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The mechanistic (or mammalian) target of rapamycin, mTOR, is a highly conserved PI3K-related kinase that regulates cell growth, proliferation, survival, transcription, and protein synthesis. It forms two different complexes, mTOR complex-1 (mTORC1) and -2 (mTORC2). Rapamycin is an allosteric inhibitor specific to mTORC1 and extends life and health span with chronic use. Previous research from our lab shows that an enteric release formulation of rapamycin, eRapa, enhances the lifespan of mice mutated for adenomatous polyposis coli (Apc) tumor suppressor (ApcMin/+ mice), which normally develop intestinal cancer. These mice model familial adenomatous polyposis (FAP) in humans. eRapa restored a normal (and in 60% of the mice longer) life span in female ApcMin/+ mice. Mutations in Apc, a part of the Wnt/β-catenin pathway, accelerate the initiation of the adeno-carcinoma in FAP, resulting in numerous colorectal polyps at a young age, and if left untreated these polyps progress to colorectal cancer.eRapa treatment prevents polyposis in ApcMin/+ mice but the precise mechanism of action remains to be determined. We used a cross sectional and survival approach to test the preventive effects of eRapa and gain mechanistic insights. Four-week-old male and female animals were put on a diet containing either 42 ppm of eRapa or empty microcapsules. A subset of the animals was sacrificed to harvest tissue for analysis at 16 weeks of age and the rest were allowed to live out their lifespans.eRapa prevented polyposis in the small intestine of ApcMin/+ mice with only a few to no tumors in both males and females. In addition to adenomas in the intestine, ApcMin/+ mice also suffer from severe anemia. Hematocrits from the animals showed anemia in the control mice, however, eRapa treated animals did not have anemia. There was a reduction in phosphorylation of ribosomal protein S6 in the small intestine tissue, showing suppression of mTORC1 activity with eRapa. Histological analysis revealed that this suppression occurred in the Paneth cells of the small intestinal crypt. The Paneth cells, along with the stem cells at the base of the crypt form a niche and signaling between this niche maintains homeostasis in the crypt. Although the polyps are believed to originate from stem cells, our results suggest involvement of Paneth cells in tumor prevention perhaps by signaling changes in the niche. These are novel effects of rapamycin and help define its mechanisms of cancer prevention. Citation Format: Manish Parihar, Sherry G. Dodds, Paul Hasty, Zelton Dave Sharp. mTORC1 suppression in Paneth cells prevents tumors in a mouse model of intestinal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 671.

The High Mobility Group A1 (HMGA1) gene is overexpressed in most poorly differentiated cancers and high levels portend adverse clinical outcomes, although the molecular mechanisms through which it functions are poorly understood. HMGA1 encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which modulate gene expression by bending chromatin and orchestrating the assembly of transcription factor complexes to DNA. HMGA1 is highly expressed during embryogenesis, but silenced in adult, differentiated tissues. Postnatally, HMGA1 expression is maintained in adult stem cells, such as intestinal stem cells (ISCs); however, its role in this setting has been unknown. Here, we report that Hmga1 overexpression in ISCs of transgenic mice drives expansion in the ISC compartment leading to hyperproliferation, aberrant crypt formation, and polyposis. Surprisingly, Hmga1 transgenic mice also exhibit marked expansion in terminally differentiated Paneth cells, which comprise an epithelial cell niche for ISCs. To dissect the mechanisms mediating these phenotypes, we generated three-dimensional (3D) intestinal organoids with varied expression of Hmga1. Strikingly, silencing Hmga1 in wildtype crypt cells disrupts their ability to organize into functional 3D organoids with bud formation, while crypt cells expressing ectopic Hmga1 exhibit enhanced organoid formation with increased ISC number, proliferation, and bud development. Because Wnt/β-catenin signaling is central to ISC function, we determined whether Hmga1 modulates this pathway. β-catenin protein is increased in the crypts of the Hmga1 transgenic mice and organoids. Hmga1 amplifies Wnt/β-catenin signaling by inducing both genes that encode Wnt cell surface receptors and target genes downstream of Wnt/β-catenin. Hmga1 also directly up-regulates Sox9, which is required for terminal differentiation to Paneth cells. This is the first example of Hmga1 fostering terminal differentiation to establish a stem cell niche. In human intestinal epithelium, HMGA1 and SOX9 are highly correlated (P = 0.008), and both become up-regulated in carcinogenesis. These results reveal a novel role for Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment and suggest that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis. Citation Format: Lingling Xian, Tait Huso, Amy Belton, David Huso, Linda M. S. Resar. High mobility group A1 chromatin remodeling protein expands the intestinal stem cell compartment and Paneth cell niche through Wnt/β-catenin signaling and Sox9. [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 1704.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that it represents the stem cell of the small intestine and colon. Similar obserations were made in hair follicles and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now been developed for the Lgr5+ve stomach stem cells. Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept. Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. Citation Format: Hans Clevers. Wnt signaling, Lgr5 stem cells, and cancer. [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 PL01-04. doi:10.1158/1538-7445.AM2013-PL01-04

