The Mechanisms And Roles Of Abnormally Activated IGF2/IGF-1R/IRS1 Signaling In Herceptin Resistance Of Breast Cancer

Background and Objective: HER2 / Erb B2 as a members of the EGFR(epidermal growth factor receptor,epidermal growth factor receptor)family.HER2 is overexpressed in about 20% ～ 30% breast cancer and is associated with breast cancer development and prognosis.Herceptin is an antibody drug that targets HER2 for the treatment of breast cancer,but no more than 35% of HER2 positive breast cancer patients are sensitive to Herceptin,and these sensitive patients in the treatment process is easy to form secondary resistance.Therefore,it is important to elucidate the mechanism of Herceptin resistance in HER2 positive breast cancer.The study suggests that IGF-1R activation is associated with Herceptin resistance in breast cancer cells.our prophase research also found that IGF-1R could mediate Herceptin resistance in breast cancer cells with HER2 and HER3,also found the expression of IGF2 protein in the Herceptin resistance cell culture medium was increased and the expression of IRS1 protein in the resistance cells was upregulated.Insulin like growth factor 2(IGF2)play an important role in individual growth and tumor formation by binding to the cell surface specific receptor IGF-1R(insulin-like growth factor-1 receptor).IGF-1R,is a tyrosine kinase growth factor receptor,combines with its ligand,through IRS1(subsequently phosphorylates insulin receptor substrate 1)activates downstream effect signaling pathway to inhibit cell apoptosis,promote cell cycle progression and angiogenesis.The purpose of this study is to explore the role and mechanism of IGF2/IGF-1R/IRS1 signaling pathway in the Herceptin treatment resistance of HER2 positive breast cancer with the model of HER2 positive breast cancer parental cells SKBR3 and BT474 and their corresponding Herceptin resistant cells SKBR3/pool2 and BT474/HR20 as models.Methods(1)HER2 positive breast cancer parental cells SKBR3,BT474 and Herceptin resistant cells SKBR3/pool2,BT474/HR20 were used as models,Cell assay combined with vivo assay were used to demonstrate the sensitivity to Herceptin of related cell lines.q RT-PCR combined with ELISA and western blot assay were performed to detect gene expression and its phosphorylation expression level.(2)The sensitivity to Herceptin of cells after treatment with related inhibitors or IRS1 was knockdowned,overexpression of IRS1 in cells and cells were treated with rh IGF2,were detected by MTS assay.(3)Bioinformatics analysis combined with luciferase reporter assay,western blot assay and ELISA assay were performed to measure the transcriptional regulation of mi RNAs on IRS1 and IGF2.The expression of mi RNAs in SKBR3/pool2 and BT474/HR20 cells,were detected by q RT-PCR.(4)The effects of overexpression IRS1 and mi RNAs,the effects of IRS1 expression interference and IGF2 treatment and mi RNAs inhibitor on sensitivity of related cell lines to Herceptin were detected by MTS assay.(5)The expression of mi RNAs in SKBR3/pool2 and BT474/HR20 cells pre-treated with m TOR inhibitor and then FOXO3 a expression interference were detected by q RT-PCR,Bioinformatics analysis combined with Ch IP-q PCR were used to detect the transcriptional regulation of FOXO3 a on mi RNAs.(6)The sensitivity to Herceptin in vivo of tumors formed by SKBR3/pool2 with IRS1 knockdown were monitored,The IGF2 protein level in serum was determined by ELISA assay,Immunohistochemistry was used to detect the IRS1 and p-FOXO3 a level in breast cancer tissue samples,The correlation between protein level and response to Herceptin was analyzed,The correlation between the expression of IRS1 and p-FOXO3 a was analyzed.Results(1)In vitro cell experiments found that the sensitivity of breast cancer resistant cells SKBR3/pool2 and BT474/HR20 to Herceptin was significantly lower than that of the corresponding parental cells;In vivo nude mouse tumorigenesis further validated the tolerance of Herceptin to resistant cells.The expression levels of IGF-1R,Akt,S6 K and FOXO3 a in drug-resistant cells and parental cells were not significantly different,but their phosphorylation levels and IRS1 protein levels were significantly higher in the drug-resistant cells than in the parental cells.There was no significant difference of IGF1,IGF2 and IRS1 m RNA levels between cell lines,but SKBR3/pool2 and BT474/HR20 cells contained higher level of IGF2 protein in CM detected by ELISA.