Loss Of Smad4 Research Articles

Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors.

Non-small cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer, however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC datasets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms including mutation, homozygous deletion, and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4−/− mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage while Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4 deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4 deficient tumors can be exploited by specific therapeutic strategies.

Smad4 loss in esophageal adenocarcinoma is associated with an increased propensity for disease recurrence and poor survival.

Previously regarded as a rare neoplasm, the incidence of esophageal adenocarcinoma has risen rapidly in recent decades. It is often discovered late in the disease process and has a dismal prognosis. Current prognostic markers including clinical, radiographic, and histopathologic findings have limited utility and do not consider the biology of this deadly disease. Genome-wide analyses have identified SMAD4 inactivation in a subset of tumors. Although Smad4 has been extensively studied in other gastrointestinal malignancies, its role in esophageal adenocarcinoma remains to be defined. Herein, we show, in a large cohort of esophageal adenocarcinomas, Smad4 loss by immunohistochemistry in 21 of 205 (10%) tumors and that Smad4 loss correlated with increased postoperative recurrence (P=0.040). Further, patients whose tumors lacked Smad4 had shorter time to recurrence (TTR) (P=0.007) and poor overall survival (OS) (P=0.011). The median TTR and OS of patients with Smad4-negative tumors was 13 and 16 months, respectively, as compared with 23 and 22 months, respectively, among patients with Smad4-positive tumors. In multivariate analyses, Smad4 loss was a prognostic factor for both TTR and OS, independent of histologic grade, lymphovascular invasion, perineural invasion, tumor stage, and lymph node status. Considering Smad4 loss correlated with postoperative locoregional and/or distant metastases, Smad4 was also assessed in a separate cohort of 5 locoregional recurrences and 43 metastatic esophageal adenocarcinomas. In contrast to primary tumors, a higher prevalence of Smad4 loss was observed in metastatic disease (44% vs. 10%). In summary, loss of Smad4 protein expression is an independent prognostic factor for TTR and OS that correlates with increased propensity for disease recurrence and poor survival in patients with esophageal adenocarcinoma after surgical resection.

Role of stomatin-like protein 2 in colorectal cancer tumorigenesis via alteration of TGF-β1/Smad4 signaling

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death in the US, resulting in over a half-million deaths annually. Stomatin-like protein 2 (SLP-2) is a novel member of the stomatin superfamily and is found in several types of human tumors. However, whether it is expressed in human CRC is unknown. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted.TGF-beta signaling occurs through Smads 2/3/4, which translocate to the nucleus to regulate transcription; TGF-beta has tumor-suppressive effects in some tumor models and pro-metastatic effects in others. In patients with colorectal cancer (CRC), mutations or reduced levels of Smad4 have been correlated with reduced survival. A total of 93 patients with CRC was examined. In the present study, we aimed to explore the diagnostic value of SLP-2 in patients with CRC and to investigate whether CRC expression is regulated by transforming growth factor-β (TGF-β)/SMAD4 signaling. The expression of SLP-2 mRNA and protein was examined by immunohistochemistry (IHC) and qPCR, respectively. A standard immunohistochemical method was applied to stain the slides for hematoxylin and eosin (H&E), Tunnel, Phospho-Smad, E-cadherin, and β-catenin. Our data, revealed for the first time the role of SLP-2 overexpression CRC through downregulation of Smad4. Thus, the loss of Smad4 in colorectal cancer appears to switch TGF-β from tumor suppressor to a tumor promoter, and this group of patients could potentially benefit from SLP-2 inhibitor therapy. Furthermore; present study suggest that SLP-2 may be considered as a useful diagnostic marker for metastatic CRC.

Clinical outcome and expression of mutant P53, P16, and Smad4 in lung adenocarcinoma: a prospective study.

