ODC/Polyamines/c-Myc调控谷氨酰胺代谢重编程促进骨肉瘤细胞转移的作用机制研究

Osteosarcoma (OS) metastasis is characterized by complex molecular events. The role of metabolic alterations, or “metabolic reprogramming”, that may similarly contribute to metastasis. Indeed, our previous investigations in OS identified metabolic changes uniquely linked to metastasis. By comparing the identity and level of the metabolites between high/low metastatic cells, we found the polyamines pathway was identified to be differentially activated by LC-MS. In highly metastatic cells, ornithine levels were significantly diminished in both species. Ornithine is an important intermediary in the urea cycle and the production of polyamines. Western blot results showed ODC, the limited enzyme of polyamines, was overexpressed in highly metastatic cells. Then, we found inhibition of ODC/polyamines pathway could modestly suppress OS cells growth and metastasis. To observe if the cellular metabolism of highly metastatic cells was affected by dysregulation of the polyamines pathway, untargeted Metabolomics were measured with NMR, after ODC inhibitor DMFOM exposure. The results showed the glutaminolysis pathway was markly changed by DFMO, which may be associated with c-Myc gene. On the basis of these observations, we will analyze the mechanisms of ODC/Polyamiens in regulation of glutaminolysis metabolism in OS and how this pathway might influence of the growth and metastasis of OS. In this study, we plan to systemetically elucidate ODC, polyamiens, and glutamine biological functions of proliferation and metastasis in OS in vitro and in vivo. In addition, we will investigate the combination effects of ODC inhibitor and glutaminolysis metabolism inhibitor in OS. We will further demonstrate our findings to evaluate the role of ODC signaling as a potential target for OS treatment as well as a diagosis or prognosis marker in OS.

转移是骨肉瘤治疗失败主要原因之一，代谢重编程是促进转移的重要机制。课题组前期利用临床标本发现骨肉瘤患者中多胺（polyamines）水平升高，肺转移过程中代谢通路异常激活，但具体机制不清。本课题对4组高低转移性不同的骨肉瘤细胞系进行代谢组学研究，发现高转移细胞系中多胺的关键限速酶ODC上升；干扰ODC可以抑制骨肉瘤生长和转移，可以调控谷氨酰胺通路中代谢物和代谢基因；进一步分析这些基因可能受到c-Myc调控，实验证实抑制ODC可以下调c-Myc表达；联用ODC抑制剂和谷氨酰胺代谢抑制剂可以显著抑制荷瘤小鼠生长和转移。因此我们提出“ODC/Polyamines/c-Myc调控谷氨酰胺代谢重编程促进骨肉瘤细胞生长和转移”的假设。本课题拟阐明ODC和谷氨酰胺代谢通路在骨肉瘤中的作用，揭示两者之间交联的机制，研究联合抑制上述两条代谢通路后对骨肉瘤的治疗作用。本课题研究期望为骨肉瘤的诊治提供新的数据。

骨肉瘤恶性程度高，容易肺转，预后差，需要开发新的有效治疗。多胺在肿瘤发生发展中具有重要作用，靶向多胺代谢作为潜在的抗癌治疗策略越来越受到关注。本课题，我们发现多胺代谢促进骨肉瘤的成瘤。然而，多胺抑制剂α-二氟甲基鸟氨酸 (DFMO) 单药对骨肉瘤具有中等的抗肿瘤作用。使用高通量化合物筛选，我们发现 CIL56 (CA3) 是一种铁死亡诱导剂和 YAP 抑制剂，在多种骨肉瘤细胞模型、异种移植模型和 PDX 模型中，发现CIL56可以增敏DFMO抗骨肉瘤作用。代谢组学和转录组学分析明，DFMO 通过提高来自转硫途径的 GSH 水平来负向调节铁死亡。此外，DFMO 处理激活了 YAP 信号通路，以响应骨肉瘤细胞中谷氨酰胺分解的增加。这些发现可能会提供新信息，解释 DFMO 单一疗法在癌症治疗中的有限疗效，并为开发基于多胺抑制剂的联合疗法以促进其在骨肉瘤中的应用引入新思路。