Lentiviral-mediated Smad4 RNAi induced anti-proliferation by p16 up-regulation and apoptosis by caspase 3 down-regulation in hepatoma SMMC-7721 cells

Transforming growth factor-beta (TGF-beta)-Smad signaling pathway participates in the regulation of a variety of cellular activities. Unlike the high incidences of Smad4 mutation or deletion in pancreatic cancer and gastrointestinal cancers, Smad4 gene is seldom mutated or deleted in hepatocellular carcinoma (HCC). The role of TGF-beta-Smad4 signaling pathway in leading to carcinogenesis of liver cells remains unknown. In this study, we succeeded in silencing Smad4 using lentiviral-mediated Smad4 RNA interference (RNAi). We investigated the role of Smad4 in TGF-beta1-induced cell proliferation and apoptosis of HCC cell line SMMC-7721. We determined cell proliferation, apoptosis, and expression of p21, p16, p53 and caspase 3. Results showed that TGF-beta1 not only had a significant anti-proliferation effect but also induced cellular apoptosis in SMMC-7721 cells. These effects induced by TGF-beta1 were almost completely blocked by the knockdown of Smad4. Western blot analysis revealed that p16 was up-regulated and caspase 3 was activated by silencing of Smad4, and the expression of p21 and wild-type p53 were not affected. These results suggest that TGF-beta1-induced cell growth inhibition by up-regulating p16 expression and cellular apoptosis by activating caspase 3 was Smad4-dependent. Additionally, the knock down of a specific gene using lentiviral-mediated RNAi appears to be a promising tool and strategy for analyzing endogenous gene function.     

Induction of Smad1 by MT1-MMP contributes to tumor growth

MT1-MMP is a key integral membrane protease, which regulates tumor growth by cleaving extracellular matrix components, activating growth factors and receptors, and consequently, triggering downstream signals. To study what genes or pathways are mediated by endogenous MT1-MMP during tumor growth in vivo, we stably suppressed endogenous MT1-MMP in human tumor cells using RNA interference (RNAi). Tumor growth was significantly reduced in tumors derived from MT1-MMP-suppressed cells relative to control cells; the effect was rescued in cells engineered to re-express MT1-MMP expression. Gene expression profiling of cultured and tumor-derived cells by DNA microarray and real-time RT-PCR revealed that Smad1 expression was upregulated in MT1-MMP-expressing cells and rapidly growing tumors; this was confirmed in 4 additional tumor cell lines. Furthermore, tumor growth of MT1-MMP-expressing cells was reduced when Smad1 was suppressed by RNAi. We also found that the active form, but not the latent form, of TGF-beta was capable in promoting Smad1 expression and 3D cell proliferation in MT1-MMP-suppressed cells. In addition, a dominant-negative form of the TGF-beta Type II receptor reduced Smad1 expression in MT1-MMP-expressing cells. Thus, we propose that MT1-MMP functions, in part, to promote tumor growth by inducing the expression of Smad1 via TGF-beta signaling.     

Restoration of transforming growth factor Beta signaling by functional expression of smad4 induces anoikis

Smad proteins transduce signals carried by the transforming growth factor beta (TGF-beta) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF-beta signaling was restored as determined by TGF-beta-dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad-Smad4 in the presence of TGF-beta1 also resulted in an altered cell morphology that coincided with enhanced beta1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF-beta-dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization revealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF-beta signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy.     

Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.     

Chemical enhancers of cytokine signaling that suppress microfilament turnover and tumor cell growth

The transforming growth factor-beta (TGF-beta) family of cytokines regulates cell proliferation, morphogenesis, and specialized cell functions in metazoans. Herein, we screened a compound library for modifiers of TGF-beta signaling in NMuMG epithelial cells using a cell-based assay to measure Smad2/3 nuclear translocation. We identified five enhancers of TGF-beta signaling that share a core structure of diethyl 2-(anilinomethylene)malonate (DAM), and D(50) values of 1 to 4 micromol/L. Taking advantage of the Mgat5 mutant phenotype of accelerated receptor loss to endocytosis, we determined that DAM-1976 restored the sensitivity of Mgat5(-/-) carcinoma cells to both TGF-beta and epidermal growth factor (EGF). In Mgat5 mutant and wild-type carcinoma cells, DAM-1976 enhanced and prolonged TGF-beta- and EGF-dependent Smad2/3 and Erk activation, respectively. DAM-1976 reduced ligand-dependent EGF receptor endocytosis, actin microfilament turnover, and cell spreading, suggesting that the compound attenuates vesicular trafficking. Hyperactivation of intracellular signaling has the potential to suppress tumor cell growth and, in this regard, DAM-1976 represents a new pharmacophore that increases basal activation of Smad2/3 and Erk, inhibits microfilament remodeling, and suppresses carcinoma cell growth.     