BackgroundWhole-exome sequencing has shown that lung adenocarcinoma (LAC) can be driven by mutant genes, including TP53, P16, and Smad4. The aim of this study was to clarify protein alterations of P53, P16, and Smad4 and to explore their correlations between the protein alterations and clinical outcome.MethodsWe investigated associations among P53 mutant (P53Mut) expression, and P16 and Smad4 loss-of-expression, with clinical outcome in 120 LAC patients who underwent curative resection, using immunohistochemical (IHC) methods.ResultsOf the 120 patients, 76 (63.3%) expressed P53Mut protein, whereas 54 (45.0%) loss of P16 expressed and 75 (62.5%) loss of Smad4 expressed. P53Mut expression was associated with tumor size (P = 0.041) and pathological stage (P = 0.025). Loss of P16 expression was associated with lymph node metastasis (P = 0.001) and pathological stage (P < 0.001). Loss of Smad4 expression was associated with tumor size (P = 0.033), lymph node metastasis (P = 0.014), pathological stage (P = 0.017), and tumor differentiation (P = 0.022). Kaplan-Meier survival analysis showed that tumor size (P = 0.031), lymph node metastasis (P < 0.001), pathological stage (P < 0.001), P53Mut protein expression (P = 0.038), and loss of p16 or Smad4 expression (P < 0.001) were significantly associated with shorter overall survival(OS), whereas multivariate analysis indicated that lymph node metastasis (P = 0.014) and loss of p16 or Smad4 expression (P < 0.001) were independent prognostic factors. Analysis of protein combinations showed patients with more alterations had poorer survival (P < 0.001). Spearman correlation analysis showed that loss of Smad4 expression inversely correlated with expression of P53Mut (r = −0.196, P = 0.032) and positively with lost P16 expression (r =0.182, P = 0.047).ConclusionsThe findings indicate that IHC status of P53Mut, P16, and Smad4 may predict patient outcomes in LAC.

Smad4 Loss Synergizes with TGFα Overexpression in Promoting Pancreatic Metaplasia, PanIN Development, and Fibrosis

AimsWhile overexpression of TGFα has been reported in human pancreatic ductal adenocarcinoma (PDAC), mice with overexpressed TGFα develop premalignant pancreatic acinar-to-ductal metaplasia (ADM) but not PDAC. TGF-β signaling pathway is pivotal to the development of PDAC and tissue fibrosis. Here we sought to investigate the interplay between TGFα and TGF-β signaling in pancreatic tumorigenesis and fibrosis, namely via Smad4 inactivation.MethodsThe MT-TGFα mouse was crossed with a new Smad4 conditional knock-out mouse (Smad4flox/flox;p48-Cre or S4) to generate Smad4flox/flox;MT-TGFα;p48-Cre (STP). After TGFα overexpression was induced with zinc sulfate water for eight months, the pancreata of the STP, MT-TGFα, and S4 mice were examined for tumor development and fibrotic responses. PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC). Qualitative analysis of fibrosis was analyzed by Trichrome Masson and Sirius Red staining, while vimentin was used for quantification. Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR.ResultsOur STP mice exhibited advanced ADM, increased fibrosis, increased numbers of PanIN lesions, overexpression of chronic pancreatitis-related marker Muc6, and elevated expression of desmoplasia-associated marker Col1A1, compared to the MT-TGFα mice. The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas. The phenotype of the STP mice represents a transient state from ADMs to PanINs, closely mimicking the interface area seen in human chronic pancreatitis associated with PDAC.ConclusionWe have documented a novel mouse model, the STP mice, which displayed histologic presentations reminiscent to those of human chronic pancreatitis with signs of early tumorigenesis. The STP mice could be a suitable animal model for interrogating the transition of chronic pancreatitis to pancreatic cancer.

ErbB2 Pathway Activation upon Smad4 Loss Promotes Lung Tumor Growth and Metastasis.

SUMMARYLung cancer remains the leading cause of cancer death. Genome sequencing of lung tumors from patients with squamous cell carcinoma has identified SMAD4 to be frequently mutated. Here, we use a mouse model to determine the molecular mechanisms by which Smad4 loss leads to lung cancer progression. Mice with ablation of Pten and Smad4 in airway epithelium develop metastatic adenosquamous tumors. Comparative transcriptomic and in vivo cistromic analyses determine that loss of PTEN and SMAD4 results in ELF3 and ErbB2 pathway activation due to decreased expression of ERRFI1, a negative regulator of ERBB2 in mouse and human cells. The combinatorial inhibition of ErbB2 and Akt signaling attenuate tumor progression and cell invasion, respectively. Expression profile analysis of human lung tumors substantiated the importance of the ErbB2/Akt/ELF3 signaling pathway as both a prognostic biomarker and a therapeutic drug target for treating lung cancer.

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...

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC.

Background:Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the β-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the β-catenin paradox.Methods:We analysed the expression patterns of SMAD4, p53 and β-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity.Results:Eighty-four percent of CRCs with high nuclear β-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner.Conclusions:The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

Deletion and down-regulation of SMAD4 gene in colorectal cancers in a Chinese population.

