KrasG12D-LOH介导的AMPK和REDD1对胰腺癌能量代谢和生物学行为的影响及其机理研究

Pancreatic ductal adenocarcinoma (PDAC) constitutes a major cancer entity of pancreatic cancer which is featured by an aggressive tumor biology and early metastasis. In particular, the majority of PDACs contain oncogenic KRASG12D mutations. And the loss of heterozygosity (LOH) mainly occurs in putative tumor suppressor genes (e.g. p53, Lkb1), but rarely affects oncogenes. In our preliminary work, we observed a LOH (a loss of wild-type allele) in KrasG12D locus in murine PDAC cell lines isolated from genetically engineered mouse models of pancreatic cancer, furthermore, the KrasG12D-LOH renders these cancer cells more invasive than those without LOH. The following pathway analysis suggested that the KrasG12D-LOH did not affect either Kras activity or downstream molecules, but its occurrence was specifically associated energy metabolism. In line, we observed that the energy metabolism-related genes-p-AMPK and REDD1 were up-regulated in murine cancer cell lines with KrasG12D-LOH. Notably, the expression of p-AMPK and REDD1 can be detected in both human primary and metastatic PDAC tissues. Hence, we hypothesize that the KrasG12D-LOH-mediated expressions of p-AMPK and REDD1 function to reprogram energy metabolism of PDAC cancer cells by which process various malignant aspects (e.g. proliferation and invasions) of tumor cells were simultaneously altered. In the current project, we aim to investigate the impact of the KrasG12D-LOH-mediated p-AMPK and REDD1 on energy metabolism and biological behaviors of PDAC cells using a set of experimental methods including measurement of energy metabolism, siRNA, PCR, Western Blot and syngeneic transplantation models. These data will provide a theoretical basis for the early diagnosis and treatmet of PDAC.

胰腺导管腺癌(PDAC)是胰腺癌的主要类型，常伴有原癌基因KRASG12D突变。杂合性缺失(LOH)多见于抑癌基因中，很少发生于原癌基因。我们在KrasG12D转基因小鼠中发现KrasG12D-LOH使肿瘤细胞侵袭性增强，其改变与KrasG12D突变基因或蛋白活性无关，而与能量代谢相关。我们观察到人原发和转移性导管腺癌灶存在REDD1特异性表达，人胰腺导管腺癌、KrasG12D转基因鼠细胞株中伴p-AMPK、REDD1表达，且KrasG12D-LOH株p-AMPK、REDD1表达水平较无LOH株更高。我们设想：KrasG12D-LOH通过介导AMPK、REDD1通路使细胞能量代谢发生重排，获得快速增殖、转移侵袭能力。本课题拟通过体内和体外实验探索KrasG12D-LOH介导的AMPK、REDD1以及mTOR通路对胰腺癌细胞能量代谢和生物学行为的影响及其机理，为胰腺癌的早期诊治提供理论依据。

胰腺癌是一种恶性程度极高，预后较差的恶性肿瘤之一，其发生发展与Kras基因具有密切联系。已有研究证实突变型KrasG12D的等位基因状态在多种肿瘤的发生、发展、浸润、转移过程中具有重要作用，但是机制尚不明确。本项目结合体内体外研究发现，无论常氧还是缺氧条件下，KrasG12D-LOH都能够赋予肿瘤细胞更强的恶性生物学行为。进一步研究发现AMPK通路在其中发挥重要的作用，尤其是在缺氧条件下，抑制缺氧诱导激活的AMPK能够同时明显抑制KrasG12D和KrasG12D-LOH胰腺癌细胞的增殖与侵袭能力。同时，在缺氧条件中沉默REDD1基因表达也能够降低肿瘤细胞能量代谢水平、抑制肿瘤生长和浸润等，且这一现象在伴有KrasG12D-LOH的胰腺癌中更为明显。上述研究提示KrasG12D-LOH或可通过AMPK通路和REDD1通路调控能量代谢从而影响胰腺癌发生发展。此外，本研究率先提出KrasG12D-LOH与肿瘤能量代谢之间具有相关性，为胰腺癌的早期诊断治疗提供了新思路。