Cancer cells undergo chromatin remodeling and epigenetic reprogramming to hijack stem cell transcriptional networks and drive tumorigenesis, although the molecular underpinnings remain unclear. High Mobility Group A (HMGA1) chromatin remodeling proteins are architectural transcription factors that bind AT-rich sequences where they “open” chromatin and recruit transcriptional complexes to modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis, but silenced postnatally in differentiated tissues. HMGA1 is re-expressed in aggressive cancers where high levels portend adverse clinical outcomes. In colorectal cancer, HMGA1 is among the genes most overexpressed compared to normal colon epithelium. We previously found that HMGA1 induces genes involved in EMT and drives tumor progression in colon cancer models. Hmga1 transgenic mice develop aberrant proliferation and polyposis in the small intestine and colon. In intestinal stem cells, Hmga1 amplifies Wnt signals to enhance self-renewal. Surprisingly, Hmga1 also induces Sox-9 to drive Paneth cell differentiation and “build” a stem cell niche. To determine how HMGA1 functions in the colon, we examined mouse models with varied levels of Hmga1. Hmga1 overexpression in colon stem cells leads to expansion of the colon stem cell pool. Both goblet cells and colonic mucous are increased. In DSS models of inflammatory bowel disease, Hmga1 enhances repair and tissue regeneration, while Hmga1 overexpression promotes aberrant proliferation and polyposis with advancing age. To determine how HMGA1 is induced during carcinogenesis, we examined mice harboring a heterozygous Apc mutation (Min+/-) following colonization with the human symbiotic bacteria, enterotoxigenic Bacterioides fragilis (ETBF). ETBF triggers increased Wnt signaling by inducing E-cadherin cleavage with release of membrane bound β-catenin, leading to enhanced distal colon tumorigenesis in mice. Further, ETBF colonization in humans is linked to colon cancer. ETBF colonization in mice induces Hmga1 and increases stem cell self-renewal in preliminary studies. Inflammatory cytokine genes are also induced. Bacteroides fragilis toxin (BFT) induces Hmga1 in wildtype and Min+/- mouse colon organoids. HMGA1 also increases modestly in human HT29/C1 cells after exposure to BFT. Studies are underway to identify transcriptional networks and epigenetic alterations governed by HMGA1 in this setting. This work not only provides new insights into the role of HMGA1 in colon epithelial homeostasis by maintaining both the stem cell pool and promoting regeneration following injury, but also suggests that HMGA1 can be aberrantly induced by signals from the microbiome to promote tumorigenesis. Our results also highlight the interactions between the microbiome and HMGA1 as a potential therapeutic opportunity in colon carcinogenesis. Citation Format: Linda M S Resar, Lingling Xian, Jawara Allen, Shaoguang Wu, Lionel Chia, Cynthia Sears. HMGA1 is induced by procarcinogenic bacteria within the microbiome where it drives expansion in the colon stem cell pool and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2601.