Colorectal cancer (CRC) is one of the most common types of human cancers. As a tumor suppressor, SMAD4 plays a key role in colorectal carcinogenesis and invasiveness. Copy number variations (CNVs) of the SMAD4 gene have been reported to be associated with cancer pathogenesis in array-based studies in different populations. Here we aimed to investigate the CNVs of the SMAD4 gene in a relatively large number of CRC patients from China. In the present study, we collected 147 Chinese CRC tumors as well as self-paired normal control tissues. Quantitative PCR was carried out to examine the copy number as well as the mRNA expression of the SMAD4 gene. Our results showed that the copy number deletions of SMAD4 were frequent in a relatively high percentage of CRC samples (34.7%, 51 out of 147). There was a positive correlation between the copy number decrease of SMAD4 and tumor progression in CRCs. Furthermore, copy number loss of SMAD4 was correlated with decreased mRNA expression. These findings suggested that the copy number deletions of SMAD4 were frequent in CRC patients from China and had the potential to serve as a diagnostic indicator, alone or in combination with other markers, for CRC.

Abstract 2963: Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development

Abstract Background: Inactivation of the TGF-β signaling occurs in 50-75% of all colorectal cancer cases. Down-regulation of Smad4, the central mediator of TGF-β signaling, occurs in &amp;gt;50% of stage III patients. How Smad4 contributes to normal intestinal homeostasis and functions as a tumor suppressor are incompletely understood. We reported that β-catenin mRNA expression is inhibited by BMP-mediated Smad4 signaling, and loss of Smad4 signaling results in biologically significant amplification of the Wnt/β-catenin signal. Here, we report on the consequences of Smad4 loss for intestinal homeostasis. Methods: We generated mice genetically engineered to undergo inducible, tissue-specific deletion of Smad4 within intestinal epithelial cells. K19-CreERT x Smad4 flx/flx and Lrig1-CreERT x Smad4 flx/flx mice were treated with vehicle or tamoxifen to induce recombination at the Smad4 locus in the intestinal stem cell compartment. Intestines from sacrificed mice were cleansed for enteroid isolation, Ussing chamber studies, RNA-seq analysis and preservation by formalin fixation and paraffin embedding (FFPE). We validated Smad4 status via immunohistochemistry and stained FFPE sections for different cell populations within the crypt. To evaluate inflammatory cytokines, we performed Luminex cytokine array analysis on colonic mucosa from control and tamoxifen treated animals. To determine the role for Smad4 in intestinal response to injury, we also treated control and tamoxifen treated animals with dextran sodium sulfate (DSS). We utilized RNA-Seq analysis to determine an intestinal Smad4 loss gene signature. Results: Smad4 deficient crypts exhibited an expansion of the Ki67 positive cells in the proliferative zone and fewer Carbonic anhydrase II staining cells suggesting fewer mature enterocytes. We found that loss of Smad4 in the intestinal crypt is accompanied by an accumulation of pericryptal CD3+ lymphocytes and F4/80+ macrophages and is associated with increased intestinal permeability as measured by low molecular weight FITC Dextran using the Ussing chamber. Cytokine analysis revealed Macrophage Inflammatory Protein 3a (MIP3a) to be up-regulated in Smad4 knockout mice. Guided by our RNA-Seq analysis, we observe increased levels of pErk in Smad4 deplete tissue. We also observed that mice with intestinal epithelial Smad4 depletion developed advanced mucinous adenocarcinoma after recovery from an inflammatory stimulus induced by DSS. Conclusions: These results suggest that Smad4 loss disrupts intestinal homeostasis by enabling persistently high levels of Erk signaling to interfere with cell maturation, thereby contributing to increased inflammation. We postulate that the increased inflammation observed in Smad4 knockout mice leads to a tumorigenic environment. Citation Format: Tanner J. Freeman, Jillian Pope, Josh Smith, Daniel Sharbel, Kay Washington, Xi Chen, Rupesh Chaturvedi, Keith Wilson, Noah Shroyer, Punita Dhawan, Anna Means, Natasha G. Deane, R. Daniel Beauchamp. Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development. [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 2963. doi:10.1158/1538-7445.AM2014-2963

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

Smad4 acts as tumor suppressor by antagonizing lymphangiogenesis in colorectal cancer

