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Epithelial metaplasia is a common adaptation to chronic inflammatory processes and can be associated with increased risk of dysplasia and cancer. The distal colon of patients with inflammatory bowel disease (IBD) commonly shows crypt architectural distortion and Paneth cell metaplasia (PCM), and IBD patients also carry increased risk of colitis-associated dysplasia and cancer (CAC). Loss of SATB2 expression (Special AT-rich binding 2 protein, a colon-restricted chromatin remodeler) has recently been shown to distinguish colitis-associated dysplasia and CAC from sporadic disease. Here we report non-diffuse heterogeneous patterns of SATB2 loss across non-dysplastic distal colon biopsies from IBD patients (n=20). This cohort was specifically curated to include biopsies with well-developed histologic features of villiform growth and PCM. Notably, CDX2 was strongly expressed and P53 showed a wild-type immunolabeling pattern across our non-dysplastic cohort, regardless of SATB2 immunolabeling pattern. Our findings fit with recent murine studies in which colon-specificSatb2deletion resulted in histologic conversion of colonic mucosa to small intestinal-like mucosa, including emergence of villi and Paneth cells. Taken together, we show that SATB2 loss is associated with a preneoplastic metaplastic response to chronic injury in human IBD and chronic colitis, reframing PCM more broadly as small intestinal metaplasia. We propose that inflammation-associated SATB2 loss mediates a remodeled chromatin landscape permissive for dysplasia and CAC.

Although much is known about the gene mutations required to drive colorectal cancer (CRC) initiation, the tissue-specific selective microenvironments in which neoplasia arises remains less characterized. Here, we determined whether modulation of intestinal stem cell niche morphogens alone can exert a neoplasia-relevant selective pressure on normal colonic epithelium. Using adult stem cell-derived murine colonic epithelial organoids (colonoids), we employed a strategy of sustained withdrawal of EGF and EGFR inhibition to select for and expand survivors. EGFR-signaling-independent (iEGFR) colonoids emerged over rounds of selection and expansion. Colonoids derived from a mouse model of chronic mucosal injury showed an enhanced ability to adapt to EGFR inhibition. Whole-exome and transcriptomic analyses of iEGFR colonoids demonstrated acquisition of deleterious mutations and altered expression of genes implicated in EGF signaling, pyroptosis, and CRC. iEGFR colonoids acquired dysplasia-associated cytomorphologic changes, an increased proliferative rate, and the ability to survive independently of other required niche factors. These changes were accompanied by emergence of aneuploidy and chromosomal instability; further, the observed mitotic segregation errors were significantly associated with loss of interkinetic nuclear migration, a fundamental and dynamic process underlying intestinal epithelial homeostasis. This study provides key evidence that chromosomal instability and other phenotypes associated with neoplasia can be induced ex vivo via adaptation to EGF withdrawal in normal and stably euploid colonic epithelium, without introducing cancer-associated driver mutations. In addition, prior mucosal injury accelerates this evolutionary process.Key definitionsColonoids: adult stem cell-derived colonic epithelial organoidsiEGFR: in vitro selective conditions devoid of EGF (epidermal growth factor) and including an EGFR (EGF receptor) inhibitor 1iEGFR colonoids: colonoids tolerant to iEGFR culture conditions with growth and survival similar to unselected passage-matched controlsINM: Interkinetic nuclear migration

