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, 36 (4), 459-68

Aberrantly Expressed Fra-1 by IL-6/STAT3 Transactivation Promotes Colorectal Cancer Aggressiveness Through Epithelial-Mesenchymal Transition

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Aberrantly Expressed Fra-1 by IL-6/STAT3 Transactivation Promotes Colorectal Cancer Aggressiveness Through Epithelial-Mesenchymal Transition

Hong Liu et al. Carcinogenesis.

Abstract

The pro-inflammatory cytokine interleukin-6 (IL-6) in tumor microenvironment has been suggested to promote development and progression of colorectal cancer (CRC). However, the underlying molecular mechanisms remain elusive. In this study, we demonstrate that fos-related antigen-1 (Fra-1) plays a critical role in IL-6 induced CRC aggressiveness and epithelial-mesenchymal transition (EMT). In CRC cell lines, the expression of Fra-1 gene was found significantly upregulated during IL-6-driven EMT process. The Fra-1 induction occurred at transcriptional level in a manner dependent on signal transducer and activator of transcription 3 (STAT3), during which both phosphorylated and acetylated post-translational modifications were required for STAT3 activation to directly bind to the Fra-1 promoter. Importantly, RNA interference-based attenuation of either STAT3 or Fra-1 prevented IL-6-induced EMT, cell migration and invasion, whereas ectopic expression of Fra-1 markedly reversed the STAT3-knockdown effect and enhanced CRC cell aggressiveness by regulating the expression of EMT-promoting factors (ZEB1, Snail, Slug, MMP-2 and MMP-9). Furthermore, Fra-1 levels were positively correlated with the local invasion depth as well as lymph node and liver metastasis in a total of 229 CRC patients. Intense immunohistochemical staining of Fra-1 was observed at the tumor marginal area adjacent to inflammatory cells and in parallel with IL-6 secretion and STAT3 activation in CRC tissues. Together, this study proposes the existence of an aberrant IL-6/STAT3/Fra-1 signaling axis leading to CRC aggressiveness through EMT induction, which suggests novel therapeutic opportunities for the malignant disease.

