生长激素释放激素受体（GHRH-R）拮抗剂通过NF-κB–PFKM代谢通路抑制食管鳞癌转移的机制

The molecular mechanism of metastasis in esophageal squamous cell carcinoma (ESCC) remains unclear, and there are no effective drugs owing to a lack of therapeutic targets. Our preliminary data showed that growth hormone-releasing hormone receptor (GHRH-R) was upregulated in ESCC, and GHRH-R antagonist MIA-602 inhibited glycolysis and invasion of ESCC cells. Bioinformatic analysis and in vitro studies indicated that the effect of MIA-602 on invasion was dependent on phosphofructokinase, muscle type (PFKM), a key glycolytic enzyme. Moreover, we showed that NF-κB p65 could increase the expression of PFKM. Further, MIA-602 was found to suppress the expression of p65. More importantly, overexpression of p65 reversed the inhibitory effect of MIA-602 on the expression of PFKM. We reasoned that MIA-602 represses ESCC metastasis through regulation of PFKM in a p65 dependent manner. We thus first set to clarify whether MIA-602 regulates metastasis via glycolysis, using cultured cells and mouse models. Second, we will verify that PFKM mediates MIA-602's effect on glycolysis and metastasis of cancer cells, using both in vitro and in vivo models. Third, we will elucidate that MIA-602 suppresses the expression of PFKM through downregulation of p65. The project aims to explore the regulatory mechanism, which GHRH-R antagonist uses to inhibit glycolysis-induced metastasis, and is favorable to design the novel therapeutic strategy against metastasis.

食管鳞癌转移机制不明，缺乏有效治疗靶点和药物。前期发现生长激素释放激素受体（GHRH-R）在食管鳞癌中高表达，且GHRH-R拮抗剂MIA-602抑制肿瘤细胞侵袭和糖酵解。生物信息分析和细胞实验发现糖酵解限速酶PFKM可能介导MIA-602对肿瘤细胞侵袭的抑制。进一步分析，发现NF-κB p65上调PFKM表达，而MIA-602下调p65表达，且过表达p65逆转MIA-602对PFKM的调节。假说：GHRH-R拮抗剂通过p65调节PFKM，进而调控肿瘤转移。本研究拟①应用细胞和小鼠模型，验证GHRH-R拮抗剂抑制肿瘤代谢和转移；②在体内外水平，验证PFKM介导GHRH-R拮抗剂调控肿瘤糖酵解和转移；③在分子水平探讨p65调节PFKM的机制，验证p65是介导GHRH-R拮抗剂调节PFKM表达的关键机制。本课题旨在探讨GHRH-R拮抗剂调控肿瘤糖代谢-转移的分子机制，为治疗转移提供新思路。

食管鳞癌是消化系统中常见恶性肿瘤之一，其治愈率低、复发率高和预后差。了解其恶性进展机理，有助于开发新靶点和治疗药物。本项目旨在研究生长激素释放激素受体GHRH-R(growth hormone releasing hormone receptor)在食管鳞癌中的临床意义和功能及靶向其抑制肿瘤的潜能。课题组发现GHRH-R在多中心食管鳞癌病人组织中表达上调，且其表达与多个临床病理学参数密切相关。接着分析发现DNA扩增和去甲基化是GHRH-R表达失调的潜在机制。生存分析显示GHRH-R低甲基化的病人预后更差；COX回归分析显示GHRH-R甲基化可作为食管癌病人总生存的独立预后因子。基于GHRH-R甲基化构建了预测食管病人预后的模型。进一步分析发现PFKM(Phosphofructokinase, Muscle)是GHRH/GHRH-R通路下游，PFKM在多中心食管鳞癌病人中表达上调，其高表达与肿瘤转移密切相关，且其DNA低甲基化预示病人预后不良。使用GHRH-R拮抗剂靶向GHRH-R可抑制肿瘤细胞糖代谢和转移潜能，PFKM则可逆转这一过程。机理分析显示NFκB通路可激活PFKM表达；靶向GHRH-R则可通过抑制NFκB通路阻断PFKM表达。进一步分析显示GHRH-R在肿瘤来源的基质细胞中表达上调，且其与免疫通路和多种免疫细胞浸润密切相关；靶向其下调PD-L1(programmed death-ligand 1)的表达，提示GHRH-R可能参与肿瘤免疫微环境调节。拓展研究发现：(1)GHRH-R潜在上游MTA3(metastasis-associated protein 3)在食管鳞癌中表达具有重要临床意义，并发挥调节食管鳞癌细胞干性和转移的作用，机制分析显示MTA3通过抑制SOX2OT/SOX2轴，进而执行上述功能；(2)GHRH-R潜在下游PTPRO (protein tyrosine phosphatase receptor type O)在肾透明细胞癌中表达失调预示病人预后不良，且其表达与免疫细胞浸润密切相关；(3)天然免疫信号通路调节子STING (stimulator of interferon genes)表达和DNA甲基化在肿瘤中具有重要临床价值，并基于其甲基化构建了预测病人预后的模型。上述研究结果为肿瘤防治提供了新的依据和线索。