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TNF

Recently, epithelial-mesenchymal transition (EMT) has been reported to contribute to tissue fibrosis through enhanced transforming growth factor (TGF)-beta1 signaling. tumor necrosis factor (TNF)-alpha has also been implicated in tissue fibrosis. Therefore, the authors investigated whether TNF-alpha affected TGF-beta1-induced EMT. Cultured alveolar epithelial cells (A549 cells) were stimulated with TGF-beta1 (5 ng/mL), with/without TNF-alpha (10 ng/mL). TGF-beta1 induced EMT of A549 cells, with loss of E-cadherin and acquisition of vimentin. Combination of TNF-alpha with TGF-beta1 enhanced EMT, causing morphological changes, while quantitative polymerase chain reaction (PCR) showed suppression of E-cadherin mRNA and expression of vimentin mRNA. In addition, the gel contraction method revealed that cells that had undergone EMT acquired cell contractility, which is a feature of mesenchymal cells. Stimulation with TGF-beta1 induced cell contraction, as did TNF-alpha. Moreover, costimulation with TGF-beta1 and TNF-alpha enhanced the cell contraction. Although IFN-gamma suppressed spontaneous cell contraction, it did not suppress cell contraction, which was induced by TGF-beta1. In conclusion, TNF-alpha enhances not only EMT but also cell contraction induced by TGF-beta1. EMT might contribute to tissue fibrosis through induction of cell contraction.

Id1 (inhibitor of differentiation 1) is a member of the bHLH protein family. Consistent with its role in promoting proliferation and inhibiting differentiation, Id1 expression is low or negligible in normal prostate epithelial cells but is high in prostate cancer. Ectopic expression of Id1 in normal prostate epithelial cells could therefore provide a model for understanding early events involved in initiation of prostate cancer. Over-expression of Id1 immortalized but did not transform ventral prostate epithelial cells (Id1-RPE). Immortalization was associated with decreased Cdkn2a, Cdkn1a, androgen receptor and increased Tert expression. Gene expression profiling over successive doublings was used to identify transcriptomic changes involved during immortalization (Tieg, Jun, alpha actin, Klf10, Id2) and in maintaining the immortalized phenotype (Igfbp3, Igfbp5, Mmp2, Tgfb3). Network analysis indicated that Id1 promotes cancer/tumor morphology, cell cycle and epithelial to mesenchymal transition by influencing AP1, tnf, tgfbeta, PdgfBB and estradiol pathways. During immortalization, the expression of majority of differentially expressed genes reduced over progressive doublings suggesting a decline in transcriptional regulatory mechanisms. The associated molecular/gene expression profile of Id1-RPE cells provides an opportunity to understand the molecular pathways associated with prostate epithelial cell survival and proliferation.

epithelial-mesenchymal transition (EMT), characterized by loss of epithelial adhesion and gain of mesenchymal features, is an important mechanism to empower epithelial cells into the motility that occurs during embryonic development and recurs in cancer and fibrosis. Whether and how EMT occurs in wound healing and fibrosis in human skin remains unknown. In this study we found that migrating epithelial cells in wound margins and deep epithelial ridges had gained mesenchymal features such as vimentin and FSP1 expression. In hypertrophic scars, EMT-related genes were elevated along with inflammatory cytokines, indicating a causal relationship. To reconstitute EMT in vitro, normal human skin and primary keratinocytes were exposed to cytokines such as tumor necrosis factor-alpha (TNF-alpha), resulting in expression of vimentin, FSP1, and matrix metalloproteinases. Moreover, TNF-alpha-induced EMT was impaired by antagonists against bone morphogen proteins (BMP) 2/4, suggesting that BMP mediates the TNF-alpha-induced EMT in human skin. Indeed, TNF-alpha could induce BMP-2 and its receptor (BMPR1A) in human skin and primary keratinocytes, and BMP2 could induce EMT features in skin explants and primary keratinocytes. In summary, we uncovered EMT features in both acute and fibrotic cutaneous wound healing of human skin. Moreover, we propose that the mesenchymal induction in wound healing is motivated by TNF-alpha, in part, through induction of BMP.

