title: Restoration of Smad4 in BxPC3 pancreatic cancer cells attenuates proliferation without altering angiogenesis.
abstract: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which the transforming growth factor beta (TGF-beta) signal transducer, Smad4, is commonly mutated or deleted. BxPC3 human pancreatic cancer cells exhibit a homozygous deletion of the Smad4 gene, yet are growth inhibited by TGF-beta1. In the present study, we sought to determine whether reintroduction of Smad4 into BxPC3 cells alters their behavior in vitro and in vivo. Sham transfected and Smad4 expressing BxPC3 cells exhibited similar responses to TGF-beta1 with respect to p21 upregulation, hypophosphorylation of the RB protein, Smad2 phosphorylation, and Smad2/3 nuclear translocation. TGF-beta1 did not alter p27 expression, and silencing of p21 with an appropriate siRNA markedly attenuated TGF-beta1-mediated growth inhibition. Nonetheless, the presence of Smad4 was associated in vitro with a more prolonged doubling time, enhanced sensitivity to the growth inhibitory actions of exogenous TGF-beta1, and a more flattened cellular morphology. In vivo, Smad4 expression resulted in delayed tumor growth and decreased cellular proliferation, without effects on either apoptosis or angiogenesis. These findings indicate that, in spite of the absence of Smad4, growth inhibition in BxPC3 cells by TGF-beta1 is dependent on p21 upregulation and maintenance of RB in a hypophosphorylated, active state. Moreover, the presence of a functional Smad4 attenuates the capacity of BxPC3 cells to proliferate in vivo. However, this effect is transient, indicating that Smad4 growth inhibitory actions are circumvented in the later stages of pancreatic tumorigenicity.
authors.affiliation: Department of Medicine and Pharmacology, Dartmouth-Hitchcock Medical Center and Dartmouth Medical School, Hanover, USA.
authors.forename: Michiya
authors.initials: M
authors.lastname: Yasutome
authors.forename: Jason
authors.initials: J
authors.lastname: Gunn
authors.forename: Murray
authors.initials: M
authors.lastname: Korc
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D021441:Carcinoma, Pancreatic Ductal / Q000098:blood supply / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic; D010190:Pancreatic Neoplasms / Q000098:blood supply / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D016160:Retinoblastoma Protein / Q000378:metabolism; D051899:Smad2 Protein / Q000032:analysis / Q000378:metabolism; D051900:Smad3 Protein / Q000032:analysis / Q000378:metabolism; D051901:Smad4 Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D050760:Cyclin-Dependent Kinase Inhibitor p27