Targeting endogenous transforming growth factor beta receptor signaling in SMAD4-deficient human pancreatic carcinoma cells inhibits their invasive phenotype1

Transforming growth factor-beta (TGF-beta) 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-beta-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-beta type I (TbetaR-I) or type II (TbetaR-II) receptors. Although minimal amounts of free bioactive TGF-beta1 and TGF-beta2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-beta3 specific) TGF-beta-neutralizing antibody and with anti-alpha(V)beta(6) integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-beta-inducible reporter gene in neighboring cells, respectively. Thus, activation of TGF-beta at the cell surface was responsible for the increased autocrine endogenous and paracrine signaling. Blocking TbetaR-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-beta 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-beta signaling. Therefore, targeting the TbetaR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.     

High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma

Transforming growth factor beta (TGF-beta) regulates growth of various cells, and inactivation of the TGF-beta signaling pathway contributes to tumor progression. Smad2 is phosphorylated and activated by TGF-beta, resulting in the antiproliferative effects of TGF-beta signaling. Smurf2 (Smad ubiquitination regulatory factor 2) was identified as the Smad ubiquitin ligase that induces the ubiquitination and degradation of Smad2. This study was undertaken to elucidate the relationships between Smurf2 expression and the clinicopathological characteristics of patients with esophageal squamous cell carcinoma (SCC) and the correlation between Smurf2 and Smad2 expression. Surgical specimens obtained from 80 patients with esophageal SCC were subjected to immunohistochemical staining. Our data indicated that high-level expression of Smurf2 correlated with depth of invasion, lymph node metastasis, and a poor survival rate. We also found an inverse correlation between the expression of Smurf2 and Smad2. Western blotting analysis of esophageal SCC-derived cell lines revealed similar inverse correlations. We demonstrated that high-level expression of Smurf2 appears to correlate with tumor development and poor prognosis in patients with esophageal SCC and that alteration of Smad2 expression in the TGF-beta signaling pathway may be induced by enhancement of Smad2 degradation mediated by high-level expression of Smurf2.     

Involvement of programmed cell death 4 in transforming growth factor-beta1-induced apoptosis in human hepatocellular carcinoma

The programmed cell death 4 (PDCD4) gene was originally identified as a tumor-related gene in humans and acts as a tumor-suppressor in mouse epidermal carcinoma cells. However, its function and regulatory mechanisms of expression in human cancer remain to be elucidated. We therefore investigated the expression of PDCD4 in human hepatocellular carcinoma (HCC) and the role of PDCD4 in human HCC cells. Downregulation of PDCD4 protein was observed in all HCC tissues tested compared with corresponding noncancerous liver, as revealed by Western blotting or immunohistochemical staining. Human HCC cell line, Huh7, transfected with PDCD4 cDNA showed nuclear fragmentation and DNA laddering characteristic of apoptotic cells associated with mitochondrial changes and caspase activation. Transforming growth factor-beta1 (TGF-beta1) treatment of Huh7 cells resulted in increased PDCD4 expression and occurrence of apoptosis, also concomitant with mitochondrial events and caspase activation. Transfection of Smad7, a known antagonist to TGF-beta1 signaling, protected cells from TGF-beta1-mediated apoptosis and suppressed TGF-beta1-induced PDCD4 expression. Moreover, antisense PDCD4 transfectants were resistant to apoptosis induced by TGF-beta1. In conclusion, these data suggest that PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, and a possible tumor suppressor in hepatocarcinogenesis.     

Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-beta

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.     

