[Overexpression of HER2/neu downregulates wild p53 protein expression via PI3K and Ras/Raf/MEK/ERK pathways in human breast cancer cells].
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.
Zheng
Li
L
Department of Pathology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Ren
Jia-qiang
JQ
Zhang
Lin
L
Chen
Qi
Q
Zhu
Hong-guang
HG
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002070:Butadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D015536:Down-Regulation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics
D002070:Butadienes; D002867:Chromones; D048049:Extracellular Signal-Regulated MAP Kinases; D009025:Morpholines; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D016159:Tumor Suppressor Protein p53; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one