CAFs Enhance Paclitaxel Resistance By Inducing EMT Through IL-6/JAK2/STAT3 Pathway

Bachgroud:Epithelial ovarian cancer(EOC)is the third most common cancer in female reproductive system,which has the highest mortality rate with a 5-year survival rate of only approximately 30%-40%.About 20%of patients in the initial chemotherapy may experience chemotherapy resistance.After treatment with the first-line chemotherapy regimen,about 80%of patients may experience tumor recurrence within 2 years and are prone to resistance after relapse,leading to treatment failure.Therefore,it is very important to inhibit metastasis and chemotherapy resistance of ovarian cancer.The tumor microenvironment consists of tumor cells,fibroblasts,smooth muscle cells,nerves,blood vessels and a variety of inflammatory/immune cells.These cells and inflammatory cytokines constitute the inflammatory tumor microenvironment,which may promote the proliferation and metastasis of tumor.As the main components of mesenchymal cells in tumor microenvironment,fibroblasts are known as tumor-associated fibroblasts(CAFs).They are activated fibroblasts in the stroma of the tumor,which express α-smooth muscle actin(a-SMA)and exhibit the characteristics of myofibroblasts.It has been shown that CAFs can secrete various inflammatory cytokines via an autocrine or paracrine mechanism,such as IL-6,COX-2,CXCL12 and HIF-1α.CAFs can also regulate the sensitivity of tumor cells to chemotherapy,playing important roles in improving the efficacy of chemotherapy therapy and reversing drug resistance.Johansson et al.have found that co-culture of CAFs and head and neck squamous cell carcinoma(HNSCC)cells can up-regulate the expression of MMP-1,thereby decreasing the sensitivity of HNSCC cells to cephalosporin.Yu et al found that miR21 is transferred from cancer-associated adipocytes(CAAs)or CAFs to the cancer cells,where it suppresses ovarian cancer apoptosis and induces chemoresistance by binding to its direct novel target,APAF1.As an important inflammatory factor,interleukin-6(IL-6)binds to its receptor IL-6R on the cell membrane and activates several downstream pathways,such as the JAK2/STAT3 and P13K/AKT pathways.The JAK2/STAT3 pathway is a signal transduction pathway from the membrane to the nucleus.The activation of JAK2 protein kinase can catalyze the phosphorylation of STAT3 protein into the nucleus,which can regulate the expressions of EMT-related genes and other genes.At present,it has been found that over-activation of IL-6/JAK2/STAT3 pathway can promote EMT of tumor cells.Recent studies have suggested that EMT is closely associated with chemotherapy resistance by promoting apoptosis resistance.In the present study,we aimed to investigate the effect of CAF-derived IL-6 on EMT in ovarian cancer cells via JAK2/STAT3 pathway.Our findings further elucidated the role of CAFs in the development of chemotherapy resistance in the tumor microenvironment.Methods:1.Primary ovarian cancer cells,CAFs and normal fibroblasts(NFs)were isolated from fresh cancer tissue and cultured for immunohistochemistry studies.IL-6 mRNA was detected by RT-PCR.ELISA was used to detect the expression of IL-6 in the culture supernatants of these cells.The expression of IL-6 in ovarian cancer tissue was determined using an immunofluorescence assay in both tissue sections and cell lobes.2.OVCAR3 cells were treated with the culture supernatants collected from CAFs and NFs.IL-6 monoclonal antibody was employed to neutralize IL-6.The expression of phosphorylated STAT3 was assessed.Changes in EMT,proliferation,invasion and proapoptotic protein expression were also examined.3.Flow cytometry was performed to detect the changes in apoptosis resistance of OVCAR3 cells.The JAK2/STAT3 pathway-specific inhibitor AG490 was used to block this pathway,and the β-TGF inhibitor was used to inhibit EMT.Between January 2009 and June 2014,269 patients underwent initial treatment at the Department of Gynecological and Oncology of Shandong Cancer Hospital and Institute.Patients with FIGO stage Ⅱa-Ⅳ ovarian cancer underwent cytoreductive surgery(The scope of surgery included uterus,double accessory,omentum,appendix with or without pelvic or abdominal metastases).The patients has complete clinical data and pathological paraffin blocks.Follow-up was performed using the following three methods:medical records,telephone follow-up,and outpatient review.Follow-up deadline was 2016.06.30.Pathological section reading:The positive results is that the cytoplasm and nucleus of fibroblasts were stained yellow in the stroma of cancer tissues.The two observers determined the result based on the expression of interstitial IL-6.The proportion of positive cells was assessed by 5 visual fields,without considering the staining intensity.IL-6 expression was divided into two grades.High expression group,in which at least 50%stromal fibroblasts were positive;low expression group,in which positive stromal fibroblasts were less than 50%.Statistical analysis:all statistical analyses were performed using SPSS 17 software.The relationship between the expression of interstitial IL-6 and other clinical features and treatment response was detected by chi-square test.Results:1.Purified CAFs and NFs were identified by immunofluorescence staining using two antibodies as follows:1)anti-Vimentin antibody;and 2)anti-α-SMA antibody.Expression of Vimentin was detected in both CAFs and NFs.However,α-SMA was almost not expressed in NFs,while it was over-expressed in CAFs..Next,we detected the expression of IL-6 in CAFs and NFs.IL-6 was highly expressed in CAFs expressing the-activity-marker α-SMA.Moreover,α-SMA and IL-6 were co-localized in CAFs.In contrast,the IL-6 expression was weak in NFs with sparse a-SMA expression.Similarly,IHC staining of ovarian epithelial carcinoma revealed that IL-6 was also mainly expressed