Loss Of SMAD4 Expression Research Articles

Smad4 inactivation predicts for worse prognosis and response to fluorouracil-based treatment in colorectal cancer

AimsTo determine whether expression of Smad4, a tumour suppressor found to be absent in 10% of colorectal cancer (CRC), is associated with outcomes in patients with CRC.MethodsTumour samples from 241...

Prognostic significance of p16 and Smad4 deleted expression in patients with lung adenocarcinoma

Objective To investigate the loss of p16 and Smad4 expression in human lung adenocarcinoma and their relationship with the clinicopathological characteristics and prognosis.Methods The protein expression of p16 and Smad4 in 120 cases of lung adenocarcinoma tissues was detected.The relationship between the expression of these proteins and clinicopathological features was analyzed,and the application of p16 and Smad4 in disease prognosis was evaluated.Results The loss frequency of p16 and Smad4 in lung adenocarcinoma tissues was 45.0％ and 62.5％,respectively.Loss of p16 expression was associated with lymph node metastasis (P ＜ 0.01) and clinicopathological stage (P ＜ 0.01).Loss of Smad4 expression was associated with tumor size (P ＜ 0.05),lymph node metastasis (P ＜ 0.05),and tumor differentiation (P ＜ 0.05).Loss of p16 and Smad4 expression was negatively correlated with overall survival,and the overall survival time in patients with two genetic loss was shortest (P ＜ 0.01).The Spearman correlation analysis showed that the expression of p16 had a positive correlation with the expression of Smad4 (r =0.182,P ＜ 0.05).Conclusion The loss of p1 6 and Smad4 expression in lung adenocarcinoma was associated with malignant biological behaviors and poor prognosis. Key words: p16 gene ; Smad4 gene ; Lung neoplasms ; Prognosis

Abstract 2474: Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma

Abstract Introduction: Cetuximab is a monoclonal antibody that increases survival in head and neck squamous cell carcinoma (HNSCC) patients when used in combination with radiation or chemotherapy. However, this EGFR targeted-agent only has modest activity when administered as a monotherapy and a majority of patients develop therapeutic resistance. Previous work has established the TGF-beta signaling pathway has a critical role in HNSCC development and progression. Modulation of this pathway, and more specifically loss of Smad4 expression, is a common means of developing drug resistance in many cancers. To determine the role of Smad4 loss in HNSCC development, metastasis, and cetuximab resistance, Smad4 was knocked down or reexpressed in HNSCC cell lines and evaluated in vitro and in vivo. Methods: Smad4 was knocked-down in SCC61 following stable introduction of Smad4-targeting shRNA. Using stable lentiviral transduction, Smad4 was reexpressed in FaDu, a HNSCC cell line which lacks de novo expression of Smad4. Following modulation, changes in cetuximab sensitivity were determined using Matrigel colony formation assays. The effects of Smad4 modulation on various signal transduction pathways were determined by Western blot analysis with total and phospho-specific antibodies. Changes in metastatic potential were evaluated using an orthotopic in vivo model. The subsequent development of lymph node metastases were monitored utilizing luciferin-based imaging. Key proteins associated with metastasis were also assayed using ELISA and protein antibody arrays. Results: Smad4 loss increased the growth of SCC61 and FaDu and Smad4 status correlated with cetuximab resistance in both cells. Western blot analysis demonstrated Smad4 loss activates JNK and MAPK signaling. Consequently, co-treatment of Smad4 knockdown cells with cetuximab and a JNK inhibitor reversed the previously observed therapeutic resistance. Smad4 knockdown also increased lymph node metastasis in the orthotopic in vivo model. Protein array analysis determined Smad4 knockdown increased the expression of amphiregulin, FGF2, and VEGF, pathways capable of promoting metastasis. Expression of these Smad4-sensitive targets was inhibited with the use of a MAPK inhibitor. Conclusion: We determined Smad4 loss enhances cetuximab resistance and in vivo metastasis development in HNSCC. Additionally, JNK and MAPK pathway activation play an important role in mediating this process. Our results suggest Smad4 downregulation plays a critical role for prognosis of HNSCC patients, and JNK and MAPK inhibition may contribute to improve therapeutic outcome of those patients. Citation Format: Hiroyuki Ozawa, Haixia Cheng, Elana J. Fertig, Jason D. Howard, Ana Markovic, Robert Hughes, Jimena Perez, Harry Quon, Christine H. Chung. Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2014-2474

