| OBJECTIVE: Previous studies indicated that IL-6/IL-6R signaling pathway may affect the sensitivity of HER2-positive breast cancer to trastuzumab treatment,while the sources of IL-6,the regulation of IL-6R expression and the specific mechanism of them mediating trastuzumab is not clear.This study aims to clarify the relationship between IL-6/IL-6R signaling pathway and the sensitivity of HER2-positive breast cancer to trastuzumab treatment,and to explore their source and regulation mechanism.Methods: Cell viability assay,colony formation assay and flow cytometry apoptosis assay were used to evaluate the sensitivity of tumor cells to trastuzumab treatment;enzymelinked immunosorbent assay was used to assess the concentration of IL-6 in cell culture supernatant.Real-time PCR,Western Blot and immunohistochemical staining were used to detect the expression level of the gene of interest.Immunofluorescence experiments were used to assess the expression and distribution of protein in the cell.Glucose assays are used to assess tumor cell glucose uptake rates.Chromatin immunoprecipitation experiments are used to determine the binding and regulation of transcription factors to target gene promoters.The correlation and expression of related genes and their relationship to prognosis were evaluated using the GEPIA and KM Plotter online databases,respectively.RESULTS: IL-6 treatment significantly increased the survival rate and colony formation of HER2-positive breast cancer cells treated with trastuzumab.All of tumor cells,normal fibroblasts,and cancer-associated fibroblasts could secrete IL-6,but IL-6 secreted by tumor-associated fibroblasts(934.6-3804.0 pg/ml)is significantly higher than tumor cells(50.7-96.6 pg/ml)and normal fibroblasts(317.4-381.9 pg/ml).Besides,the IL-6 secretion capacity of cancer-associated fibroblasts is not affected by trastuzumab treatment.Trastuzumab treatment inhibited glucose uptake by tumor cells.The glucose uptake rates at low and high concentrations of trastuzumab were respectively 67.0 % and 40.7 % for BT474 and 80.7 % and 32.1 % for SKBR3 compared to the control group.While IL-6treatment could promote glucose uptake of tumor cells by up-regulating GLUT3,making the glucose uptake increase by 2.5 nmol/ul and 1.9 nmol/ul(P < 0.05),respectively.Downstream signaling pathway detection showed that IL-6 activated the JAK2/STAT3 signaling pathway;phosphorylated STAT3 entries into the nucleus and up-regulates the transcriptional level of GLUT3 by binding to its promoter.Online database and in vitro experiments showed that the expression level of transcription factor CHD1 L was positively correlated with IL-6R.As a transcription factor,Ch IP-seq experiments showed that CHD1 L can target the promoter region of IL-6R.Overexpression of CHD1 L in HER2-positive breast cancer cell lines reduces the sensitivity of trastuzumab treatment;whereas the application of IL-6 neutralizing antibody reverses CHD1L-mediated trastuzumab resistance.Conversely,knockdown of CHD1 L or treatment with IL-6 increased the sensitivity of tumor cells to trastuzumab treatment.In HER2-positive patients who received trastuzumab,CHD1 L overexpression was associated with poor prognosis in patients.Conclusion: Trastuzumab-mediated reduction of tumor cell glucose uptake is one of the important mechanisms for its tumor suppressive effect.IL6 was highly secreted from Cancer-associate-fibroblasts.IL-6 up-regulates GLUT3 expression by activating the JAK2/STAT3 signaling pathway,promoting tumor cell uptake of glucose and resistance to trastuzumab treatment.The expression level of IL-6R in tumor cells is regulated by CHD1 L.Overexpression of CHD1 L up-regulates IL-6R mediating trastuzumab resistance. |
PDF Full Text Request