Emerging evidence suggests that cancer cells undergo chromatin remodeling and epigenetic reprogramming to co-opt stem cell properties and drive tumor progression. The HMGA1 chromatin remodeling protein is an architectural transcription factor that binds to DNA at AT-rich sequences where it “opens” chromatin, recruits transcriptional complexes, and modulates gene expression. The HMGA1 gene is highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in differentiated tissues. HMGA1 becomes re-expressed in most high-grade cancers and high levels portend adverse clinical outcomes. In colon cancer, HMGA1 is among the genes most highly overexpressed compared to normal intestinal epithelium. We previously reported that HMGA1 drives tumor progression in colon cancer by inducing stem cell genes involved in an epithelial-mesenchymal transition. We also discovered that Hmga1 transgenic mice develop marked proliferative changes and pre-malignant polyposis in the intestinal epithelium. To determine how Hmga1 functions in the intestines during tissue homeostasis and carcinogenesis, we examined in transgenic mice and organoid models. Here, we uncover a novel role for Hmga1 in maintaining the intestinal stem cell (ISC) pool and Paneth cell niche. Hmga1 is required by ISCs to organize into three-dimensional organoids in vitro; silencing Hmga1 disrupts organoid formation and bud development. Conversely, overexpression of Hmga1 increases organoid formation, bud development, and replating efficiency, suggesting that Hmga1 enhances ISC function and/or number. We therefore crossed the Hmga1 transgenic mice onto the Lgr5-EGFP background to enumerate ISCs and found that Hmga1 expands the ISC compartment. To determine how this occurs, we performed in vivo imaging and discovered that Hmga1 enhances self-renewal of ISCs. Mechanistically, we found that Hmga1 amplifies Wnt/β-catenin signaling by inducing genes encoding both Wnt agonist receptors and downstream Wnt target genes. Surprisingly, Hmga1 also expands the Paneth cell niche, which is comprised of terminally differentiated crypt cells that secrete Wnt to support ISCs. Because Paneth cells require Sox9 for development, we determined whether Hmga1 regulates its expression. Hmga1 binds directly to the Sox9 promoter at 2 AT-rich sites to activate its expression. In human colonic epithelium, HMGA1 and SOX9 are positively correlated, and both become markedly up-regulated in colon carcinogenesis. This work not only provides new insights into the role of Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment, but also suggests that deregulated Hmga1 perturbs this equilibrium during polyposis and carcinogenesis. Our results also highlight the HMGA1-WNT-SOX9 pathway as rational therapeutic target in colon carcinogenesis. Citation Format: Lingling Xian, Dan Georgess, Li Luo, Lionel Chia, Qihua Gu, Tait Huso, Amy Belton, David Huso, Andrew Ewald, Linda M.S. Resar. HMGA1 amplifies Wnt signaling and expands the intestinal stem cell compartment to drive premalignant polyposis in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5019. doi:10.1158/1538-7445.AM2017-5019

In the precision medicine era, multi-parametric interrogation of tumors in cancer patients has enabled rationally refined clinical trials and considerably improved response rates. Nevertheless, incomplete mass obliteration and late relapse invariably emerge. This also relates to metastatic colorectal cancer (mCRC), as massive tumor regression upon anti-EGFR therapy with cetuximab is relatively infrequent. Clinical evidence suggests that tumor recurrence may be endorsed by a population of lingering cancer cells that survive therapy and fuel residual disease through reversible, non-mutational cues, thus installing a “poised” state of drug tolerance that anticipates tumor relapse. The mechanisms underlying this resilience, and the strategies to tackle it, remain to be explored. In this study, we capitalized on different exploratory resources, including a proprietary collection of patient-derived xenografts, as well as CRC cell lines and primary cultures, to explore the mechanisms of cetuximab tolerance. Biochemical characterization of cetuximab persisters evidenced a dichotomous output, with full MAPK neutralization but persistent PI3K/AKT activity, suggesting that PI3K is engaged as a competitor route. Consistently, concomitant EGFR/PI3K blockade specifically unleashed apoptosis in cell lines, as opposed to the cytostatic activity of cetuximab alone, and prevented colosphere growth recovery after drug washout. In vivo, evaluation of residual cellularity confirmed the superior efficacy of dual EGFR/PI3K deactivation. Hence, both the number and fitness of tolerant cells were thwarted by EGFR/PI3K interception. Integrative morphologic analysis, immunohistochemistry, gene-expression profiling and reporter assays in vitro put forward increased beta-catenin activity and Paneth cell-like reprogramming as distinctive traits of residual cells surviving cetuximab treatment. These features are likely related to cetuximab-induced biochemical rewiring, as relieve of ERK-mediated beta-catenin repression in conjunction with persistent PI3K-AKT activity may converge on Wnt-dependent Paneth cell-like differentiation. Importantly, PI3K inhibition dampened cetuximab-triggered Paneth cell specification, thus intercepting a putative mechanism of drug-tolerance. Future studies will extricate the relative contribution of PI3K, MAPK, and Wnt-dependent networks to sustaining the reservoir of persisters, and how Paneth-like traits may foster residual disease. Citation Format: Barbara Lupo, Paolo Gagliardi, Francesco Sassi, Eugenia Rosalinda Zanella, Francesca Cottino, Andrea Bertotti, Livio Trusolino. Targeting the PI3K pathway to intercept cetuximab tolerance in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2017-LB-293

