Z Gastroenterol 2006; 44 - P338
DOI: 10.1055/s-2006-950942

The effect of SMAD4 expression on angiogenesis of pancreatic adenocarcinoma

A Schrödel 1, R Jesnowski 1, I Molema 2, M Löhr 3
  • 1dkfz, Molecular Gastroenterology, Heidelberg, Germany
  • 2University of Groningen, Groningen, Netherlands
  • 3II. Med. Klinik, Mannheim, Germany

Aims: SMAD proteins have been implicated as downstream effectors of TGFβ/BMP signaling. TGFß activates receptors on the cell surface, stimulating the phosphorylation of receptor-regulated SMAD proteins, which then form complexes with SMAD4 that accumulate in the nucleus and activate TGFß responsive gene transcription (e.g. Collagen-I, WAF-1). SMAD4 is inactivated in 50% of the pancreatic adenocarcinomas.

Methods: We compared BxPC3 cells with and without SMAD4 reconstitution in vitro regarding proliferation, invasion, migration and gene expression. Moreover we analyzed the vessel density in tumors derived from these cells

Results: The reconstituted cell-line showed a stable SMAD4 expression which was independent on TGFß1 stimulation. Invasion and migration of the tumor cells was markedly inhibited after SMAD4 reconstitution in vitro. As expected, the SMAD4 reconstituted cells in vitro grow slower than the parental cells. Using gene chip analysis several differentially expressed genes were identified. We detected 34 genes which were (>2x) upregulated on basal level in the SMAD4 reconstituted cells as TSP-1 and 27 genes which were (>2x) downregulated after SMAD4 reconstitution like Interleukin 8 and VEGF.

Additional results arose from analyses of selected genes by RT-PCR and Westernblot. The phosphorylation of SMAD2 was elevated in SMAD4(-) cells on basal levels. Stimulation with TGFß1 resulted in a 2.6-fold induction of SMAD2 phosphorylation in these cells. In contrast, in SMAD4(+) cells TGFß stimulation resulted in an 5.7-fold induction of SMAD2 phosphorylation, thus reaching comparable levels in both cell lines after TGFß stimulation.

Furthermore we analyzed the vessel density in tumor xenografts. There was no difference between SMAD4(+) and SMAD4(-) tumors regarding vessel density, co-localisation of Pericytes and EC or vessel distribution detectable.

Conclusion: We got several hints for anti-angiogenic and anti-tumorigenic effects due to SMAD4 reconstitution in BxPC3 cells in vitro, but we haven't got any direct influence on angiogenesis in vivo. The loss of SMAD4 leads to changes in the overall signaling network of the cell because of manifold regulatory mechanisms of TGFß signaling.