Loss of SMAD4 Protein Expression is Associated With High Tumor Grade and Poor Prognosis in Disseminated Appendiceal Mucinous Neoplasms

The distinction between low-grade and high-grade disseminated appendiceal mucinous neoplasms is of critical importance in assessing prognosis and guiding patient therapy. SMAD4 encodes a protein that is a central component of the TGFβ signal transduction pathway, and loss of SMAD4 expression has been associated with poor prognosis in carcinomas of the gastrointestinal tract. We reviewed the clinicopathologic and molecular features of 109 disseminated appendiceal mucinous neoplasms identified over an 8-year period at our institution in an attempt to: (1) correlate SMAD4 immunohistochemical expression with tumor grade; and (2) assess the prognostic significance of SMAD4 expression in predicting overall survival. Compared with tumors demonstrating preserved SMAD4 expression, tumors with loss of SMAD4 expression more frequently exhibited high cytologic grade (85% vs. 50%, P=0.035), high cellularity (100% vs. 45%, P<0.001), and destructive invasion (100% vs. 55%, P=0.001). SMAD4 expression significantly correlated with overall tumor grade (P=0.003): all 13 tumors with loss of SMAD4 expression were high grade, whereas all 42 low-grade tumors displayed preserved SMAD4 expression. A significantly higher proportion of tumors with loss of SMAD4 immunohistochemical expression demonstrated loss of heterozygosity at chromosome 18q (38%) compared with tumors with preserved SMAD4 expression (11%) (P=0.049), suggesting that loss of SMAD4 expression is due to genomic deletion in a high proportion of cases. Patients with SMAD4-negative tumors had significantly worse overall survival in comparison with patients with preserved SMAD4 expression (log rank P=0.023). However, our multivariable analysis found that SMAD4 expression was not independent of overall tumor grade in predicting overall survival. Our results indicate that loss of SMAD4 immunohistochemical expression is associated with loss of heterozygosity at chromosome 18q and is always associated with aggressive histologic features in disseminated appendiceal mucinous neoplasms. SMAD4 immunohistochemistry may be a useful ancillary study in select cases of disseminated appendiceal neoplasia, in which the distinction between low-grade and high-grade tumors is difficult.

Pancreatic ductal adenocarcinoma with autoimmune pancreatitis-like histologic and immunohistochemical features

Autoimmune pancreatitis (AIP) often manifests as a mass lesion causing obstructive jaundice, clinically mimicking pancreatic carcinoma. A diagnosis of AIP may obviate the need for surgical resection, as most patients respond to steroid treatment. However, it is not clear whether these 2 conditions can coexist. In this study, 105 specimens resected for pancreatic ductal adenocarcinoma (PDAC) that also have changes of chronic pancreatitis were examined for features considered to be characteristic of AIP. Of 105 cases of PDAC with changes of chronic pancreatitis, 10 (9.5%) exhibited histologic features of AIP, including exuberant fibrosis, lymphoplasmacytic infiltration, obliterative phlebitis, or granulocytic epithelial lesions. Of these 10 cases, 7 had more than 20 immunoglobulin G4+ plasma cells per high-power field. Of these 7 cases, 5 were analyzed for Kirsten rat sarcoma viral oncogene mutation and SMAD4 expression. Three cases showed K-ras mutation and/or loss of SMAD4 expression in benign AIP-like areas. These findings suggest 2 possibilities: first, AIP-like lesions may occur in a small but significant portion of PDAC cases; second, some PDACs may arise in a background of AIP. Therefore, caution is necessary when making a diagnosis of AIP by needle biopsy of a mass lesion, and patients with a tentative AIP diagnosis should be closely followed up clinically.

Disruption of Smad4 Expression in T Cells Leads to IgA Nephropathy-Like Manifestations

The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (Smad4co/co;Lck-cre). Loss of Smad4 expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that Smad4co/co;Lck-cre mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the β1, 4-galactosyltransferases (β4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced β4GalT2 and β4GalT4 mRNA levels in B cells. These findings indicate that Smad4co/co;Lck-cre mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype.