Epithelial metaplasia, or the histologically evident replacement of one differentiated somatic cell type with another, offers insights into mucosal homeostasis as an adaptation to diverse inflammatory pathogenic stimuli. The cellular framework for metaplasia can be created by coordination of tissue-specific transcription factors (TFs) with epigenetic regulators of chromatin remodeling, allowing altered TF binding of inflammation-associated exposed genomic loci. For example, intestinal metaplasia (aberrant goblet cell differentiation and CDX2 expression, a pan-intestinal TF) is a common metaplastic response to chronic injury in the upper gastrointestinal tract, associated with increased risk of epithelial dysplasia and cancer. Inflammatory bowel disease (IBD) is an etiologically complex chronic inflammatory disease characterized by relapsing cycles of intestinal injury and healing. IBD patients carry increased risk of colitis-associated dysplasia and colorectal cancer (CAC) proportional to disease duration, extent, and severity. SATB2, or special AT-rich binding protein 2, is a chromatin organizing protein with homeostatic roles in osteoblastic, neural, and colonic differentiation. Intriguingly, others have recently shown that SATB2 is uniquely lost in human IBD dysplasia and CAC, in contrast to sporadic CRC where its expression is typically retained. (By contrast, CDX2 shows retained expression in both CAC and CRC.) Other recent work has shown that colon-specific Satb2 knockout in mice results in conversion of colon to small intestine, including emergence of villi and Paneth cells. Colonic mucosal biopsies from chronic IBD patients show histologic features reminiscent of small intestine with so-called Paneth cell metaplasia (PCM) and villiform, distorted growth. The molecular mediators of this epithelial remodeling in IBD are unknown. We hypothesized that SATB2 loss could result in a form of “small intestinal metaplasia” in human IBD colon. To investigate this, we selected a cohort of 20 IBD distal colon mucosal biopsies with marked crypt architectural changes and PCM and performed SATB2 immunostaining (n=7 Crohn’s disease, n=13 ulcerative colitis). Indeed, 19 of 20 such samples showed striking evidence of SATB2 loss. The pattern of SATB2 loss was heterogeneous; some tissue fragments showed complete epithelial loss as expected in small intestine, whereas others showed patchy loss in alternating crypts, even some showing heterogeneous expression within the same crypt. As positive and negative controls, we included normal distal colon and terminal ileum samples, observing expected intact and negative SATB2 expression, respectively. Of note, CDX2 was strongly expressed across the entire cohort, even in IBD tissue with loss of SATB2 expression. We propose that SATB2 loss is a pre-neoplastic metaplastic response to chronic injury in human colon. Future work will investigate how SATB2 loss enables a plasticity state permissive for emergence of TP53 mutations, dysplasia, and CAC. Citation Format: Maged Zeineldin, Reem Abu-Shamma, Tatianna Larman. Implicating SATB2 loss in a metaplasia-dysplasia sequence in human inflammatory bowel disease [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B026.

Neuroblastoma is a leading cause of cancer-related death in children. Accumulated data suggest that differentiation arrest of the neural-crest-derived sympathoadrenal lineage contributes to neuroblastoma formation. The developmental arrest of these cell types explains many biological features of the disease, including its cellular heterogeneity, mutational spectrum, spontaneous regression, and response to drugs that induce tumor cell differentiation. In this review, we provide evidence that supports the notion that arrested neural-crest-derived progenitor cells give rise to neuroblastoma and discuss how this concept could be exploited for clinical management of the disease.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX–histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma., In most cancers, mutations that lead to oncogene activation and tumor suppressor inactivation synergize to promote tumorigenesis. However, in neuroblastomas, MYCN amplification and ATRX mutations are mutually exclusive and incompatible.

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.

SUMMARYAggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX–histone chaperone complex and that induced by MYCN-mediated metabolic reprogramming leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.

Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.

Mutations and structural alterations of the SWI/SNF-like chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation, X-linked) have been reported at high frequency in a number of adult and pediatric tumors. However, the consequences of ATRX mutations in cancer and their impact on the epigenome remain ill defined. Particularly intriguing are the large N-terminal deletions of ATRX in neuroblastoma that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase domain. We hereby demonstrate that ATRX IFF proteins have distinct genomic distribution compared to wild type (WT) ATRX and are absent from H3K9me3-enriched chromatin, yet bound to active promoters. We find REST (RE-1 Silencing Transcription Factor) as one such ATRX IFF target that is activated and promotes silencing of neuronal differentiation genes. Notably, we uncover that ATRX IFF cells display exquisite sensitivity to EZH2 inhibitors (EZH2i) in vitro and in vivo, due in part to derepression of neurogenesis genes, including a subset of REST targets. Examination of the epigenomic landscape of neuroblastoma tumor specimens harboring ATRX IFFs reveals that H3K27me3 occupies a subset of neurogenesis genes that are transcriptionally silenced and sensitive to EZH2i in our cell-based assays. In summary, this study demonstrates that ATRX structural alterations are not loss-of-function as predicted, and supports EZH2i as a key therapeutic strategy for ATRX IFF neuroblastoma.

Adenomatous polyposis coli (APC) is best known for its crucial role in colorectal cancer suppression. Rodent models with various Apc mutations have enabled experimental validation of different Apc functions in tumors and normal tissues. Since the development of the first mouse model with a germline Apc mutation in the early 1990s, 20 other Apc mouse and rat models have been generated. This article compares and contrasts currently available Apc rodent models with particular emphasis on providing potential explanations for their reported variation in three areas: (i) intestinal polyp multiplicity, (ii) intestinal polyp distribution, and (iii) extraintestinal phenotypes. Cancer Res; 73(8); 2389–99. ©2013 AACR.

