Prognostic significance of the expression of Smad4 and Smad7 in human gastric carcinomas.

BACKGROUND: Transforming growth factor-beta (TGF-beta) modulates the growth and function of many cells, including those with malignant transformation. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. PATIENTS AND METHODS: To clarify the correlations between clinicopathologic profiles and the patient's survival, the expression of common mediator Smad (Smad4) and inhibitory Smad (Smad7) were evaluated immunohistochemically in 304 consecutive gastric carcinomas using the tissue array method. RESULTS: Positive Smad4 expression was observed in 266 (87.5%) tumors and positive Smad7 expression in 98 (32.2%) tumors. The prognosis of patients with a Smad4-positive tumor was significantly better than that of the patients with a negative tumor. The survival rate was significantly higher in patients with negative Smad7 expression than those with positive Smad7 expression. In subgroup analysis according to TNM (tumour-node-metastasis) stage, both Smad4 and Smad7 showed most significant prognostic differences in stage I gastric cancer patients. Multivariate analysis indicated that tumor size, depth of invasion, lymph node metastasis and Smad7 expression were independent prognostic factors. CONCLUSION: Enhanced expression of the TGF-beta signaling inhibitor Smad7 may present one of the novel mechanisms of TGF-beta resistance in human gastric carcinomas.     

Expression of Smad proteins in human colorectal cancer.

Escape from transforming growth factor-beta (TGF-beta)-induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common-mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts.     

No mutations of the Smad2 gene in human sporadic gastric carcinomas

BACKGROUND: The majority of cancer cells escape from TGF-beta-mediated growth control. However, the mechanism of resistance to the growth inhibitory effects by TGF-beta is not clear. TGF-beta signaling is initiated when the type I receptor phosphorylates the SMAD proteins, Smad2 and Smad3. Recently, mutations of Smad2 have been detected in human colon and lung cancers. Mutation of coding sequences of Smad2 in gastric carcinomas has not yet been elucidated adequately. METHODS: PCR-SSCP analysis of the entire coding region of Smad2 in 35 human sporadic gastric cancers and eight gastric cancer cell lines was performed using 11 sets of intron-based primers. RESULTS: No mutations of Smad2 were detected in any tumor or cell line. CONCLUSIONS: The results suggest that mutation of Smad2 does not play a key role in human stomach carcinogenesis.     

The role of PRAJA and ELF in TGF-beta signaling and gastric cancer

Emerging research has shown that the transforming growth factor-beta (TGF-beta) pathway plays a key role in the suppression of gastric carcinoma. Biological signals for TGF-beta are transduced through transmembrane serine/threonine kinase receptors, which in turn signal to Smad proteins. Inactivation of the TGF-beta pathway often occurs in malignancies of the gastrointestinal system, including gastric cancer. Yet, only a fraction of sporadic gastric tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be mediated through E3-mediated ubiquitination of Smad4/adaptor protein complexes. The regulation of the TGF-beta pathway through a PRAJA, a RING finger (RING-H2) protein, and ELF, a beta-Spectrin adaptor protein, both which were originally identified in endodermal stem/progenitor cells committed to foregut lineage, could play a pivotal role in gastric carcinogenesis. PRAJA, which functions as an E3 ligase, interacts with ELF in a TGF-beta-dependent manner in gastric cancer cell lines. PRAJA is increased five-fold in human gastric cancers, and inactivates ELF. This is particularly significant since ELF, a Smad4 adaptor protein, possesses potent anti-oncogenic activity and is frequently seen to be inactivated in carcinogenic gastric cells. Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. The alteration of ELF and/or Smad4 expression and function in the TGF-beta signaling pathway may be induced by enhancement of ELF degradation, which is mediated by the high level expression of PRAJA in gastrointestinal cancers. These studies reveal a mechanism for gastric tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.     

The TGF-beta signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer.

Transforming growth factor-beta (TGF-beta) signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaRII) with the type I TGF-beta receptor (TbetaRI). Activated TbetaRI then mediates TGF-beta 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 TbetaRI 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 full-length Smad7 construct leads to complete loss of the growth inhibitory response to TGF-beta1, without altering TGF-beta1-mediated induction of PAI-I. Furthermore, Smad7 transfected COLO-357 cells display enhanced anchorage-independent growth and accelerated growth in nude mice. These findings point to a previously unrecognized mechanism for selective suppression of TGF-beta-mediated growth inhibition in cancer cells that allows for continued activation of the PAI-I promoter by TGF-beta1, which may act to enhance the tumorigenicity of certain cancer cells.     

Smad7 sensitizes tumor necrosis factor induced apoptosis through the inhibition of antiapoptotic gene expression by suppressing activation of the nuclear factor-kappaB pathway

Although tumor necrosis factor (TNF) induces apoptosis and cell death in many tumor cells, some cancer cells are still resistant to the TNF-induced death signal. In this report, we showed that Smad7, an inhibitory Smad of transforming growth factor-beta (TGF-beta) signaling, can overcome the TNF resistance in human breast and gastric cancer cells. Overexpression of Smad7 induces the degradation of poly(ADP-ribose) polymerase and the activation of caspase cascade. Although c-Jun NH2-terminal kinase (JNK) signaling is involved in TNF-induced cell death, the expression of Smad7 does not synergize the activation of JNK. However, the activation of nuclear factor-kappaB (NF-kappaB), the cell survival factor, is markedly decreased in Smad7-stable cells. Furthermore, the expression of antiapoptotic target genes of NF-kappaB is significantly reduced in accordance with the level of Smad7. In addition, Smad7 mediates the inhibitory activity of TGF-beta on TNF-induced NF-kappaB activation and the synergistic activity of TGF-beta on TNF-induced apoptosis. These findings suggest that Smad7 sensitizes the tumor cells to TNF-induced apoptosis through the inhibition of expression of antiapoptotic NF-kappaB target genes.     