Inactivation of Smad4 is a prognostic factor in intrahepatic cholangiocarcinoma

Background Smad4 is found mutated in many cancers. It acts as a tumor suppressor in the regulation of TGF-β 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 &lt;0.05). The detection of Smad4 may be helpful in determining the degree of malignancy and prognosis of ICC.

Failure Patterns in Resected Pancreas Adenocarcinoma

To determine whether SMAD4 expression is associated with recurrence pattern after resection for pancreatic ductal adenocarcinoma (PDA). SMAD4 expression status has been reported to be associated with patterns of failure in PDA, but studies have not examined recurrence patterns after resection. A tissue microarray was constructed including 127 patients with resected PDA and either short-term (<12 months) or long-term (>30 months) survival. SMAD4 expression was evaluated by immunohistochemistry and categorized as present or lost in tumor cells. Conventional pathologic features (lymph node metastases, positive resection margin, poor grade, and tumor size) were recorded, and disease-specific outcomes (eg, recurrence pattern and early cancer-specific mortality) were determined. Loss of SMAD4 expression in pancreatic adenocarcinoma was identified in 40 of 127 patients (32%). SMAD4 loss occurred in 27% of patients who experienced isolated local recurrence, 33% of patients with a distant recurrence, 33% of patients who experienced local and distant site recurrences, and 25% of patients who were without evidence of recurrence (Fisher exact, P = 0.9). In a multivariate analysis, the presence of regional lymph node metastases was the only factor associated with the development of distant metastases (odds ratio = 4.7, P = 0.02). SMAD4 was neither associated with recurrence pattern (odds ratio = 0.9, P = 0.9) nor associated with early death (odds ratio = 0.5, P = 0.15). Primary tumor SMAD4 expression status was not a predictor of recurrence pattern in a large cohort of patients with resected PDA.

SMAD4 gene mutation and prognosis of pancreatic adenocarcinoma.

180 Background: Pancreatic adenocarcinoma remains resistant to many key cytotoxic chemotherapeutic agents and novel targeted therapies. The molecular heterogeneity of this cancer may account for therapy failures to date, although the growing arsenal of novel targeted agents could translate into patient survival. A better understanding of the cellular and molecular features of advanced disease will afford new opportunities for investigation, therapeutic intervention and clinical management of patients afflicted with pancreatic cancer. Methods: Data of 46 patients with pancreatic adenocarcinoma were analyzed. The clinicopathological parameters, the histologic features were determined and correlated to the stage at initial diagnosis and patterns of failure (locally advanced v metastatic disease). Using tissue microarray, we assessed the relationship of SMAD4 expression with the overall survival of patients. Results: Among the 46 treated patients, 32 underwent pancreaticoduodenectomy. 40% of patients died with metastatic disease and 15 % died with locally advance evolution. Loss of SMAD4 expression was found in 22% of patients and seems to be correlated with a high grade histologic features and progression to metastasis disease. Complementary data about correlation between the mutational status of SMAD4 and survival will be presented during congress. Conclusions: SMAD4 gene inactivation seems to be associated with poorer prognosis and disease progression. Prospective validation of SMAD4 as a predictive biomarker may personalize treatment strategies for patients with pancreatic cancer.

Contribution of CXCR4 and SMAD4 in predicting disease progression pattern and benefit from adjuvant chemotherapy in resected pancreatic adenocarcinoma

Prognosis of patients with pancreatic adenocarcinoma is poor. Many prognostic biomarkers have been tested, but most studies included heterogeneous patients. We aimed to investigate the prognostic and/or predictive values of four relevant biomarkers in a multicentric cohort of patients. A total of 471 patients who had resected pancreatic adenocarcinoma were included. Using tissue microarray, we assessed the relationship of biomarker expressions with the overall survival: Smad4, type II TGF-β receptor, CXCR4, and LKB1. High CXCR4 expression was found to be the only independent negative prognostic biomarker [hazard ratio (HR) = 1.74; P < 0.0001]. In addition, it was significantly associated with a distant relapse pattern (HR = 2.19; P < 0.0001) and was the strongest prognostic factor compared with clinicopathological factors. In patients who did not received adjuvant treatment, there was a trend toward decrease in the overall survival for negative Smad4 expression. Loss of Smad4 expression was not correlated with recurrence pattern but was shown to be predictive for adjuvant chemotherapy (CT) benefit (HR = 0.59; P = 0.002). CXCR4 is a strong independent prognostic biomarker associated with distant metastatic recurrence and appears as an attractive target to be evaluated in pancreatic adenocarcinoma. Negative SMAD4 expression should be considered as a potential predictor of adjuvant CT benefit.