Strictly defined architecture of intestinal epithelial cells and the gradient of signaling cues along the villus-crypt axis ensure the maintenance of adult intestinal stem cells, as well as proper regeneration and differentiation of all intestinal epithelial cell types. The Rho family small GTPase Cdc42 controls the polarized division of intestinal stem cells, and affects subsequent fate-determination of the progenitor cells. As we previously described in Cdc42 Villin-Cre knockout mice, loss of Paneth cells from crypt bottom and emergence of Goblet cell-like intermediate cells at the villus indicate possible compromise of the canonical Wnt signaling during Paneth cell differentiation. Here we further characterized the underlying molecular mechanism of Cdc42 regulating intestinal stem cell survival, proliferation, and differentiation. IHC of the intestinal tissue in Cdc42 Villin-Cre knockout mice suggests greatly reduced Olfm4 level in the stem cells, which are abnormally positioned in the crypts, along with a reduction of both transcription co-factors beta-Catenin and YAP. However, the Notch signaling, which is required for stem cells and the differentiation of absorptive linage, seems intact. The autonomous regulation of Cdc42 on Yap signaling is probably through its specific role in stabilizing cell-cell junction and actin filaments in the epithelium, both of which are known regulatory inputs of the Hippo-YAP pathway. Both Wnt/beta-Catenin and Yap signaling are required for the initiation and progression of colorectal cancer, and the regeneration of stem cells post radiation. Given that Cdc42 is overexpressed in multiple types of cancers, especially colorectal cancer, our finding that Cdc42 regulates both Wnt and YAP pathways in the homeostasis of adult intestinal tissue sheds new light on the molecular mechanism of its pathological involvement, and potential therapeutic target. Citation Format: Xiao Zhang, Nan Gao. Cdc42 is crucial for intestinal stem cells survival by regulating Wnt and YAP signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2017-918

The stability of several oncoproteins, including c-Myc, is regulated by ubiquitin-dependent degradation mediated by the SCF(Fbw7) ubiquitin ligase. This activity is antagonized by the deubiquitinase Usp28, which is highly expressed in murine and human intestinal cancers. Usp28 was previously shown to interact with its substrates via a “piggyback” interaction with Fbw7, which suggested that Fbw7 is required for Usp28 activity. Unexpectedly, we found that genetic deletion of Usp28 rescued the lethality of Fbw7-deficient primary fibroblasts. Moreover, Usp28 inactivation in the intestine (Usp28ΔIEC) ameliorated the hyperproliferation and the impaired goblet and Paneth cell differentiation observed in Fbw7ΔIEC mice. The aggressive intestinal tumor formation of APCMin/+; Fbw7ΔIEC mice was restrained when Usp28 was inactivated concomitantly. In both fibroblasts and intestinal cells, Usp28 deficiency corrected the accumulation of SCF(Fbw7) substrate proteins, including NICD1, c-Jun, and c-Myc. These findings suggested that Usp28 function does not depend on the presence of Fbw7, but instead independently recognizes and deubiquitylates the same substrates as SCF(Fbw7). Fbw7 binds to a phosphorylated motif termed the phosphodegron and we found that Usp28 also interacted with this same motif, but only when it is unphosphorylated, offering a mechanistic explanation for identical substrate selection by Fbw7 and Usp28. Our results indicate an unusually direct antagonism between an E3 ligase and a deubiquitinase, Fbw7 and Usp28, in modulating intestinal homeostasis and cancer. Cancer Res; 75(7); 1181–6. ©2015 AACR.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that it represents the stem cell of the small intestine and colon. Similar obserations were made in hair follicles and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now been developed for the Lgr5+ve stomach stem cells. Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept. Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. Citation Format: Hans Clevers. Lgr5 stem cells in self-renewal and cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA1.