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt-signaling pathway by targeting the proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription cofactor for genes required for cell proliferation such as cyclin-D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and the cytoplasm, with nuclear APC implicated in the inhibition of Wnt target gene expression. Adopting a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals (NLSs) of Apc (Apc(mNLS)). Apc(mNLS/mNLS) mice are viable and fractionation of mouse embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc as compared with Apc(+/+) MEFs. The levels of Apc and β-catenin protein were not significantly altered in small intestinal epithelia from Apc(mNLS/mNLS) mice. Compared with Apc(+/+) mice, Apc(mNLS/mNLS) mice showed increased proliferation in epithelial cells from the jejunum, ileum and colon. These same tissues from Apc(mNLS/mNLS) mice showed more mRNA from three genes upregulated in response to canonical Wnt signal, c-Myc, axin-2 and cyclin-D1, and less mRNA from Hath-1, which is downregulated in response to Wnt. These observations suggest a role for nuclear Apc in the inhibition of canonical Wnt signaling and the control of epithelial proliferation in intestinal tissue. Furthermore, we found Apc(Min/+) mice, which harbor a mutation that truncates Apc, to have an increased polyp size and multiplicity if they also carry the Apc(mNLS) allele. Taken together, this analysis of the novel Apc(mNLS) mouse model supports a role for nuclear Apc in the control of Wnt target genes, intestinal epithelial cell proliferation and polyp formation.

Neuroblastoma is a clinically heterogeneous tumor responsible for 15% of cancer-related deaths in children. Older age at diagnosis and MYCN amplification are associated with poor prognosis in neuroblastoma. Previously, sequencing stage 4 neuroblastoma tumors revealed recurrent mutations in the chromatin remodeler ATRX that are significantly associated with older age and mutually exclusive with MYCN amplification. Here, we expanded our genetic analysis of neuroblastoma across all ages and stages. We sequenced 475 neuroblastoma tumors and found that the incidence of ATRX mutations is correlated with older age at diagnosis and advanced stages of the disease. We also directly studied the mutual exclusivity between ATRX mutations and MYCN amplification using both cultured cells and orthotopic mouse models. We found that targeting ATRX using shRNA or CRISPR-Cas-9 reduces cell viability in MYCN-amplified cells. Additionally, induction of MYCN overexpression results in cell death in ATRX-mutant neuroblastoma cells. To characterize the molecular basis of the ATRX-mutations/MYCN-amplification incompatibility, we studied epigenetic, metabolic, and replicative changes associated with MYCN amplification in these cells. We discovered that induction of MYCN overexpression provokes mitochondrial abnormalities, metabolic reprograming, and replicative stress in ATRX mutant cells. Collectively, these data show synthetic lethality between ATRX mutations and MYCN amplification and suggest that the chromatin remodeler ATRX is important in reducing replicative stress associated with MYCN amplification in neuroblastoma. These data will also help in identifying therapeutic targets, which will reduce the morbidity and mortality associated with high-risk neuroblastoma. Citation Format: Maged Zeineldin, Sara M. Federico, Lyra Griffith, Jongyre Jeon, Xiang Chen, John Easton, Jianrong Wu, Shenghua Mao, Yanling Liu, Arlene Naranjo, Alberto S. Pappo, Michael D. Hogarty, Michael A. Dyer. Synthetic lethality between ATRX mutations and MYCN amplification in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR11.

Age and stage of disease at diagnosis are associated with outcome in neuroblastoma. A previous study identified ATRX point mutations and in-frame deletions in older patients with stage 4 disease. To validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables, we collaborated with the Children’s Oncology Group and sequenced the entire ATRX genomic locus in 475 neuroblastoma tumor samples to validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables. Older age at diagnosis, stage 4 disease, and unfavorable histology were significantly associated with mutations in the ATRX gene; MYCN amplification was mutually exclusive from ATRX mutations. To directly test if ATRX mutations and MYCN amplification are incompatible in neuroblastoma, we mutated the ATRX gene in MYCN amplified neuroblastoma cell lines and ectopically expressed MYCN in ATRX mutant neuroblastoma cell lines. Both approaches showed that ATRX and MYCN amplification are incompatible in neuroblastoma in culture and in vivo. To gain a better understanding of the underlying molecular and cellular mechanisms of this incompatibility, we performed a series of experiments including telomere analysis, epigenetic profiling, electron microscopic analysis of dying cells, and metabolic profiling. We discovered that induction of MYCN expression in ATRX mutant neuroblastoma cells leads to metabolic reprogramming, mitochondrial dysfunction, increased reactive oxygen species, and replicative stress. We propose that this leads to synthetic lethality in ATRX mutant neuroblastomas because of the underlying replicative stress in those cells as a result of dysfunction of this essential histone chaperone complex. Citation Format: Maged Zeineldin, Sara Federico, Xiang Chen, Beisi Xu, Elizabeth Stewart, Arlene Naranjo, Michael D. Hogarty, Michael A. Dyer. MYCN amplification and ATRX mutations are incompatible in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA18.