Metastasis induced by chronic inflammation has been considered as a major challenge during cancer therapy. epithelial-mesenchymal transition (EMT) is associated with cancer invasion and metastasis promoted by pro-inflammatory cytokine TNFalpha. However, the mechanisms underlying TNFalpha-induced EMT in prostate cancer cells is not entirely clear. Here we showed that EMT induced by longstanding stimulation with TNFalpha in prostate cancer PC3 cells is mediated by up-regulation of the transcriptional repressor Snail. TNFalpha-mediated EMT was characterized by acquiring mesenchymal fusiform morphology, increasing the expression of Vimentin and decreasing the expression of E-cadherin. Exposure to TNFalpha increased the expression of transcription factor Snail via post-transcriptional regulation process and induced Snail nuclear localization in PC3 cells. Moreover, overexpressed Snail in PC3 cells induced EMT. Conversely, suppressing Snail expression abrogated TNFalpha-induced EMT, suggesting that Snail plays a crucial role in TNFalpha-induced EMT in prostate cancer cells. Finally, we showed that TNFalpha time-dependently activated NF-kappaB, AKT, ERK, p38 MAPK signaling pathways, and elevated Snail stability by activating AKT pathway that subsequently inhibited GSK-3beta activity. Taken together, these results reveal that stabilization of Snail via AKT/GSK-3beta signaling pathway is required for TNFalpha-induced EMT in prostate cancer cells. This study offers a better understanding of TNFalpha-induced metastasis and provides an effective therapeutic strategy for prostate cancer treatment.CI - Copyright (c) 2013 Elsevier B.V. All rights reserved.

Cluster of differentiation 14 (CD14) protein functions as a co-receptor with either the Toll-like receptor TLR4 or MD-2 in the detection of bacterial lipopolysaccharide (LPS) and plays a role in the innate immune system. Recently, it was shown to have effects on the regulation of epithelial-mesenchymal transition (EMT). Thus, the present study investigated the effects of CD14 knockdown on the regulation of gastric cancer cell EMT and invasive capacity following treatment with or without LPS. Knockdown of CD14 expression using CD14 shRNA in MGC-803 cells significantly enhanced E-cadherin expression, but reduced N-cadherin and vimentin expression in both LPS-treated and untreated cells. Morphologically, the phenotype of LPS-treated CD14-knockdown cells was altered to a sporadic long spindle shape. Moreover, TNF-alpha-treated cells were further elongated, connections between cells were reduced, the gap between the cells was increased and the cells were transformed into mesenchymal cells. Furthermore, the invasive capacity of CD14-knockdown cells was significantly lower compared to that of the negative control shRNA-transfected MGC-803 cells. LPS-treated CD14-knockdown cells had significantly lower levels of tumor cell invasive ability when compared to the LPS-treated parental MGC-803 cells. However, addition of TNF-alpha to LPS-treated CD14-knockdown cells significantly increased tumor cell invasion. This study demonstrated that CD14 promoted tumor cell EMT and invasion through TNF-alpha, whereas knockdown of CD14 expression inhibited gastric cancer cell invasion and EMT.

Lymph node metastasis is an important factor determining the outcome of colorectal cancer. Although epithelial-to-mesenchymal transition (EMT), TNF-alpha and microRNA (miRNA) have been found to play important roles in lymph node metastasis, the underlying molecular mechanism remains unclear. Here we reported that high expression of microRNA-19a (miR-19a) was associated with lymph node metastasis and played an important role in TNF-alpha-induced EMT in colorectal cancer (CRC) cells. We analyzed miR-19a expression in surgical tissue specimens from 11 CRC patients and 275 formalin-fixed, paraffin-embedded CRC patients. We found that miR-19a was up-regulated in CRC tissues and high expression of miR-19a was significantly associated with lymph node metastasis. We further analyzed miR-19a lymph node metastasis signature in an external validation cohort of 311 CRC cases of the TCGA. MiR-19a was found to be significantly associated with lymph node metastasis in rectal cancer. In vitro, we showed that overexpression of miR-19a in human CRC cell lines promoted cell invasion and EMT. Furthermore, miR-19a was up-regulated by TNF-alpha and miR-19a was required for TNF-alpha-induced EMT and metastasis in CRC cells. Collectively, miR-19a played an important role in mediating EMT and metastatic behavior in CRC. It may serve as a potential marker of lymph node metastasis.

