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DOI: 10.1055/s-2005-920091
Erk-independent Regulation of Growth in Pancreatic Carcinoma via Rap1
Aims: For the small GTPase Rap1 it has been shown that it inhibits growth in pancreatic cancer cells. The underlying mechanism has not yet been fully clarified. Interestingly this effect of Rap1 maynot be due to interference to Ras effector function. There is evidence for additional functions of endogenous Rap1 in cellular differentiation, adhesion and cell migration.The aim of the present study was to explore the influence of endogenous Rap1 activation on pancreatic cell growth.
Methods: Activation of Rap1 or Ras proteins in MiaPaCa or Panc1 pancreatic carcinoma cell lines was detected by affinity precipitation assays (ARIA). MAP-Kinase activation was determined using antibodies against phERK1/2. Expression of Epac1 (GEF) mRNA was determined by real time PCR. Inhibition of PKA was achieved using PKA siRNA and the PKA inhibitor (H–89). Endogenous Rap1 was activated through the specific Epac1 activator 8-pCPT–2'-O-Me-cAMP. Cell proliferation was measured through MTT-assay. Expression of integrins in Jurkat cells was quantified through FACS analysis.
Results: We have shown that cAMP activates the endogenous GTPase Rap1 in MiaPaCa and Panc1 cells. Epac1, a specific GTP-exchange factor for Rap1 that is activated by cAMP, is expressed in human pancreatic carcinoma and cell lines. A specific activator of Epac1, 8-pCPT–2'-O-Me-cAMP, activates endogenous Rap1 in pancreatic carcinoma cells. Administration of 8-pCPT–2'-O-Me-cAMP leads to growth retardation similar to the effect of nonspecific cAMP analogues. This effect was not mediated through the inhibition of ERK-activity. Transfection studies in Jurkat cells have shown that Rap1 leads to the upregulation of αLß2 (CD11a/CD18) integrin. α4ß1 (CD49d) integrin expression was also induced.
Conclusions: These findings describe a growth regulatory pathway in pancreatic carcinoma cells mediated through the GTPase Rap1. The cAMP/Epac1/Rap1-signaling pathway modulates cell growth through an ERK-independent pathway. This might be mediated through increased integrin-mediated cell adhesion. In other studies it has been shown that Rap1 also regulates the formation of E-cadherin-based cell-cell contacts. In cancer cells down-regulation of both integrins and cadherins can correlate with tumour dedifferentiation.
Keywords: Epac1, PKA, Rap1, cAMP