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.     

A role for human MUC4 mucin gene, the ErbB2 ligand, as a target of TGF-beta in pancreatic carcinogenesis

MUC4: encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-beta, a pleiotropic cytokine, regulates numerous genes involved in pancreatic carcinogenesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-beta in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4+/Smad4-), CAPAN-2 (MUC4+/Smad4+) and PANC-1 (MUC4-/Smad4+), were used. By RT-PCR, transfection assays and immunohistochemistry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-beta, (ii) Smad2 positively cooperates with Smad4 to activate the promoter, (iii) activation of Smad4 by exogenous TGF-beta induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-beta activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-beta is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-beta as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.     

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.     

Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling

Members of the TGF-beta family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-beta signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-beta-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-beta receptor type I (TbetaRI), whereas YAP65(1-301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-beta signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TbetaRI, and enhances the inhibitory activity of Smad7 against TGF-beta signaling.     

Correlation of glypican-1 expression with TGF-beta, BMP, and activin receptors in pancreatic ductal adenocarcinoma

Glypican1 (GPC1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors as well as for members of the TGF-beta family. GPC1 plays a role in pancreatic cancer by regulating growth factor responsiveness. In view of the importance of members of the TGF-beta family in pancreatic cancer, in the present study, the role of GPC1 in TGF-beta, BMP and activin signaling was analyzed. Quantitative RT-PCR and immunohistochemistry were utilized to analyze GPC1 and TGF-beta, BMP and activin receptor expression levels. Panc-1 and T3M4 pancreatic cancer cells were transfected in a stable manner with a GPC1 antisense expression construct. Anchorage-dependent and -independent growth was determined by MTT and soft agar assays. TGF-beta1, activin-A and BMP-2 responsiveness was determined by MTT assays and immunoblotting with p21, p-Smad1, and p-Smad2 antibodies. QRT-PCR demonstrated increased GPC1 mRNA levels in pancreatic ductal adenocarcinoma (PDAC) compared to normal pancreatic tissues (NPT), as described previously. There was a significant correlation between GPC1 mRNA levels and TbetaRII, act-R1a, act-R1b, act-R2a, BMP-R1a, and BMP-R2 mRNA expression in NPT. In contrast, GPC1 mRNA expression correlated directly with act-R1a and BMP-R1a in N0 PDAC cases and with act-R2a and BMP-R1a in lymph node positive cases. Down-regulation of GPC1 resulted in increased doubling time in Panc-1 but not in T3M4 cells, and decreased anchorage-independent growth in both cell lines. GPC1 down-regulation resulted in a slightly altered response towards TGF-beta1, activin-A and BMP-2 in terms of growth, p21 induction and Smad2 phosphorylation. In conclusion, enhanced GPC1 expression correlates with BMP and activin receptors in pancreatic cancer. GPC1 down-regulation suppresses pancreatic cancer cell growth and slightly modifies signaling of members of the TGF-beta family of growth factors.     

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.     

Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells

Transforming growth factor-beta (TGF-beta) signals through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, TGF-beta activates intracellular Smad proteins and regulates proliferation and apoptosis in various cell types. To demonstrate the effects of TGF-beta/Smad signal on growth and apoptosis of human embryonal rhabdomyosarcoma (RMS) cells, a strategy of RNAi-mediated 'gene silencing' of Smad4 was used to interrupt endogenous TGF-beta/Smad signaling in an RMS cell line, RD, and the regulation of exogenous TGF-beta1 to growth and apoptosis of the cells was also determined. Physiologically, TGF-beta/Smad signaling was essential for the normal growth of RD. The interruption of endogenous TGF-beta/Smad signaling by RNAi significantly suppressed the growth of RD cells and dramatically induced apoptosis of RD cells. Exogenous TGF-beta1 also inhibited the growth of RD cells, but had no effect on apoptosis. It also partially counteracted the growth inhibition and apoptosis induced by Smad4 silencing in RD cells. These findings provide a new insight into how TGF-beta/Smad signaling regulates the growth and apoptosis of cancer cells. Moreover, as a powerful tool, shRNA interference suppresses endogenous Smad4 gene expression and subsequently modulates cell growth and apoptosis, which may provide a novel basis for the development of rational intervention strategies in RMS therapy.     

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.     

Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.     

Smad2 mediates Erk1/2 activation by TGF-beta1 in suspended, but not in adherent, gastric carcinoma cells

Integrin-mediated cell adhesion enables cells to respond to extracellular stimuli for diverse cellular functions including proliferation, leading to differential biological activities from cells in suspension. Integrins can transduce signals (directly) to intracellular molecules and also collaborate with other membrane receptor-mediated signal pathways, including TGF-beta1 pathway. TGF-beta1 induces growth inhibition in epithelial cells and is known to transduce intracellular signaling in Smad-dependent or -independent manner. Currently effects of cell adhesion status on the TGF-beta1-mediated Erk1/2 regulation and on its Smad-(in)dependency are not known. In this study, we examined effects of cell adhesion status on the TGF-beta1-mediated Erk1/2 regulation, and roles of Smad proteins on the cell adhesion-mediated effects, using a gastric carcinoma cell variant. First, we found that cell adhesion-dependent Erk1/2 activation responded differentially to TGF-beta1, depending on cell adhesion status; TGF-beta1 treatment resulted in activation of Erk1/2 in suspended cells, whereas a decrease was noted in adherent cells. This activation of Erk1/2 by TGF-beta1 in suspension was more enhanced by an overexpression of Smad2, but not of other Smads 2, 4, and 7, but abolished by a Smad2 reduction via an introduction of its siRNA. In contrast, PKB/Akt regulation by TGF-beta1 was not different in suspension or in adhesion, and Smad7, but not the other Smads, activated PKB/Akt phosphorylation on TGF-beta1 treatment, indicating a specificity of Smad2-mediated and cell adhesion status-dependent activation of Erk1/2 activity.     

Mutational analysis of TGF-beta type II receptor,Smad2, Smad3, Smad4, Smad6 and Smad7 genes in colorectal cancer

Transforming growth factor-beta(TGF-beta) is known to play an important role in controlling embryonal development, cell proliferation and homeostasis. The purpose of this study is to elucidate the involvement of the TGF-beta pathway in colorectal carcinogenesis. DNA was extracted from 100 patients with colorectal cancer. Then, all coding regions of the TGF-beta type II receptor (TRII) and the genes for Smad2, Smad3, Smad4, Smad6, and Smad7 were analyzed by PCR-SSCP and direct sequencing. Also, a LOH analysis of 18q21, where the Smad2 and Smad4 genes are located, was performed. We detected 11 cases of frameshift mutation in the TRII gene (11%) and 5 cases of point mutations in the Smad4 gene (5.0%); LOH at 18q21 was detected with 33% frequency. No abnormalities were found in the genes for Smad2, Smad3, Smad6, and Smad7. These results suggest that the abnormalities of TRII and Smad4 play an important role inhibiting TGF-beta signaling in colorectal carcinogenesis.