The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 miRNAs, although Let-7 independent effects have been surmised. Our past studies have shown that LIN28B expression in colorectal cancers is associated with poor prognosis and cancer recurrence, and that LIN28B promotes migration, invasion, and metastasis of CRC cell lines in mouse xenograft models. Considering these studies, and the observation that LIN28B is more frequently up-regulated in a variety of other cancers, we sought to elucidate the molecular mechanisms of LIN28B function in intestinal homeostasis and colorectal cancer. We report that intestine-targeted expression of Lin28b in mice causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Lgr5-positive crypt base columnar cells were not expanded, nor compromised by the depletion of Paneth cells. However, Lin28b fostered intestinal polyp and adenocarcinoma formation. We observed that higher Lin28b levels also correlated with increased incidence of adenocarcinoma. This is consistent with human epidemiological data illustrating that increased LIN28B levels are observed at early stages of CRC, with high expression at both stages I and II disease correlating inversely with patient survival and higher probability of recurrence. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-Seq) to identify direct RNA targets. This revealed that LIN28B protein binds a substantial number of mRNAs, many of which are specific to epithelial cells. LIN28B modestly augmented protein levels of these target mRNAs, in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. Previous reports have linked Let-7 to the regulation of the PI3K-Akt-mTOR signaling pathway via regulation of Insr, Igf1r, and Irs2 mRNAs, yet we found that Lin28b did not have a significant impact on these mRNAs or insulin-PI3K-mTOR signaling. Kras and Myc mRNAs, which are also reported to be Let-7 targets, were likewise unaffected. Instead, we found profound up-regulation of Hmga2, Igf2bp1, Igf2bp2, and E2f5 in the context of Let-7 depletion or Lin28b over-expression. Our study reveals that Let-7 miRNAs are critical for repressing tissue growth and promoting Paneth cell differentiation. These results highlight the critical nature of LIN28B and Let-7 dosage, where a stepwise reduction of Let-7 levels has a direct effect on target mRNAs and adenocarcinoma risk. These Let-7 dependent effects of LIN28B appear to supersede any Let-7 independent effects. In sum, LIN28B can definitively act as an oncogene in the absence of canonical genetic alteration in intestinal carcinogenesis. This abstract is also presented as poster A42. Citation Format: Blair Madison, Qi Liu, Xue Zhong, Christopher M. Hahn, Nan Lin, Matthew Emmet, Ben Z. Stanger, Anil K. Rustgi. LIN28B promotes growth and tumorigenesis of the intestinal epithelium primarily via Let-7 repression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR04.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that it represents the stem cell of the small intestine and colon. Similar obserations were made in hair follicles and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now been developed for the Lgr5+ve stomach stem cells. Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept. Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. 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 PL02-02. doi:1538-7445.AM2012-PL02-02

