Tumor Necrosis Factor-α Stimulates the Epithelial-to-Mesenchymal Transition of Human Colonic Organoids

Abstract

An epithelial-mesenchymal transition (EMT) characterizes the progression of many carcinomas and it is linked to the acquisition of an invasive phenotype. Given that the tumor microenvironment is an active participant in tumor progression, an important issue is whether a reactive stroma can modulate this process. Using a novel EMT model of colon carcinoma spheroids, we demonstrate that their transforming-growth factor-␤1 (TGF-␤)-induced EMT is accelerated dramatically by the presence of activated macrophages, and we identify tumor necrosis factor-␣ (TNF-␣) as the critical factor produced by macrophages that accelerates the EMT. A synergy of TNF-␣ and TGF-␤ signaling promotes a rapid morphological conversion of the highly organized colonic epithelium to dispersed cells with a mesenchymal phenotype, and this process is dependent on enhanced p38 MAPK activity. Moreover, exposure to TNF-␣ stimulates a rapid burst of ERK activation that results in the autocrine production of this cytokine by the tumor cells themselves. These results establish a novel role for the stroma in influencing EMT in colon carcinoma, and they identify a selective advantage to the stromal presence of infiltrating leukocytes in regulating malignant tumor progression. INTRODUCTIONMany epithelial tumors undergo an epithelial-mesenchymal transition (EMT) that facilitates their invasion. The EMT is also an essential component of embryonic development, tissue remodeling, and wound repair (reviewed in Arias, 2001;Thiery, 2002). During this transition, the epithelial phenotype, characterized by strong cell-cell junctions and polarity, is replaced by a mesenchymal phenotype, with reduced cell-cell interactions, a fibroblastic morphology and increased motility. Given the importance of the EMT in carcinoma progression, there is considerable interest in understanding the mechanisms that contribute to this complex process. Although the exact mechanisms that underlie the EMT have not been elucidated, TGF-␤ has been implicated as a key inducer of the process (Oft et al., 1998;Portella et al., 1998;Lehmann et al., 2000;Bhowmick et al., 2001a;Ellenrieder et al., 2001;Fujimoto et al., 2001). TGF-␤ stimulates proliferation of many cell types, particularly those of mesenchymal origin, and it is also a potent inhibitor of epithelial cell proliferation. Contrary to an early ascribed role as a tumor suppressor (Markowitz and Roberts, 1996), TGF-␤ has been found to be abundantly expressed in many epithelial tumors and it acts in both an autocrine manner on the tumor cells themselves and as a paracrine modulator of the stroma (reviewed in Gold, 1999;de Caestecker et al., 2000;Yue and Mulder, 2001). In colon carcinoma, TGF-␤ also acts differently depending on the differentiation stage of the tumor, in general by switching from an early inhibitor of proliferation to a stimulator of growth and invasion during tumor progression (Hsu et al., 1994).TGF-␤ has been implicated as a major factor in the EMT, but this is likely to be a "multifactorial" process. Moreover, the strom...

Frizzled-7 dictates three-dimensional organization of colorectal cancer cell carcinoids

“…Consistent with these findings in carcinoma tissues, decreased E-cadherin and cell proliferation, and increased SNAI2, laminin a3 and g2 chains, and MMP7 defined spontaneous EMT of the carcinoids. Thus, different variations of the LIM1863 cell line can model the complete EMT associated with single-cell dissociation from the tumour mass (Bates and Mercurio, 2003;Bates et al, 2005) as well as the initial stages of tubular de-construction (incomplete EMT) and construction (carcinoid assembly) as we show in the current study. Moreover, the LIM1863-Mph cells recapitulate the dynamic nature of these transitions.…”

“…In addition, it has been known for some time that TGFb induces EMT in LIM1863 cells (Hayward et al, 1995). More recently, Bates and Mercurio (2003) have shown that tumour necrosis factor (TNF) a accelerates TGFb-mediated EMT, and that combined TGFb/TNFa treatment of LIM1863 cells is an excellent model of integrin modulation during CRC metastasis . This cytokine-induced EMT of LIM1863 cells described by Bates et al (2005) induces complete EMT, as defined by loss of E-cadherin expression.…”

“…This cytokine-induced EMT of LIM1863 cells described by Bates et al (2005) induces complete EMT, as defined by loss of E-cadherin expression. Although some single cells are detected, most of the cells maintain cell-cell contact via intact adherens junctions, despite this loss of E-cadherin (Bates and Mercurio, 2003). Further analysis revealed that the cells remain in contact because E-cadherin loss is compensated by the expression of another adherens junction protein, N-cadherin (Bates, 2005).…”

“…Assembly of carcinoids was associated with cell proliferation and was inhibited in the presence of the DNA synthesis inhibitor mitomycin-C. As anticipated, monolayer cells were resistant to mitomycin-C and remained viable without proliferation (Figure 4a and b). Carcinoid assembly and cell proliferation were decreased after treatment with transforming growth factor (TGF) b, a cytokine known to induce EMT of LIM1863 cells (Hayward et al, 1995;Bates and Mercurio, 2003), but was not inhibited by treatment with LiCl ( Figure 4a and b). LiCl mimics canonical Wnt signalling (Kao and Elinson, 1998), which we confirmed here in the context of LIM1863-Mph using the b-catenin/TCF transcription reporter TOPflash (Korinek et al, 1997) (Figure 4c).…”

Enhancement of active MMP release and invasive activity of lymph node metastatic tongue cancer cells by elevated signaling via the TNF-α-TNFR1-NF-κB pathway and a possible involvement of angiopoietin-like 4 in lung metastasis

“…The increase of TNF-α release brings about further NF-κB activation via TNFR1 signaling pathway in the cells and resultant elevation of MMPs secretion. As an autocrine TNF-α from tumor cells stimulated by this cytokine was reported (38), TNF-α release from the highly metastatic cells is possibly amplified in an autocrine mannaer. These findings suggest an autoactivation mechanism for TNF-α-triggered NF-κB activation in these cells.…”