Mutation of tumor suppressor adenomatous polyposis coli (APC) initiates most colorectal cancers and chronic colitis increases risk. APC is a nucleo-cytoplasmic shuttling protein, best known for antagonizing Wnt signaling by forming a cytoplasmic complex that marks β-catenin for degradation. Using our unique mouse model with compromised nuclear Apc import (Apc(mNLS)), we show that Apc(mNLS/mNLS) mice have increased susceptibility to tumorigenesis induced with azoxymethane (AOM) and dextran sodium sulfate (DSS). The AOM-DSS-induced colon adenoma histopathology, proliferation, apoptosis, stem cell number and β-catenin and Kras mutation spectra were similar in Apc(mNLS/mNLS) and Apc(+/+) mice. However, AOM-DSS-treated Apc(mNLS/mNLS) mice showed more weight loss, more lymphoid follicles and edema, and increased colon shortening than treated Apc(+/+) mice, indicating a colitis predisposition. To test this directly, we induced acute colitis with a 7 day DSS treatment followed by 5 days of recovery. Compared with Apc(+/+) mice, DSS-treated Apc(mNLS/mNLS) mice developed more severe colitis based on clinical grade and histopathology. Apc(mNLS/mNLS) mice also had higher lymphocytic infiltration and reduced expression of stem cell markers, suggesting an increased propensity for chronic inflammation. Moreover, colons from DSS-treated Apc(mNLS/mNLS) mice showed fewer goblet cells and reduced Muc2 expression. Even in untreated Apc(mNLS/mNLS) mice, there were significantly fewer goblet cells in jejuna, and a modest decrease in colonocyte Muc2 expression compared with Apc(+/+) mice. Colonocytes from untreated Apc(mNLS/mNLS) mice also showed increased expression of inflammatory mediators cyclooxygenase-2 (Cox-2) and macrophage inflammatory protein-2 (MIP-2). These findings reveal novel functions for nuclear Apc in goblet cell differentiation and protection against inflammation-induced colon tumorigenesis.

Mutation of tumor suppressor gene Adenomatous polyposis coli (APC) is an initiating step in most colon cancers. This review summarizes Apc models in mice and rats, with particular concentration on those most recently developed, phenotypic variation among different models, and genotype/ phenotype correlations.

The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In this protocol, epithelial cell adhesions are weekend by chelating Ca +2 ions followed by mechanical separation of the cells by vortexing. Analysis of protein levels and gene expression patterns in isolated epithelial cells versus whole GI tissue minimizes the potential for confounding contributions from contaminating stromal cells.

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide. As with other cancers, CRC is a genetic disease, however, several risk factors including diet and chronic colitis predispose to the disease. Mutations in the tumor suppressor adenomatous polyposis coli (APC) initiate most cases of CRC. Recent data from mouse models suggest that APC mutations and colitis are not completely independent factors in colorectal carcinogenesis. Here, we review the evidence supporting an interaction between APC muta- tions and chronic colitis. We will also discuss possible pathophysiologic mechanisms behind this interaction.

Mutation of the tumor suppressor gene Adenomatous Polyposis Coli (APC) is considered an initiating event in the development of most intestinal tumors. Although much effort has been spent determining functions of the APC protein, to date little is known about the mechanisms that regulate cellular APC levels. Here we report that in cultured cells, induction of a heat-shock response, via heat or compounds such as the HSP90 inhibitor 17-AAG, resulted in increased levels of APC. A novel non-toxic small molecule that we developed, KN1, also induced a heat-shock response and led to increased APC levels in both cultured cells and mice. To investigate the effect of heat-shock response induction and elevation of Apc level in intestinal tumorigenesis, we performed a series of experiments treating mice with either KN1 or 17-AAG. We tested these compounds on two mouse models with different germline Apc mutations, ApcMin/+ and Apc1322T/+. In both cases, a moderate dose of either drug did not change tumor burden, but surprisingly altered the distribution of intestinal polyps. In a third mouse model, colonic tumors were induced via administration of the mutagen azoxymethane (AOM) and colon irritant dextran sodium sulfate (DSS) rather than by germline Apc mutation. In AOM-DSS-treated mice, KN1 reduced tumor incidence, multiplicity, and size. Moreover, KN1-treated mice lost significantly less weight and had smaller spleens than vehicle-treated mice, suggesting KN1 may also suppress colitis. We conclude that induction of the heat-shock response affects intestinal tumorigenesis in germline Apc-mutant and colitis-induced tumor models. Citation Format: Maged Zeineldin, William McGuinness, Brian Blagg, Roger Rajewski, Kristi L. Neufeld. Induction of the heat-shock response upregulates the tumor suppressor APC and alters intestinal tumorigenesis in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2728. doi:10.1158/1538-7445.AM2013-2728