Concordant repression and aberrant methylation of transforming growth factor-beta signaling pathway genes occurs early in gastric cardia adenocarcinoma

The loss of transforming growth factor-β (TGF-β) response due to the dysregulation of TGF-β receptor type I (TGFBR1), type II (TGFBR2) and Smad4 is well known for its contribution to oncogenesis, although the role of the genes of TGF-β/Smad signalling pathway in gastric cardia adenocarcinoma (GCA) is poorly understood. In the present study, the methylation status and expression of TGF-β receptor type I (TGFBR1), type II (TGFBR2), and Smad4 was investigated in GCA and dysplasia. MSP approach was used to detect the methylation status of TGFBR1, TGFBR2, and Smad4. Immunohistochemistry and quantitative RT-PCR methods were used respectively to examine the protein and mRNA expression of them in tissues. The methylation frequency of TGFBR1 and TGFBR2 in the tissues of high grade dysplasia and GCA was significantly higher than that in corresponding normal tissues (p < 0.01) and was significantly associated with mRNA and protein expression of the two genes (p < 0.05). The methylation frequency of Smad4 in the 30 ~ 171 sites was higher than that in the -248 ~ 26 sites and was associated with the loss of Smad4 expression. The decreased expression of TGFBR1, TGFBR2 and Smad4 was correlated with increased expression of TGF-β1 in GCA. In all, these data suggest that methylation of TGFBR1, TGFBR2 and Smad4 may exist in the gastric cardia dysplasia stages and plays an important role in these genes silencing and subsequently affect the TGF-β/Smad signaling pathway.

Concordant Promoter Methylation of Transforming Growth Factor-Beta Receptor Types I and II Occurs Early in Esophageal Squamous Cell Carcinoma

IntroductionTransforming growth factor-β (TGF-β) is a pleiotropic growth factor with multiple functions through its type I (TGFBR1) and type II (TGFBR2) receptors. A reduction or loss of expression of TGFBRs enables cancer cells to escape the growth inhibitory effect of TGF-β and to gain a growth advantage. ObjectiveThe promoter methylation status and expression of TGFBR1, TGFBR2 and Smad4 gene were investigated in esophageal squamous cell carcinoma (ESCC). DesignMethylation-specific polymerase chain reaction approach was used to detect the methylation status. Immunohistochemistry and reverse transcription-polymerase chain reaction method were used to examine the protein and messenger RNA expression, respectively. ResultsBoth the ESCC and the high-grade dysplastic tissues showed hypermethylation of TGFBR1 and TGFBR2, and the hyper-methylation of TGFBR1 and TGFBR2 in ESCC tissues was significantly associated with decreased messenger RNA and protein expression (P < 0.05). When stratified for tumor lymph node metastasis stages, TGFBR1 and TGFBR2 gene methylation was more frequent in stage III and stage IV tumor tissues than that in stage I and stage II tumor tissues (P < 0.05). Simultaneous methylation of the 2 receptors was progressively increased along with the increasing of tumor lymph node metastasis stage and decreasing of histological differentiation. Smad4 hypermethylation was only detected in 7 ESCC tumor tissues and associated with the loss of Smad4 expression. The decreased protein expression of TGFBR1, TGFBR2 and Smad4 was correlated with increased expression of TGF-β1 in ESCC. ConclusionsThese data suggest that promoter methylation of TGFBR1 and TGFBR2 may exist in the early stage of ESCC and play important roles in TGFBR1 and TGFBR2 gene silencing.

Abstract 4491: Transforming growth factor (TGF) ≤ pathway and clinical outcome of pancreatic cancer

