Fra-1Mediates IL-6Induced Aggressiveness And Epithelial-mesenchymal Transition In Human Colorectal Cancer

Fra-1(Fos related antigen-1), a member of the Fos family, is aberrantly expressed in several types of human cancers, but its precise functional roles and regulatory mechanisms are still not well understood. In this study, we investigated the significance of Fra-1expression in human CRC (colorectal cancer). IHC (Immunohistochemistry) within CRC specimens from229patients revealed that Fra-1was specifically expressed in the nuclei of tumor cells but not in neighbouring normal tissues. Fra-1levels were positively correlated to the tumor invasion depth, lymph node metastasis, pathological stage and metastatic liver lesions. Intense IHC staining was observed at the tumor invasive front adjacent to inflammatory cells and associated with the proinflammatory cytokine IL-6(Interleukin-6) secretion in CRC tissues. Exogenous recombinant human IL-6administration in.CRC cell lines significantly upregulated Fra-1expression at both mRNA and protein levels. By employing small molecular inhibitors and siRNAs, we identified a crucial role of STAT3(signal transducer and activator of transcription3) in the process of Fra-1induction. Promoter analysis and luciferase reporter assays demonstrated that2contiguous STAT-inducible elements located in the-720/-476region of Fra-1promoter mediated the response to IL-6. ChIP (chromatin immunoprecipitation) assays confirmed the direct binding of STAT3to the Fra-1promoter. In the early stage after IL-6treatment, phosphorylated and acetylated PTMs (post-translational modifications) of STAT3occurred simultaneously. Reintroducing wild-type or PTM site mutants Y705F and K685R into STAT3null PC3cells was carried out to analyze the regulation and function of STAT3PTMs. The mutated sites blocked the PTMs of themselves respectively without impact on each other, indicating that the phosphorylation and acetylation of STAT3were stimulated by IL-6in an independent manner. ChIP, DNA pull-down and luciferase reporter assays revealed that either of the mutations impaired the activities of STAT3in Fra-1gene promoter binding and the reporter gene induction. Chemical inhibition of JAK2and HAT attenuated STAT3phosphorylation and acetylation respectively, and both prevented IL-6induced Fra-1upregulation. On the contrary, HDAC inhibitor further enhanced IL-6mediated STAT3acetylation and Fra-1elevation. The positive correlation between Fra-1and p-STAT3or Ac-STAT3was also manifested by IHC staining in CRC serial sections. Besides, we demonstrated that Fra-1mediated IL-6function in endowing CRC cells with morphological and molecular properties of EMT (epithelial-mesenchymal transition), an essential step for tumor progression and metastasis. RNA interference-based attenuation of STAT3or Fra-1prevented IL-6induced EMT, whereas ectopic expression of Fra-1markedly reversed the inhibitory effect of STAT3-knockdown on the EMT process. Wound healing and Matrigel transwell assays showed that Fra-1and STAT3were also required in IL-6induced cell migration and invasion. Fra-1stably transfected in CRC cells caused EMT inducing transcription factors elevation and MMPs activation, which might account for Fra-1driven tumor invasion and metastasis. Collectively, This study uncovered the existence of an aberrant IL-6/STAT3/Fra-1signaling axis leading to EMT and aggressiveness in colorectal cancer and suggested novel biomarkers and therapeutic opportunities for the diagnosis and treatment of the malignant disease.