UNLABELLED: The airway epithelium is a semi-impermeable barrier whose disruption by growth factor reprogramming is associated with chronic airway diseases of humans. Transforming growth factor beta (TGFbeta)-induced epithelial mesenchymal transition (EMT) plays important roles in airway remodeling characteristic of idiopathic lung fibrosis, asthma and chronic obstructive pulmonary disease (COPD). Inflammation of the airways leads to airway injury and tumor necrosis factor alpha (TNFalpha) plays an important pro-inflammatory role. Little systematic information about the effects of EMT on TNFalpha signaling is available. Using an in vitro model of TGFbeta-induced EMT in primary human small airway epithelial cells (hSAECs), we applied quantitative proteomics and phosphoprotein profiling to understand the molecular mechanism of EMT and the impact of EMT on innate inflammatory responses. We quantified 7925 proteins and 1348 phosphorylation sites by stable isotope labeling with iTRAQ technology. We found that cellular response to TNFalpha is cell state dependent and the relative TNFalpha response in mesenchymal state is highly compressed. Combined bioinformatics analyses of proteome and phosphoproteome indicate that the EMT state is associated with reprogramming of kinome, signaling cascade of upstream transcription regulators, phosphor-networks, and NF-kappaB dependent cell signaling. BIOLOGICAL SIGNIFICANCE: epithelial mesenchymal transition and inflammation have important implications for clinical and physiologic manifestations of chronic airway diseases such as severe asthma, COPD, and lung fibrosis. Little systematic information on the interplay between EMT and innate inflammation is available. This study combined quantitative proteomics and phosphorproteomics approach to obtain systems-level insight into the upstream transcription regulators involved in the TGFbeta-induced EMT in primary human small airway epithelial cells and to elucidate how EMT impacts on the TNFalpha signaling pathways. The proteomics and phosphoproteomics analysis indicates that many signaling pathways involved in TGFbeta-induced EMT and EMT has profound reprogramming effects on innate inflammation response.CI - Copyright (c) 2016 Elsevier B.V. All rights reserved.

Substantial evidence has shown that epithelial-mesenchymal transition (EMT) plays critical roles in colorectal cancer (CRC) development and prognosis. To uncover the pivotal regulators that function in the cooperative interactions between cancer cells and their microenvironment and consequently affect the EMT process, we carried out a systematic analysis and evaluated prognosis in CRC specimens. tumor buds and their surrounding stroma were captured using laser microdissection. We used gene expression profiling, bioinformatics analysis and regulatory network construction for molecular selection. The clinical significance of potential biomarkers was investigated. We identified potential EMT biomarkers, including BGN, MMP1, LGALS1, SERPINB5, and TM4SF4, all of which participated in the integrated pathway of TGFbeta/Snail with TNFalpha/NFkappaB. We also found that BGN, MMP1, LGALS1, SERPINB5 and TM4SF4 were related to CRC patient prognosis. Patients with higher expression of these individual potential biomarkers had poorer prognosis. Among the identified biomarkers, BGN and TM4SF4 are reported, for the first time, to probably be involved in the EMT process and to predict CRC prognosis. Our results strongly suggest that the integrated pathway of TGFbeta/Snail with TNFalpha/NFkappaB may be the principal axis that links cancer cells to their microenvironment during the EMT process and results in poor prognosis in CRC patients.

TNF-alpha is a central proinflammatory cytokine contributing to malignant tumor progression in tumor microenvironment. In this study, we found the upregulation of miR-105 in colorectal cancer was associated with aggressive phenotype, and the enhanced expression of miR-105 was required for TNF-alpha-induced epithelial-mesenchymal transition (EMT). The expression of miR-105 was remarkably stimulated by TNF-alpha in a time-dependent manner using real-time qPCR analysis. Inhibition of miR-105 remarkably weakened the aggressive effects of TNF-alpha through preventing the activation of NF-kappaB signaling and the initiation of EMT. Furthermore, miR-105 was demonstrated directly targeted on the 3'-UTRs of RAP2C, a Rap2 subfamily of small GTP-binding protein. Consistently, suppression of RAP2C stimulated the role of miR-105, which dramatically promoted the invasion and metastasis of CRC cells. Thalidomide, a TNF-alpha and NF-kappaB inhibitor, significantly weakened the metastasis and homing capacity of miR-105-overexpressed CRC cells in nude mice. Our investigation initiatively illustrated the modulatory role of miR-105 in TNF-alpha-induced EMT and further CRC metastasis. We also offer a better understanding of TNFalpha-induced metastasis and suggest an effective therapeutic strategy against CRC metastasis.