4089

SMAD4

SMAD family member 4;SMAD4;SMAD family member 4

MAD homolog 4|SMAD, mothers against DPP homolog 4|deleted in pancreatic carcinoma locus 4|deletion target in pancreatic carcinoma 4|mothers against decapentaplegic homolog 4|mothers against decapentaplegic, Drosophila, homolog of, 4

18q21.1

protein-coding

Distant metastases represent the major cause of death after curative surgery of colorectal cancer. The aim of this study was to evaluate the role of Smad4 and KRAS genetic alterations in colorectal metastases taking into account both the site (hepatic versus extrahepatic) and the time (synchronous versus metachronous) of recurrence. We examined the immunohistochemical expression of Smad4 and frequency of KRAS mutation in primary colorectal tumors and in their corresponding metastatic tissues. Loss of Smad4 expression was noted in 37% (26/71) of the primary tumors and the corresponding metastases. Absence of Smad4 protein was more frequently observed in hepatic metastases, whether they were metachronous or synchronous, than in extrahepatic metastases (p<0.005). The frequency of KRAS mutations was high in the synchronous and extrahepatic metachronous metastases (68-80%), but was significantly lower in the hepatic metachronous metastases (11%). Our results indicate that absence of Smad4 expression correlated significantly with liver metastases regardless of the time of their occurrence and represents a promising new biomarker to predict liver metastasis in colorectal cancer patients. Therefore, this group of patients could benefit from a specific and appropriate pre- and/or post-operative therapy.

Recent studies have reported that Smad4 has a TGF-beta-independent function as a tumor suppressor in cooperating with beta-catenin/Lef to regulate target gene expression. The objective of this research was to study the role of Smad4 in colon cancer migration and its potential mechanism. In our study, stable colon cancer cells that have increased Smad4 expression were created using a eukaryotic vector containing Smad4-pcDNA3.1(+). Smad4 restoration directly suppressed the Wnt/beta-catenin signal activity in SW480 cells by down-regulating beta-catenin expression and altering its localization from cytoplasm and nucleus to the plasma membrane. Up-regulation of Smad4 also increased the expression of E-cadherin and decreased the transcriptional activity of beta-catenin/Tcf target genes, such as claudin-1 and MMP-7. SW480 cells with increased Smad4 expression had decreased in vitro cell migration. The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer migration and metastasis.

The transforming growth factor beta (TGF-beta) signaling pathway has been considered both a tumor suppressor and a cancer promoter. Additionally, downregulation of cell adhesion molecules such as E-cadherin plays an important role in the metastatic potential of colorectal cancer (CRC). The aim of the present study was to evaluate TGF-beta, TGF-beta type I receptor (TGF-betaR1), TGF-beta type II receptor (TGF-betaR2), Smad4, pSmad2/3, and E-cadherin expression in colorectal carcinoma and to correlate the obtained data with other standard prognostic parameters, such as disease stage, metastases, and patient survival. TGF-beta, TGF-betaR1, TGF-betaR2, Smad4, pSmad2/3, and E-cadherin expression was evaluated immunohistochemically in 195 unrelated CRC specimens and the results subjected to various statistical analyses. TGF-beta was expressed in 71.28%, TGF-betaR1 in 61.0%, TGF-betaR2 in 54.4%, Smad4 in 61.5%, pSmad2/3 in 71.3%, and E-cadherin in 50.26% of the colorectal carcinoma samples tested. The correlation of immunoexpression with the clinicopathological parameters of CRC revealed that the high expression of TGF-beta and low expression of TGF-betaR1, TGF-betaR2, Smad4, pSmad2/3, and E-cadherin were correlated with tumor-node-metastasis (TNM) stage of disease. High TGF-beta expression and low TGF-betaR1, TGF-betaR2, Smad4, and pSmad2/3 expression were also correlated with lymph node metastasis. The Kaplan-Meier survival curves demonstrated a clear association of cancer-specific overall survival with high TGF-beta, as well as low TGF-betaR1, TGF-betaR2, Smad4, pSmad2/3, and E-cadherin expression. Our results suggest that TGF-beta, TGF-betaR1, TGF-betaR2, Smad4, pSmad2/3, and E-cadherin are closely related to TNM stage of CRC. Moreover, TGF-beta, TGF-betaR2, Smad4, pSmad2/3, and E-cadherin emerge as valuable independent biomarkers of prognosis in CRC patients.