in interstitial cells of ovarian cancer and co-localized with a-SMA in interstitial CAFs.Detected by RT-PCR,the expression of IL-6 mRNA in CAFs was significantly higher than that in NFs,primary cancer cells and OVCAR3.Since IL-6 is a secretory protein,we further detected the expression of IL-6 in the culture supernatants of CAFs,NFs,primary cancer cells and OVCAR3 cells using an ELISA.The expression of IL-6 in CAFs was significantly higher than that in NFs,primary cancer cells and OVCAR3.This result also suggested that the secretion of IL-6 in ovarian cancer was mainly from interstitial CAFs.2.OVCAR3 cells were incubated with the culture supernatants collected from CAFs and NFs.CCK8 proliferation assay showed that the culture supernatant collected from CAFs could significantly enhance the proliferation of OVCAR3 cells.Similarly,the invasive potential of OVCAR3 cells treated with culture supernatant from CAFs was significantly higher than that of cells treated with culture supernatant from NFs.However,with the addition of IL-6 mAb,the effect of CAFs’ supernatant on the proliferation and invasion of OVCAR3 cells was significantly suppressed.It suggested that CAF-derived IL-6 was an important factor in promoting the proliferation and invasion of OVCAR3 cells.EMT is an important step to obtain malignant biological behaviour in ovarian cancer cells.We then examined the EMT markers of OVCAR3 cells after the treatment of the culture supernatant of CAFs.The results showed that culture supernatant of CAFs significantly up-regulated interstitial markers N-cadherin and Vimentin and decreased the expression of epithelium marker E-cadherin in OVCAR3 cells,suggesting that the EMT of OVCAR3 cells was significantly enhanced.However,the effect of CAFs’ supernatant on the expression of EMT markers in OVCAR3 cells was significantly attenuated by the addition of IL-6 mAb.There was no significant change in EMT markers of OVCAR3 cells after treatment with NFs’ supernatant,suggesting that CAFs-derived IL-6 promoted the EMT of OVCAR3 cells.In the present study,we treated OVCAR3 cells with culture supernatants of CAFs and NFs.The phosphorylation level of JAK2 and STAT3 protein in OVCAR3 cells treated with culture supernatant from CAFs was significantly higher than that of cells treated with culture supernatant from NFs.After addition of IL-6 mAb,the phosphorylation level of JAK2 and STAT3 protein was decreasedAG490,the JAK2/STAT3 signalling pathway specific inhibitor,was applied to OVCAR3 cells treated with the culture supernatant from CAFs.Then,the interstitial markers N-cadherin and Vimentin was decreased and the expression of epithelium marker E-cadherin was increased in OVCAR3 cells..This result suggested that CAF-derived IL-6 could mediate the EMT in OVCAR3 cells via the JAK2/STAT3 pathway.3.0VCAR3 cells were treated with the culture supernatants collected from CAFs and NFs.After 24 h,the cells were treated with paclitaxel to induce apoptosis,which was confirmed by flow cytometry.We found that the number of apoptotic cells was decreased in OVCAR3 cells treated with CAFs’ supernatant.After the addition of IL-6 mAb,paclitaxel resistance was reduced and paclitaxel-induced apoptosis was promoted.Furthermore,we assessed the expression of apoptotic-related protein in OVCAR3 cells by Western blotting analysis.Our result indicated that the expression of pro-apoptotic protein was decreased and the expression of apoptosis-suppressing protein was enhanced in cells treated with CAFs’ supernatant compared with the cells treated with NFs’ supernatant.Suggesting that CAF-derived IL-6 was involved in paclitaxel resistance of OVCAR3 cells.SB431542,the inhibitor of EMT-inducible factor β-TGF,was applied to the culture system.Moreover,EMT in OVCAR3 cells treated with CAFs’ supernatant was inhibited,resulting in the down-regulation of interstitial markers N-cadherin and Vimentin as well as the up-regulation of epithelium marker E-cadherin.Moreover,the number of apoptotic cells treated with paclitaxel was increased after the addition of SB431542 and AG490.These findings suggested that CAFs could induce EMT of cancer cells via IL-6/JAK2/STAT3 pathway,resulting in increased apoptosis resistance,increased expression of pro-apoptotic protein and decreased expression of apoptosis-suppressing protein.Finally,it might lead to pactitaxel resistance.Chi-square test and Fisher’s exact test showed that the expression of IL-6 in ovarian interstitial cells was closely related to pathological type(P = 0.014)and CA125(P<0.001),but not correlated with age,pathological grade,FIGO staging,neoadjuvant chemotherapy and the satisfaction of cytoreductive surgery(P>0.05).Between January 2009 and June 2013,255 patients underwent cytoreductive surgery and chemotherapy with TP(docetaxel plus cisplatin or carbopatin)at the Department of Gynecological and Oncology of Shandong Cancer Hospital and Institute.They were included in the study.We divided the patients into chemotherapy-sensitive group(n = 160)and chemotherapy-resistant group(n=95),according to whether or not the tumor relapsed within 6 months after the last chemotherapy.The patients were divided into low expression group(n = 176)and high expression group(n = 79)according to the staining of IL-6 protein in interstitial cells.The results showed that the sensitivity rate of chemotherapy in patients with low expression of IL-6 was 69.3%,and the sensitivity of chemotherapy in high expression group was 48.1%.There was a significant difference between the two groups(P = 0.01).Conclusion:CAFs could highly secrete IL-6 and promote β-TGF-mediated EMT of ovarian cancer via the JAK2/STAT3 pathway,leading to promoted proliferation and invasion of ovarian cancer cells as well as inhibited apoptosis and paclitaxel resistances,which might be a new therapeutic target for ovarian cancer.