Abstract
BackgroundInterleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.MethodsSTAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors.ResultsOur results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype.ConclusionIL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.
Highlights
Interleukin-27 signaling is mediated by the JAK-Signal Transducer and Activator of Transcription (STAT) pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively
Total STAT1 (T-STAT1) and STAT3 (T-STAT3) levels were not significantly affected by IL-27 exposure. To validate this concept in other histological subtypes of non-small cell lung carcinoma (NSCLC), seven additional human lung cancer cell lines (H1703, H292, H157, H1437, H460, H1650, and H358) were exposed to IL-27 for 24 hours and phosphorylated STAT1 (P-STAT1) and P-STAT3 protein levels were analyzed by Western blot
IL-27 inhibition of in vitro cell migration is mediated by a STAT3-independent and STAT1-dependent pathway To further evaluate phenotypic changes associated with IL-27- epithelial marker expression beyond morphologic appearance, we examined in vitro cell migration, a defining feature of the mesenchymal phenotype, by creating a scratch or wound in a confluent monolayer of NSCLC cells and evaluating wound closure as a result of cell migration
Summary
Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. Activated STAT1 signaling has tumor suppressive roles by inhibiting angiogenesis, tumor growth and metastasis as well as promoting apoptosis [12,16]. The STAT3 pathway has been shown to be constitutively activated in many human cancers and has been implicated in oncogenic transformation and progression [17,18,19,20,21]. The JAK-Signal Transducer and Activator of Transcription (STAT) signaling pathway, which was initially identified as a critical process in normal cellular processes, has been implicated in tumor initiation and malignant progression. The STAT transcriptional factors, which are phosphorylated by the JAKs, dissociate from the receptor and dimerize followed by nuclear translocation [23]
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