Transforming growth factor alpha activates Ha-Ras in human pancreatic cancer cells with Ki-ras mutations

Background & Aims: The aim of this study was to identify signaling pathways that mediate cell proliferation in response to a Pas-activating growth fac tor, transforming growth factor (TGF)-alpha, in two pancreatic cancer cell lines with constitutively active Ki-Ras, MiaPaCa-2, and Pane-1. Methods: ER K1/-2- and p90(rsk) activation were determined by immune complex kinase ass ays. AP-1 and E74 activation were assessed in transient transfections using luciferase reporter plasmids. Ha-Ras activation was determined using a glu tathione S-transferase fusion protein comprising the Pas-binding domain of Raf and by immunocytochemistry, growth by DNA synthesis and colony formatio n in softagar. Results: TGF-alpha stimulated activation of ERK1/-2, which w as dependent on MEK-1, but independent of PKC activity. TGF-alpha-induced a ctivation of an AP-1 reporter plasmid also required MEK-1 and Pas activity, Using an E74 reporter plasmid, we demonstrate that TGF-alpha indeed activa tes Pas in both cell lines. In particular, TGF-alpha induced membrane trans location and activation of the Pas isoform Ha-Ras. Finally, TGF-alpha-stimu lated DNA synthesis and clonal growth in soft agar were prevented by treatm ent of cells with a MEK-1 inhibitor or a Pas farnesyl transferase inhibitor . Conclusions: The Ha-Ras-ERK cascade plays an important role in TGF-alpha- induced growth of pancreatic cancer cells with activating Ki-ras mutations. Inhibitors of this cascade could constitute novel anticancer agents for pa ncreatic tumors.