Transforming growth factor-beta suppresses nonmetastatic colon cancer through Smad4 and adaptor protein ELF at an early stage of tumorigenesis

Although transforming growth factor-beta (TGF-beta) is both a suppressor and promoter of tumorigenesis, its contribution to early tumor suppression and staging remains largely unknown. In search of the mechanism of early tumor suppression, we identified the adaptor protein ELF, a beta-spectrin from stem/progenitor cells committed to foregut lineage. ELF activates and modulates Smad4 activation of TGF-beta to confer cell polarity, to maintain cell architecture, and to inhibit epithelial-to-mesenchymal transition. Analysis of development of colon cancer in (adult) elf+/-/Smad4+/-, elf+/-, Smad4+/-, and gut epithelial cells from elf-/- mutant mouse embryos pinpoints the defect to hyperplasia/adenoma transition. Further analysis of the role of ELF in human colorectal cancer confirms reduced expression of ELF in Dukes' B1 stage tissues (P < 0.05) and of Smad4 in advanced colon cancers (P < 0.05). This study indicates that by modulating Smad 4, ELF has a key role in TGF-beta signaling in the suppression of early colon cancer.     

Transforming growth factor-beta suppresses the ability of Ski to inhibit tumor metastasis by inducing its degradation

c-Ski is an important corepressor of transforming growth factor-beta (TGF-beta) signaling through its ability to bind to and repress the activity of the Smad proteins. It was initially identified as an oncogene that promotes anchorage-independent growth of chicken and quail embryo fibroblasts when overexpressed. Although increased Ski expression is detected in many human cancer cells, the roles of Ski in mammalian carcinogenesis have yet to be defined. Here, we report that reducing Ski expression in breast and lung cancer cells does not affect tumor growth but enhances tumor metastasis in vivo. Thus, in these cells, Ski plays an antitumorigenic role. We also showed that TGF-beta, a cytokine that is often highly expressed in metastatic tumors, induces Ski degradation through the ubiquitin-dependent proteasome in malignant human cancer cells. On TGF-beta treatment, the E3 ubiquitin ligase Arkadia mediates degradation of Ski in a Smad-dependent manner. Although Arkadia interacts with Ski in the absence of TGF-beta, binding of phosphorylated Smad2 or Smad3 to Ski is required to induce efficient degradation of Ski by Arkadia. Our results suggest that the ability of TGF-beta to induce degradation of Ski could be an additional mechanism contributing to its protumorigenic activity.     

The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells

Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.     

Critical interactions between TGF-beta signaling/ELF, and E-cadherin/beta-catenin mediated tumor suppression

Inactivation of the transforming growth factor-beta (TGF-beta) pathway occurs often in malignancies of the gastrointestinal (GI) system. However, only a fraction of sporadic GI tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Here, we show a wide range of GI tumors, including those of the stomach, liver and colon in elf+/- and elf+/- / Smad4+/- mutant mice. We found that embryonic liver fodrin (ELF), a beta-Spectrin originally identified in endodermal stem/progenitor cells committed to foregut lineage, possesses potent antioncogenic activity and is frequently inactivated in GI cancers. Specifically, E-cadherin accumulation at cell-cell contacts and E-cadherin-beta-catenin-dependent epithelial cell-cell adhesion is disrupted in elf+/- / Smad4+/- mutant gastric epithelial cells, and could be rescued by ectopic expression of full-length elf, but not Smad3 or Smad4. Subcellular fractionation revealed that E-cadherin is expressed mainly at the cell membrane after TGF-beta stimulation. In contrast, elf+/- / Smad4+/- mutant tissues showed abnormal distribution of E-cadherin that could be rescued by overexpression of ELF but not Smad3 or Smad4. Our results identify a group of common lethal malignancies in which inactivation of TGF-beta signaling, which is essential for tumor suppression, is disrupted by inactivation of the ELF adaptor protein.     

Preserved Smad4 expression in the transforming growth factor beta signaling pathway is a favorable prognostic factor in patients with advanced gastric cancer.

The signals of the transforming growth factor beta (TGF-beta) superfamily are conveyed through cell surface serine/threonine kinase receptors to the intracellular mediators known as Smads. Activation of Smads causes their translocation from the cytoplasm to the nucleus, where they function to control gene expression. The present study analyzed the expression of Smad4 and TGF-beta1 to determine their prognostic significance in advanced gastric cancer. Of 249 cases of advanced gastric cancer, 41 had invaded the muscular layer, 114 had invaded the subserosal layer, and 94 had invaded the serosa. Anti-Smad4 and TGF-beta1 antibodies were used for immunohistochemical staining. Reduced expression of Smad4 was 75.1%, whereas positive expression of TGF-beta1 was 39.6% in gastric cancer. Smad4 expression was related to the depth of tumor invasion (P < 0.05), and TGF-beta1 expression correlated with tumor gross type (P < 0.05). Postoperative survival analysis indicated that patients who had a tumor with reduced Smad4 expression had a poorer clinical outcome than those with preserved expression (P < 0.05). Furthermore, in patients with TGF-beta1-positive tumors, survival rate was significantly better in patients with preserved Smad4 expression than in those with reduced Smad4 expression (P < 0.05). According to multivariate analysis, Smad4 expression acted as an independent prognostic factor. Smad4 expression, particularly in the TGF-beta pathway, is an effective predictor of outcome for patients with advanced gastric cancer.