Abstract Background: TGFβ acts both as a tumor suppressor or tumor promoter depending upon cellular context and stage of tumor progression. TGFβ exerts its effects through the TGFβR1/ R2 receptors and the SMAD transcription regulators. We investigated the prognostic value of TGFβ signaling biomarkers in pancreatic cancer. Methods: We measured plasma TGFβ1 level using Meso Scale Discovery Multi-array® Human TGFβ1 Assay in 643 pancreatic cancer patients. TGFβR2 (Novus) and SMAD4 (Proteintech) protein expression were measured in 86 biopsies using immunohistochemisty (IHC). IHC intensity was scored 0-3. Percentage IHC-positive cancer cells were 0-3 (0: 0%+, 1: 1-10%+, 2: 10-25%+, 3: &amp;gt;25%+). Final IHC score was measured as intensity × percentage. We also genotyped 3 SNPs of TGFB1 gene using the Taqman assay. Kaplan-Meier and log-rank tests were used to analyze overall survival (OS) by TGFβ1 level. Multivariate Cox proportional hazards models with relevant clinical covariates were used to assess the relationship between IHC score and OS. Results: In patients with locally advanced and metastatic disease (n = 355), there was a significant association of plasma TGFβ1 with OS. OS of the top quartile of TGFβ1 levels (&amp;gt;19.05 ng/mL) vs. those with low level was 27.7 weeks vs. 40 weeks, respectively [log-rank p = 0.0125, adjusted for baseline CA 19-9 and performance status (PS)]. No significant association between plasma TGFβ1 and OS was noted in surgical patients (n = 288). In the multivariate Cox model, after adjusting for baseline stage, CA 19-9, PS, age and TGFβR2 expression, complete loss of SMAD4 expression (IHC score 0) was significantly associated with lower OS [HR (95% CI): 1.85 (1.06-3.23), p = 0.03]. Progressive disease on gemcitabine-based therapy was more likely in SMAD4 IHC 0 group as compared with higher score (46.5% vs. 38.1% progressed; chi square p = 0.069). TGFB1 –1346T&amp;gt;C CT/TT genotype correlated with shorter OS [HR (95% CI): 1.21 (1.02-1.45), p = 0.03] after adjusting for clinical variables. Conclusions: This large retrospective study suggests an important stage-dependant role of the TGFβ pathway in pancreatic cancer. Targeting this pathway in advanced pancreatic cancer may have therapeutic relevance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4491. doi:1538-7445.AM2012-4491

Transforming growth factor (TGF) beta pathway and clinical outcome of pancreatic cancer.

201 Background: TGF-beta acts both as a tumor suppressor or tumor promoter depending upon cellular context and stage of tumor progression. TGF-β exerts its effects through the TGF-beta-R1/ R2 receptors and the SMAD transcription regulators. We investigated the prognostic value of TGFβ signaling biomarkers in pancreatic cancer. Methods: We measured plasma TGF-beta-1 level using Meso Scale Discovery Multi-array Human TGF-beta-1 Assay in 643 pancreatic cancer patients. TGF-beta-R2 and SMAD4 protein expression were measured in 86 biopsies using immunohistochemisty (IHC). IHC intensity was scored 0-3. Percentage IHC-positive cancer cells were 0-3 (0: 0%+, 1: 1-10%+, 2: 10-25%+, 3: &gt;25%+). Final IHC score was measured as intensity x percentage. We also genotyped 3 SNPs of TGFB1 gene using the Taqman assay. Kaplan-Meier and log-rank tests were used to analyze overall survival (OS) by TGFβ1 level. Multivariate Cox proportional hazards models with relevant clinical covariates were used to assess the relationship between IHC score and OS. Results: In patients with locally advanced and metastatic disease (n = 355), there was a significant association of plasma TGF-beta-1 with overall survival (OS). OS of the top quartile of TGF-beta-1 levels (&gt;19.05 ng/mL) vs. those with low level was 27.7 weeks vs. 40 weeks, respectively [log-rank p = 0.0125, adjusted for baseline CA 19-9 and performance status (PS)]. No significant association between plasma TGF-beta-1 and OS was noted in surgical patients (n=288). In the multivariate Cox model, after adjusting for baseline stage, CA 19-9, PS, age and TGF-beta-R2 expression, complete loss of SMAD4 expression (IHC score 0) was significantly associated with lower OS (HR: 1.85, 95% CI: 1.06-3.23; p = 0.03). Progressive disease on gemcitabine-based therapy was more likely in SMAD4 IHC 0 group as compared with higher score (46.5% vs. 38.1% progressed; chi square p = 0.069). TGFB1 -1346T&gt;C CT/TT genotype correlated with shorter OS [HR (95% CI): 1.21 (1.02-1.45), p=0.03] after adjusting for clinical variables. Conclusions: This large retrospective study suggests an important stage-dependant role of the TGF-beta pathway in pancreatic cancer. Targeting this pathway in advanced pancreatic cancer may have therapeutic relevance.

