Pim-3通过ASS1介导参与精氨酸代谢进而促进胰腺癌发生发展的机制解析

Dysregulation of cellular metabolism is a key event in cancer development and progression and contributes to many hallmarks of cancer. Several lines of evidence demonstrated that the glutamine, cysteine, and lysine promote the proliferation and invasion of human pancreatic cancer cell. However, the roles of amino acid metabolism in human pancreatic cancer development have not been fully characterized. We have performed the yeast two hybrid screening to identify the gene(s) that can interact with Pim-3, a proto-oncogene with serine/threonine kinase activity. Through these studies, we identified the enzyme, argininosuccinate synthetase (ASS1), a recognized rate-limiting step in arginine synthesis, as the gene product interacting with Pim-3. We further obtained a preliminary result to show that Pim-3 directly interacts and regulates ASS1 expression in human pancreatic cancer cells. However, the functional relevance still remains elusive. Based on these observations, we assume that Pim-3 can participate in arginine metabolism through ASS1 in human pancreatic cancer and can eventually promote pancreatic cancer development and progression. In order to address this hypothesis, we will investigate the roles of Pim-3-induced ASS1 expression in arginine metabolism in human pancreatic cancer cells by using in vitro and in vivo. For these purposes, we will establish human pancreatic cancer in the orthotopic nude mouse model and arginine auxotrophy mouse model. As dysregulated metabolic pathway can become targets for anti-cancer treatment, we believe that this project will elucidate the molecular mechanisms of Pim-3 in pancreatic cancer development and progression from a new perspective on the amino acid metabolism and will establish the theoretical basis for developing Pim-3 molecule target therapy.

细胞代谢失调是肿瘤发生发展的重要原因。现已有报道谷氨酸，半胱氨酸和赖氨酸促进胰腺癌细胞的增殖和侵袭。但是，绝大多数氨基酸代谢在胰腺癌中的作用及分子机制尚未得到充分解析。申请者应用酵母双杂交技术筛选出的精氨酸代琥珀酸合成酶（ASS1）是精氨酸代谢过程中的限速酶，能够与具有丝氨酸/苏氨酸激酶活性的原癌基因的Pim-3发生相互作用。预实验结果显示，Pim-3与ASS1直接结合并调节ASS1的表达。因此我们提出假说：胰腺癌细胞中Pim-3可能通过ASS1参与精氨酸代谢，促进胰腺癌的发生发展。本申请项目将通过裸小鼠胰腺原位移植瘤模型及精氨酸营养缺陷模型，利用体外和体内实验，从分子、细胞、组织以及动物整体水平等多方面验证上述假说，明确Pim-3通过ASS1参与胰腺癌细胞精氨酸代谢机制。从氨基酸代谢这个新视点，完善Pim-3参与胰腺癌发生发展的分子机制，为开发Pim-3分子靶向治疗药物充实理论基础。

[背景]胰腺癌是全球范围内常见恶性肿瘤，5年生存率小于5%。细胞代谢失调是肿瘤发生发展的重要原因。精氨酸代琥珀酸合成酶（ASS1）是一种肿瘤相关蛋白，参与多种肿瘤细胞代谢，并与原癌基因Pim-3有直接相互作用。在ASS1低表达肿瘤组织中，内源性精氨酸合成障碍，并应用精氨酸脱亚氨酶（ADI）降解外源性的精氨酸，会诱导肿瘤细胞发生凋亡。现已有部分降解外源性精氨酸的药物用于黑色素瘤和肝癌的临床试验阶段。[研究目的及内容]本课题旨在从分子、细胞、组织以及动物整体水平等多方面阐明在胰腺癌中Pim-3通过ASS1介导参与精氨酸代谢,导致胰腺癌精氨酸剥夺治疗耐药的机制。[重要结果及关键数据] 1. 在胰腺癌细胞中，Pim-3 可以与ASS1直接结合；并通过抑制ASS1蛋白的泛素化水平，抑制ASS1蛋白酶体降解，增加ASS1的蛋白稳定性，上调其蛋白表达。2. 精氨酸剥夺显著抑制胰腺癌细胞的增殖；高表达Pin-3通过ASS1介导，逆转由精氨酸剥夺诱导的细胞生长抑制；敲低Pim-3的表达，可以促进精氨酸剥夺诱导的细胞增殖抑制。3. 裸鼠皮下胰腺癌移植瘤模型体内实验结果显示与体外细胞实验相一致，精氨酸剥夺明显抑制肿瘤生长。并且敲低Pim-3的表达，进一步抑制体内肿瘤的生长。4. Pim-3通过ASS1介导，来调节细胞内精氨酸，谷氨酸及转运体蛋白CAT等氨基酸的物质代谢，从而参与精氨酸剥夺耐药；并且Pim-3 可以保护由精氨酸剥夺导致的细胞线粒体功能障碍，从而妨碍了精氨酸剥夺的治疗效果。5.临床胰腺癌147例标本组织免疫化学染色及kaplan-meier统计学分析结果显示低表达ASS1与不良预后显著相关。[结论及其科学意义]在胰腺癌精氨酸营养缺陷型肿瘤中，Pim-3参与了精氨酸剥夺治疗耐药。利用Pim-3抑制剂和ADI-PEG20联合治疗比单用精氨酸剥夺的疗法更能抑制胰腺肿瘤生长，更有前途。