The conventional practice of analyzing overall age-adjusted cancer mortality rates heavily emphasizes the experience of older, higher mortality age groups. This may conceal shifts in lifetime cancer mortality experience emerging first in younger age groups. We examined age-specific cancer mortality rates and birth cohort–specific cancer mortality rates in U.S. mortality data recorded since 1955 to assess the effects of age, period, and cohort in secular mortality trends. Cancer mortality and population data were obtained from WHO Statistical Information System. Age-specific cancer mortality rates have been steadily declining in the United States since the early 1950s, beginning with children and young adults and now including all age groups. During the second half of the 20th century, each successive decade of births from 1925 to 1995 experienced a lower risk of cancer death than its predecessor at virtually every age for which such a comparison can be made. A major decline in cancer mortality has been occurring in the United States for the past 50 years, affecting birth cohorts born as long as 80 years ago. Excepting lung cancer, much of this decline has occurred despite relatively stable cancer incidence. These findings suggest that improvements in cancer detection, treatment, and/or prevention have reduced the risk of cancer death across the life span for individuals born in the last three quarters of the 20th century. [Cancer Res 2009;69(16):6500–5]

Znf131 is a relatively uncharacterized member of the POZ-ZF family of transcription factors with suspected roles in both vertebrate development and carcinogenesis. Previous studies from our lab identified Znf131 as a specific binding partner of another POZ-ZF protein, Kaiso, which has been implicated as a regulator of the canonical Wnt signaling pathway. Our lab successfully generated an intestinal-specific Kaiso overexpressing (KaisoTg/+) mouse model, which was found to exhibit enlarged crypts and mild hyperplasia in the small intestine. Several lines of evidence implicate Znf131 in cell proliferation, but whether Znf131 expression increases or decreases cell proliferation has yet to be elucidated. To determine whether Znf131 expression correlates with increased proliferation in hyperplastic KaisoTg/+ crypts, the expression and localization of Znf131 was correlated with the proliferation marker Ki-67 in KaisoTg/+ and wildtype (WT) mouse intestinal tissues. Znf131 nuclear expression was localized to the Paneth cell region of the crypts in both the KaisoTg/+ and WT small intestinal tissues, but decreased Znf131 expression was observed in the KaisoTg/+ model. Intriguingly, Ki-67 was localized to the progenitor cell region of the crypts and was excluded from Znf131-positive nuclei. In contrast to our hypothesis, these results suggest that Znf131 negatively correlates with cell proliferation. Since Znf131 has also been implicated as a regulator of ERα, we repeated these experiments using human breast tumor tissues to determine whether the observed results were tissue specific. We detected decreased nuclear expression of Znf131 in breast tumor tissues compared to matched normal tissues, and we observed a negative correlation between the nuclear expression of Znf131 and the histologic grade and stage of the tumor. Collectively, our results from both murine intestinal and human breast tumor tissues suggest a negative correlation between Znf131 nuclear expression and cell proliferation and raise the possibility that Znf131 may act as a tumor suppressor. Citation Format: Joseph Longo, Shaiya Robinson, Roopali Chaudhary, Juliet M. Daniel. Expression of the Kaiso-binding partner Znf131 negatively correlates with cell proliferation in murine intestinal and human breast tumor tissues. [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 1817. doi:10.1158/1538-7445.AM2013-1817

Introduction: In experimental models, Mucin-depleted foci (MDF), formed by dysplastic crypts devoid of mucin production have been recognized to be correlated with colorectal carcinogenesis and to serve as preneoplastic lesions of colorectal cancer (CRC). In humans, there is only one report of identification of MDF mainly in patients with familial adenomatous polyposis, however the histological characteristics of human MDF was not discussed sufficiently in this report. Therefore, we conducted this study to identify MDF in sporadic CRC patients and to evaluate the histological characteristics of human MDF. Materials and Methods: Colonic samples from 53 patients of sporadic CRC were stained with Alcian blue and examined for the presence of MDF. Subsequently, the samples were also examined for the presence of aberrant crypt foci (ACF) by methylene blue staining. We classified MDF into two categories, namely, flat-MDF and protruded-MDF (having the characteristics of both ACF and MDF), and analyzed their histological characteristics. Results: We found a total of 354, 41 and 19 colonic mucosal lesions with a mean multiplicity of 44, 38.9 and 66.9 crypts (ACF, flat-MDF and protruded-MDF, respectively). The density of MDF was 0.0082 lesions/cm2. ACF identified in sporadic CRC patients corresponded to hyperplastic or non-dysplasic lesions. On the other hand, MDF (both of flat-MDF and protruded-MDF) identified in these patients corresponded to low-grade dysplasia. In addition, we found that Paneth cell metaplasia and inflammatory cell infiltration were specific histological features of MDF. These histopathological characteristics have been reported to be associated with the development of CRC. Conclusions: This is the first report of a study conducted to investigate the density and histopathological characteristics of MDF in a large sample of sporadic CRC patients. Our results indicate that MDF might represent preneoplastic lesions in human colorectal carcinogenesis. 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 4418. doi:1538-7445.AM2012-4418