(2)IGF-1R inhibitor PPP(Picropodophyllin),Akt inhibitor MK-2206 2HCl,and m TOR inhibitor WAY-600 treated SKBR3/pool2 and BT474/HR20 cells can increase the sensitivity of drug-resistant cells to Herceptin,respectively.Interfering with IRS1 also increased the sensitivity of drug-resistant cells to Herceptin.In parental cells,the overexpression of IRS1 or IGF2 treatment had no significant effect on sensitivity of SKBR3 and BT474 cells to Herceptin,However,IGF2 treatment dramatically enhanced the effect of ectopic IRS1 expression.(3)According to online software predicts that mi RNAs may be targeted to regulate IRS1 and IGF2.The expression levels of mi RNAs were downregulated in SKBR3/pool2 and BT474/HR20 cells.Overexpression of mi R-128-3p and mi R-30a-5p in SKBR3/pool2 and BT474/HR20 cells remarkably decreased IRS1 expression,while inhibiting mi R-128-3p and mi R-30a-5p did the opposite.Overexpression of mi R-193a-5p in SKBR3/pool2 and BT474/HR20 cells remarkably decreased IGF2 expression,the opposite results were got in SKBR3/pool2 and BT474/HR20 cells with inhibit mi R-193a-5p.Luciferase reporter assay comfirmed that mi R-128-3p and mi R-30a-5p can direct target regulation IRS1 expression and mi R-193a-5p can direct target regulation IGF2 expression in SKBR3/pool2 and BT474/HR20 cells.(4)Results indicated that ectopic overexpression of mi R-128-3p and mi R-30-5p synergistically sensitized SKBR3/pool2 cells to Herceptin,while overexpression of IRS1 in SKBR3/pool2 cells simultaneously overexpression of mi R-128-3p and mi R-30a-5p showed remarkably attenuated the sensitivity of SKBR3/pool2 cells to Herceptin.In parental cells,IGF2 treatment can significantly increase the tolerance to Herceptin after the inhibition of mi R-128-3p and mi R-30a-5p function,and the interference of IRS1 expression can reverse the synergistic resistance of IGF2 and mi RNAs inhibitors.(5)Treatment SKBR3/pool2 and BT474/HR20 cells with m TORC1 and m TORC2 inhibitor showed that WAY-600 remarkably upregulated mi R-128-3p,mi R-30a-5p and mi R-193a-5p expression,but treatment of SKBR3/pool2 and BT474/HR20 cells with m TORC1 inhibitor suggested no significant changes in mi R-128-3p,mi R-30a-5p and mi R-193a-5p expression.Knockdown of FOXO3 a in SKBR3/pool2 and BT474/HR20 cells simultaneously m TOR inhibitor treatment,q RT-PCR results showed that the expression of mi R-128-3p,mi R-30a-5p and mi R-193a-5p was significantly upregulated.Bioformatics predicts that there are FOXO3 a binding sites on the mi RNAs prometor,Ch IP-q PCR experiments comfirmed that FOXO3 a was significantly enriched for the promoters of mi R-128-3p,mi R-30a-5p and mi R-193a-5p in SKBR3 and BT474 cells,compared to in that SKBR3/pool2 and BT474/HR20 cells.Luciferase reporter assay that FOXO3 a can transcriptionally regulate the expression of mi RNAs.Experimental results indicated that the m TOR inhibitor treatment promoted the enrichment of FOXO3 a at mi R-128-3p,mi R-30a-5p and mi R-193a-5p promoter in SKBR3/pool2 and BT474/HR20 cells.(6)In vivo of tumors formed experimental results showed the interference of IRS1 could increase the sensitivity of drug-resistant cells to Herceptin.ELISA assay indicated the protein levels of IGF2 in the serum from patient group with poor response were much higher than those from patient group with good response.The protein levels in tissues were detected by immunohistochemical staining,tissues from patients with poor response had higher IRS1 and p-FOXO3 a,The p-FOXO3 a level was positively correlated with IRS1 level.Conclusions(1)IGF-1R/Akt/m TORC signal mediated by IGF2 and IRS1 is constitutively continuously activated in Herceptin resistant breast cancer cells and mediates Herceptin resistance.(2)mi R-128-3p and mi R-30a-5p regulates IRS1 expression,and mi R-193a-5p regulates IGF2 expression.(3)m TORC2 downregulated expression of mi R-128-3p,mi R-30a-5p and mi R-193a-5p in Herceptin resistance cells by inhibiting FOXO3 a activity.(4)IGF2/IRS1/Akt/m TORC/FOXO3a/mi RNAs positive feedback signal loop mediates Herceptin resistance in HER2+ breast cancer cells.