To Investigate The Role Of EGFR To FGFR2 Switch In Drug Resistance Of Triple-negative Breast Cancer

ObjectivesThe incidence of breast cancer has increased as the most frequently diagnosed cancer among women.It is estimated that about 70 thousand Chinese women will die from breast cancer every year.The number of deaths ranks sixth in female tumors and ranks first in gynecological cancers.The survival rate has been significantly improved with the use of targeted drugs and new hormone therapy drugs.Triple-Negative Breast Cancer(TNBC)is a heterogeneous subtype of breast cancer,and the prognosis of TNBCs is very poor due to the lack of molecular targeting drugs and they are also not suitable for endocrine treatment.Chemotherapy is the major strategy for treating TNBC in addition to surgery.However,the occurrence of drug resistance is the main cause of treatment failure and death for TNBC patients.The detailed mechanism of chemotherapy resistance remains unclear.Therefore,it is critical to investigate the molecular mechanism of TNBC drug resistance.In this study,we proposed to establish a drug-resistant cell model for investigating the special phenotype of TNBC drug resistant cells.Then,we will also employ a series of molecular biology and cell biology research techniques to explore the potential mechanism of drug resistance in TNBC cells.Methods1)Two TNBC drug resistance cell lines were induced by a stepwise long-term exposure to increasing concentration of chemotherapeutic drugs methods.2)The IC50 and the RI was analzed by CCK-8 method.The expression of drug resistance genes was examined by Western blot and immunostaining methods.3)These two drug-resistant cells were cultured in drug-free condition for 12 consecutive weeks.Then the maintenance of MDR phenotype were further examined.4)The cell proliferation ability was analyzed by CCK-8 and colony formation assay.The cell motility capacity were investigated by wound healing and Transwell assay.5)The expression of EGFR/FGFR1/FGFR2 and activation of the key downstream signaling in parental and drug-resistant cells was examined by Western blot methods.6)Treatment of drug-resistant cells with small molecule inhibitors of FGFR2,then the expression of P-gp was investiaged.7)Small molecule inhibitors or siRNAs were used to analyze the effect of Erk1/2 and STAT3 signaling on expression of P-gp in drug-resistant cells.8)Small molecule inhibitors and western blot method was used to investigate whether PI3K/Akt/mTOR signaling regulates P-gp expression in drug-resistant cells.Results1)Two TNBC multidrug resistance cell lines were established and these two cell lines exhibited cross resistance to a variety of chemotherapy drugs.2)Perinuclear expression of P-gp and BCRP was detected in drug resistance cells,however,no apparent membrane distribution was observed.3)The drug resistance cells displayed a significant decrease in cell proliferation,and an obvious increase in cell migration and invasion ability.4)The expression of Erk1/2 was decreased,whereas the phosphorylation of Erk1/2 and Akt was upregulated in drug-resistant cells than that in parental cells.5)Drug-resistant cells showed a significant kinase switch from EGFR to FGFR2.6)Inhibition of FGFR2 decrease the expression of P-gp in drug-resistant cells,whereas knockdown of EGFR has no significant effect on P-gp expression.7)Blocking of Erk1/2 signaling or knockdown of STAT3 does not affect P-gp expression in drug-resistant cells.8)PI3K/Akt/mTOR signaling regulates P-gp expression in drug resistance cells.ConclusionsTwo TNBC-resistant cell lines with stable multidrug resistance phenotype were established.Perinuclear expression of P-gp and BCRP was detected in drug-resistant cells,whereas few membrane distribution was observed.TNBC resistant cells showed decreased proliferation index and enhanced invasion ability.Moreover,drug-resistant cells showed a kinase switch from EGFR to FGFR2 along with the activation of Erk1/2 and Akt.Inhibition of Erk1/2 does not affect P-gp expression,while blocking of PI3 K or mTOR signaling inhibited P-gp expression,indicating that the EGFR to FGFR2 transition is responsible for acquisition of drug resistance via activation of PI3K/AKT/mTOR signal pathway.