清胰化积方通过靶向Rho GTP酶激活蛋白抑制胰腺癌侵袭和转移的机制研究

Invasion and metastasis are main reasons of death for pancreatic cancer. Treatment for invasion and metastasis of pancreatic cancer is of great difficulty in clinical practice. The research group put forward that accumulation of damp, heat, and toxicity is the core pathogenesis of pancreatic cancer and the basis of invasion and metastasis. Clinical researches indicated that the anti-tumor effect of Qingyihuaji (QYHJ) formula in combination with conventional treatment such as chemotherapy or radiotherapy was significantly better than the latter alone. Experimental studies revealed that QYHJ formula could inhibit the growth of high liver metastasis potential human pancreatic cancer cells, and decrease the formation of liver metastatic lesions. Rho GTPase activating protein (ARHGAP) family is involved in signaling pathways such as tumor invasion, cell cycle, and TGF-β pathway, etc. The family is highly correlated with the evolution of tumor metastasis. However, the relationship between invasion or metastasis of pancreatic cancer and ARHGAP family is not well understood. Our preliminary research indicated the following facts: 1. High throughput screening experiment combined with the third party database authentication showed that the expression of ARHGAPs in pancreatic cancer tissue was highly different from that of the para-carcinoma tissue. 2. Gene Set Enrichment Analysis illustrated that ARHGAP family may participate in multiple cell signaling pathways in correlation with invasion and metastasis of pancreatic cancer. 3. After treatment of Qingyihuaji formula, the expression of ARHGA4 in peripheral blood of the pancreatic patients was significantly increased. Is the inhibition of invasion and metastasis in pancreatic cancer caused by QYHJ formula related to regulation of ARHGAP family? What is the mechanism underlying? To answer the questions, the project plans to investigate the effect of QYHJ formula in vivo and in vitro, and the regulation of Rho GTPase-activating protein family and its downstream biological molecular signals. The aim of the research is to explore the mechanism of QYHJ formula in inhibition of invasion and metastasis of pancreatic cancer.

侵袭和转移是胰腺癌主要死因和治疗难点，课题组提出"湿热毒聚"是胰腺癌发病的核心病机和侵袭转移内因。研究证实，针对该病机组方的清胰化积方治疗转移性胰腺癌临床疗效显著优于常规化放疗；该方可抑制胰腺癌高转移细胞系的体内生长和肝转移灶形成。Rho GTP酶激活蛋白(ARHGAP)家族参与肿瘤侵袭、细胞周期、TGF-β等通路，与肿瘤转移密切相关。然而该家族与胰腺癌关系尚未阐明。课题组预实验中:1.高通量筛选实验证实胰腺癌与癌旁组织ARHGAP表达差异显著;2.基因集富集分析提示ARHGAP4可能参与胰腺癌侵袭转移多条通路;3.清胰化积方治疗后胰腺癌患者外周血ARHGA4表达升高。清胰化积方是否通过ARHGAP抑制胰腺癌侵袭转移？其参与的机制如何？本研究拟从体内外实验观察清胰化积方抑制胰腺癌侵袭转移、调控ARHGAP和下游转移相关信号通路的作用，探索清胰化积方抗胰腺癌侵袭转移的作用机制。

胰腺癌转移是临床治疗难点。本研究通过高通量筛选实验及临床病理资料分析，证实胰腺癌患者肿瘤组织ARHGAP表达下降，并与患者肿瘤血管侵袭及预后不良相关。而清胰化积中药作用后胰腺癌患者外周血ARHGAP4表达上调。体内外实验研究提示，ARHGAP4可抑制胰腺癌细胞增殖、侵袭和转移。清胰化积方含药血清可提高胰腺癌细胞中ARHGAP4表达，使胰腺癌细胞的增殖和侵袭转移能力下降；动物实验证实清胰化积中药可使裸鼠胰腺癌移植瘤生长减慢，肺转移减少。为进一步探索其作用机制，我们采用基因集富集分析，发现ARHGAP4可能参与胰腺癌侵袭转移多条通路，其中β-连环蛋白参与的Wnt信号通路可介导多种细胞过程，是细胞迁移和侵袭的关键分子。HDAC2在β-catenin结合的Wnt靶基因启动子区域保持去乙酰化状态的去乙酰化酶，负调节β-catenin激活。我们的研究证实，ARHGAP4作为β-catenin途径的一个新的调节因子，在体外调节胰腺癌细胞的侵袭和迁移以及下游效应因子MMP2和MMP9的表达。进一步研究发现，ARHGAP4与HDAC2相互作用并泛素化HDAC2，后者反过来抑制β-连环蛋白活化。此外，XAV939治疗可减弱ARHGAP4沉默对胰腺癌细胞的影响。从而证明ARHGAP4是HDAC2/β-catenin途径的一个新的调节因子，在肿瘤生长和转移中起着关键作用。接着，我们以清胰化积方含药血清对胰腺癌细胞进行干预，发现清胰化积方可通过ARHGAP4参与β-catenin通路，并对其下游的MMP2和MMP9等效应分子具有调节作用，从而使胰腺癌细胞的侵袭和转移下降。本研究采用体内外实验，证实了清胰化积方抑制胰腺癌转移的作用，并从调节转移相关关键分子及其下游靶基因角度对其作用机制进行了深入探讨。本研究成果为进一步优化清胰化积方组方和完善清胰化积方为主的胰腺癌综合治疗方案、改善这一临床难治性恶性肿瘤的疗效奠定基础，并对阐明中医药抗肿瘤转移的机制研究提供新思路和新方法。相关研究成果已形成多篇论文，并被SCI期刊录用，在国内外进行交流。