Human pancreatic cancers overexpress the epidermal growth factor (EGF) receptor (EGFR) and all 5 ligands that bind to this receptor, including amphiregulin. It is not known, however, whether amphiregulin contributes in an autocrine manner to enhance pancreatic cancer cell growth. Therefore, we used an amphiregulin antisense oligonucleotide (AR-AS) to suppress amphiregulin expression in T3M4 human pancreatic cancer cells. These cells express high levels of EGFR and amphiregulin. AR-AS abolished amphiregulin immunoreactivity in T3M4 cells, decreased amphiregulin release into the medium and inhibited cell growth in a dose-dependent manner. Exogenous amphiregulin reversed AR-AS-mediated growth inhibition. A random oligonucleotide (AR-R) did not alter either cell growth or cellular amphiregulin immunoreactivity. AR-AS also increased cellular EGFR protein levels and enhanced the growth-inhibitory actions of TP40, a chimeric protein consisting of transforming growth factor-a coupled to Pseudomonas exotoxin that internalizes into cells via EGFR. These findings indicate that there is an important EGFR/ amphiregulin autocrine loop in T3M4 cells and raise the possibility that modalities aimed at abrogating amphiregulin action may prove useful in pancreatic cancer, especially when used in conjunction with EGFR-targeted therapy. Int. J. Cancer 72:512-517, 1997.r 1997 Wiley-Liss, Inc.Excessive expression of growth factors and their receptors may lead to aberrant activation of growth signaling pathways and neoplastic transformation (Aaronson, 1991). The epidermal growth factor (EGF) receptor EGFR possesses intrinsic tyrosine kinase activity, and its overexpression is associated with malignant transformation (Schlessinger and Ullrich, 1992). In addition to binding EGF, EGFR binds transforming growth factor-a (TGF-a), amphiregulin, heparin-binding EGF-like growth factor (HB-EGF) and betacellulin (Shoyab et al., 1989;Thorne and Plowman, 1994). Amphiregulin is a positively charged Lys-Arg-rich polypeptide that possesses a hydrophilic region at its amino-terminus (Shoyab et al., 1989). It binds to DNA, is present in the nuclei of normal human pancreatic ductal cells and may exert direct regulatory effects on the expression of certain genes (Ebert et al., 1994;Thompson et al., 1996).Pancreatic cancer is a devastating disease with an extremely poor prognosis. These cancers also often overexpress amphiregulin, EGFR, EGF, TGF-a, HB-EGF and betacellulin (Ebert et al., 1994;Korc et al., 1992;Kobrin et al., 1994; Yokoyama et al., 1995a and b). Furthermore, the presence of amphiregulin in the cancer cells is associated with an increased frequency of local lymph node involvement (Yokoyama et al., 1995a), and the concomitant expression of EGFR and either EGF or TGF-a correlates with a decrease in patient survival (Yamanaka et al., 1993). These observations suggest that excessive EGFR activation contributes to pancreatic cancer aggressiveness and its propensity to metastasize.Previous studies have demonstrated that antisense oligonucleotides...
