Transcription Factor AP-1 is required for the proliferation of pancreatic cancer cells and its constitutive activation is dependent on PI 3-kinase-mediated signals

Abstract

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Our recent studies have suggested that the transcription factor activator protein-1 (AP-1) is constitutively activated in pancreatic cancer cells. Gel mobility shift assays revealed strong AP-1-binding activities in various serum-starved pancreatic cancer cell lines suggesting that AP-1 activation is constitutive and independent of serum factors. Through a combination of immunoblotting and super-shift analyses, we determined that AP-1 specifically contained phospho-c-Jun (serines 63 and 73), Jun D, Jun B, Fra 1 and Fra 2. Little or no c-Fos or ATF-2 was detectable in the AP-1/DNA complexes. Consistent with these results, two different AP-1-dependent reporter genes were highly induced in pancreatic cancer cells as determined by transient transfection assays. Through stable transfection of a transactivation domain mutant of c-Jun (TAM67), we demonstrate that AP-1 is critical for the proliferation of pancreatic cancer cells both in anchorage-dependent, and -independent conditions. An investigation of the signaling pathways that might be responsible for AP-1 activation in pancreatic cancer cells indicated that multiple signals could be involved. Indeed, while the Jun N-terminal kinase (JNK) was constitutively activated in these cells and responsible for the phosphorylation of c-Jun on serines 63 and 73, phosphatidylinositide 3-kinase (PI 3-kinase) and its mediator Akt were also essential. PI 3-kinase function was required for the transactivation potential of AP-1 but not for its DNA-binding activity. The mechanism by which the transcription factor is regulated by PI 3-kinase in pancreatic cancer cells did not appear to involve changes in the expression or phosphorylation of various AP-1 proteins. Induction of the AP-1 reporter gene could be inhibited, however, by cotransfection with a CREB construct mutated on an Akt-regulated site (Ser133Ala), suggesting that PI 3-kinase regulated AP-1 activity through transcription factor CREB. These and other observations suggest that AP-1 is constitutively activated through specific PI 3-kinase-controlled mechanisms, and that it is essential for the proliferation of pancreatic cancer cells.