[CANCER RESEARCH 60, 2007–2017, April 1, 2000] Insulin-like Growth Factor-I Protects Colon Cancer Cells from Death Factor- induced Apoptosis by Potentiating Tumor Necrosis Factor -induced Mitogen-activated Protein Kinase and Nuclear Factor B Signaling Pathways 1 Maryse M. Remacle-Bonnet, Franc ¸oise L. Garrouste, Sara Heller, Fre ´de ´ric Andre ´, 2 Jacques L. Marvaldi, and Gilbert J. Pommier 3 Unite ´ 6032, “Interactions entre Syste `mes Prote ´iques et Diffe ´renciation dans la Cellule Tumorale,” Centre National de la Recherche Scientifique, Faculte ´ de Me ´decine, 13385 Marseille Cedex 5, France ABSTRACT Resistance of cancer cells against apoptosis induced by death factors contributes to the limited efficiency of immune- and drug-induced de- struction of tumors. We report here that insulin and insulin-like growth factor-I (IGF-I) fully protect HT29-D4 colon carcinoma cells from IFN- /tumor necrosis factor- (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 B (NF-B) activity by preventing degradation of the inhibitor of NF-B (IB-) 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-B 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-B 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-B, 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 simul- taneously. INTRODUCTION Instructive apoptosis is a kind of apoptosis in which death factors play a central role. It is essential in maintaining tissue homeostasis and eliminating deleterious cells (1–3). When this system under- or over- functions, it contributes to the pathogenesis of a number of human diseases (4). In the intestinal mucosa, recent evidence shows that an excess of cell death is associated with inflammatory bowel diseases, whereas increased cell survival contributes to the outgrowth of colon cancer cells (5, 6). The best characterized death factors Fas ligand and TNF 4 bind to ubiquitously expressed members of the TNFR superfamily. Fas ligand binds to Fas/CD95/APO-1 receptor, and TNF binds to two receptors, p55 (TNFR1) and p75 (TNFR2), that do not share any homology within their cytoplasmic domain. Both Fas and TNFR1 can activate apoptotic signaling pathways through a similar mechanism, recruiting directly or indirectly Fas-associated death domain protein and pro- caspase 8 (1–3). However, both TNFR1 and TNFR2 also associate with molecules that do not interact with Fas, especially the TRAF family of adaptor proteins and receptor-interacting protein. These molecules activate additional signaling pathways including NF-B and the MAPK cascades (especially, JNK and p38), these latter being involved in the stimulation of AP-1 activity (7–9). TNF recruitment of both NF-B and AP-1 transcription factors is pivotal to regulate many genes, especially those involved in expression of inflammatory cyto- kines and cell survival (10 –14). Thus, TNF transmits one signal eliciting cell death and another that protects against cell death, this latter being closely linked to the proinflammatory signaling. In con- trast, Fas-mediated signal appears to be simpler and does not lead to direct and efficient NF-B and AP-1 activation. This may explain why Fas activation generally results in a more efficient apoptotic response (15). The phenomenon of resistance by tumor cells to death factor- induced apoptosis is of major concern in cancer therapy. It contributes in a great part to the limited effectiveness of naturally occurring as well as peptide/cytokine-driven antitumor immune response generally observed in cancer patients. Moreover, this resistance may also an- tagonize the efficiency of chemotherapeutic drugs because many of them induce apoptosis of tumor cells by activating death factor/ receptor systems, particularly the Fas/Fas ligand system (16, 17). Several growth factors have been identified as regulators of cell survival (18), and among them IGF-I, IGF-II, and insulin have been reported to have a potent ability to protect a broad range of cells from a variety of proapoptotic challenge (19). The biological functions of the IGFs and insulin are pleiotropic and mediated by specific mem- brane receptors designated IGF-IR and IR, respectively. These recep- tors are heterotetrameric proteins with a highly homologous intracel- lular tyrosine kinase domain. An earliest step in signal transduction by both IGF-IR and IR is the extensive tyrosine phosphorylation of Received 8/9/99; accepted 2/3/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work has been partially supported by a grant from Association pour la Recher- che sur le Cancer. 2 Present address: Laboratory of Experimental Cancerology, University Hospital, B 9000 Gent, Belgium. 3 To whom requests for reprints should be addressed, at Unite ´ 6032, Centre National de la Recherche Scientifique, Faculte ´ de Me ´decine, 27 Boulevard J. Moulin, 13385 Marseille Cedex 5, France. Phone: 33-4-91-32-44-14; Fax: 33-4-91-25-89-70; E-mail: pommier@medecine.univ-mrs.fr. 4 The abbreviations used are: TNF, tumor necrosis factor ; TNFR, TNF receptor; TRAF, TNFR-associated factor; MAPK, mitogen-activated protein kinase; ERK, extra- cellular signal-regulated kinase; JNK, c-Jun NH 2 -terminal kinase; IGF, insulin-like growth factor; IGF-IR, IGF-I receptor; IR, insulin receptor; IRS-1, insulin receptor substrate-1; PI3'K, phosphatidylinositol 3'-kinase; PKC, protein kinase C; PKB/AKT, protein kinase B/product of the oncogene v-akt; pp70 S6K , pp70 ribosomal protein S6 kinase; AP-1, activator protein-1; NF-B, nuclear factor B; IB, inhibitor of NF-B; FAK, focal adhesion kinase; mAb, monoclonal antibody; PI, propidium iodide; CHX, cycloheximide; BIM, bisindolylmaleimide; CPHC, calphostin C; WMN, wortmannin; IL, interleukin; IFN, IFN-. 2007 Research. on January 18, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from