BACKGROUND & AIMS: SMAD4 frequently is lost from colorectal cancers (CRCs), which is associated with the development of metastases and a poor prognosis. SMAD4 loss is believed to alter transforming growth factor beta signaling to promote tumor progression. However, SMAD4 is also a central component of the bone morphogenetic protein (BMP) signaling pathway, implicated in CRC pathogenesis by human genetic studies. We investigated the effects of alterations in BMP signaling on the invasive and metastatic abilities of CRC cells and changes in members in this pathway in human tumor samples. METHODS: We activated BMP signaling in SMAD4-positive and SMAD4-negative CRC cells (HCT116, HT-29, SW480, and LS174T); SMAD4 was stably expressed or knocked down using lentiviral vectors. We investigated the effects on markers of epithelial-mesenchymal transition and on cell migration, invasion, and formation of invadopodia. We performed kinase activity assays to characterize SMAD4-independent BMP signaling and used an inhibitor screen to identify pathways that regulate CRC cell migration. We investigated the effects of the ROCK inhibitor Y-27632 in immunocompromised (CD-1 Nu) mice with orthotopic metastatic tumors. Immunohistochemistry was used to detect BMPR1a, BMPR1b, BMPR2, and SMAD4 in human colorectal tumors; these were related to patient survival times. RESULTS: Activation of BMP signaling in SMAD4-negative cells altered protein and messenger RNA levels of markers of epithelial-mesenchymal transition and increased cell migration, invasion, and formation of invadopodia. Knockdown of the BMP receptor in SMAD4-negative cells reduced their invasive activity in vitro. SMAD4-independent BMP signaling activated Rho signaling via ROCK and LIM domain kinase (LIMK). Pharmacologic inhibition of ROCK reduced metastasis of colorectal xenograft tumors in mice. Loss of SMAD4 from colorectal tumors has been associated with reduced survival time; we found that this association is dependent on the expression of BMP receptors but not transforming growth factor beta receptors. CONCLUSIONS: Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.

Smad signaling pathway plays an important role in tumorigenesis and progression in cancer (Halder, S.K., Rachakonda, G., Deane, N.G., Datta, P.K., 2008. Smad7 induces hepatic metastasis in colorectal cancer. Br. J. cancer 99, 957-965). The protein level of Smad is associated with growth, inhibition, and metastasis in different cancers. It is unclear if the differentiation, metastasis and apoptosis are reduced by Smad expression pattern in gastric cancer. To determine the effect of Smad on gastric cancer cells, we investigated the relationship of Smad4/Smad7 expression, and differentiation, metastasis, and apoptosis in different gastric cancer. The results show that Smad4 expression in the gastric cancer tissue was dramatically lower than that in the peritumoral tissue. A lower expression of Samd4 was significantly lower in the poorly differentiated tissue than that in the well and middle differentiated tissues (P<0.01). In contrast, Smad7 expression in gastric cancer tissues was significantly higher than that in the peritumoral tissue. Smad7 was overexpressed in poorly differentiated tissue, also higher than those in the middle, and well differentiated tissues (P<0.05). The Smad4 or Smad7 expression obviously related with the lymphatic metastasis in gastric cancer. There were 45 cases with lymphatic metastasis in all 78 patients. Smad4 expression in the cases with lymphatic metastasis was lower than the cases without metastasis (P<0.01), whereas Smad7 expression in the cases with lymphatic metastasis was much higher than the case without metastasis (P<0.01). To better understand the mechanisms involved in tumorigenesis of gastric cancer, we established SGC7901 gastric cancer cell lines transduced with Smad4 or Smad7 plasmid DNA. Apoptosis and survival of cancer cells was induced after Smad4 and Smad7 transduction. This effect is concentration and time dependent. Thus, this study provides a mechanism by which a balance between Smad4 and Smad7 in human gastric cancer is critical for differentiation, metastasis, and apoptosis of tumor cells.

