乳腺癌细胞过表达HER2/neu时经PI3K和Ras/Raf/MEK/ERK信号通路下调野生型p53蛋白含量

Overexpression of HER2/neu downregulates wild p53 protein expression via PI3K and Ras/RaE/ MEK/ERK pathways in human breast cancer cells

目的 研究HER2/neu基因过表达对乳腺癌细胞野生型p53蛋白含量的影响并探讨其分子机制。方法 脂质体介导的HER2/neu基因转染MCF7细胞,G418筛选阳性克隆。Western印迹法鉴定HER2/neu蛋白表达,并同时检测p53及信号转导分子Akt,p-Akt,p-Raf,p-MEK,p-ERK蛋白含量,以及P13K信号通路抑制剂LY294002和MEK抑制剂U0126处理对p53蛋白含量的影响。逆转录-聚合酶链反应(RT-PCR)检测p53 mRNA水平。结果 成功建立稳定过表达HER2/neu的MCF7细胞(MCF7-neu3),其HER2/neu蛋白表达量是对照组MCF7细胞的13倍。MCF7-neu3中p-Akt,p-Raf,p-MEK,P-ERK蛋白含量分别较MCF7细胞升高了2.5倍、2倍、1.6倍和1.6倍(P<0.01),p53蛋白含量仅为对照组细胞的40％(P<0.01),但p53mRNA表达无明显差异。在经PI3K信号通路抑制剂LY294002及MEK抑制剂U0126处理24 h后,MCF7-neu3细胞p53蛋白含量分别上升了1.7和1.5倍(P<0.01),在LY294002和U0126处理48 h后,p53蛋白含量分别升高了4.7倍和5.3倍(P<0.01)。LY294002和U0126处理对MDA-MB-453细胞突变型p53蛋白含量不产生影响。结论 乳腺癌细胞中HER2/neu基因的过表达能够通过激活P13K和Ras/Raf/MEK/ERK通路导致野生型p53蛋白含量减少,可能是HER2/neu过表达乳腺癌患者预后不良及对治疗产生抗性的分子机制。

Objective To evaluate the effect of HER2/neu overexpression on wild p53 protein expression and to delineate the related signal pathways. Methods Lipofectin method was used to transfer HER2/neu into human breast tumor cell line MCF7. Overexpression of HER2/neu was then determined by Western blot. Western blot was also used to detect the quantity of p53, Akt, p-Akt, p-Raf, p-MEK, p-ERK protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect p53 mRNA expression. PI3K pathway inhibitor LY294002 and MEK inhibitor U0126 were used to block the two pathways. The subsequent effect on p53 protein expression was then determined. Results HER2/neu-overexpressed MCF7 clone (MCF7-neu3) was successfully established, in which the amount of HER2/neu protein was 13 times more than that in parental MCF7 cells. The amount of p53 protein in MCF7-neu3 was 40% of that in parental MCF7 cells (P < 0. 01) , while there was no difference on p53 mRNA level. There were 2.5, 2.0, 1.6 and 1.6 fold increase in the amount of p-Akt, p-Raf, p-MEK, p-ERK protein respectively in MCF7-neu3 to that in parental MCF7 cells (P <0. 01). When treated with LY294002 or U0126 for 24 hours, the amount of wild p53 protein in MCF7-neu3 cells was 1. 7 or 1. 5 times higher than those in DMSO treated cells. There were 4. 7 or 5. 3 times increase in the p53 protein when MCF7-neu3 cells were treated with LY294002 or U0126 for 48 hours (P <0. 01). Similar results were not seen in MDA-MB-453 cells which contained mutant p53. Conclusions HER2/neu overexpression can activate PI3K and Ras/Raf/MEK/ERK pathways, resulting in reduction of wild p53 protein expression. This may be the molecular mechanism responsible for the poor prognosis and therapeutic non-responsiveness in HER2/neu- overexpressed breast cancer patients.