Cancer-associated Fibroblasts induce epithelial-mesenchymal transition via the Transglutaminase 2-dependent IL-6/IL6R/STAT3 axis in Hepatocellular Carcinoma.
ABSTRACT: Cancer-associated fibroblasts (CAFs) play crucial roles in enhancing cell survival, proliferation, invasion, and metastasis. We previously showed that hepatocellular carcinoma-derived CAFs (H-CAFs) promoted proliferation of hepatocellular carcinoma (HCC) cells. This study aimed to further explore the role of CAFs in HCC epithelial-mesenchymal transition (EMT) and the underlying mechanism. High CAF density was significantly associated with liver cirrhosis, inferior clinicopathologic characteristics, elevated EMT-associated markers, and poorer survival in human HCC. Within HCC cells, EMT was induced after co-culture with H-CAFs. Secretomic analysis showed that IL-6 and HGF were the key EMT-stimulating cytokines secreted by H-CAFs. Proteomic analysis revealed that TG2 was significantly upregulated in HCC cells with EMT phenotypes. Overexpression of TG2 promoted EMT of HCC cells, and knockdown of TG2 remarkably attenuated the H-CAF-induced EMT. Furthermore, during EMT, TG2 expression was enhanced after HCC cells were stimulated by IL-6, but not HGF. Inhibition of the IL-6/STAT3 signaling decreased TG2 expression. The principal TG2 transcription control element and a potential STAT3 binding site were identified using promoter analysis. Hence, H-CAFs facilitates HCC cells EMT mediated by IL-6, which in turn activates IL-6/IL6R/STAT3 axis to promote TG2 expression.
Project description:BACKGROUND:Local resident normal fibroblasts (NFs) are the major source of cancer-associated fibroblasts (CAFs), which are distinguishable from NFs by their tumor-supportive properties. However, the mechanism and the effects underlying the transition of NFs to CAFs in oral squamous cell carcinoma (OSCC) remain unclear. METHODS:Five pairs of matching primary NFs and CAFs derived from OSCC patients were sent for RNA sequencing. Epiregulin (EREG) expression was analyzed by IHC in fibroblasts from OSCC patients. The role of EREG in the NF-CAF transition and the consequential effects on OSCC progression were examined by upregulation/downregulation of EREG in NFs/CAFs both in vitro and in vivo. RESULTS:Here, we identified epiregulin (EREG) as the most remarkably upregulated gene in CAFs. High EREG expression in CAFs correlated with higher T stage, deeper invasion and inferior worst pattern of invasion (WPOI) in OSCC patients and predicted shorter overall survival. Overexpression of EREG in NFs activated the CAF phenotype. Mechanistically, the JAK2/STAT3 pathway was enhanced by EREG in parallel with increased IL-6 expression, which could be inhibited by the JAK2 inhibitor AG490. Recombinant IL-6 upregulated the JAK2/STAT3/EREG pathway in a feedback loop. Moreover, EREG-induced CAF activation promoted the epithelial-mesenchymal transition (EMT) necessary for migration and invasion, which was dependent on JAK2/STAT3 signaling and IL-6. In vivo, EREG expression in stroma fibroblasts promoted tumor growth with high stromal α-SMA, phospho-JAK2/STAT3, and IL-6 expression and upregulated EMT in HSC3 cells. CONCLUSIONS:EREG is essential for the NF-CAF transformation needed to induce EMT of tumor cells in a JAK2-STAT3- and IL-6-dependent manner in OSCC.
Project description:Although carcinoma-associated fibroblasts (CAFs) in tumor microenvironments have a critical role in immune cell modulation, their effects on the generation of regulatory dendritic cells (DCs) are still unclear. In this study, we initially show that CAFs derived from hepatocellular carcinoma (HCC) tumors facilitate the generation of regulatory DCs, which are characterized by low expression of costimulatory molecules, high suppressive cytokines production and enhanced regulation of immune responses, including T-cell proliferation impairment and promotion of regulatory T-cell (Treg) expansion via indoleamine 2,3-dioxygenase (IDO) upregulation. Our findings also indicate that STAT3 activation in DCs, as mediated by CAF-derived interleukin (IL)-6, is essential to IDO production. Moreover, IDO inhibitor, STAT3 and IL-6 blocking antibodies can reverse this hepatic CAF-DC regulatory function. Therefore, our results provide new insights into the mechanisms by which CAFs induce tumor immune escape as well as a novel cancer immunotherapeutic approach (for example, targeting CAFs, IDO or IL-6).