BACKGROUND: Smad4 is found mutated in many cancers. It acts as a tumor suppressor in the regulation of TGF-beta signaling pathway. The objective of this work was to study the expression of Smad4 in intrahepatic cholangiocarcinoma (ICC) and its relationship with the biological behavior and prognosis of the disease. METHODS: Forty-nine paraffin-embedded ICC specimens and nine normal liver tissues were analyzed by immunohistochemical methods using Smad4 monoclonal antibodies. The expression of Smad4 was compared with the clinical pathological characteristics of the patients. RESULTS: The expression of Smad4 was 100% positive in normal liver tissues, which was higher than that in the ICC (44.9%). Negative labeling of the Smad4 protein was found in 26.1% (6/23) of well-differentiated ICCs and 61.5% (16/26) of poorly to moderately differentiated ICCs, and 34.3% (12/35) and 71.4% (10/14) showed negative Smad4 labeling (P = 0.018) of ICC at pathological tumor Node metastasis (pTNM) stage I-II and pTNM stage III-IV separately. Furthermore, 72% (8/11) of lymph node metastatic ICCs and 73.3% (11/15) of intrahepatic metastatic ICCs showed negative labeling of the Smad4 protein. The loss of Smad4 expression in those metastatic ICCs was significantly more severe compared with non-metastatic ICCs (P = 0.000). CONCLUSIONS: The expression of Smad4 was associated with the histological grade, clinical stage, and metastasis of ICC (P < 0.05). The detection of Smad4 may be helpful in determining the degree of malignancy and prognosis of ICC.

Pancreatic adenocarcinoma is a genetic disease showing somatic mutations of multiple genes, including SMAD4. SMAD4 is a tumor suppressor gene that is inactivated in a sub-set of pancreatic adenocarcinoma, either by the intragenic mutation of one allele in combination with the loss of the other allele or by homozygous deletion of both alleles. This study examines SMAD4 expression in fine-needle aspiration cell blocks from patients with pancreatic adenocarcinoma, as well as a variety of human cancers, in order to assess its viability as a tumor marker. A total of 100 patients with pancreatic adenocarcinoma, with diagnostic material from fine-needle aspiration cell blocks were selected for this study. In addition cancers from different sites were examined in multitumor tissue microarrays, which included two tissue cores from neoplastic surgical resection specimens. cancers studied included endometrium (n = 100), colon (n = 100), ovary (n = 100), lung (n = 100), breast (n = 100), and malignant melanoma (n = 100). The sections were immunostained with SMAD4 using pressure cooker antigen retrieval labeled polymer horseradish peroxidase (DAKO), and the DAKO autostainer. Immunohistochemical expression was scored as negative, 1+, 2+, 3+. Only 2+ and 3+ staining was considered as positive staining. SMAD4 staining was nuclear and the results for tumor cell positivity for primary sites studied are as follows: Pancreas (80/100; 80%), endometrium (0/100; 0%), colon (0/100; 0%), ovary (3/100; 3%), lung (0/100; 0%), breast (2/100; 2%), and malignant melanoma (4/100; 4%). This study suggests that SMAD4 is an important marker for confirming a diagnosis of pancreatic adenocarcinoma as a primary tumor, as well as when it presents as a metastatic tumor on small fine-needle aspirate samples.

The DPC4 influences tumourigenesis and tumor progression by various mechanisms, including angiogenesis. The aim of this study was to determine whether the expression of DPC4 is related to the angiogenesis in lung cancer and whether it could be involved in its clinical behaviour. Immunohistochemistry revealed that DPC4 was expressed at high level in normal broncho-tracheal epithelium, but at low level in tumor tissues, and closely correlated with tumor lymph node metastasis. This result was further confirmed by Western blot analysis. Furthermore, carcinomas with high DPC4 expression demonstrated low VEGF expression and low MVD (microvessel density) labelled with CD34. In addition, DPC4 siRNA in A549 cells also showed that DPC4 could decrease VEGF protein and mRNA expression, and increase TSP1 protein and mRNA expression. Our findings indicated that DPC4 might be an important biomarker for malignant transformation and be involved in preventing the tumor metastasis by inhibiting tumor angiogenesis.

