Insulin promotes invasion and migration of KRAS(G12D) mutant HPNE cells by upregulating MMP-2 gelatinolytic activity via ERK- and PI3K-dependent signalling
Insulin promotes invasion and migration of KRAS(G12D) mutant HPNE cells by upregulating MMP-2 gelatinolytic activity via ERK- and PI3K-dependent signalling
复制标题
胰岛素通过 ERK 和 PI3K 依赖性信号传导上调 MMP-2 明胶分解活性，促进 KRAS(G12D) 突变型 HPNE 细胞的侵袭和迁移
DOI：
10.1111/cpr.12575
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发表时间：
2019
影响因子：
8.5
通讯作者：
Lu Zipeng
中科院分区：
文献类型：
--
作者：
Wang Guangfu;Yin Lingdi;Peng Yunpeng;Gao Yong;Gao Hao;Zhan Jingjing;Lv Nan;Miao Yi;Lu Zipeng
目的高胰岛素血症是胰腺癌的危险因素，但胰岛素在胰腺癌发生发展中的作用尚不清楚，因此本研究旨在阐明胰岛素在胰腺癌早期的致癌作用及其与KRASs突变的协同作用。材料与方法用一对永生化的人胰管来源细胞hTERT-HPNE E6/E7/st(HPNE)及其致癌基因KRASG12D变异体hTERT-HPNE E6/E7/KrasG12D/st(HPNE-MUT-KRAS)研究胰岛素的作用。用细胞计数试剂盒8和Transwell检测细胞增殖、迁移和侵袭能力。用Western blotting和/或qRT-PCR检测E-钙粘蛋白、N-钙粘蛋白、波形蛋白和基质金属蛋白酶(MMP2、MMP7、MMP9)的表达。用明胶酶谱法检测条件培养液中明胶酶活性。结果突变的KRASs基因导入HPNE细胞后，细胞的迁移和侵袭能力增强，MMP2的表达增强，同时胰岛素的作用进一步增强。利用MMP2siRNA证实MMP2siRNA参与了细胞侵袭的调控。此外，在胰岛素治疗后，明胶酶活性有浓度和时间依赖性的增加，这可以被胰岛素受体酪氨酸激酶抑制剂(HNMPA-(AM)3)逆转。此外，胰岛素显著增强PI3K/AKT、p38、JNK和ERK1/2 MAPK通路的磷酸化，Wortmannin或PI3K特异性抑制剂LY294002和MEK1特异性抑制剂PD98059显著抑制胰岛素诱导的MMP2明胶溶解活性的增加。结论以上结果提示，胰岛素通过PI3K/AKT和ERK1/2激活诱导HPNE和HPNE-mut-KRAS的迁移和侵袭，其中MMP2明胶溶解活性在此过程中起重要作用。这些发现可能为预防高胰岛素血症背景下胰腺癌的发生和演变提供新的治疗靶点。
ObjectivesHyperinsulinemia is a risk factor for pancreatic cancer, but the function of insulin in carcinogenesis is unclear, so this study aimed to elucidate the carcinogenic effects of insulin and the synergistic effect with theKRASmutation in the early stage of pancreatic cancer.Materials and methodsA pair of immortalized human pancreatic duct‐derived cells, hTERT‐HPNE E6/E7/st (HPNE) and its oncogenicKRASG12Dvariant, hTERT‐HPNE E6/E7/KRASG12D/st (HPNE‐mut‐KRAS), were used to investigate the effect of insulin. Cell proliferation, migration and invasion were assessed using Cell Counting Kit‐8 and transwell assays, respectively. The expression of E‐cadherin, N‐cadherin, vimentin and matrix metalloproteinases (MMP‐2, MMP‐7 and MMP‐9) was evaluated by Western blotting and/or qRT‐PCR. The gelatinase activity of MMP‐2 and MMP‐9 in conditioned media was detected using gelatin zymography. The phosphorylation status of AKT, GSK3β, p38, JNK and ERK1/2 MAPK was determined by Western blotting.ResultsThe migration and invasion ability of HPNE cells was increased after the introduction of the mutatedKRASgene, together with an increased expression of MMP‐2. These effects were further enhanced by the simultaneous administration of insulin. The use of MMP‐2 siRNA confirmed that MMP‐2 was involved in the regulation of cell invasion. Furthermore, there was a concentration‐ and time‐dependent increase in gelatinase activity after insulin treatment, which could be reversed by an insulin receptor tyrosine kinase inhibitor (HNMPA‐(AM)3). In addition, insulin markedly enhanced the phosphorylation of PI3K/AKT, p38, JNK and ERK1/2 MAPK pathways, with wortmannin or LY294002 (a PI3K‐specific inhibitor) and PD98059 (a MEK1‐specific inhibitor) significantly inhibiting the insulin‐induced increase in MMP‐2 gelatinolytic activity.ConclusionsTaken together, these results suggest that insulin induced migration and invasion in HPNE and HPNE‐mut‐KRASthrough PI3K/AKT and ERK1/2 activation, with MMP‐2 gelatinolytic activity playing a vital role in this process. These findings may provide a new therapeutic target for preventing carcinogenesis and the evolution of pancreatic cancer with a background of hyperinsulinemia.