Project description:Cancer-associated fibroblasts (CAFs) are important components of tumor stroma and play a key role in tumor progression. CAFs involve in crosstalk with tumor cells through various kinds of cytokines. In the present study, we screened hepatocyte growth factor (HGF) as a cytokine predominantly originating from CAFs. CAFs-derived HGF was found to promote MET-unamplified gastric cancer (GC) proliferation, migration, and invasion through the activation of HGF/c-Met/STAT3/twist1 pathway. It also activated interleukin (IL)-6/IL-6R/JAK2/STAT3/twist1 pathway by up-regulating IL-6R expression. As IL-6 was also found to upregulate c-Met expression, we identified the cooperation of HGF and IL-6 in enhancing the characteristics of CAFs. In vivo experiments revealed that CAFs-derived HGF promoted tumorigenesis and metastasis of MET-unamplified GC. Gene set enrichment analysis (GSEA) was performed to confirm our findings. Our study found that the increased expression of HGF in CAFs induced by MET-unamplified GC contributed to the malignant phenotype of both MET-unamplified GC and CAFs in tumor microenvironment.
Project description:BACKGROUND:Cancer stem cells (CSCs) require stromal signals for maintaining pluripotency and self-renewal capacities to confer tumor metastasis. Resolvin D1 (RvD1), an endogenous anti-inflammatory lipid mediator, has recently been identified to display anti-cancer effects by acting on stroma cells. Our previous study reveals that hepatic stellate cells (HSCs)-derived cartilage oligomeric matrix protein (COMP) contributes to hepatocellular carcinoma (HCC) progression. However, whether RvD1 inhibits paracrine of cancer-associated fibroblasts (CAFs)-derived COMP to prevent epithelial-mesenchymal transition (EMT) and cancer stemness in HCC remains to be elucidated. METHODS:CAFs were isolated from HCC tissues. Direct and indirect co-culture models were established to analyze the interactions between HCC cells and CAFs in the presence of RvD1 in vitro. The transwell and tumor sphere formation assays were used to determine invasion and stemness of HCC cells. The subcutaneous tumor formation and orthotopic liver tumor models were established by co-implantation of CAFs and HCC cells to evaluate the role of RvD1 in vivo. To characterize the mechanism of RvD1 inhibited paracrine of COMP in CAFs, various signaling molecules were analyzed by ELISA, western blotting, reactive oxygen species (ROS) detection, immunofluorescence staining, dual luciferase reporter assay and chromatin immunoprecipitation assay. RESULTS:Our data revealed that RvD1 treatment can impede the CAFs-induced cancer stem-like properties and the EMT of HCC cells under co-culture conditions. In vivo studies indicated that RvD1 intervention repressed the promoting effects of CAFs on tumor growth and metastasis of HCC. Furthermore, RvD1 inhibited CAF-induced EMT and stemness features of HCC cells by suppressing the secretion of COMP. Mechanistically, formyl peptide receptor 2 (FPR2) receptor mediated the suppressive effects of RvD1 on COMP and forkhead box M1 (FOXM1) expression in CAFs. Notably, RvD1 impaired CAF-derived COMP in a paracrine manner by targeting FPR2/ROS/FOXM1 signaling to ultimately abrogate FOXM1 recruitment to the COMP promoter. CONCLUSION:Our results indicated that RvD1 impaired paracrine of CAFs-derived COMP by targeting FPR2/ROS/FOXM1 signaling to repress EMT and cancer stemness in HCC. Thus, RvD1 may be a potential agent to promote treatment outcomes in HCC.
Project description:In this study, we investigated the role of cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC) progression. We showed that CAFs secrete high levels of IL-6, which promoted stem cell-like properties in HCC cells by activating Notch signaling through STAT3 Tyr705 phosphorylation. These effects were abolished by Notch1 shRNA knockdown in HCC cells or treatment with an IL-6 neutralizing antibody or the p-STAT3 (Tyr705) inhibitor cryptotanshinone. Xenografted liver tumors were larger in nude mice injected with HCC cells and CAFs than in those receiving HCC cells alone. Moreover, immunohistochemical analysis of liver tissue specimens from 88 HCC patients revealed that high nuclear Notch intracellular domain (nNICD) levels in HCC cells correlated with poor prognosis in patients. These findings suggest that IL-6 secreted by CAFs promotes stem cell-like properties in HCC cells by enhancing STAT3/Notch signaling.
