Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-β (original) (raw)
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Biochemical and Biophysical Research Communications, 2004
Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-b (TGF-b) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-b-induced growth inhibition, there seems to be a Smad4-independent TGF-b signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-b-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-b stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were upregulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-b. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-b signal pathway that did not involve Smad4.
Induction of Inhibitory Smad6 and Smad7 mRNA by TGF-β Family Members
Biochemical and Biophysical Research Communications, 1998
propagates the signal through phosphorylation of Smad6 and Smad7 function as intracellular antagopathway-restricted SMADs, that oligomerize with nists in transforming growth factor-b (TGF-b) signalcommon-mediator Smad4 and translocate to the nuing. Here we report the isolation of human Smad6, cleus where they affect transcription of target genes which is closely related to Smad7. Smad6 and Smad7 (5). Inhibitory SMADs have been identified that in-mRNAs were differentially expressed in lung cancer terfere with the activation of pathway-restricted cell lines and were rapidly and directly induced by SMADs by preventing their interaction with and TGF-b1, activin and bone morphogenetic protein-7. phosphorylation by type I receptors (6 -8) or by inhib-
Cancer research, 2004
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 (TR-I) or type II (TR-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 TR-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 Smad4deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF- signaling. Therefore, targeting the TR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.
2004
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 (TR-I) or type II (TR-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 TR-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 Smad4deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF- signaling. Therefore, targeting the TR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.
Cancer Research, 2004
Transforming growth factor-β (TGF-β) 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-β-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-β type I (TβR-I) or type II (TβR-II) receptors. Although minimal amounts of free bioactive TGF-β1 and TGF-β2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-β3 specific) TGF-β-neutralizing antibody and with anti-αVβ6 integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-β-inducible reporter gen...
Oncotarget, 2016
Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/ AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.
Oncogene, 2003
We have investigated the mechanism whereby tumor cells become resistant to the antiproliferative effects of transforming growth factor (TGF)-b, while maintaining other responses that can lead to increased malignancy and invasiveness. TGF-b signaling results in nuclear accumulation of active Smad complexes which regulate transcription of target genes. Here we show that in two pancreatic carcinoma cell lines, PT45 and Panc-1, that are resistant to TGF-b-induced growth arrest, the TGF-b-Smad signaling pathway is attenuated compared with epithelial cells that are sensitive to the antiproliferative effects of TGF-b (HaCaT and Colo-357). In PT45 and Panc-1 cells, active Smad complexes remain nuclear for only 1-2 h compared with more than 6 h in HaCaT and Colo-357 cells. The attenuated pathway in PT45 and Panc-1 cells correlates with low levels of TGF-b type I receptor and results in an altered expression profile of TGF-b-inducible genes required for cell cycle arrest. Most significantly, expression of the CDK inhibitor, p21 Cip1/WAF1 , which is required for TGF-b-induced growth arrest in these cells, is not maintained. Moreover, we show that artificially attenuating the TGF-b-Smad signaling pathway in HaCaT cells is sufficient to prevent TGF-b-induced growth arrest. Our results demonstrate that the duration of TGF-b-Smad signaling is a critical determinant of the specificity of the TGF-b response.
The TGF-β signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer
Oncogene, 1999
Transforming growth factor-beta (TGF-b) signaling is dependent on the heterodimerization of the type II TGFb receptor (TbRII) with the type I TGF-b receptor (TbRI). Activated TbRI then mediates TGF-b signals by inducing the phosphorylation of Smad2 and/or Smad3, which separately hetetorodimerize with Smad4 and translocate to the nucleus. Phosphorylation of Smad2/ Smad3 by activated TbRI is inhibited by two newly discovered members of the Smad family, Smad6 and Smad7. We now report that Smad7 mRNA levels are increased in human pancreatic cancer by comparison with the normal pancreas, and that by in situ hybridization, Smad7 is over-expressed in the cancer cells within the tumor mass. Stable transfection of COLO-357 human pancreatic cancer cells with a fulllength Smad7 construct leads to complete loss of the growth inhibitory response to TGF-b1, without altering TGF-b1-mediated induction of PAI-I. Furthermore, Smad7 transfected COLO-357 cells display enhanced anchorage-independent growth and accelerated growth in nude mice. These ®ndings point to a previously unrecognized mechanism for selective suppression of TGF-b-mediated growth inhibition in cancer cells that allows for continued activation of the PAI-I promoter by TGF-b1, which may act to enhance the tumorigenicity of certain cancer cells.
Differential regulation of TGF-β signaling through Smad2, Smad3 and Smad4
Oncogene, 2003
Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-b (TGF-b) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-b-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-b-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiationcompetent cells by using improved antisense molecules. We found that TGF-b mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-b-induced cell cycle arrest and to induce or suppress endogenous cell cycle regulators. Inhibition of Smad4 expression exhibited a partial effect, whereas inhibition of Smad2 expression had no effect. By gene expression profiling, we identified TGF-b-dependent genes that are differentially regulated by Smad2 and Smad3 under regular growth conditions on a genome-wide scale. We show that Smad2, Smad3 and Smad4 contribute to the regulation of TGF-b responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.