EGFR-dependent pancreatic carcinoma cell metastasis through Rap1 activation.

Tyrosine kinase receptors have an essential role in various aspects of tumor progression. In particular, epidermal growth factor receptor (EGFR) and its ligands have been implicated in the growth and dissemination of a wide array of human carcinomas. Here, we describe an EGFR-mediated signaling pathway that regulates human pancreatic carcinoma cell invasion and metastasis, yet does not influence the growth of primary tumors. In fact, ligation/activation of EGFR induces Src-dependent phosphorylation of two critical tyrosine residues of p130CAS, leading to the assembly of a Crk-associated substrate (CAS)/Nck1 complex that promotes Ras-associated protein-1 (Rap1) signaling. Importantly, GTP loading of Rap1 is specifically required for pancreatic carcinoma cell migration on vitronectin but not on collagen. Furthermore, Rap1 activation is required for EGFR-mediated metastasis in vivo without impacting primary tumor growth. These findings identify a molecular pathway that promotes the invasive/metastatic properties of human pancreatic carcinomas driven by EGFR.

Oncogenic Ras/Src cooperativity in pancreatic neoplasia.

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5-8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDA's indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation.

Epithelial-mesenchymal transformation in the atrioventricular (AV) cushion of the tubular heart is a critical step in the formation of the valves and membranous septa. Transforming growth factor beta (TGFbeta) ligands are a primary signal of this transformation. To investigate the expression and function of specific Type I TGFbeta receptors during AV cushion transformation, we cloned and characterized the chicken homologues of two mammalian activin receptor-like kinases (ALK), ALK2 and ALK5, and generated specific, polyclonal antibodies against the extracellular binding domains of each. Both the chicken ALK2 (ChALK2) and the chicken ALK5 (ChALK5) cDNAs encode proteins that bind TGFbeta1 in the presence of the Type II TGFbeta receptor. However, as expected, only ChALK5 stimulated the TGFbeta-responsive PAI-1 promoter. These data establish that ChALK2 and ChALK5 are the chicken homologues of the mammalian receptors ALK2 and ALK5. Both ChALK2 and ChALK5 are expressed by AV endocardial cells. AV cushion explants harvested from stage 13-18 embryos were incubated with antisera to ChALK2 or ChALK5. Anti-ChALK2 antisera inhibited mesenchyme formation by 34-50% while neutralizing anti-ChALK5 antisera were without effect. These data demonstrate that ChALK2 can mediate transformation in the AV cushion.

Conservation of an insulin response unit between mouse and human glucose-6-phosphatase catalytic subunit gene promoters: transcription factor FKHR binds the insulin response sequence.

Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, we have investigated whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. We have previously shown that the accessory factor binding region A is hepatocyte nuclear factor-1, and we show here that the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B.