Project description:BACKGROUND:Cancer-associated fibroblasts (CAFs), activated by tumour cells, are the predominant type of stromal cells in cancer tissue and play an important role in interacting with neoplastic cells to promote cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. However, the mechanism by which CAFs induce EMT program in bladder cancer cells remains unclear. METHODS:To investigate the role of CAFs in bladder cancer progression, healthy primary bladder fibroblasts (HFs) were induced into CAFs (iCAFs) by bladder cancer-derived exosomes. Effect of conditioned medium from iCAFs (CM iCAF) on EMT markers expression of non-invasive RT4 bladder cancer cell line was determined by qPCR and Western blot. IL6 expression in iCAFs was evaluated by ELISA and Western blot. RT4 cell proliferation, migration and invasion were assessed in CM iCAF +/- anti-IL6 neutralizing antibody using cyQUANT assay, scratch test and transwell chamber respectively. We investigated IL6 expression relevance for bladder cancer progression by querying gene expression datasets of human bladder cancer specimens from TCGA and GEO genomic data platforms. RESULTS:Cancer exosome-treated HFs showed CAFs characteristics with high expression levels of ?SMA and FAP. We showed that the CM iCAF induces the upregulation of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ß-catenin expression in non-invasive RT4 cells. Moreover, EMT transcription factors SNAIL1, TWIST1 and ZEB1 were upregulated in CM iCAF-cultured RT4 cells compared to control. We also showed that the IL-6 cytokine was highly expressed by CAFs, and its receptor IL-6R was found on RT4 bladder cancer cells. The culture of RT4 bladder cancer cells with CM iCAF resulted in markedly promoted cell growth, migration and invasion. Importantly, inhibition of CAFs-secreted IL-6 by neutralizing antibody significantly reversed the IL-6-induced EMT phenotype, suggesting that this cytokine is necessary for CAF-induced EMT in the progression of human bladder cancer. Finally, we observed that IL6 expression is up-regulated in aggressive bladder cancer and correlate with CAF marker ACTA2. CONCLUSIONS:We conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6.
Project description:Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the molecular mechanisms underlying the tumor-promoting properties of CAFs in gastric cancer remain unclear. Here, we show that CAFs isolated from gastric cancer produce significant amounts of interleukin-6 (IL-6). CAFs enhances the migration and EMT of gastric cancer cells through the secretion of IL-6 that activates Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in gastric cancer cells, while deprivation of IL-6 using a neutralizing antibody or inhibition of JAK/STAT3 pathway with specific inhibitor AG490 markedly attenuates these phenotypes in gastric cancer cells induced by CAFs. Moreover, silencing IL-6 expression in CAFs or inhibiting JAK2/STAT3 pathway in gastric cancer cells impairs tumor peritoneal metastasis induced by CAFs in vivo. Taken together, these results suggest that CAFs in the tumor microenvironment promote the progression of gastric cancer through IL-6/JAK2/STAT3 signaling, and IL-6 targeted therapy could be a complementary approach against gastric cancer by exerting their action on stromal fibroblasts.
Project description:Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGF?1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGF?1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGF?1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGF?1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGF?1-ZEB2NAT-ZEB2 axis.
Project description:Cancer-associated fibroblasts (CAFs) promote tumor growth and progression, and increase drug resistance through several mechanisms. We have investigated the effect of CAFs on the p53 response to doxorubicin in prostate cancer cells. We show that CAFs produce interleukin-6 (IL-6), and that IL-6 attenuates p53 induction and upregulation of the pro-apoptotic p53 target Bax upon treatment with doxorubicin. This is associated with increased levels of MDM2 mRNA, Mdm2 protein bound to p53, and ubiquitinated p53. IL-6 also inhibited doxorubicin-induced cell death. Inhibition of JAK or STAT3 alleviated this effect, indicating that IL-6 attenuates p53 via the JAK/STAT signaling pathway. These results suggest that CAF-derived IL-6 plays an important role in protecting cancer cells from chemotherapy and that inhibition of IL-6 could have significant therapeutic value.