Transforming growth factor alpha activates Ha-Ras in human pancreatic cancer cells with Ki-ras mutations.
OBJECTIVE
The aim of this study was to identify signaling pathways that mediate cell proliferation in response to a Ras-activating growth factor, transforming growth factor (TGF)-alpha, in two pancreatic cancer cell lines with constitutively active Ki-Ras, MiaPaCa-2, and Panc-1.


METHODS
ERK1/-2- and p90(rsk) activation were determined by immune complex kinase assays. AP-1 and E74 activation were assessed in transient transfections using luciferase reporter plasmids. Ha-Ras activation was determined using a glutathione S-transferase fusion protein comprising the Ras-binding domain of Raf and by immunocytochemistry, growth by DNA synthesis and colony formation in softagar.


RESULTS
TGF-alpha stimulated activation of ERK1/-2, which was dependent on MEK-1, but independent of PKC activity. TGF-alpha-induced activation of an AP-1 reporter plasmid also required MEK-1 and Ras activity. Using an E74 reporter plasmid, we demonstrate that TGF-alpha indeed activates Ras in both cell lines. In particular, TGF-alpha induced membrane translocation and activation of the Ras isoform Ha-Ras. Finally, TGF-alpha-stimulated DNA synthesis and clonal growth in soft agar were prevented by treatment of cells with a MEK-1 inhibitor or a Ras 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 pancreatic tumors.
Seufferlein
T
T
Department of Internal Medicine I, University of Ulm, Ulm, Germany. thomas.seufferlein@medizin.uni-ulm.de
Van Lint
J
J
Liptay
S
S
Adler
G
G
Schmid
R M
RM
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D004789:Enzyme Activation / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D011905:Genes, ras / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D009154:Mutation / Q000502:physiology*; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D010957:Plasmids / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000502:physiology; D016211:Transforming Growth Factor alpha / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D018808:Transcription Factor AP-1; D016211:Transforming Growth Factor alpha; D019893:Ribosomal Protein S6 Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases