SHMT2调控AMPK/mTOR通路诱导自噬促进结肠癌化疗耐药的机制研究

Autophagy can induce tumor cells to escape apoptosis, resulting in resistance to chemotherapy. Activation of AMPK/mTOR signaling is an important molecular events of autophagy. Our previous study found that SHMT2 was upregulated in colon cancer and was associated with tumor prognosis and resistance to chemotherapy. Overexpression of SHMT2 inhibited apoptosis induced by 5-FU and enhanced autophagy in colon cancer cells. Besides, AMPK/mTOR was found to be the downstream target of SHMT2. Thus we suggested SHMT2 may promote phosphorylation of AMPK, then suppress the activity of mTOR directly or indirectly, inducing autophagy in colon cancer cells. Autophagy mediated by SHMT2 can eliminate the harmful products such as damaged organelles, thereby resulting in cell apoptosis tolerance and reducing chemotherapy sensitivity. To validate the hypothesis, in this project, CRISP/Cas9 knockout methods is used to elucidate the relationship between SHMT2 and chemotherapy sensitivity; Transmission electron microscopy and flow cytometry are used to investigate the drug resistance function of SHMT2 in regulating apoptosis tolerance by inducing autophagy; confocal laser scanning technology is applied to explore the underlying molecular mechanism of autophagy caused by SHMT2 through AMPK/mTOR signaling pathway; public databases are mined to verify the association between SHMT2 and autophagy, and the relationship between AMPK/mTOR and prognosis within chemotherapy; a large sample of tissue is used to verify their clinical significance. All these together would provide a new approach to reversal the resistance to chemotherapy.

自噬可诱导肿瘤细胞凋亡逃避，致使化疗耐药。AMPK/mTOR激活是自噬发生的重要分子事件。课题组发现SHMT2在结肠癌中表达上调，并与肿瘤预后及化疗耐药相关。过表达SHMT2抑制5-FU引发的凋亡，增强自噬; 此外发现AMPK/mTOR是SHMT2潜在的下游靶标。我们推测SHMT2可能通过促进AMPK磷酸化直接或间接抑制mTOR活性诱导自噬；自噬清除化疗产生的破损细胞器等有害成分，导致化疗诱导的凋亡耐受，从而降低化疗敏感性。本项目拟采用CRISP/Cas9等基因操作技术，验证SHMT2在结肠癌化疗敏感性中的作用；透射电镜及流式细胞仪等观察SHMT2通过诱导自噬抵抗凋亡影响肿瘤耐药；激光共聚焦等技术探讨SHMT2调控AMPK/mTOR增强自噬的分子机制；公共数据库验证SHMT2与自噬的相关性及AMPK/mTOR与化疗预后的联系，结合大样本组织验证其临床意义，为逆转肿瘤化疗耐受提供新思路。

肿瘤细胞耐药是导致化疗失败、患者生存率降低的主要原因。研究肿瘤耐药发生机制和寻找逆转耐药的关键节点是当前亟待探索的重要课题。近年来,自噬与肿瘤耐药之间的密切联系日益倍受关注，自噬可诱导肿瘤细胞逃避凋亡致使化疗耐药。探讨肿瘤细胞耐药中自噬的调控机制,有助于合理调节自噬及制定有效的肿瘤化疗新策略。课题组首先利用GEO和TCGA数据库分析确定了与结直肠癌（CRC）进展相关的SHMT2等基因。SHMT2位于线粒体,催化丝氨酸生成甘氨酸,是细胞一碳单位的最大来源,从而为肿瘤细胞分裂增殖提供物质基础。Nature 报道, SHMT2 在脑组织缺血区的脑胶质瘤细胞中表达水平最高,低氧环境中 SHMT2 可降低耗氧速率,使肿瘤细胞更适合于在缺血微环境中生存,避免细胞死亡。 然而,SHMT2 与肿瘤耐药的关系及其作用机制鲜有报道。课题组选择患有TNM II或III期疾病（n = 378）的CRC患者探讨SHMT2基于5-氟尿嘧啶（5-FU）为基础的化疗中的功能，发现低表达SHMT2可诱导5-FU化学耐药并与CRC的预后不良有关。本项目利用质谱技术确定了SHMT2在大肠癌细胞中的结合蛋白P53。我们发现SHMT2通过结合细胞质p53抑制自噬。 SHMT2通过抑制p53和HDM2的结合来防止细胞质p53降解。在5-FU处理下，SHMT2的耗竭促进自噬并抑制细胞凋亡。自噬抑制剂CQ在体内外降低了SHMT2诱导的5-FU耐药性。最后，在患者PDX和CRC细胞移植模型中通过自噬抑制剂增强了5-FU对CRC细胞的杀伤力。我们的发现确定了低SHMT2诱导的CRC对5-FU耐药的机制。综上所述，SHMT2-p53可作为一种新型的癌症治疗靶点，并提供了降低化疗药物耐药的潜在治疗机会。