PAI-1 Induces Src Inhibitor Resistance Via CCL5 In HER2-positive Breast Cancer Cells

Background and objectiveBreast cancer is the most frequent and common malignancy among female according to the latest data.In different subtypes of breast cancer,human epidermal growth factor receptor2(HER2)-positive breast cancer is a type of breast cancer with overexpression or amplification of HER2,accounting for 15-20%of all breast cancer,which exhibits aggressive behavior and is regarded as a refractory disease.Although series of clinical trials confirmed that addition of trastuzumab to chemotherapy has strikingly declined mortality rate of HER2-positive breast cancer and emerged as a first-line treatment strategy for patients with HER2-positive breast cancer.However,primary and/or acquired resistance occurs in majority of patients receiving targeted therapy and eventually leads to the failure of treatment.It is crucial to search new therapeutic strategies for improving the prognosis of HER2-positive breast cancer patients.Src protein was encoded by gene c-Src,which is a member of the tyrosine kinase family and is highly expressed in a variety of tumors,regulating various cell processes,including cell proliferation,motility,invasion and metastasis in tumor.In approximately 60%of breast cancers,Src protein expression is significantly elevated with aberrant activation.Therefore,Src kinase is an emerging target for the treatment of breast cancer in recent years.Saracatinib,as a selective inhibitor of Src kinase,numbers of studies in vitro and in vivo have found that it can effectively inhibit the growth of breast cancer cells.Recent studies have even found that saracatinib can reverse the resistance of tumor cells to fulvestrant,trastuzumab,lapatinib,and gemcitabine.Unfortunately,the potency of Src inhibitor is limited by the development of drug resistance.However,the specific mechanism of resistance is still not fully understood.To explore molecular mechanism of resistance and feasible regulatory targets,we established a Src inhibitor saracatinib-resistant breast cancer cell line(SKBR-3/SI)for the first time and provide reliable evidence that the overexpression of PAI-1 contributes to Src inhibitor resistance and promotes cell proliferation and cell migration in breast cancer via upregulating CCL5.It may also provide new strategy for the treatment of local advanced or metastasis HER2-positive breast cancer.Materials and MethodsHER2-positive breast cancer cell line SKBR-3 and saracatinib-resistant breast cancer cell subline SKBR-3/SI were used in our study.1.The difference of SKBR-3/SI and SKBR-3 was detected by CCK8 assay,Colony formation assay,Flow cytometric analysis and Western blot analysis,and confirmed that saracatinib-resistant cell line SKBR-3/SI was successfully established.2.To explore whether certain factor conferred to resistance of saracatinib,we evaluated mRNA expression profile between parental cell line SKBR-3 and resistant cell line SKBR-3/SI.On the basis of our laboratory's previous study results,we further found that mRNA expression of PAI-1 was increased in the resistant cell line.To our interest,gene ontology analysis of genes with over 2-fold change between SKBR-3/SI and SKBR-3 revealed an enrichment of factors involved in positive regulation of monocyte chemotaxis.Hence,we chose PAI-1 for further research and confirmed that expression of PAI-1 in SKBR-3/SI was relatively higher in mRNA and protein levels than in SKBR-3.3.The effects of target mRNA(PAI-1)on saracatinib sensitivity,cell proliferation,apoptosis,and invasive of SKBR-3/SI cells were investigated by CCK8,colony formation,flow cytometry,and cell migration assays.4.On basis of our laboratory's previous study results,mRNA expression profiles,and literature reports,we speculated whether PAI-1 conferred Src inhibitor resistance was via CCL5.Subsequently,to explore the correlation between PAI-1 and CCL5,we treat with PAI-1 and PAI-1 antagonists PAI-039 or small interfering RNAs(siPAI-1)in SKBR-3 and SKBR-3/SI cells.5.The effects of CCL5 on saracatinib sensitivity,cell proliferation,apoptosis,and invasive of SKBR-3/SI cells were investigated by CCK8,colony formation,flow cytometry,wound healing and cell migration assays.6.Conducted nude mice xenograft models to analysize the difference of SKBR-3/SI tumors and SKBR-3 tumors.Immunohistochemistry staining was performed in tissue microarray to analysize the expression of PAI-1,CCL5 and Ki67.Results1.The cell proliferation of SKBR-3 was significantly inhibited by saracatinib,while SKBR-3/SI cell line was not suppressed.As for cell apoptosis,saracatinib markedly increased the early and later apoptosis rate of SKBR-3,but had no effect on SKBR-3/SI,suggesting the stable proliferation of resistant breast cancer cells SKBR-3/SI.Breast cancer resistance protein(BCRP/ABCG2)was upregulated in SKBR-3/SI compared with SKBR-3.These data indicated that the saracatinib-resistant cell line was successfully established.2.The expression of PAI-1 in SKBR-3/SI was relatively higher in mRNA and protein levels than in SKBR-3.3.Recombinant human PAI-1(rPAI-1),which can mimic the function of PAI-1,decreased SKBR-3 cell sensitivity to saracatinib,promoted cell proliferation and cell migration,while PAI-039,inhibiting PAI-1 activity,led to increased SKBR-3/SI cell sensitivity to saracatinib,suppress cell proliferation and cell migration.4.PAI-1 up-regulates the expression of CCL5 in saracatinib resistant cell line SKBR-3/SI.5.Recombinant human CCL5 decreased SKBR-3 cell sensitivity to saracatinib,promoted cell proliferation and cell migration,whereas CCL5 ab induced increased SKBR-3/SI cell sensitivity to saracatinib,inhibited cell proliferation and cell migration.Taken together,our observations suggested that overexpressed PAI-1 might result in resistance to Src inhibitor in SKBR-3 by upregulating of CCL5.6.SKBR-3/SI had a bigger tumor size compared to SKBR-3 group.SKBR-3/SI tumors showed higher PAI-1 and CCL5 expression than SKBR-3 tumor.Besides,Ki67 stain of SKBR-3/SI tumors was also stronger than SKBR-3 tumors,indicating the higher cell proliferation.ConclusionIn conclusion,our findings provide reliable evidence that the overexpression of PAI-1 contributes to Src inhibitor resistance and promotes cell proliferation and cell migration in breast cancer via upregulating CCL5.Meanwhile,PAI-1 inhibitor or neutralizing CCL5 antibody could reverse saracatinib resistance in saracatinib-resistant cells.To make full use of Src inhibitor,it might be a choice to combine agents targeting PAI-1 or CCL5 with Src inhibitor.