靶向LDH-A治疗非小细胞肺癌耐药的分子机制研究

Higher rate of glycolysis has been long observed in cancer cells and returned to the battlefield of fighting against cancer recently. Accumulating evidence shows that inhibition of lactate dehydrogenase A (LDH-A), one of key enzymes in glycolysis, induces apoptosis though oxidative stress in cancer cells. However, we found that in A549 cells with Kras mutation，LDH-A inhibition induced autophagy instead of apoptosis, as well as G0/G1 arrest, accompanying with the drug resistance, but the involving mechanism remains unclear. It is well known that Kras and PI3K/Akt signaling pathways play important roles in both glycolysis and autophagy, based on the data above, we proposed a hypothesis that Kras and PI3K/Akt signaling pathways also played a role in the autophagy and resistance induced by LDH-A inhibition in non-small cell carcinoma cells. In the present study, oxamate sodium, and shRNA are used to inhibit LDH-A expression in NSCLC cells, then MTT assay, gene mutation analysis, flow cytometry, MDC/PI double staining, transmission electron microscopy, mRFP-GFP-LC3 immunofluorescence and nude mouse model are employed to study the effect of LHD-A inhibition on NSCLC cells both in vitro and in vivo, the molecular mechanisms and regulating pathways are explored by using western blot, RT-PCR, immunohistochemical staining and co-immunoprecipitation. Our study will deepen the understanding of the connection and crosstalk among glycolysis, autophagy and apoptosis, and provide novel targets for anti-cancer treatment.

能量代谢是目前肿瘤领域的研究热点。研究显示，靶向乳酸脱氢酶-A（LDH-A）可通过活性氧爆发促使细胞凋亡，是肿瘤治疗的有效手段。本课题组前期发现，在非小细胞肺癌中，不同细胞系对乳酸脱氢酶抑制剂的敏感性明显不同，且对部分细胞，靶向抑制LDH-A并没有引起细胞凋亡，而是明显诱导耐药发生。进一步研究提示，该耐药性与自噬活动密切相关。然而，其具体分子机制尚不清楚。鉴于Kras在糖酵解及自噬中的重要作用，本课题拟以Kras及下游PI3K/Akt信号通路为中心，通过体外细胞实验及移植瘤模型，采用RNA干扰、基因过表达、Western blotting、RT-PCR、免疫荧光等技术研究靶向LDH-A耐药的分子机制，探讨靶向LDH-A治疗NSCLC的可行性，并寻找预测LDH-A抑制剂疗效的分子标志物。本课题将有助于更好地理解肿瘤细胞能量代谢的调控机制，为联合靶向治疗提供科学依据。

乳酸脱氢酶A作为糖酵解的关键限速酶，及肿瘤瓦博格效应的驱动因素，在多种肿瘤中高表达，并且是不良预后的标志之一。大量文献表明，靶向LDHA可产生良好的抗肿瘤效应，并与其他肿瘤治疗产生协同作用。我们前期工作表明不同肺癌细胞对LDHA抑制剂敏感性不同。进一步通过基因测序及生物信息学分析，我们发现LDHA耐药的机制较为复杂，当肿瘤细胞糖代谢受到抑制后，细胞内脂肪酸及其他代谢通路可激活代偿，同时可启动G0/G1周期阻滞及诱导自噬发生，PI3K-Akt通路活性升高及KRAS突变与LDHA敏感性具有一定相关性，联合其它代谢抑制剂可能是靶向糖酵解治疗肿瘤的较好策略。此外，研究发现，靶向LDHA可显著增加NSCLC放射敏感性，其机制可能与ROS爆发造成放疗后DNA损伤加重，以及ATP耗竭影响DNA修复有关。目前LDHA抑制剂仍不能转化为临床应用，主要与其特异性不强及毒性有关。通过跨学科合作，我们发现纳米化碳点甘露糖在细胞内具有良好的吸收性及分布，且作用更强，更具有靶向性。本研究有助于寻找糖酵解抑制剂的疗效预测分子标志物，更好地理解肿瘤细胞能量代谢的调控机制，并为联合治疗及靶向LDHA放疗增敏提供科学依据。