Tanner J. Freeman

Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of β-catenin.

BACKGROUND & AIMS Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor β and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of β-catenin messenger RNA (mRNA) and Wnt signaling. METHODS We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APC(Δ1638/+)) and (APC(Δ1638/+)) × (K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection. RESULTS In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc(APC(Δ1638/+)), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes compared with adenomas from APC(Δ1638/+) mice that expressed Smad4. CONCLUSIONS Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor β, BMP, and Wnt signaling pathways in progression of CRC.

XIAP Monoubiquitylates Groucho/TLE to Promote Canonical Wnt Signaling

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.

Selenoprotein P influences colitis-induced tumorigenesis by mediating stemness and oxidative damage

Patients with inflammatory bowel disease are at increased risk for colon cancer due to augmented oxidative stress. These patients also have compromised antioxidant defenses as the result of nutritional deficiencies. The micronutrient selenium is essential for selenoprotein production and is transported from the liver to target tissues via selenoprotein P (SEPP1). Target tissues also produce SEPP1, which is thought to possess an endogenous antioxidant function. Here, we have shown that mice with Sepp1 haploinsufficiency or mutations that disrupt either the selenium transport or the enzymatic domain of SEPP1 exhibit increased colitis-associated carcinogenesis as the result of increased genomic instability and promotion of a protumorigenic microenvironment. Reduced SEPP1 function markedly increased M2-polarized macrophages, indicating a role for SEPP1 in macrophage polarization and immune function. Furthermore, compared with partial loss, complete loss of SEPP1 substantially reduced tumor burden, in part due to increased apoptosis. Using intestinal organoid cultures, we found that, compared with those from WT animals, Sepp1-null cultures display increased stem cell characteristics that are coupled with increased ROS production, DNA damage, proliferation, decreased cell survival, and modulation of WNT signaling in response to H2O2-mediated oxidative stress. Together, these data demonstrate that SEPP1 influences inflammatory tumorigenesis by affecting genomic stability, the inflammatory microenvironment, and epithelial stem cell functions.

Elevated ALCAM Shedding in Colorectal Cancer Correlates with Poor Patient Outcome

Molecular biomarkers of cancer are needed to assist histologic staging in the selection of treatment, outcome risk stratification, and patient prognosis. This is particularly important for patients with early-stage disease. We show that shedding of the extracellular domain of activated leukocyte cell adhesion molecule (ALCAM) is prognostic for outcome in patients with colorectal cancer (CRC). Previous reports on the prognostic value of ALCAM expression in CRC have been contradictory and inconclusive. This study clarifies the prognostic value of ALCAM by visualizing ectodomain shedding using a dual stain that detects both the extracellular and the intracellular domains in formalin-fixed tissue. Using this novel assay, 105 patients with primary CRCs and 12 normal mucosa samples were evaluated. ALCAM shedding, defined as detection of the intracellular domain in the absence of the corresponding extracellular domain, was significantly elevated in patients with CRC and correlated with reduced survival. Conversely, retention of intact ALCAM was associated with improved survival, thereby confirming that ALCAM shedding is associated with poor patient outcome. Importantly, analysis of patients with stage II CRC showed that disease-specific survival is significantly reduced for patients with elevated ALCAM shedding (P = 0.01; HR, 3.0), suggesting that ALCAM shedding can identify patients with early-stage disease at risk of rapid progression.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer

Background & Aims Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor β (TGFβ) family signaling through SMAD4 in colonic epithelial cells. Methods The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFβ1 and bone morphogenetic protein 2. Results Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis–associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis–associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFβ signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions TGFβ suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

Abstract 4429: CCL20 regulation via TGFB /BMP signaling pathway in murine immortalized colonocytes and colonic epithelium

