IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE 2 synthesis in Caco-2 human colon carcinoma cells Anna Di Popolo 1 , Annamaria Memoli 1 , Anna Apicella 1 , Concetta Tuccillo 1,2 , Antonella di Palma 1 , Paolo Ricchi 1 , Angela M Acquaviva* ,1 and Raaele Zarrilli 1 1 Dipartimento di Biologia e Patologia Cellulare e Molecolare `L Califano', Centro di Endocrinologia ed Oncologia Sperimentale `G. Salvatore' del Consiglio Nazionale delle Ricerche, Universita Á `Federico II', Napoli, 80131 Italy; 2 Dipartimento di Internistica Clinica e Sperimentale ± Divisione di Gastroenterologia, II Ateneo di Napoli, Napoli, 80131 Italy Nonsteroidal anti-in¯ammatory drugs reduce the risk of colon cancer and this eect is mediated in part through inhibition of type 2 prostaglandin endoperoxide synthase/ cyclo-oxygenase (COX-2). In the present study, we demonstrate that COX-2 expression and PGE 2 synthesis are up-regulated by an IGF-II/IGF-I receptor autocrine pathway in Caco-2 colon carcinoma cells. COX-2 mRNA and PGE 2 levels are higher in proliferating cells compared with post-con¯uent dierentiated cells and in cells that constitutively overexpress IGF-II. Up-regula- tion of COX-2 expression by IGF-II is mediated through activation of IGF-I receptor because: (i) treatment of Caco-2 cells with a blocking antibody to the IGF-I receptor inhibits COX-2 mRNA expression; (ii) transfec- tion of Caco-2 cells with a dominant negative IGF-I receptor reduces COX-2 expression and activity. Also, the blockade of the PI3-kinase, that mediates the proliferative eect of IGF-I receptor in Caco-2 cells, inhibits IGF-II-dependent COX-2 up-regulation and PGE 2 synthesis. Moreover, COX-2 expression and activity inversely correlate with the increase of apoptosis in parental, IGF-II and dominant-negative IGF-I receptor transfected cells. This study suggests that induction of proliferation and tumor progression of colon cancer cells by the IGF-II/IGF-I receptor pathway may depend on the activation of COX-2-related events. Oncogene (2000) 19, 5517 ± 5524. Keywords: IGF-II; IGF-Ir; COX-2; PGE 2 ; colon cancer Introduction The turnover of the gastrointestinal epithelium is very rapid and is completed over a 3- to 5-day period. The mammalian intestinal mucosa undergoes a process of continual renewal characterized by active proliferation of stem cells in the crypts, progression of these cells up the crypt-villus axis with cessation of proliferation and subsequent dierentiation. The dierentiated entero- cytes then undergo a process of programmed cell death (i.e., apoptosis) and extrusion into the gut lumen (Simon and Gordon, 1995). Aberrant regulation of cell proliferation, dierentiation and apoptosis is thought to play an important role in the establishment of gut neoplasia (Kinzler and Vogelstein, 1996). Prostaglandins (PG) are arachidonic acid derivates that mediate important functions involved in the regulation of intestinal epithelial cell homeostasis (Eberhart and DuBois, 1995). PGs synthesis depends on the activity of a constitutively expressed and an inducible PG endoperoxide synthase/cyclo-oxygenase (COX-1 and COX-2, respectively) (Williams and DuBois, 1996). Mounting evidence indicates that COX-2 may play an important role as an early event in colorectal carcinogenesis (Williams et al., 1997; Prescott and Fitzpatrick, 2000). In fact, COX-2 is overexpressed in 80 ± 90% of colorectal adenocarcino- mas and in 40 ± 50% of premalignant adenomas (Williams et al., 1997), and inactivation of the COX- 2 gene in mice is associated with decreased intestinal tumorigenesis (Oshima et al., 1996). Reduced prosta- glandin biosynthesis through the inhibition of COX-2 activity is thought to be the molecular basis for the chemopreventive eects of nonsteroidal anti-in¯amma- tory drugs (NSAIDs) on colorectal tumorigenesis in both human and rodents (Williams et al., 1997; Prescott and Fitzpatrick, 2000). COX-2 may facilitate colon cancer progression by stimulating cell prolifera- tion and survival (Tsujii and DuBois, 1995; Sheng et al., 1998), tumor cell invasiveness (Tsujii et al., 1997) and the production of angiogenic agents in colon cancer cells (Tsujii et al., 1998). Polypeptide growth factors are also known to regulate gastro-intestinal homeostasis by aecting cell proliferation, dierentiation and apoptosis (Podolsky, 1993). In particular, insulin-like growth factors (IGF-I and IGF-II), are important modulators of growth in normal and transformed intestinal epithelial cells (Humbel, 1990; Singh and Rubin, 1993). IGF-I and IGF-II are small peptides structurally related to insulin and both exert their mitogenic activity through type I IGF receptor (IGF-Ir) (Humbel, 1990). A large percentage of tumors express IGF-II (Singh and Rubin, 1993; Zhang et al., 1997), and almost all primary human colon cancers and colon cancer cell lines are positive for IGF-I receptor (Guo et al, 1992; Singh and Rubin, 1993). Also, experimental evidences from our and other laboratories indicate that an autocrine IGF-II/IGF-Ir signal transduction pathway stimulates proliferation and inhibits dierentiation of cultured colon cancer cells (Pommier et al., 1992; Garrouste et al., 1997; Zarrilli et al., 1994, 1996, 1999a). Oncogene (2000) 19, 5517 ± 5524 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: AM Acquaviva, Dipartimento di Biologia e Patologia Cellulare e Molecolare `L Califano', UniversitaÁ `Federico II', Via S. Pansini 5, Napoli, 80131 Italy Received 2 June 2000; revised 18 September 2000; accepted 22 September 2000