CSE介导的氧化应激调控在KRAS突变型胰腺癌恶性进展中的机制研究

Pancreatic cancer is a highly malignant tumor. Hypoxic microenvironment causes metabolic changes in pancreatic cancer, resulting in excessive ROS. Kras mutation pancreatic cancer strictly regulated ROS levels and protected tumor cells from oxidative stress injury through the activation of Nrf2 anti-oxidation process. In this study, we found that cystathionine--lyase (CSE) was highly expressed in Kras mutant pancreatic cancer by high-throughput transcriptome sequencing. Our further data showed that CSE expression was significantly related to Nrf2. Prediction of transcription factor binding sites suggested that there were Nrf2 binding sites in CSE promoter, and co-immunoprecipitation analysis indicated the interaction between CSE and Nrf2 in nuclear. Accordingly, we deduced that Kras mutation up-regulated CSE expression by activation of Nrf2, and Kras mutation promoted the entry to nuclear of CSE to interact with Nrf2, which mediated the transcription of antioxidant genes. We will elucidate the mechanism of CSE-mediated the process of Nrf2 resistance to oxidative stress. In conclusion, our project may reveal a novel molecular mechanism of Kras mutation pancreatic cancer resistance to oxidative stress injury, thus providing new targets for the treatment of pancreatic cancer.

胰腺癌是高度恶性的肿瘤。乏氧微环境引起胰腺癌代谢改变，产生过量ROS。Kras突变胰腺癌通过激活Nrf2抗氧化过程，严格调控ROS水平，保护肿瘤细胞免受氧化应激损伤。本研究通过高通量转录组测序发现胱硫醚-gamma-裂解酶(CSE)在Kras突变的胰腺癌中显著高表达，并降低了细胞ROS水平。进一步研究发现，CSE表达明显与Nrf2相关；预测分析发现CSE启动子区存在Nrf2结合位点；质谱及免疫共沉淀分析CSE入核与Nrf2结合。据此推断 Kras突变激活Nrf2，上调CSE表达；同时Kras突变促使CSE入核与Nrf2结合，共同介导抗氧化基因转录。本课题在明确Kras突变促进CSE转录，并促进CSE转移入核基础上，拟阐明CSE介导Nrf2抵抗氧化应激作用的分子机制。通过本课题研究，有望揭示Kras突变胰腺癌抵抗氧化应激损伤的新机制，为胰腺癌的治疗提供理论依据。

KRAS G12D 突变是胰腺癌中最常见的突变，促进胰腺癌发生发展。肿瘤生长产生过量的胞内ROS，这对胰腺癌是不利的。然而，我们在 KRAS G12D 突变胰腺癌中并没有观察到过量的 ROS。 KRAS 突变胰腺癌对细胞内 ROS 水平的调节不清楚。我们建立了表达野生型和G12D突变 KRAS的 BxPC3 稳转细胞，突变 KRAS 细胞的糖酵解和增殖活力显著高于 野生型 KRAS 细胞，但ROS 水平并没有显著变化。 我们发现体内关键的硫化氢 (H2S) 生成酶胱硫醚-γ-裂解酶 (CSE) 在突变的 KRAS 细胞中显着上调。用siRNA敲低 CSE，KRAS突变的细胞内 ROS 水平升高，细胞糖酵解和增殖活性降低。Nrf2 被突变的KRAS 激活，促进 CSE 转录。我们在CSE启动子区发现两个潜在的Nrf2结合位点。通过荧光素酶报告基因活性检测结合ChIP检测，我们证实Nrf2结合(-49/-37bp)位点，促进CSE转录。在 Nrf2 被siRNA敲低或被 brusatol 抑制后，过表达CSE或添加 NaHS，升高的 ROS 水平被显著抑制，降低的细胞增殖活性也得到恢复。总之，我们的研究揭示了 KRAS 突变胰腺癌细胞通过Nrf/CSE/H2S调节ROS 水平，促进胰腺癌生长的潜在机制，为胰腺癌治疗提供了潜在的靶点。