Figures

Figure 1.
Figure 1.
IL-6 induces EMT changes and Fra-1 expresison in CRC cells. (A) Morphology of HT-29 cells treated with or without 50ng/ml IL-6 for 72h under phase contrast microscopy. Scale bar, 20 μm. (B) Immunofluorescent staining for E-cadherin and vimentin in HT-29 cells treated with IL-6 for 72h (nuclei stained with DAPI). Scale bar, 20 μm. (C) Western blots of E-cadherin and vimentin for HT-29 cells incubated with gradient concentrations of IL-6 for 72h. (D) Western blots of EMT markers with specific antibodies in SW480 cells exposed to IL-6 for 72h. (E) Relative mRNA levels of EMT regulators in HT-29 cells treated with IL-6 for 24h (normalized to GAPDH). *P < 0.05. (F) Western blots of Fra-1 and GAPDH from whole-cell lysates extracted from HT-29 and SW480 cells treated with 50ng/ml IL-6 for the indicated times.
Figure 2.
Figure 2.
Fra-1 is transcriptionally regulated by STAT3 in response to IL-6 stimulation. (A) Western blots with the indicated antibodies from HT-29 cells pretreated for 1h with LY294002, U0126, or Stattic (specific inhibitors of PI3K, MEK and STAT3, respectively) and exposed to IL-6 for 12h. (B) Western blots of Fra-1 and STAT3 from HT-29 cells transfected with control or STAT3 siRNAs and incubated with IL-6 for 12h (GAPDH was used as a loading control). (C) Schematic representation of Fra-1 promoter with seven potential SIEs and the primer pair used in ChIP-PCR assays. The reporter construct Fra-1-Luc and its truncated and mutated derivatives are also shown. (D) Transcription activity in response to IL-6 treatmemt for 6h measured by luciferase assay in 293T cells with a series of deletion mutants of Fra-1-luc (internal control, pRL-TK). *P < 0.05. (E) Relative luciferase activity 6h after IL-6 incubation in 293T cells transfected with the wild-type or SIE mutated Fra-1 promoter reporter construct. *P < 0.05. (F) Chromatin prepared from HT-29 cells stimulated with IL-6 for 1h was immunoprecipitated with the indicated antibodies; PCR was performed on immunoprecipitated DNAs or soluble chromatin using specific primer pair for the Fra-1 promoter.
Figure 3.
Figure 3.
Acetylation and phosphorylation are both required for STAT3 activation to transactivate the Fra-1 gene. (A) Western blots of STAT3 and its PTM status with site-specific antibodies in serum-starved HT-29 cells treated with 50ng/ml IL-6 for the indicated times. (B) Western blots for the STAT3-null PC3 cells transfected with empty vector (EV), wild-type (WT) or mutated STAT3 and stimulated with IL-6 for 1h. (C) Western blots with specific antibodies from whole-cell extracts of HT-29 cells pretreated with AG490, anacardic acid or trichostatin A (inhibitors of JAK2, histone acetyltransferases and histone deacetylases, respectively) and further stimulated with IL-6 for 12h. (D) Relative luciferase activity in PC3 cells co-transfected with wild-type or mutated STAT3, Fra-1 promoter reporter (−720/+173) and an internal control reporter pRL-TK and administered with IL-6 24h later. *P < 0.05. (E) Upon IL-6 stimulation for 1h, ChIP assays were performed in PC3 cells reintroduced with wild-type STAT3 or derived mutants using the indicated antibodies. The Fra-1 promoter region containing STAT3 binding sites was amplified by PCR. (F) PC3 cells transfected with STAT3-WT or its mutant were treated with IL-6 for 1h, the binding ability of STAT3 to the biotin-labeled Fra-1 promoter probe (−760/−524) was analyzed by DNA pull-down assay. Unlabeled Fra-1 promoter probe (cold-probe) was used for competitive inhibition. Ku80 served as a control.
Figure 4.
Figure 4.
Fra-1 plays a critical role in IL-6 induced EMT and cell mobility. (A) Morphology of HT-29 cells with siRNAs against Fra-1 or scrambled control and stimulated with 50ng/mL of IL-6 for 72h. Scale bar, 20 μm. (B) Western blots of 72h IL-6 treated HT-29 cells receiving the indicated siRNAs using specific antibodies. (C) Western blots of HT-29 cells transfected with Fra-1 expression vector or empty vector and indicated siRNAs and incubated with IL-6 for 72h afterwards. (D) Left panels: images from scratch assays with HT-29 cells transfected with indicated siRNAs. Scale bar, 200 μm. Right panel: percentage wound closure 24h after stimulation with IL-6. *P < 0.05. (E) Left panels: representative images of HT-29 tumor cells penetrating the Matrigel in invasion assays. Scale bar, 100 μm. Right panel: numbers of invasive cells transfected with the indicated siRNAs and exposed to IL-6 for 48h. *P < 0.05.
Figure 5.
Figure 5.
Fra-1 facilitates cell migration and invasion via EMT-promoting factors. (A) Left panels: images from scratch assays with Fra-1 or EV stably transfected HT-29 cells. Scale bar, 200 μm. Right panel: percentage wound closure 24h after scratch. *P < 0.05. (B) Left panels: representative images of stably transfected HT-29 cells invading the Matrigel. Scale bar, 100 μm. Right panel: numbers of invasive cells stably transfected with Fra-1 or EV. *P < 0.05. (C) The mRNA levels of EMT-TFs and MMPs in Fra-1 stably transfected HT-29 cells. *P < 0.05. (D) Left panels: western blots of total proteins from stably transfected HT-29 cells (equal protein loading confirmed by GAPDH expression). Right panels: gelatin zymography for MMPs activity in conditioned medium of stably transfected HT-29 cells.
Figure 6.
Figure 6.
Expression of Fra-1 is upregulated coincident with IL-6 secretion in advanced CRC. (A) Representative images of Fra-1 immunostaining in CRC tissues and adjacent non-cancerous tissues. Scale bar, 50 μm. (B) Expression of Fra-1 protein in primary tumors versus pathological parameters. *P < 0.05. (C) Fra-1 levels in the primary colorectal and liver metastatic tumors. *P < 0.05 (D) Representative images of Fra-1 immunostaining in tumor cells at the marginal area neighboring inflammatory infiltrations. Scale bar, 50 μm. (E) Left panels: representative images of concurrent expression of Fra-1 and IL-6 in consecutive sections of CRC tissues. Scale bar, 50 μm. Right panel: linear regression between immunostaining intensity of Fra-1 and IL-6.

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References

    1. Center M.M., et al. (2009). International trends in colorectal cancer incidence rates. Cancer Epidemiol. Biomarkers Prev., 18, 1688–1694. - PubMed
    1. Andre N., et al. (2005). Chemoradiotherapy for colorectal cancer. Gut, 54, 1194–1202. - PMC - PubMed
    1. Ullman T.A., et al. (2011). Intestinal inflammation and cancer. Gastroenterology, 140, 1807–1816. - PubMed
    1. Grivennikov S., et al. (2009). IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell, 15, 103–113. - PMC - PubMed
    1. Bollrath J., et al. (2009). gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell, 15, 91–102. - PubMed

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