Vascular endothelial growth factor-C (VEGF-C) is considered a vital promoter to tumor lymphangiogenesis. Smad4 is an important downstream mediator of transforming growth factor beta (TGF-β) signaling pathway. Smad4 is regarded as a tumor suppressor in colon cancer, but its role in lymphangiogenesis in colorectal cancer is still unclear. In the present study, we investigated the potential inhibiting effect of Smad4 in lymphangiogenesis, and its correlation with VEGF-C secretion in colon cancer. We investigated endogenous Smad4 expression in 7 human colorectal cancer cell lines, and found that six of colon cancer cell lines existed with loss of Smad4. Expression of Smad4 in SW480 cell line is extremely low. SW480 cells were used to establish a Smad4 over-expression model. We investigated the relationship between Smad4 and VEGF-C expression in SW480 cells and found that Smad4 can inhibit the expression of VEGF-C. In an in vitro assay, we found that overexpression of Smad4 can reduce migration and invasion ability in SW480 cells, but had no effect on cell proliferation. Interestingly, Smad4 definitely inhibited the growth in an in vivo assay, and the volumes of transplanted tumors which originated from Smad4 overexpressing SW480 cells in nude mice were smaller than those of normal SW480 cells. In addition, we also demonstrated that Smad4 can inhibit lymphangiogenesis in vivo by immunohistochemistry. In summary, our findings provide molecular evidence that Smad4 may reduce lymphangiogenesis of colon cancer cell by attenuating VEGF-C secretion and act as tumor suppressor by inhibiting migration, invasion and tumorigenicity.

Abstract 3831: Clinical significance of SMAD4 expression in resectable pancreatic cancer: correlation with tumor progression and recurrence pattern

Abstract [Background] SMAD4 is well recognized as contributing to pancreatic carcinogenesis. The recent studies suggest that SMAD4 expression pattern in pancreatic cancer is associated with tumor progression and eventual failure pattern. The SMAD4 protein expression is thought to be associated with a locally predominant progression pattern, whereas the loss of SMAD4 associated with distant metastases. In the current study, we validated the clinical significance of SMAD4 expression status as a biomarker of tumor progression and failure pattern in a large cohort of patients with resectable pancreatic cancer. [Materials and Methods] Surgical specimens were obtained from 174 pancreatic cancer patients who underwent resection at Nagoya University Hospital, Japan, during the period from April 2004 to March 2010. SMAD4 immunohistochemistry was performed in 174 patients and classified into SMAD4(+); staining or SMAD4(-); no staining. The correlation between clinicopathological parameters, failure pattern and prognosis was statistically analyzed. [Results] 1. 59.8% of patients was SMAD4(-) (104/174), whereas, 40.2% of patients was SMAD4(+) (70/174). The disease-specific survival in SMAD4(+) patients was significantly better than that in SMAD4(-) patients (median survival time; 36.2 months vs. 14.3 months, P &amp;lt; 0.001). SMAD4(-) had significantly more portal vein invasion, lymphatic invasion and neural invasion (P = 0.046, P = 0.029 and P = 0.035, respectively). Importantly, multivariate analysis revealed that SMAD4(-) was the most significant independent prognostic factor for pancreatic cancer. 2. In terms of the local spread pattern, the ratio of SMAD4(-) was 49.4% in the patients without vascular invasion, 61.9% in those with portal vein invasion, 76.5% in those with common hepatic artery invasion and 80.8% in those with superior mesenteric artery invasion, which showed the statistical significance (P = 0.013). 3. For postoperative failure pattern, the ratio of SMAD4(-) was 59.0% in the patients with local recurrence, 66.7% in those with both local and distant recurrence and 73.7% in those with distant recurrence, which showed that the patients who lost SMAD4 expression were more likely to have distant metastasis (P = 0.318). [Discussion] Our study demonstrated that the SMAD4 expression in resectable pancreatic cancer was a prognostic factor. The pancreatic cancer with SMAD4(+) was more likely to remain in local invasion, whereas, that with SMAD4(-) could develop to distant metastases. Preoperative assessment of SMAD4 expression in patients with resectable pancreatic cancer would support a treatment paradigm, where the local treatment like chemoradiation would be favored in patients harboring tumors with retained SMAD4 expression. On the contrary, systemic chemotherapy would be appropriate for the patients with absent SMAD4 expression. Citation Format: Suguru Yamada, Tsutomu Fujii, Mitsuro Kanda, Hiroyuki Sugimoto, Shuji Nomoto, Yasuhiro Kodera. Clinical significance of SMAD4 expression in resectable pancreatic cancer: correlation with tumor progression and recurrence pattern. [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 3831. doi:10.1158/1538-7445.AM2014-3831