OBJECTIVE: The aim of this study was to investigate the expression of transforming growth factor-beta1 (TGF-beta1) and its signaling pathway molecules in oral squamous cell carcinoma (OSCC) and analyze the association between these factors and genesis and metastasis of OSCC. METHODS: The express of TGF-beta1, TbetaRI, TbetaRII and Smad4, a pivotal downstream molecule of its signaling, in 10 normal oral mucosa tissues and 108 OSCC was detected by SP immunohistochemistry, and thier correlation with genesis and metastasis of OSCC were assessed. RESULTS: The expressions of TbetaRII and Smad4 were lower in the tumors (34.3%, 38.9%) than those in the normal oral epithelium (80.0%, 100.0%, P < 0.05). The positive expression rates of TGF-beta1 and TbetaRI in the normal oral epithelium and OSCC were not significantly different (P > 0.05). There was an inverse correlation between TGF-beta1, Smad4, TbetaRII, TbetaRI expression and clinical stages (P < 0.01). The expression of TGF-beta1 was related with histological differentiation and tumor localization (P < 0.05). There was a relationship beteween Smad4 expression and histological differentiation and lymph node metastasis (P < 0.05). The expression of TbetaRII in the samples with lymph node metastasis was less than that in the ones without lymph node metastasis (P < 0.01), although there was no association between expression of TbetaRII and lymph node metastasis status. CONCLUSION: There is an important relationship between the abnormal TGF-beta1/Smad4 signal pathway and genesis and development of OSCC, while the low expressed Smad4 and TbetaRII may promote the metastasis of OSCC.

Smad4 is involved in cancer progression and metastasis. Using a pair of human syngeneic epithelial ovarian cancer cells with low (HO-8910) and high (HO-8910PM) metastatic abilities, we aimed to reveal the role of Smad4 in ovarian cancer metastasis in vitro. Smad4 was down-regulated in HO-8910PM cell line relative to HO-8910 by implicating Smad4 was probably a potential tumor suppressor gene for ovarian cancer. Re-expression of Smad4 decreased the migration ability and inhibited the invasion capacity of HO-8910PM, while promoted the cell adhesion capacity for HO-8910PM. The stable expression of Smad4 increased the expression of E-cadherin, reduced the expression of plasminogen activator inhibitor-1 (PAI-1) and slightly down-regulated the expression of VEGF. Smad4 suppresses human ovarian cancer cell metastasis potential through its effect on the expressions of PAI-1, E-cadherin and VEGF. Results from current work implicate Smad4 might suppress the invasion and metastasis of human ovarian tumor cells through a TGF-Beta/Smad-mediated pathway.

The virtual lack of well-characterized metastatic pancreatic cancer tissues for study has limited systematic studies of the metastatic process of this deadly disease. To address this important issue, we have instituted a rapid autopsy protocol for the collection of high quality tissues from patients with metastatic pancreatic cancer, called the Gastrointestinal cancer Rapid Medical Donation Program (GICRMDP). At the time of preparation of this manuscript, 20 patients with metastatic pancreatic cancer and one patient with metastatic colon cancer have undergone a rapid autopsy in association with the GICRMDP. The average time interval achieved for these 21 patients was 8.0 hours, with more than 500 individual samples of matched high quality primary and metastatic pancreatic cancer tissues, peritoneal/pleural fluid and blood obtained so far. For the first four patients in which the autopsy was performed in <6 hours, we have successfully xenografted the primary tumor and/or two to four independent matched metastases from a variety of target organ sites, with a take rate of almost 60% for the first 26 xenografted tumors attempted. In an initial survey of KRAS2, TP53 and DPC4 genetic status in lethal metastatic pancreatic cancers, activating KRAS2 mutations were detected in 82% of cases and inactivating TP53 mutations in 55% of cases, consistent with rates of genetic alteration of these genes in early stage pancreatic cancers. However, DPC4 inactivation was found in 75% of patients analyzed, suggesting that genetic inactivation of the DPC4 tumor suppressor gene continues to be selected for with growth at the primary site and metastatic spread to other organs. The invaluable tissue resources generated by the success of the GICRMDP will provide an unparalleled resource for study of metastatic pancreatic cancer and of the metastatic process in general.

