Abstract 4006: Smad6 upregulation provides an alternative mechanism for BMP inactivation in SMAD4 wild type pancreatic cancers

Introduction: Genetic inactivation of SMAD4, a central mediator of TGF-β superfamily signaling, is significantly correlated with metastatic behavior in pancreatic cancer patients. However, because some patients with pancreatic cancer have genetically intact TGF-β and BMP pathway components, we investigated the role of alternative mechanisms of inactivation in promoting pancreatic cancer metastasis. Methods: Eighteen cell lines for which the genetic status of all members of the TGF-β pathway were known were used in this study. TGF-β signaling levels were analyzed in each cell line using a luciferase reporter system under the control of a Smad Binding Element (SBE). Immunoblotting was performed on protein lysates from pancreatic cancer cell lines of known genotype. Cell lines that express high levels of Smad6 were transiently transfected with shRNA constructs targeting Smad6; cell lines that express low levels of Smad6 were transiently transfected with a construct to constitutively express Smad6. The effects of Smad6 modulation were assessed by proliferation assay, migration assay, and invasion assay. Immunohistochemistry was performed on primary and metastatic pancreatic cancer tissues and staining intensity correlated to clinical data. Results: Functional TGF-β and BMP signaling were eliminated in cell lines with known SMAD4 inactivation, as well as in several cell lines in which these pathways remain intact. Immunoblotting for known TGF-β superfamily antagonists in these cell lines revealed differential expression of Smad6, an inhibitory Smad. Modulation of Smad6 levels in vitro suggests that Smad6 overexpression contributes to increased levels of proliferation, migration, and invasion in SMAD4-intact cell lines. Overexpression of Smad6 did not restore TGF-β signaling, but did increase BMP response. Immunohistochemistry for Smad6 in patient samples revealed a nuclear localization pattern, suggesting that the pro-oncogenic roles of Smad6 are mediated by its ability to act as a transcription factor. High Smad6 levels correlated with worse prognosis and metastatic behavior among SMAD4-intact pancreatic cancers. Conclusions: Smad6, an inhibitory Smad, is differentially expressed in pancreatic cancer, both in cell lines and patient samples. High levels of Smad6 in patients at autopsy associate with widespread metastasis, irrespective of SMAD4 status. Preliminary studies in vitro support a metastasis-promoting function of Smad6: Smad6 overexpression is associat