Abstract 2258: Tempol diet provides chemoprotection against UV-induced skin cancer in mice with epidermal Smad4 deletion

Abstract Solar ultraviolet (UV) radiation is the primary cause of skin cancer. Non-melanoma skin cancer, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is the most common form of skin cancer and accounts for 2-3 million worldwide cases of cancer each year. Smad4 loss occurs frequently in human skin squamous cell carcinoma (SCC), and we have shown that keratinocyte specific Smad4 deletion in K14.Smad4-/- mice leads to reduced expression of DNA repair genes, increased DNA damage and increased susceptibility to UV-induced SCC. Since UV-irradiation leads to oxidative damage to DNA, and unrepaired DNA damage may lead to cancer, so we tested if administration of an antioxidant, 4-hydroxy-tempo (tempol) in the diet of WT and K14.Smad4-/- mice reduces UV-induced skin carcinogenesis. In the present study, we divided WT and K14.Smad4-/- mice into two groups each, one group was fed tempol chow while the other group was fed control chow. All mice were exposed to chronic UV irradiation for 38 weeks from 3 months of age. Indeed, the tempol diet led to delayed onset and reduced number of skin tumors in K14.Smad4-/- mice. Tempol diet led to reduced 8-hydroxyguanosine staining, indicative of reduced oxidative DNA damage, as well as reduced cox-2 expression in the skin of K14.Smad4-/- mice compared to K14.Smad4-/- mice on control diet. It is plausible that reduced inflammation in these mice led to reduced carcinogenesis, these and other mechanisms are being investigated. Citation Format: Doyel Mitra, Bian Li, Qinghong Zhang, James B. Mitchell, Xiao-Jing Wang. Tempol diet provides chemoprotection against UV-induced skin cancer in mice with epidermal Smad4 deletion. [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 2258. doi:10.1158/1538-7445.AM2014-2258

Cooperativity of E-cadherin and Smad4 loss to promote diffuse-type gastric adenocarcinoma and metastasis.

Loss of E-cadherin (CDH1), Smad4, and p53 has been shown to play an integral role in gastric, intestinal, and breast cancer formation. Compound conditional knockout mice for Smad4, p53, and E-cadherin were generated to define and compare the roles of these genes in gastric, intestinal, and breast cancer development by crossing with Pdx-1-Cre, Villin-Cre, and MMTV-Cre transgenic mice. Interestingly, gastric adenocarcinoma was significantly more frequent in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice than in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(+/+) mice, demonstrating that Cdh1 heterozygosity accelerates the development and progression of gastric adenocarcinoma, in combination with loss of Smad4 and p53. Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice developed gastric adenocarcinomas without E-cadherin expression. However, intestinal and mammary adenocarcinomas with the same genetic background retained E-cadherin expression and were phenotypically similar to mice with both wild-type Cdh1 alleles. Lung metastases were identified in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, but not in the other genotypes. Nuclear β-catenin accumulation was identified at the invasive tumor front of gastric adenocarcinomas arising in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. This phenotype was less prominent in mice with intact E-cadherin or Smad4, indicating that the inhibition of β-catenin signaling by E-cadherin or Smad4 downregulates signaling pathways involved in metastases in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. Knockdown of β-catenin significantly inhibited the migratory activity of Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) cell lines. Thus, loss of E-cadherin and Smad4 cooperates with p53 loss to promote the development and metastatic progression of gastric adenocarcinomas, with similarities to human gastric adenocarcinoma. This study demonstrates that inhibition of β-catenin is a converging node for the antimetastatic signaling pathways driven by E-cadherin and Smad4 in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, providing novel insights into mechanisms for gastric cancer metastasis.