metastatic mucinous carcinomas in the ovary are readily recognized when they show characteristic features, including bilateral involvement, only moderate tumor size, surface and superficial cortical involvement, nodular growth, and an infiltrative pattern. However, it is well established that some metastatic mucinous carcinomas can simulate primary ovarian mucinous tumors grossly and microscopically. metastatic pancreaticobiliary tract adenocarcinomas present a particular diagnostic challenge due to their ability to exhibit borderline-like and cystadenomatous growth patterns, which can be misinterpreted as underlying primary ovarian precursor tumors and can be erroneously used to support interpretation of the carcinomatous components as arising from these purported precursors within the ovary. Thirty-five cases of metastatic pancreaticobiliary tract adenocarcinomas were analyzed. The mean patient age was 58 years (median, 59 y; range, 33 to 78 y). In 15 cases (43%), the pancreaticobiliary tract and ovarian tumors presented synchronously and in 2 cases (6%) the ovarian tumors presented earlier as the first manifestation of the disease. Ovarian tumors were bilateral in 31 cases (89%). Mean and median tumor sizes were 10.6 and 9.5 cm, respectively (range, 2.5 to 21.0 cm). Nodularity was present in 22 cases (63%) and surface involvement was identified in 14 cases (40%). An infiltrative growth pattern was present at least focally in 28 cases (80%), accompanied by borderline-like and/or cystadenomatous areas in 17 (49%) cases and as the exclusive pattern in 11 cases (31%). Conversely, borderline-like and cystadenomatous patterns were identified in 24 cases (69%) and as the exclusive patterns (either pure or combined with one another) in 7 cases (20%). Dpc4 expression was lost in 20 of 33 tumors analyzed (61%). Of 25 patients with follow-up, 23 patients had died of disease (mean/median time, 9/6 mo; range, 1 to 39) and 2 patients were alive with disease (at 1 and 25 mo). Frequent bilateral ovarian involvement, moderate tumor size, nodularity, and infiltrative patterns are useful features for identifying these ovarian tumors as metastatic. However, many tumors exhibit borderline-like and cystadenomatous patterns that, when dominant and combined with synchronous presentation, make recognition as metastases an ongoing challenge. Loss of Dpc4 expression provides the most useful immunohistochemical evidence for establishing the pancreaticobiliary tract as the most likely source of these metastatic mucinous carcinomas in the ovary.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified alpha-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with alpha-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that regulates cell growth, differentiation, migration, apoptosis and extracellular matrix remodeling. TGF-beta1 transduces signals from the cell membrane to the cell nucleus through serine/threonine kinase receptors and their downstream effectors, Smad molecules. Although many studies have been focused on TGF-beta1-Smad signaling pathway, the role of TGF-beta1/Smad in tongue squamous cell carcinoma is not fully understood. In the present study, we used a series of cell function assays to examine the role of TGF-beta-Smad4 signaling in tongue squamous cell carcinoma. We observed the effects of TGF-beta1 on the growth and metastatic potential of the tongue squamous cell carcinoma cell line Ts, which expresses lower level of Smad4 protein. We found that Smad4 could decrease TGF-beta1-induced cell proliferation, and that Smad4 overexpression promoted Ts cell apoptosis. In Ts vector control cells, TGF-beta1 increased the expression of TbetaRII, as well as MMP-2, and enhanced cell invasion through the basement membrane, and then induced cell metastasis. However in Ts cells stably expressing Smad4, Smad4 mediated TGF-beta1-induced p21 expression promoted cell apoptosis and inhibited cell proliferation, delayed MMP-2 expression, and decreased cell metastasis. Therefore, TGF-beta1 plays distinct roles in the Smad4-dependent and -independent signaling pathways.