Novel Role Of MiR-375in Trastuzumab-resistance And Metastasis Of HER2-positive Breast Cancer

【Backgroud】Breast cancer remains the most common malignant disease in women, with onemillion new cases diagnosed worldwide per year and causing400,000deaths.Given thatthe overexpression of ErbB2/HER2occurs in20to25%of human breast cancers and isassociated with poor prognosis, the humanized HER2antibody, trastuzumab (Herceptin),has been successfully used for therapy of HER2-positive early-stage and metastatic breastcancers.However, the response rate of patients with HER2-positive breast cancers totrastuzumab monotherapy is less than35%, which is slightly increased to approximately40%when combined with microtubule stabilizing drugs. Furthermore, most patientsresponsive to the initial trastuzumab treatment develop resistance within a year. Therefore,clarifying the mechanisms underlying trastuzumab resistance and its crosstalk withmetastasis will provide great impetus for the development of new treatment of breastcancer.As a new family of gene regulators, miRNAs are involved in modulating multiplecellular pathways, including cell proliferation, differentiation, metastasis and apoptosis,and thus may function as oncogenes or tumor suppressor genes.In this study, we established a Trastuzumab-resistant cell line by continually exposingHER2+breast cancer cells to Trastuzumab. we demonstrated that miR-375expression was down-regulated and its function was elevated in HER2(+) breast cancer cells. Theseresults are in line with previous validation of the tumor-suppressive roles of miR-375since decreased expression of miR-375was observed in primary esophageal squamouscell cancer, gastric carcinoma, and tamoxifen-resistant breast cancer cells.We demonstratein the following sections that miR-375confers properties significant for breast cancer cellsmetastasis and drug resistance in vitro and in vivo, and is a useful therapeutic target forfuture breast cancer therapy.【Objectives】We evaluated the role of miR-375in regulating Trastuzumab resistance andmetastasis in HER2-positive breast cancer in multi-levels, and we primarily investigatedthe possible mechamisms of miR-375in Trastuzumab resistance.【Methods】1.Resistant cells (Trastuzumab Resistance) were developed by culturing parental cells(WT) in the presence of5μg/ml Trastuzumab for6months;2.Using soft agar colonyformation assays and transwell assays to detect the aggressive feature in resistancecells(SK-BR-3R) compared with parental cells;3. Using microassay and QRT-PCR todetect differentially expressedmiRNAs;4. Construction of pre-miR-375plasmid, miR-375inhibitor and controls, transfection of resistance cells(SK-BR-3R) compared with parentalcells,QRT-PCR-PCR assays for detecting their expression;5. MTT was preformed toexamine the sensitivity of infected and transfected cells to Trastuzumab;6. Flowcytometry was carried out to evaluate the effcts of miR-375on cell apoptosis；7.Woud-healing assay and transwell assay for detecing migration and invasion of cells;8.Nude tumorigenesis and metastasis models were used to evaluate in vivo miR-375effectsin relation to chemotherapy;9.Candidate target gene of miR-375, IGF1R was identified byreporter gene assay, Western blot, PCR, and MTT assays.[Results】1. Trastuzumab resistant cells were more aggressive and have lower expression ofmiR-375compared with parental cellsHuman breast cancer SK-BR-3cells, which overexpress HER2, were cultured continuously for6month in the presence of5μg/ml trastuzumab, resulting in theacquisition of trastuzumab resistance in the surviving cell population. Compared with theparental cells, these SK-BR-3cells showed dramatically increased colony formation onthe agar plates, and much higher invasion capacity in a transwell assay (p<0.05,).Therefore, trastuzumab-resistant cells are more aggressive than the parentalHER2-positive breast cancer cells.To investigate the roles of miRNA in trastuzumab resistance of breast cancers, weperformed microarray analysis to compare the miRNA profiles betweentrastuzumab-resistant and parental SK-BR-3cells. Using a cutoff of>2-fold changespoint in miRNAexpression, we identified20differentially expressed miRNAs. Among theabove miRNAs, we focused particularly on miR-375, which showed the second largestabsolute fold change in the miRCURYLNAarray (p<0.001).2.miR-375modulates trastuzumab resistance in breast cancer cellsTo further investigate the role of miR-375in trastuzumab resistance of breastcancers, we altered the levels of miR-375in both parental and trastuzumab-resistant cells.Introduction of pre-miR-375via a lentiviral vector in trastuzumab-resistant cellssignificantly increased the cellular levels of this miRNA, and resulted in greatly enhancedsensitivity to trastuzumab (p<0.05). Conversely, transfection of parental SK-BR-3cellswith the miR-375antisense caused dramatic downregulation of miR-375, and conferredresistance in these cells when treated with increasing concentrations of trastuzumab(p<0.01,). Enforced expression of pre-miR-375significantly suppressed in vitro colonyformation of trastuzumab-resistant cells (p<0.01). We next examined the apoptosis of cellsafter treatment with trastuzumab. Pre-miR-375expression caused a marked increase inapoptosis of trastuzumab-refractory cells (p<0.05), and inhibition of miR-375by antisenseRNA in the parental SK-BR-3cells caused significantly suppressed apoptosis (p<0.01),indicating that miR-375modulates trastuzumab resistance in breast cancer cells.3. miR-375reduced trastuzumab-resistant cell invasion and migrationThe effect of miR-375expression on invasion and metastasis of breast cancer cellswas also explored. SK-BR-3cells were infected with pre-miR-375-expressing or controllentiviruses, followed by treatment with trastuzumab for48hrs. As a result, miR-375expression could suppress the mobility of breast cancer cells in a wound-healing assay,and dramatically impair the invasion capacity of cells on a matrigel (p<0.01,). Conversely,loss of miR-375expression in parental SK-BR-3cells promoted cell motility and invasion. These results indicated that restored miR-375expression plays a suppressive role inmetastasis of trastuzumab-resistant breast cancers.4. miR-375inhibits resistance of trastuzumab and modulates metastasis in HER2-positivebreast cancer cells by targeting IGF1RRe-expression of miR-375sensitized resistant cells to trastuzumab and partlysuppressed migration of these cells. Knockdown of IGF1R partially phenocopied theeffects of miR-375on the sensitivity to trastuzumab and the reversal of migration.Luciferase activity was reduced in miR-375-expressing cells compared with the control(p<0.05), and enforced expression of pre-miR-375in trastuzumab-resistant SK-BR-3cellscaused reduced IGF1R level. In addition, we observed that phosphorylated AKT levelsdecreased in parallel with the downregulation of IGF1R by pre-miR-375intrastuzumab-resistant cells. Therefore, miR-375might mediate resistance of trastuzumabthrough modulating the expression of IGF1R in breast cancer.【Conclusion】This study demonstrates that miR-375has a strong tumour-suppressive effect throughinhibiting the expression of IGF1R. The downregulation of miR-375is one of themolecular mechanisms involved in the development and progression of trastuzumabresistance and miR-375exhibits therapeutic potential by sensitizing the malignancy toanti-HER2treatment.