MYPT1调控c-Myc通路抑制前列腺癌Warburg效应的机制研究

c-Myc signaling is a key pathway that regulates the Warburg effect of cancer metabolism, which is an important hallmark of rapid proliferation of cancer cells. But the mechanism of its regulation in prostate cancer cell is still not fully understood. Our previous study have proved that Myosin phosphatase target subunit 1(MYPT1) is downregulated in PCa(Mol Cancer. 2017) and its low expression is accosiated with poor prognosis of PCa and high expression of LDHA phosphorylated protein, which is a key enzyme of glycolysis. We further found that MYPT1 inhibited Warburg effect in prostate cancer cells and negatively regulated the activity of c-Myc by enhancing its dephosphorylation. Therefore, we hypothesized that “MYPT1 might negatively regulates c-Myc pathway by regulating the dephosphorylation of its proteins, and inhibits glycolysis metabolism in PCa ”. We intend to construst overexpressing and knock-out PCa cell lines, and tumor xenografts induced by the construsted PCa cells, use binding site analysis, CO-IP and GST pull-down experiment to verify the mechanism of MYPT1 inhibited Warburg effect by negatively regulating the c-Myc pathway. A further analysis of clinical data correlation with MYPT1 pathway is needed to assess clinical significance of MYPT1 to prostate cancer diagnosis and treatment. And we hope to find a new metabolic marker and therapeutic target with in prostate cancer.

Warburg效应是前列腺癌（PCa）细胞快速增殖的重要标志，c-Myc通路作为其中关键通路，其上游调控机制尚未明确。我们前期已证实相比癌旁良性组织，蛋白磷酸酶1调节亚基（MYPT1）在PCa组织的表达下调（Mol Cancer. 2017），并与糖酵解关键酶LDHA活性及患者临床预后都呈负相关；MYPT1可抑制PCa细胞糖酵解，还能促进c-Myc蛋白去磷酸化并抑制其通路活性。据此，我们提出“MYPT1参与蛋白去磷酸化调控，抑制c-Myc通路活性，从而抑制PCa细胞Warburg效应”的假设。我们拟构建MYPT1基因过表达和KO的PCa细胞株及裸鼠皮下种植瘤模型，采用结合位点分析、CO-IP、GST pull-down等技术揭示MYPT1通过负调控c-Myc通路抑制Warburg效应的机制，并通过临床数据相关性分析该代谢通路对PCa诊疗的临床意义，为PCa诊疗提供新的代谢标记物和治疗靶点。

Warburg效应是前列腺癌（PCa）细胞快速增殖的重要标志，c-Myc通路作为其中关键 通路，其上游调控机制尚未明确。我们前期已证实相比癌旁良性组织，蛋白磷酸酶1调节 亚基（MYPT1）在PCa组织的表达下调，并与糖酵解关键酶LDHA活性 及患者临床预后都呈负相关；MYPT1可抑制PCa细胞糖酵解，还能促进c-Myc蛋白去磷酸化 并抑制其通路活性。为了揭示MYPT1通过调控c-Myc通路负调控前列腺癌细胞Warburg效应的机制，本课题拟在前期工作的基础上，利用CRISPR/Cas9和慢病毒载体转染技术构建MYPT1稳定KO和过表达的前列腺癌细胞株以及裸鼠皮下种植瘤模型，采用糖代谢相关检测实验（乳酸、葡萄糖、细胞外酸化率和耗氧量检测等）获得MYPT1抑制Warburg效应的充分依据，发现MYTP1负调控前列腺癌细胞糖酵解，抑制肿瘤增殖。检测MYPT1与前列腺癌细胞中c-Myc和下游关键酶基因和蛋白的表达关系，并通过结合位点分析和免疫共沉淀（CO-IP）技术获得参与MYPT1调控LDHA蛋白去磷酸化的直接证据，进一步加入糖酵解抑制剂药物实验，阐明MYPT1通过调控LDHA活性影响Warburg效应，揭示MYPT1抑制前列腺癌细胞糖酵解和肿瘤增殖主要是通过抑制糖酵解关键酶LDHA活化。最后在临床样本中更深入的验证上述调控通路的临床意义，证实MYPT1和pLDHA联合可作为预测前列腺癌患者根治术后生化复发风险的独立预测指标，充实、完善对前列腺癌发病机制及其治疗的研究认识，为临床上靶向抑制前列腺癌Warburg效应、治疗前列腺癌特别是CRPC提供新思路。