FOXA1通过染色质重塑重编程SMAD顺反组差异化调节TGF-β靶基因群抑制鼻咽癌的机制研究

Abnormal activation of TGF-β/SMAD signaling pathway in nasopharyngeal carcinoma(NPC) was associated with relapse and poor prognosis. TGF-β signaling switches from tumor suppression to promoting cancer progression, invasion, and tumor metastasis during cancer development, but the molecular mechanism underlying this function switch of TGF-β in human cancer is not clear. We found the pioneering factor FOXA1 decreased in NPC samples. Re-expression of FOXA1 suppresses the malignant behaviors of NPC cells. Microarray analysis indicates that FOXA1 differentially regulates TGF-β/SMAD signaling targets expression in NPC cells. Characteristically, FOXA1 facilitates the induction of tumor suppressors such as BAMBI and PMEPA1, but attenuates the expression of that molecules with tumor promotive function induced by TGF-β, including L1CAM, PTHLH, Vimentin and HMGA2 et al. Functionally, FOXA1 expression enhances the sensitivity of NPC cells to the cytostatic effects of TGF-β. We hypothized that FOXA1 expression in NPC cells reprogrammed TGF-β stimulated SMAD cistrome via induction of chromatin remodeling on the targets locus. Loss of FOXA1 leads to the dysregulation of TGF-β/SMAD signaling pathway in NPC development. In this study, we aim to determine the tumor suppressive role of FOXA1 on NPC cells, and to determine the effect of FOXA1 on physiological functions of TGF-β. We also aim to decipher the detail mechanisms underlying the inhibition of TGF-β/SMAD activation via induction of BAMBI by FOXA1 cooperating with SMAD complex. By using ChIP-Seq and RNA-Seq，the association of FOXA1 mediated chromating remodeling with reprogramming of TGF-β stimulated SMAD cistrome and transcriptome would be studied. We also plan to measure the expression level of FOXA1 and TGF-β targets in NPC samples and to study the association of these molecules with the clinical characteristics.

鼻咽癌发生过程中TGF-β/SMAD活化水平增高，并且由抑制生长转变为促进肿瘤发生、进展，与疾病复发和不良预后相关。我们发现先导转录因子FOXA1具有抑制鼻咽癌细胞恶性行为的作用，在鼻咽癌组织中表达降低。基因芯片分析显示FOXA1影响了TGF-β/SMAD信号通路靶分子群表达。FOXA1促进了TGF-β抑瘤性靶基因BAMBI、PMEPA1等表达，抑制了TGF-β对促瘤性靶基因L1CAM、PTHLH、Vimentin、HMGA2等分子的诱导作用，并恢复了鼻咽癌细胞对TGF-β生长抑制作用的敏感性。我们推测FOXA1通过引起靶基因染色质重塑，重编程SMAD顺反组和转录组；FOXA1缺失是引起鼻咽癌细胞TGF-β/SMAD信号失常的主导因素。本项目拟深入研究FOXA1通过染色质重塑，差异化调节抑瘤性或促瘤性TGF-β靶基因群表达，抑制鼻咽癌恶性行为的机制，并探索其临床价值。

鼻咽癌是高发于我国南方和东南亚地区的恶性肿瘤。鼻咽癌发病隐匿，极易发生早期转移。该项目首次研究FOXA1蛋白在鼻咽癌中的表达与生物学作用，发现FOXA1在正常鼻咽粘膜高表达，而在鼻咽癌中显著降低，并与转移相关。通过建立过表达、knock down细胞系开展研究，发现FOXA1抑制鼻咽癌细胞增殖、侵袭转移，促进分化。在机制上，阐明了FOXA1对TGF-β1刺激的鼻咽癌转录组具有重编程效应：在FOXA1高表达的情况下，TGF-β1优先激活抑瘤性靶基因群表达；在FOXA1低表达的情况下，TGF-β1优先激活促转移性靶基因群表达（L1CAM、vimentin）。进一步以BAMBI基因为例，证明了FOXA1结合于BAMBI上游调控区的Forkhead binding motif，招募组蛋白修饰酶，提高BAMBI基因调控区染色质H3K4me1、H3K27ac水平，提高染色质开放程度，促进TGF-β1激活的SMAD2/3/4复合物结合于BAMBI启动子上的TGF-β反应元件。通过实验证明BAMBI具有抑制鼻咽癌恶性行为的作用；在过表达FOXA1的细胞中干扰BAMBI表达，引起鼻咽癌细胞恶性行为得以部分恢复。另外，还研究了FOXA1对miRNA表达的影响，发现FOXA1通过下调miR-100-5p、miR-125b-5p表达，上调RASGRP3和FOXN3发挥抑瘤效应。项目执行期间共发表标注基金资助的SCI论文16篇，其中以独立通讯或并列通讯发表SCI论文14篇；执行期间以第一完成人获得国家发明专利授权3项。项目执行期间毕业硕士研究生5名，毕业博士研究生2名。