Hepatocyte Growth Factor Induces Breast Cancer Cells Invasion Through PI3K/Akt And P38MAPK Signaling Pathways To Up-regulate Expressions Of COX-2

Objective: To investigate the effects of HGF on the invasion ofhuman breast cancer cell lines MDA-MB-231and MCF-7throughup-regulate expression of COX2and the molecular mechanism of HGFinduced invasion via cyclooxygenase2(COX2) up-regulation.Methods: MDA-MB-231and MCF-7cells were serum-deprivedovernight and then treated with various doses of HGF to detect theirinvasiveness. The effects of HGF on the invasion of breast cancer cellswere determined by transwell chamber experiment. HGF induced COX2expression in breast cancer cells and analyzed by qRT-PCR and westernblot. HGF-treated MDA-MB-231cells and MCF-7cells were transfectedwith pshRNA-HK, pshRNA-COX2, respectively. Total cell protein wascarried out Western blot analysis to detect the expressions of COX2andMMP-9protein. Cells were serum-deprived for24h followed by treatmentas indicated and extracted proteins were analyzed by Western blot to assayexpressions of Akt, p38MAPK and phosphorylated Akt and p38MAPK. MDA-MB-231and MCF-7cells were serum-deprived for24h and thentreated with signaling inhibitors for1h, followed by60ng/ml HGF for15min for p-Akt and p-p38MAPK detection, and12h for COX2detection.MDA-MB-231and MCF-7cells were incubated in the presence or absenceof60ng/ml HGF plus10μM LY294002(Akt/PI3K inhibitor) or/and10μMSB202190(p38inhibitor). Cells were carried out invasion assays.LY294002and SB202190decreased HGF-induced expression of MMP-9protein.Results:1. The positive expressions of HGF receptor c-Met and HGF-inducedincrease of phosphorylated c-Met (p-c-Met) in both breast cancer cellMDA-MB-231and MCF-7by Western blot suggested that both breastcancer cell lines could respond to HGF. The effects of HGF on the invasionof breast cancer cells were determined by transwell chamber experiment. Inthe presence of various concentrations of HGF, invasion cells of bothbreast cancer cell lines were significantly increased and HGF showed aconcentration-dependent stimulation effect.2. The quantitative real-time PCR (qRT-PCR) was performed todetermine whether HGF regulate COX2mRNA expression inMDA-MB-231and MCF-7cells. The mRNA level of COX2was markedincreased by HGF, which revealed a dose-dependent manner. Further analysis proved COX2mRNA was up-regulated by60ng/ml of HGF with atime-dependent manner. In consistent with the up-regulation of COX2mRNA transcription, the protein expression levels of COX2in Westernblot showed a similar dose-and time-dependent induction in HGFstimulation cells.3. MDA-MB-231and MCF-7cells were transfected withpshRNA-COX2, which confirmed that the pshRNA-COX2plasmid couldsilence COX2gene by western blot. Both breast cancer cell linestransfected with pshRNA-COX2plasmid showed obviously lessHGF-induced invasiveness than those of HGF and HGF+pshRNA-HKtreated cells, respectively. However, there were no significant difference ininvasiveness between untransfected cells and the pshRNA-HK transfectedcells.4. HGF-induced MMP-9up-regulation was reduced to62%and49%by COX2gene silenced in MDA-MB-231/HGF+pshRNA-COX2andMCF-7/HGF+pshRNA-COX2groups compared with MDA-MB-231/HGFand MCF-7/HGF groups, respectively (P<0.05). There was significantlydecreased expressions of MMP-9in cells transfected with pshRNA-COX2compared to either with or without HGF treatment control cells (P<0.05).5. In the concentrations of10μmol/L LY294002or SB202190(10μmol/L) partially attenuated the HGF-induced increase on invasion in both breast cancer cell lines. While simultaneously treated with LY294002and SB202190, HGF-driven increasing invasion was completely preventedin MDA-MB-231and MCF-7cells. Furthermore, MMP-9expression levelswere significantly decreased in LY294002/HGF or SB202190/HGF groupcompared with HGF group. Similarly, In the presence of both LY294002and SB202190showed that HGF-induced expression of MMP-9andincrease of invasion were completely inhibited, too.Conclusions: In this study, it has been demonstrated that HGF couldinduce the up-regulation of COX2via activating PI3K/Akt and p38MAPK signaling pathways, and result in increasing expression of matrixmetalloproteinase-9(MMP9) and invasion of breast cancer cells. Thisstudy might provide some valuable insights into the mechanism of HGFsignaling involved in the progression and development of breast cancer.