PIK3CA Mutation Induces Resistance To Chemotherapy In Triple-negative Breast Cancer Through Inhibiting Apoptosis

Background and Objective:Triple-negative breast cancer is characterized by lack of ER,PR,and Her-2.It accounts for approximately 10-20%of breast cancer patients and has a poor prognosis.Chemotherapy is still its main treatment for post-operative TNBC patients.At present,chemotherapy is still the most important adjuvant therapy after TNBC surgery.PIK3CA mutation initiates the sustained activation of Akt through the PI3K/Akt pathway,promotes cell growth and transformation,and inhibits cell apoptosis,which are closely related to the occurrence and development of many human solid tumors.The mutation frequency of PIK3CA is the second mutation in breast cancer just less than TP53 mutation,and mainly occurs in the two hotspots,helical region and kinase region.TNBC is more sensitive to chemotherapeutic drugs than other types of breast cancer,and it is easier to obtain complete pathological response.Our previous clinical studies showed that the survival rate of tumors after neoadjuvant chemotherapy was high in TNBC patients with PIK3CA mutations,and their follow-up prognosis was poor,suggesting that PIK3CA mutation may be related to TNBC chemotherapy resistance.At present,studies on the effect of PIK3CA mutation on the prognosis of breast cancer patients are mostly focused on hormone-positive breast cancer or Her-2 amplified breast cancer,but the study on the triple-negative breast cancer is relatively rare.This study aims to explore the role of PIK3CA mutation in TNBC and the molecular mechanism of TNBC resistance to chemotherapy drugs,to provide theoretical basis for the prevention and treatment of TNBC,to provide new therapeutic strategies for the screening of new drugs,and to lay a foundation for clinical application.Methods:1.In vitro experiment:TNBC cell lines with PIK3CA mutantion and wild type were established by infecting MDA-MB-231 and MDA-MB-468 with lentivirus,and the growth curves of each cell line after infection was plotted with CCK-8 reagent.Flow cytometry were performed to compare the cell cycle and apoptosis among the cell lines.Transwell invasion assays were used to compare the invasive ability of each cell line.In addition,we use Western blot,real-time quantitative PCR and immunohisto-chemistry to detect the differences of expression of p110?,akt,p-akt,mTOR,pten,xiap,Ki67,p53,Bcl-2 etc.in PI3K/Akt/mTOR signaling pathway in cell lines between pretreatment and posttreatment of epirubicin to clarify the role of PIK3CA mutation in TNBC cells and to explore the relationship between PIK3CA mutation and chemotherapy drug resistance and its mechanism.2.In vivo experiments:PIK3CA mutant and wild-type TNBC NOD-SCID mouse model was established by using the above-mentioned post-infection MDA-MB-231 cell lines.Then mice were injected with epirubicin,and their tumor growth was measured at regular intervals.Mice were killed and tumor tissues were removed.Western blot,real-time quantitative PCR and immunohistochemistry were used to examine the expression of p110?,akt,p-akt,mTOR,pten,xiap,Ki67,p53,and Bcl-2 in the PI3K/Akt/mTOR signaling pathway and to verify the role of PIK3CA mutations in TNBC cells and the relationship between PIK3CA mutations and chemoresistance and the mechanisms.3.Clinical retrospective analysis:The clinical and pathological data of 50 cases of TNBC patients undergoing surgery in our hospital were collected.And their tumor tissues were sequenced and immunohistochemically analyzed.Combined with follow-up results,the clinical pathological feature factors were analyzed to investigate the effect of PIK3CA mutation on TNBC patients between PIK3CA mutant and wild type group.Results:1.In vitro experiments:(1)The PIK3CA mutant MDA-MB-231 and MDA-MB-468 cell lines grew faster than the wild type.The proportion of S phase cells increased(P>0.05),and apoptotic cells were significantly reduced(P<0.05);(2)Transwell assay results:The number of PIK3CA-mutant cell lines passing through the Transwell chamber was significantly higher than that of the wild type(P<0.05),suggesting that the PIK3CA mutation enhanced the invasive ability of TNBC cells;(3)In PIK3CA mutant MDA-MB-231 and MDA-MB-468 cell lines,the phosphorylation of Akt was increased,and the expressions of pten,Ki67,Xiap and p-p53 were up-regulated.Oppositely,the expressions of Stat3,p-4E-BPl,Caspase3,Bcl2 and p53 were down-regulated.(4)Apoptosis experiments showed that apoptosis of PIK3CA-mutant MDA-MB-231 cell lines decreased after being treated with epirubicin(P<0.05);however,only PIK3CA over-expressing cell line decreased statistically significantly in MDA-MB-468 cell lines(P<0.05);(5)Western blot results:Akt phosphorylation was increased in PIK3CA mutant MDA-MB-231 and MDA-MB-468 cell lines treated with epirubicin,and expression of mTOR,phosphorylated mTOR,Ki67,Xiap upregulated.2.In vivo experiments:(1)The tumor of PIK3CA-mutant MDA-MB-231 cell lines grew faster than that of the wild-type cell line,and the average tumor volume was larger(P<0.05);(2)Western blot and immunohistochemistry assays showed that phosphorylation of Akt was increased in tumor tissues of PIK3CA-mutant TNBC mice,and the expression of mTOR,Xiap,Ki67,and p53 were up-regulated,while the expression of caspase3,Bcl2,and pten were down-regulated.3.Clinical retrospective analysis:(1)PIK3CA mutations occurred in 11 of the 50 patients(22%),among which 8 were in the H1047R locus and 1 was in the E545K locus;(2)There was no significant difference in age distribution,tumor size,tumor location,axillary lymph node status,histological grade,TNM stage,recurrence,tumor metastasis,postoperative survival,family history,p53,Ki67 index between PIK3CA mutant and PIK3CA wild type tumors(P>0.05).(3)In PIK3CA mutant tumor tissues,Akt,mTOR were higher expressed.Pten and Bcl-2 were higher expressed in wild type and PIK3CAE545K,but relatively lower in PIK3CAH1047R.There was no difference in expression of Xiap,Cyclin D1,Caspase 3 between wild-type and mutant tumor tissues.Conclusions:1.In vitro experiments:(1)PIK3CA mutation promotes TNBC cell growth.(2)PIK3CA mutation enhances TNBC invasiveness.(3)PIK3CA mutation inhibits TNBC cell apoptosis.(4)PIK3CA mutation increases TNBC resistance to chemotherapeutic drugs.(5)PIK3CA mutation activates the PI3K/Akt/mTOR pathway and promotes cell growth.2.In vivo experiments:(1)PIK3CA mutation promoted tumorigenesis and tumor growth in TNBC mice;(2)PIK3CA mutation promoted tumor growth by activating the PI3K/Akt/mTOR signaling pathway.3.Clinical retrospective analysis:(1)The mutation frequency of PIK3CA mutation in TNBC was 22%,and mainly happened in E545K,H1047R sites.(2)PIK3CA mutation may not be a predictor of prognosis of triple-negative breast cancer.