Alterations of Smad expression and activation in defining 2 subtypes of human head and neck squamous cell carcinoma

We postulated that disruptions of the canonical transforming growth factor-beta (TGF-β)/Smad signaling pathway might contribute to the development of head and neck squamous cell carcinoma (HNSCC). A cohort of 798 HNSCC tumor samples from 346 patients were analyzed by immunohistochemistry (IHC) to define the pattern of expression of (phospho)Smad2, (phospho)Smad3, and Smad4. We found that 19%, 40%, and 12% of HNSCC specimens failed to express pSmad2, pSmad3, or Smad4, respectively. Loss of Smad2/3 activation was observed in 8.5% of specimens. In addition, 4% of specimens failed to express only Smad4. Moreover, patients with pSmad2/3-negative tumors had a significantly better overall survival than that of those whose tumors expressed activated Smad2/3. In contrast, loss of Smad4 expression did not have prognostic significance. Our results indicate that HNSCC in which Smad2/3 are inactivated or in which Smad4 expression is lost represent 2 distinct tumor subtypes with different clinical outcomes.

Loss of Smad4 expression inhibits epithelial-mesenchymal transition in SMMC-7721 cells

Loss of Smad4 expression inhibits epithelial-mesenchymal transition in SMMC-7721 cells

High SMAD4 levels appear in microsatellite instability and hypermethylated colon cancers, and indicate a better prognosis

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in western countries. CRC are commonly divided in cancers showing microsatellite stability (MSS) or microsatellite instability (MSI). A more novel classification is dependent on promoter hypermethylation of CpG islands (the CpG island methylator phenotype, CIMP), where cancers show high, low or negative methylation status. SMAD4, located on chromosome 18q, has been thoroughly investigated during the last years. Loss of SMAD4 expression has been reported to correlate with poor CRC patient prognosis. In this study, we analyze the impact of SMAD4 expression on prognosis in relation to MSI screening status and CIMP status. Four hundred and seventy-nine paraffin-embedded specimens of CRC were examined for nuclear SMAD4 expression using immunohistochemistry. The tumors were scored loss (-), moderate (+) and high (++) expressing tumors. Loss of SMAD4 correlated significantly with decreased survival in all colon cancer patients. High SMAD4 expression, however, was significantly associated with increased survival, especially in colon cancer patients, which has undergone potential curative surgery. In addition, in MSI tumors and CIMP-high tumors, high SMAD4 expression was significantly related to increase in survival, while loss of SMAD4 resulted in a significantly poorer prognosis. SMAD4 expression was not correlated to prognosis in rectal cancer cases. We conclude, loss of SMAD4 indicates a poor prognosis in colon cancer patients. The novel findings that high SMAD4 expression predicts a better prognosis suggests that SMAD4 immunohistochemistry could constitute a prognostic marker in combination with CIMP and MSI screening status.

Smad4 May Help to Identify a Subset of Colorectal Cancer Patients with Early Recurrence after Curative Therapy

Loss of Smad4 function is associated with the acquisition of advanced colorectal cancer phenotypes. We investigated the role of Smad4 as a prognostic marker after curative therapy. Four hundred and twenty nine consecutive colorectal cancers were analyzed by tissue microarray-based immunohistochemical assay. Smad4 protein was expressed in 61.5% (24/39), 53.1% (77/145), 41.3% (78/189) and 34.8% (16/46) of stage I, II, III and IV cancers, respectively. Lymphovascular invasion and lymph node metastasis were strongly correlated with the loss of Smad4 expression (p<0.0001 and p=0.002, respectively). Disease-free survival did not differ between Smad4-positive and Smad4-negative cancers. In stage III disease, time to recurrence after curative therapy was shorter in the Smad4-negative than in the Smad4- positive cancers (20.1±15.1 vs. 34.6 ± 34.1 months, p=0.035). Smad4 protein is of no value in predicting recurrence after curative therapy in colorectal cancer, but it may be helpful in identifying a subset of patients with early recurrence after curative therapy.