Dietary factors are significant in determining probability for intestinal tumor development even in the presence of strong genetic initiation. For example, a rodent Western-style diet (WD1), modeled to reproduce intake of nutrient risk factors in the human diet associated with sporadic colon cancer, potentiates the formation of intestinal tumors in Apc1638N/+ mice, even when the genetic initiation by Apc is augmented by targeted inactivation of the p21Waf1/cip1 locus. To investigate mechanisms of this synergistic effect, we profiled gene expression along the mouse crypt-villus axis (CVA) using cells isolated as a function of their position along this axis from WT, Apc1638N/+ and p21−/− mice fed control AIN76A diet, and WT mice fed the WD1. Modulation of sequence expression by the WD1 was enriched in the villus compartment, while changes induced by targeting either genetic locus (Apc1638N/+ or p21−/−) were largely in the crypt, where the overlap in altered gene sets was greatest for the genetic models. There was greater overlap among expression changes between Apc1638N/+ and p21−/− mice, than between these genetic mice models and mice fed the WD1, in both the villus and crypt. Paneth cell markers, whose deregulation has been reported in intestinal tumors, were up-regulated in the p21−/− mouse crypt and in WD1 mouse villus. Moreover, there was attenuated expression of secretory cell lineage markers (ie goblet and enteroendocrine cells), but enhanced expression of enterocyte markers, for both the genetic and dietary models. Genes that encode tricarboxylic acid (TCA) cycle enzymes were uniformly decreased in the crypt of both genetic models, with fewer changes in this pathway altered in the WD1 mouse. Gene ontology and biological pathway analysis also showed that Wnt pathway and c-myc target genes were overrepresented in the villus of the WD1 mouse. Supplementation of WD1 with calcium and vitamin D (WD2) reversed aberrantly up-regulated cyclin D1, cyclin D2, defensins and lysozymes in the WD1 villus back to control levels. In summary, complementary changes in cell reprogramming contributed by a western style diet and targeted inactivation of the p21 gene are important in the synergy of these factors in elevating probability of intestinal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4157.

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.

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed, all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. PdgfRα is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating for the first time that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα. [G.G. and Z.K. contributed equally to this work.] Citation Format: Gediminas Greicius, Zahra Kabiri, Kristmundur Sigmundsson, Chao Liang, Ralph Bunte, Manvendra K. Singh, David M. Virshup. PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5931.

Ras is the most commonly mutated oncogene in human cancer, and is highly mutated in pancreatic, lung, and colorectal cancer. The most commonly reported canonical activating point mutations occur in codons 12, 13, and 61. Colorectal cancer, in particular, harbors a number of noncanonical K-Ras mutations, including mutations at codon 146, which occur in ~4% of patients. To understand the biologic effects of K-RasA146T expression in the intestinal epithelium and other tissues, we engineered a conditional Cre recombinase-dependent mutant allele (K-RasLSL-A146T), expressed from the endogenous KRas locus. We crossed K-RasLSL-A146T mice to Fabpl-Cre mice, which express Cre recombinase in the colonic and distal small intestinal epithelium, and compared the phenotype of Fabpl-Cre; K-RasLSL-A146T/+ mice to that of Fabpl-Cre; K-RasLSL-G12D/+ mice. Compared to K-RasG12D, expression of K-RasA146T results in a mild hyperplastic and hyperproliferative phenotype, while expression of K-RasG12D causes drastic hyperplasia and hyperproliferation. Additionally, we found that K-RasA146T expression activates Mapk signaling and that K-RasA146T-induced hyperproliferation is Mapk-dependent. Interestingly, unlike Fabpl-Cre; K-RasLSL-G12D mice, which lack Paneth cells in the small intestine, Fabpl-Cre; K-RasLSL-A146T/+ mice have intact Paneth cells. Finally, we found that although K-RasG12D expression in the pancreas causes neoplastic transformation, K-RasA146T expression has no detectable effect on pancreatic homeostasis, even after one year. Together, our results suggest that compared to the canonical K-RasG12D mutation, activating K-RasA146T mutations have a similar but milder effect on colonic homeostasis. Additionally, consistent with human data, we found that K-RasA146T mutations have tissue-specific effects on neoplastic transformation. Citation Format: Emily Poulin, Jessica Gierut, Kevin Haigis. Activating K-RasA146T mutations induce Mapk-dependent hyperproliferation in the intestinal epithelium [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B04.