Colon cancer is the second leading cause of cancer-related mortality in the United States, resulting in over 50,000 deaths annually. Approximately 80% of colon cancers begin with a mutation in the gene coding for the tumor suppressor protein Adenomatous Polyposis Coli, APC. APC protein can shuttle between the cytoplasm and the nucleus of cells, facilitated by two nuclear localization signals (NLS) and multiple nuclear export signals. To better understand functions of nuclear APC, our lab generated a “knock-in” mouse model with mutations introduced that inactivate both Apc NLS. These ApcmNLS/mNLS mice show a dramatic decrease in nuclear Apc. We previously showed increased proliferation and Wnt target gene expression in intestinal epithelial cells from ApcmNLS/mNLS mice, suggesting a role for nuclear APC in inhibition of proliferation and Wnt signaling. We also showed increased tumor number and size in ApcMin mice if they also harbor the ApcmNLS allele. Here we examine the role of nuclear Apc in intestinal epithelial differentiation and stem cell homeostasis. Paraffin-embedded tissue from the intestines of Apc+/+ and ApcmNLS/mNLS mice was sectioned and stained for markers of enterocytes (alkaline phosphatase) and goblet cells (alcian blue). Quantification of the positive cells indicated that ApcmNLS/mNLS mice had fewer differentiated enterocytes and goblet cells than their wild-type littermates. Intestines from ApcmNLS/mNLS mice were also stained for markers of quiescent and active stem cells, DCAMKL-1 and Lgr5 respectively. Quantification of the various stem cell populations will be described. Together, our data supports a role for nuclear Apc in promoting enterocyte and goblet cell differentiation, in suppression of tumors, and in inhibiting intestinal cell proliferation and Wnt signaling. Information gathered from analysis of ApcmNLS/mNLS mice will contribute to our understanding of the functions of nuclear APC in tumor suppression and ultimately the mechanism of intestinal tumorigenesis. Citation Format: Matthew A. Miller, Maged Zeineldin, Kristi L. Neufeld. Demonstrating a role for nuclear Adenomatous polyposis coli in intestinal cell differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1978. doi:10.1158/1538-7445.AM2013-1978

Because mutation of tumor suppressor gene APC is the initiating step in most colorectal cancers (CRC), understanding the full spectrum of APC functions will illuminate better diagnostic, preventive and therapeutic strategies for the disease. Although APC shuttles between the cytoplasm and nucleus, testing proposed roles for nuclear APC in the context of a whole organism was only recently made possible using a mouse model compromised for nuclear Apc which we generated by introducing germline mutations that inactivate the Apc nuclear localization signals (ApcmNLS). Our previous analysis of ApcmNLS mice revealed a role for nuclear Apc in regulation of Wnt signal transduction and intestinal cell proliferation as well as in tumor suppression. In humans, chronic colitis significantly increases CRC risk and APC mutations occur late in this cancer progression. In the current study, we show increased expression of inflammatory mediators cyclo-oxygenase-2 (Cox-2) and macrophage-inflammatory-protein-2 (MIP-2) in colon epithelial cells from ApcmNLS/mNLS mice, suggesting a role for nuclear Apc in suppressing colitis-mediated colon cancer. To test this hypothesis, we initiated colon tumors with a single injection of the mutagen, azoxymethane (AOM) and promoted the tumors with repeated oral administration of dextran sodium sulfate (DSS) to induce colonic inflammation. When treated with AOM-DSS, ApcmNLS/mNLS mice developed more colonic tumors than treated wildtype mice. Tumors from treated ApcmNLS/mNLS and wildtype mice had the same spectrum of β-catenin mutations, proliferation rates and histopathological features, consistent with the ApcmNLS allele enhancing colitis-associated tumor initiation rather than progression. ApcmNLS/mNLS mice had increased weight loss and colonic lymphoid follicles implicating nuclear Apc in suppression of AOM-DSS-induced colitis. These findings reveal novel functions for nuclear Apc and also indicate a critical protective role for Apc early in inflammation-induced colon tumorigenesis. 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 1342. doi:1538-7445.AM2012-1342