Smad4 loss is associated with fewer S100A8-positive monocytes in colorectal tumors and attenuated response to S100A8 in colorectal and pancreatic cancer cells

S100A8 and its dimerization partner S100A9 are emerging as important chemokines in cancer. We previously reported that Smad4-negative pancreatic tumors contain fewer stromal S100A8-positive monocytes than their Smad4-positive counterparts. Here, we studied S100A8/A9-expressing cells in colorectal tumors relating their presence to clinicopathological parameters and Smad4 status. Two-dimensional gel electrophoresis (n = 12) revealed variation in the levels of S100A8 protein in colorectal cancer tumors, whereas immunohistochemical analysis (n = 313) showed variation in the numbers of stromal S100A8-positive and S100A9-positive cells. Loss of Smad4 expression was observed in 42/304 (14%) colorectal tumors and was associated with reduced numbers of S100A8-positive (P = 0.03) but not S100A9-positive stromal cells (P = 0.26). High S100A9 cell counts were associated with large tumor sizes (P = 0.0006) and poor differentiation grade (P = 0.036). However, neither S100A8 nor S100A9 cell counts predicted poor survival, except for patients with Smad4-negative tumors (P = 0.02). To address the impact of environmental S100A8/A9 chemokines on tumor cells, we examined the effects of exogenously added S100A8 and S100A9 proteins on cellular migration and proliferation of colorectal and pancreatic cancer cells. S100A8 and S100A9 enhanced migration and proliferation in Smad4-positive and Smad4-negative cancer cells. However, transient depletion of Smad4 resulted in loss of responsiveness to exogenous S100A8, but not S100A9. S100A8 and S100A9 activated Smad4 signaling as evidenced by phosphorylation of Smad2/3; blockade of the receptor for the advanced glycation end products inhibited this response. In conclusion, Smad4 loss alters the tumor's interaction with stromal myeloid cells and the tumor cells' response to the stromal chemokine, S100A8.

M1941 Expression of Proteins Involved in Carcinogenic Pathways in Patients With Primary Sclerosing Cholangitis and Colitis-Associated Colorectal Cancer

Background : The molecular background of colitis-associated carcinogenesis differs from the sporadic pathway in frequency and moment of underlying genetic mutations. Patients with IBD and concurrent primary sclerosing cholangitis (PSC) have a significantly higher risk of developing colorectal cancer (CRC) than patients without PSC. Although themolecular background behind colorectal carcinogenesis is gradually elucidated, still little is known about this specific subgroup of tumors. Aim : To compare the expression of proteins involved in different carcinogenic mechanisms in PSC-IBD-related CRC with other subtypes of colorectal tumors, such as IBD-CRC, sporadic CRC and Lynch syndrome-related CRC, and to determine differences in the molecular background of inflammatoryand noninflammatory-associated CRCs. Methods : A tissue micro-array was constructed with colonic samples from 7 groups: (1) healthy subjects, (2) IBD-patients, (3) IBD-related CRC, (4) PSC-IBD-related CRC, (5) Lynch syndrome-related CRC, (6) sporadic left-sided CRC, and (7) sporadic right-sided CRC. Each group consisted of 8-20 patients. Immunohistochemistry was performed using monoclonal antibodies against β-catenin, Cylin D1, CD44V6, p53, SMAD4, and COX2. Results : Median age of all patients was 55 years (range 12-92 years), with 61% of cases being male. Of all cases in groups 2-4, ulcerative colitis was present in 53% of cases. In groups 3, 4 and 5, 29%, 60%, and 50% of CRCs, respectively, were proximally located, (p=0.3). In 55.4% of all tumors, CRC had metastasized to regional lymph nodes or distant sites. Overexpression of p53 was found in the majority of all tumors (60%), except the Lynch-CRC group (12.5%) (p=0.44). Expression of Cyclin D1 was found in 56% and 88% of the inflammatory-related tumors and sporadic tumors, respectively (p= 0.002). After correction for tumor stages and age this difference remained significant. No statistical differences were found in expression of nuclear β-catenin, CD44V6, and COX-2. Loss of expression of SMAD4 was twice as high in the inflammatory-related tumor groups compared to the sporadic tumors, 26% and 9%, respectively (p=0.09). No association was found between the expression of β-catenin andCyclin D1.Conclusion : Cyclin D1 expression is significantly less in the inflammatory-related tumor group than in the sporadic CRCs and there appears to be loss of SMAD4 expression as well: this suggests a difference in colorectal carcinogenesis between these two groups.