TGFβ and BMP signaling through receptor-mediated activation of SMAD proteins is crucial for normal homeostasis in intestinal epithelium. Alterations in this pathway contribute to the development of colorectal cancers. To understand the role of these pathways in homeostasis of the colon we conditionally deleted loxP-flanked alleles of Smad4 in adult mouse colon. We used Lrig1CreERT allele to achieve greater than 80% SMAD4 gene deletion and a CK19CreERT allele for mosaic deletion in colonic epithelium. Our data indicates that SMAD4 activity regulates multiple interactions between intestinal epithelium cells and surrounding stroma and that deletion of SMAD4 in adult mouse colonic epithelial cells alters expression of cytokines, increases immune cell infiltration and predisposes mice to tumorigenesis. Increases in the chemokine CCL20 have been reported in CRC tumors beginning at the adenoma stage and it may regulate both Tregs and Th-17 cells in an active immune response. Luminex cytokine/chemokine array analysis revealed increased levels of CCL20 protein in colonic mucosa and RNAseq data revealed that when SMAD4 is knocked out in the epithelium there is an upregulation of CCL20 and CCR6 (receptor) mRNA in the epithelium and stroma, respectively. To further explore the regulation of CCL20, we used immortalized mouse colonocytes (IMC cells) to further determine how the TGFβ and BMP signaling pathways may be involved in CCL20 expression. We found that CCL20 mRNA expression was suppressed by TGFβ1 and BMP2 and increased by inhibitors of these pathways. The inhibition of CCL20 by the TGFβ and BMP2 pathways occurs even in the presence of a known strong inducer of CCL20, such as TNFα. We have further observed in mice induced to undergo deletion of SMAD4 in the intestinal epithelium that treatment with DSS results in 100% of mice developing mucinous adenocarcinomas in the distal colon. We conclude that ligand-dependent Smad4-mediated signaling provides important negative homeostatic regulation of CCL20 expression in colonic epithelium. Loss of Smad4 results in increased CCL20 expression and we are currently testing the hypothesis that CCL20 overexpression contributes to the inflammation induced carcinogenesis in this mouse model. Citation Format: Tasia D. Brown, Anna Means, Tanner Freeman, Connie Weaver, Keith Wilson, R. Daniel Beauchamp. CCL20 regulation via TGFB /BMP signaling pathway in murine immortalized colonocytes and colonic epithelium. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4429.

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

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 >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

Correlation of elevated ALCAM shedding with patient outcome in colorectal cancer.

46 Background: ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a cell-cell adhesion protein that has been highlighted as a putative biomarker for the progression of many cancers, including colorectal cancer (CRC). Current prognosis for CRC patients predominantly rely on pathologic staging classification. Although TNM staging successfully stratifies high-risk patients, there is significant variability in the rate of disease progression within each stage. Previous studies evaluating ALCAM as a biomarker for predicting colorectal cancer patient survival have published conflicting and inconclusive results. The aim of this present study was to investigate the prognostic value of ALCAM shedding, quantified using a dual stain that detects both the extracellular and the intracellular domains in formalin-fixed tissue, in colorectal patients. METHODS A novel dual-immunohistological detection of ALCAM shedding was performed on tissue microarrays constructed from 75 colorectal primary tumors and 9 healthy control specimens. Tissue was assessed by CellProfiler image analysis to obtain quantitative estimates of ALCAM shedding, defined as the detection of the intracellular domain the absence of the extracellular domain of ALCAM. Two tailed p values ≤ 0.05 were considered to be statistically significant. RESULTS ALCAM shedding was elevated in 33/79 (41.8%) of tissue from CRC patients compared to healthy controls (p=0.0239). Univariate analysis of CRC patients, stages I-IV, revealed a statistical significance correlating high ALCAM shedding with worse overall survival (p=0.0401). Moreover, ALCAM shedding is a predictor of overall survival (p=0.432) and more notably, disease-specific survival in stage II patients (p=0.0098). CONCLUSIONS Detection of ALCAM shedding allows for stratification of patients according to their risk for poor long-term outcome. Considering that ALCAM is altered in a number of malignancies, the clinical correlation of ALCAM shedding to patient outcome is likely to extend beyond CRC to other cancers.

Abstract 499: NFATc1 is a novel invasion promoter in colorectal cancer

Colorectal carcinoma is the third leading cause of cancer-related death in the United States. In order to understand the mechanism/signaling pathways responsible for invasion, migration and metastasis in colorectal cancer, our lab successfully modeled human cancer invasion/metastasis using mouse colon cancer cells. We have developed an integrative and comparative computational approach to reveal transcriptional regulatory mechanisms underlying colon cancer progression. Applying this approach to fourteen human colorectal cancer (CRC) microarray data sets and to one microarray dataset from an immunocompetent mouse model of metastasis, we identified known and novel transcriptional regulators in CRC. Among these transcriptional regulators, the Nuclear Factor of Activated T cells (NFAT) family of transcription factors play a central role in inducible gene transcription in various signaling pathways including regulation of cell differentiation, development, adaption, immune system response, inflammation, adipocyte metabolism, and lipolysis, and carcinogenesis. We found that an NFAT-driven transcriptional program significantly correlates with disease-free survival in multiple CRC Stage II patients. Using quantitative real time RT-PCR, we validated high NFATc1 expression in a subgroup of stage II colorectal cancer patients and differential expression of predicted NFATc1 targets in the same patients. The tumor-associated NFATc1 co-regulated gene signature was associated with worse clinical survival outcomes in stage II colorectal cancer patients. In comparison with parental MC38 mouse colon cancer cells, NFATc1 expression is increased in MC38 met cells selected for invasive and metastatic ability, RNAi-based inhibition of NFATc1 expression in the MC38 met cells resulted in decreased invasiveness in a transendothelial invasion model. Our studies suggest a role for NFATc1 as a tumor promoting transcription factor that contributes to invasion in colon cancer. 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 499. doi:1538-7445.AM2012-499