Differential roles of JNK and Smad2 signaling pathways in the inhibition of c-Myc-induced cell death by TGF-beta

The transforming growth factor beta (TGF-beta) plays an important role in constraining cellular proliferation, but it is also a potent inducer of programmed cell death or apoptosis. Here, we demonstrate that TGF-beta can have an opposite effect, acting as a survival factor to prevent c-Myc-induced cell death in Rat-1 fibroblasts. However, in marked contrast to TGF-beta, Smad2, which is a critical intracellular mediator of the TGF-beta signaling pathway, functions as an antagonist to induce increased cell death. The protective activity of TGF-beta was associated with the activation of c-Jun N-terminal Kinase (JNK) and was not linked to the ability of TGF-beta to promote cell cycle progression. Expression of dominant-interfering forms of various components of the JNK signaling pathway, including Rac1, Cdc42, mitogen-activated protein kinase kinase 4 (MKK4), and c-Jun, abolished TGF-beta-mediated cell survival. Furthermore, overexpression of the constitutively activated mutant RacL61F37A, which selectively stimulates JNK cascade but not G1 cell cycle progression or actin polymerization, was sufficient to prevent apoptosis induced by c-Myc. These findings describe a differential effect of two separated signaling pathways of TGF-beta and indicate for the first time that Smad2 can act as antagonist to suppress TGF-beta-dependent cell survival. Oncogene (2000) 19, 1277 - 1287.     

Functional blockade of Smad4 leads to a decrease in beta-catenin levels and signaling activity in human pancreatic carcinoma cells

In the last several years, many laboratories have tried to unravel the complex signaling mechanisms activated by TGF-beta(1) in transformed cells. Smad proteins are the principal mediators of the transforming growth factor beta (TGF-beta) response, but this factor can also activate Smad-independent pathways in different cell types. Our previous studies in murine keratinocytes led to the identification of a cooperation between oncogenic Ras and Smad4 inactivation during malignant progression. We further investigated the function of Smad4 in human pancreatic cancer, in which loss-of-function mutations affecting Smad4 occur with a 50% frequency. Expression of a dominant-negative Smad4 construct in the adenocarcinoma cell line PANC-1 led to increased ubiquitination and proteasomal degradation of beta-catenin. Moreover, loss of Smad4 abrogated beta-catenin-signaling activity and was associated with a reduction of the tumorigenic potential of PANC-1 cells in scid mice. Although the expression of the dominant-negative Smad4 blocked TGF-beta(1)/Smad2,3-signaling activity, the above-mentioned effects of Smad4 on beta-catenin stability were independent of the TGF-beta1/Smad2,3-signaling pathway. These findings provide evidence for a cross talk between Smad4 and the Wnt/beta-catenin pathway in pancreatic carcinoma cells, suggesting a new role for Smad4 as an attenuator of beta-catenin proteasomal degradation.     

Regulation of TGF-beta family signaling by E3 ubiquitin ligases

Members of the transforming growth factor-beta (TGF-beta) family, including TGF-beta, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Alterations in their downstream signaling pathways are associated with a range of human diseases like cancer. TGF-beta family members transduce signals through membrane serine/threonine kinase receptors and intracellular Smad proteins. The ubiquitin-proteasome pathway, an evolutionarily conserved cascade, tightly regulates TGF-beta family signaling. In this pathway, E3 ubiquitin ligases play a crucial role in the recognition and degradation of target proteins by the 26S proteasomes. Smad degradation regulates TGF-beta family signaling; HECT (homologous to the E6-accessory protein C-terminus)-type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1), Smurf2, and a RING-type E3 ubiquitin ligase, ROC1-SCF(Fbw1a) have been implicated in Smad degradation. Smurf1 and Smurf2 bind to TGF-beta family receptors via the inhibitory Smads, Smad6 and Smad7, to induce their ubiquitin-dependent degradation. Arkadia, a RING-type E3 ubiquitin ligase, induces the ubiquitination and degradation of Smad7 and corepressors, c-Ski and SnoN, to enhance TGF-beta family signaling. Abnormalities in E3 ubiquitin ligases that control components of TGF-beta family signaling may lead to the development and progression of various cancers.