氨酸397位点磷酸化的FAK）与TGFβR2结合，并将TGFβR2保留在HSCs的外周质膜上，同时诱导TGFβR2酪氨酸336位点的磷酸化。相反，靶向FAK或将TGFβR2上的Y336突变为F会导致TGFβR2的溶酶体分选和降解。通过RNA测序，我们发现764个TGFβ靶基因的转录本受FAK抑制的影响，并且通过FAK，TGFβ1刺激HSCs产生一系列促肿瘤因子，包括细胞外基质重塑蛋白、生长因子和细胞因子以及免疫检查点分子PD-L1。在功能上，靶向FAK可抑制HSCs在肿瘤植入小鼠模型中的促肿瘤作用。FAK将TGFβR2靶向质膜并保护其免受溶酶体介导的降解，从而促进TGFβ介导的HSC活化。FAK是抑制HSC活化和肝肿瘤微环境的一个靶点。
