Targeting Endogenous Transforming Growth Factor β Receptor Signaling in SMAD4-Deficient Human Pancreatic Carcinoma Cells Inhibits Their Invasive Phenotype

Abstract

Transforming growth factor-␤ (TGF-␤) suppresses tumor formation by blocking cell cycle progression and maintaining tissue homeostasis. In pancreatic carcinomas, this tumor suppressive activity is often lost by inactivation of the TGF-␤-signaling mediator, Smad4. We found that human pancreatic carcinoma cell lines that have undergone deletion of MADH4 constitutively expressed high endogenous levels of phosphorylated receptor-associated Smad proteins (pR-Smad2 and pR-Smad3), whereas Smad4-positive lines did not. These elevated pR-Smad levels could not be attributed to a decreased dephosphorylation rate nor to increased expression of TGF-␤ type I (T␤R-I) or type II (T␤R-II) receptors. Although minimal amounts of free bioactive TGF-␤1 and TGF-␤2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-␤3 specific) TGF-␤-neutralizing antibody and with anti-␣ V ␤ 6 integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-␤-inducible reporter gene in neighboring cells, respectively. Thus, activation of TGF-␤ at the cell surface was responsible for the increased autocrine endogenous and paracrine signaling. Blocking T␤R-I activity using a selective kinase inhibitor (SD-093) strongly decreased the in vitro motility and invasiveness of the pancreatic carcinoma cells without affecting their growth characteristics, morphology, or the subcellular distribution of E-cadherin and F-actin. Moreover, exogenous TGF-␤ strongly stimulated in vitro invasiveness of BxPC-3 cells, an effect that could also be blocked by SD-093. Thus, the motile and invasive properties of Smad4-deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF-␤ signaling. Therefore, targeting the T␤R-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

“…We also observed that TGF␤ induced a comparable increase in Smad3 phosphorylation both in Smad4-and Smad4(⌬290 -300)-transfected CFPAC1 cells (Fig. 2C), demonstrating that these cells did respond to TGF␤ as previously described (28). To test whether Smad4⌬300 and Smad4(⌬290 -300) retain their ability to interact with Smad3 in a TGF␤-dependent manner, FLAG-tagged full-length Smad4 and Smad4 mutants were transfected into Mv1Lu cells, and the ability of the FLAG-tagged Smad4 proteins to bind to Smad3 in response to 100 pM TGF␤ was assessed.…”

EW‐7203, a novel small molecule inhibitor of transforming growth factor‐β (TGF‐β) type I receptor/activin receptor‐like kinase‐5, blocks TGF‐β1‐mediated epithelial‐to‐mesenchymal transition in mammary epithelial cells

“…Small molecule inhibitors devised to directly block the catalytic activity of TbRI, including SB-431542 (23) and SB-505124 (GlaxoSmithKline, Brentford, Middlesex, UK) (24) , SD-093 (25) and SD-208 (Scios, New Brunswick, NJ, USA) (26) , and LY-580276 (Lilly Research Laboratories, Indianapolis, IN, USA) (27) , which all act as competitive inhibitors by attacking the ATP-binding site of TbRI kinase. (28) The present study shows that TGF-b1 induces EMT in mammary epithelial cells and that this process can be effectively blocked by EW-7203 (Fig.…”

Antiangiogenic Treatment with Three Thrombospondin-1 Type 1 Repeats versus Gemcitabine in an Orthotopic Human Pancreatic Cancer Model

“…Although AsPC-1 cells and MiaPaCa-2 cells do not directly respond to TGF-h because of mutated SMAD4 (MiaPaCa-2) or TGF-h receptors (AsPC-1; refs. 39,40), TGF-h increased desmoplasia and epithelialmesenchymal transdifferentiation in pancreatic tumors (41,42). It has been shown that pancreatic cancer cells acquired che moresistance when cocultured with fibroblasts or grown on collagen-coated plates in vitro (43,44).…”