Down-regulation of MicroRNA-494 via Loss of SMAD4 Increases FOXM1 and β-Catenin Signaling in Pancreatic Ductal Adenocarcinoma Cells

Dysregulation of β-catenin and the transcriptional activator FOXM1 mediate oncogenesis, but it is not clear how these proteins become dysregulated in tumors that do not typically carry mutations in adenomatous polyposis coli (APC) or β-catenin, such as pancreatic ductal adenocarcinomas (PDACs). We searched for microRNAs that regulate levels of FOXM1 in PDAC cells and samples from patients. We identified microRNAs that affect levels of FOXM1 in PDACs using bioinformatic, genetic, and pharmacologic approaches. We altered expression of the microRNA-494 (miR-494) in PDAC cell lines (AsPC-1 and PANC-1) and examined the effects on FOXM1 and β-catenin signaling and cell proliferation and colony formation. The cells were injected into immunocompromised mice and growth of xenograft tumors and liver metastases were measured. We performed immunohistochemical analyses of 10 paired PDAC and nontumor pancreatic tissue samples collected from untreated patients during surgery. We identified miR-494 as a negative regulator of FOXM1 levels in PDAC cells, and found that levels of this microRNA were reduced in PDAC specimens, compared with nontumor tissues. Loss of response of PDAC cells to transforming growth factor β, owing to SMAD4 deficiency, reduced expression of miR-494. Transgenic expression of miR-494 in PDAC cells produced the same effects as reducing expression of FOXM1 or blocking nuclear translocation of β-catenin, reducing cell proliferation, migration, and invasion, and increasing their sensitivity to gemcitabine. Reduced expression of miR-494 correlated with PDAC metastasis and reduced survival times of patients. Loss of SMAD4 in PDAC cells leads to reduced levels of miR-494, increased levels of FOXM1, and nuclear localization of β-catenin. miR-494 might be developed as a prognostic marker for patients with PDAC or a therapeutic target.

Loss of SMAD4 Alters BMP Signaling to Promote Colorectal Cancer Cell Metastasis via Activation of Rho and ROCK

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 β 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. 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. Activation of BMP signaling in SMAD4-negative cells altered proteinand 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 invitro. 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 β receptors. 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.

Loss of SMAD4 staining in pre-operative cell blocks is associated with distant metastases following pancreaticoduodenectomy with venous resection for pancreatic cancer

Venous resection of locally advanced pancreatic cancer is associated with increased morbidity and mortality; therefore identification of patients most likely to benefit from this aggressive surgical approach is an important goal. Loss of SMAD4 staining on resected specimens has been associated with outcomes. Few studies have evaluated the prognostic significance of SMAD4 staining of pre-operative cell blocks, which would be useful in clinical decision making for patients with locally advanced disease. Clinical data were retrospectively evaluated from all patients undergoing pancreaticoduodenectomy with venous resection. Immunohistochemical staining for SMAD4 was performed on pre-operative cell blocks and subsequent post-operative resections. One hundred seventeen patients underwent pancreaticoduodenectomy with venous resection. Sixty had sufficient specimens available for SMAD4 staining. SMAD4 loss was observed in 70% of resections and was associated with earlier time to metastatic disease. Pre-operative SMAD4 loss correlated well with post-operative staining and was associated with six times higher likelihood of developing metastases. In this pilot study, preoperative SMAD4 staining showed a strong correlation with postoperative staining and predicted metastases in locally advanced cancer. Preoperative SMAD4 status may be considered as one of several factors when selecting patients most likely to benefit from aggressive en bloc venous resection.

SMAD4 Loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells

BackgroundSMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.MethodsThe AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down.ResultsHere, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.ConclusionsThis study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

Loss of Smad4 in Sertoli and Leydig Cells Leads to Testicular Dysgenesis and Hemorrhagic Tumor Formation in Mice1

As the central component of canonical TGFbeta superfamily signaling, SMAD4 is a critical regulator of organ development, patterning, tumorigenesis, and many other biological processes. Because numerous TGFbeta superfamily ligands are expressed in developing testes, there may exist specific requirements for SMAD4 in individual testicular cell types. Previously, we reported that expansion of the fetal testis cords requires expression of SMAD4 by the Sertoli cell lineage. To further uncover the role of Smad4 in murine testes, we produced conditional knockout mice lacking Smad4 in either Leydig cells or in both Sertoli and Leydig cells simultaneously. Loss of Smad4 concomitantly in Sertoli and Leydig cells led to underdevelopment of the testis cords during fetal life and mild testicular dysgenesis in young adulthood (decreased testis size, partially dysgenic seminiferous tubules, and low sperm production). When the Sertoli/Leydig cell Smad4 conditional knockout mice aged (56- to 62-wk old), the testis phenotypes became exacerbated with the appearance of hemorrhagic tumors, Leydig cell adenomas, and a complete loss of spermatogenesis. In contrast, loss of Smad4 in Leydig cells alone did not appreciably alter fetal and adult testis development. Our findings support a cell type-specific requirement of Smad4 in testis development and suppression of testicular tumors.