HOXA9调控HIF-1α及其下游糖酵解代谢在皮肤鳞癌发生发展中的功能与机制研究

Hypoxia inducible factor HIF-1α is sensitive to ultraviolet radiation and promotes glycolytic metabolism and proliferation of cancer cells. The expression level of HIF-1α is positively correlated with the malignant grades of cutaneous squamous cell carcinoma (CSCC). Yet the regulatory mechanism of HIF-1α expression and function is unclear in CSCC. Our preliminary studies showed that HOXA9 is significantly downregulated in CSCC tumors and cells. Knockdown of HOXA9 promoted the proliferation of CSCC cells, while the expression of HIF-1α and its downstream glycolytic genes were also significantly upregulated, which supports the notion that HOXA9 negatively regulates glycolysis via targeting HIF-1α. Also, tumor suppressor CRIP2 was found to be the direct interacting partner of HOXA9. We speculate that CRIP2 functions as the co-factor for the tumor-suppressive role of HOXA9. Based on the above findings, the following studies will be conducted. Firstly, HOXA9’s roles in tumor-suppression and inhibiting HIF-1α and its downstream glycolysis will be verified in CSCC. Secondly, the epigenetic mechanisms in regulating HIF-1α and its downstream glycolytic genes by HOXA9 together with its direct interacting partner CRIP2 will be clarified. Thirdly, the functions and mechanism of HOXA9 in regulating the tumorigenesis, development and glycolysis of CSCC in nude mouse xenograft model and mouse CSCC model with epidermis-specific knockout of HOXA9 will be investigated and validated. Thus, this project will clarify the regulatory roles and mechanism of HOXA9 on glycolysis and lay a solid foundation for establishing a new therapeutic regime to cure CSCC by reconstitution of HOXA9 to target glycolysis.

低氧诱导因子HIF-1α对紫外辐射敏感，促进癌细胞糖酵解代谢和增殖；其表达水平与皮肤鳞癌分级呈正相关，但调控机理不明。课题组前期发现转录因子HOXA9在皮肤鳞癌中显著下调，敲减其表达促进癌细胞增殖并显著上调HIF-1α及其下游糖酵解关键基因，提示HOXA9通过靶向HIF-1α负调控糖酵解；并观察到抑癌蛋白CRIP2与HOXA9相互作用，推测前者辅助HOXA9抑癌功能。本项目拟：①证实在皮肤鳞癌中HOXA9抑制HIF-1α及其下游糖酵解，并发挥抑癌功能；②阐明HOXA9与CRIP2互作调控HIF-1α及其下游糖酵解基因的分子机制；③在裸鼠移植瘤模型和表皮特异性敲除HOXA9的小鼠皮肤鳞癌模型中，研究和验证HOXA9调控糖酵解与皮肤鳞癌发生发展的功能与机制。本项目将阐明HOXA9对HIF-1α及其下游糖酵解的调控作用与机制，为开发以HOXA9表达重建、抑制糖酵解为手段的皮肤鳞癌新疗法奠定基础。

皮肤鳞癌恶性程度高、预后差，在全球发病率呈上升趋势，为遏制这一趋势并改善不适宜手术人群的生活质量，深入了解皮肤鳞癌进展的分子机制、开发有效的皮肤鳞癌新疗法成为当务之急。有氧糖酵解是肿瘤的标志性特征之一，关键调控因子低氧诱导因子（HIF-1α）对紫外辐射敏感，可促进皮肤鳞癌细胞糖酵解代谢和增殖，且其表达水平与皮肤鳞癌分级呈正相关。本研究针对皮肤鳞癌中HIF-1α调控机理不明的现状开展研究，发现转录因子HOXA9在皮肤鳞癌中显著下调，敲减其表达可促进癌细胞增殖并显著上调HIF-1α及其下游糖酵解关键基因，提示HOXA9可通过靶向HIF-1α负调控糖酵解。通过免疫共沉淀鉴别到抑癌蛋白CRIP2与HOXA9相互作用，通过挽救实验验证了CRIP2辅助HOXA9发挥抑癌基因功能。通过数据库预测HOXA9在HIF-1α、HK2、GLUT1和PDK1基因启动子的结合位点并经过凝胶迁移率实验（EMSA）验证。通过染色质免疫沉淀实验（ChIP）发现敲减HOXA9可导致HOXA9蛋白在HIF-1α、HK2、GLUT1和PDK1基因启动子位点的结合丰度显著下调。通过在裸鼠皮下接种皮肤鳞癌细胞建立移植瘤模型，使用siRNA敲减HOXA9表达，观察移植瘤生长并检测相关基因的表达情况。结果发现，敲减HOXA9可显著上调糖酵解代谢基因HIF-1α、HK2、GLUT1和PDK1在RNA和蛋白水平的表达，促进皮肤鳞癌移植瘤的生长。同时，在该动物模型基础上，经尾静脉注射葡萄糖类似物18F-FDG后，采用microPET/CT观察发现18F-FDG在敲减HOXA9的移植瘤内摄取和富集度显著增加。本项研究验证了，HOXA9负调控HIF-1α、HK2、GLUT1和PDK1基因以抑制糖酵解代谢。本项目阐明了HOXA9对HIF-1α及其下游糖酵解的调控机制，为开发以HOXA9抑制糖酵解为手段的皮肤鳞癌新疗法奠定了基础。