Insulin-like growth factor-I protects colon cancer cells from death factor-induced apoptosis by potentiating tumor necrosis factor alpha-induced mitogen-activated protein kinase and nuclear factor kappaB signaling pathways.

Resistance of cancer cells against apoptosis induced by death factors contributes to the limited efficiency of immune- and drug-induced destruction of tumors. We report here that insulin and insulin-like growth factor-I (IGF-I) fully protect HT29-D4 colon carcinoma cells from IFN-gamma/tumor necrosis factor-alpha (TNF) induced apoptosis. Survival signaling initiated by IGF-I was not dependent on the canonical survival pathway involving phosphatidylinositol 3'-kinase. In addition, neither pp70(S6K) nor protein kinase C conveyed IGF-I antiapoptotic function. Inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) with the MAPK/ERK kinase inhibitor PD098059 and MAPK/p38 with the specific inhibitor SB203580 partially reversed, in a nonadditive manner, the IGF-I survival effect. Inhibition of nuclear factor kappaB (NF-kappaB) activity by preventing degradation of the inhibitor of NF-kappaB (IkappaB-alpha) with BAY 11-7082 also blocked in part the IGF-I antiapoptotic effect. However, the complete reversal of the IGF-I effect was obtained only when NF-kappaB and either MAPK/ERK or MAPK/p38 were inhibited together. Because these pathways are also those used by TNF to signal inflammation and survival, these data point to a cross talk between IGF-I- and TNF-induced signaling. We further report that TNF-induced IL-8 production was indeed strongly enhanced upon IGF-I addition, and this effect was totally abrogated by both MAPK and NF-kappaB inhibitors. The IGF-I antiapoptotic function was stimulus-dependent because Fas- and IFN/Fas-induced apoptosis was not efficiently inhibited by IGF-I. This was correlated with the weak ability of Fas ligation to enhance IL-8 production in the presence or absence of IGF-I. These findings indicate that the antiapoptotic function of IGF-I in HT29-D4 cells is based on the enhancement of the survival pathways initiated by TNF, but not Fas, and mediated by MAPK/p38, MAPK/ERK, and NF-kappaB, which act in concert to suppress the proapoptotic signals. In agreement with this model, we show that it was possible to render HT29-D4 cells resistant to Fas-induced apoptosis provided that IGF-I and TNF receptors were activated simultaneously.

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells.

To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.

Expression of type I, but not type II insulin-like growth factor receptor on both undifferentiated and differentiated HT29 human colon carcinoma cell line.

The HT29 human colonic carcinoma cell line secretes insulin-like growth factor (IGF)-II. We have examined these cells for expression of IGF receptors. Competitive binding assays as affinity cross-linking experiments using 125I-IGF-II fail to reveal type II IGF receptors at the cell surface. In contrast, cross-linking studies with either 125I-IGF-I or 125I-IGF-II reveal an M(r) 135,000 protein that follows a peptide binding specificity characteristic of the alpha-subunit of the type I IGF receptor. However, 125I-IGF-II binding to this receptor is not inhibited at 4 C by alpha IR-3, a monoclonal antibody to the type I IGF receptor. Analysis of the competitive binding curves with each one of these radioligands suggests that HT29 cells express both a classical type I IGF receptor (about 6,000/cell; KdIGF-I = 0.48 nmol) and a variant one whose 125I-IGF-II binding is not blocked by alpha IR-3 (about 15,000/cell; KdIGF-II = 4.0 nmol). Endocytosis studies of specific cell-bound 125I-IGF-I or 125I-IGF-II suggest that ligand interaction with the classical, but not the variant, binding site is only able to induce receptor internalization. An identical IGF receptors pattern is observed with HT29-D4 clonal cells induced to differentiate by culture in a glucose-free medium.

Potential autocrine role of insulin-like growth factor II during suramin-induced differentiation of HT29-D4 human colonic adenocarcinoma cell line.

Suramin, a drug that binds to several types of growth factors, has been previously shown to induce the enterocyte-like differentiation of HT29-D4 human colonic adenocarcinoma cells, suggesting that growth factors are involved in such a process. Undifferentiated HT29-D4 cells release insulin-like growth factor II (IGF-II) into the culture medium that is totally complexed to heterogeneous IGF binding proteins (IGFBP) expressing high affinities for this growth factor (Kda = 0.02 nM and Kdb = 1.4 nM). These complexes do not allow IGF-II to bind to HT29-D4 cell surface type I IGF receptors, as evidenced by using 125I-IGF-II-IGFBP complexes. However, the addition of 40-100 micrograms/ml suramin, i.e., concentrations identical to the ones that are able to induce HT29-D4 cell differentiation, induces the release of IGF-II from IGF-II-IGFBP complexes, thereby allowing IGF-II to bind to the cell surface receptors. At such concentrations, suramin is indeed unable to alter IGF-II binding to HT29-D4 cells, a capacity that is observed only for concentrations higher than 200 micrograms/ml. Thus, suramin might have the unusual capacity to allow the establishment of an IGF-II autocrine loop involved in HT29-D4 cell differentiation. Consistent with this hypothesis is the fact that exogenously applied IGF-I (2.5 micrograms/ml) or agonist monoclonal antibody alpha IR-3 (2.5 micrograms/ml), which can bypass IGFBP present in the culture medium, induces part of HT29-D4 cell differentiation that is characterized by an important carcinoembryonic antigen release and the induction of numerous intercellular cysts with microvilli.