Although oral dosing is the most convenient route of drug administration, most anti-neoplastic drugs are administered intravenously due to poor bioavailability because of the epithelial barriers. A major organ involved in oral drug absorption is the small intestine where enterocytes are linked together by tight junctions (TJs). TJs regulate paracellular transport by creating a selectively permeable barrier to small molecules, ions, and water, thereby restricting the uptake of drugs. Transient modulation of the TJs is considered a potential strategy to improve drug delivery. Human small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. However, their geometry prevents access to the apical compartment of the epithelium (the outer, or luminal, surface being located within the organoid with the basal surface on the outside), making them unsuitable for the study of orally-administered drugs. Here we describe a method to generate transwell-grown cell monolayers derived from normal human small intestine, that allows direct access to the apical surface. We demonstrate that the format provides an in vivo-like cell composition, comprising of all the intestinal cellular types, including stem cells, Paneth cells, goblet cells, enteroendocrine cells and enterocytes, and respond to treatment with ‘tool drug’ TJ modulators such as sodium caprate, chitosan, EGTA and various cytokines. Briefly, for the generation of transwell-grown cell monolayers, SI organoids were dissociated into single cells and plated in matrix-coated transwell inserts. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements. Treatments with TJ modulators were applied after TEER reached a plateau (8-10 days after plating), and effects on TJs were evaluated 24-72 hours after treatment by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. In conclusion, our models provide an innovative in vitro solution to study the effect of TJ modulators on increasing paracellular transport of orally-administered, anti-neoplastic drugs. Furthermore, as anti-neoplastic drugs can also have toxic effects on the intestinal epithelium by affecting TJs, thereby creating a leaky barrier, the model is useful to predict such effects and stratify drug candidates prior to in vivo studies. The model also provides a system to test the restorative potential of molecules on barrier integrity that could be used in combination or after the administration of anti-neoplastic drugs. Citation Format: Valentina Ubertini, Frida Ponthan, Aude-Marine Bonavita, James Wilson. Transwell-grown monolayers derived from small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2754.

Transforming growth factor beta signaling has important roles in stem cell behavior, differentiation, cell motility, cell cycle and apoptosis. Here, we have utilized a novel mouse model that facilitates the sporadic inactivation of TgfβR2 via frameshift reversion of Cre in single, isolated cells and concomitant lineage tracing with a Cre-reporter. Short-term lineage tracing of TgfβR2-deficient crypts revealed reduced expansion suggesting a growth disadvantage among crypts. In contrast, long-term lineage tracing of TgfβR2-deficient crypts revealed an increased number suggesting a growth advantage within the crypt. To reconcile these apparently contradictory findings, we studied the dynamics of proliferative, Lgr5+ stem cells following TgfβR2 loss. We found that TgfβR2-deficient stem cell lineages required more time to reach monoclonality within the crypt. Using mathematical modeling of stem cell neutral drift, we found that increased “stemness” or a greater probability of a self-renewing asymmetrical outcome for TgfβR2-deficient stem cells was more consistent with our experimental results. Further analysis of TgfβR2-deficient stem cells in vivo revealed a decreased production of Paneth cells, a finding we corroborated in cultured intestinal organoids by treating with either a TgfβR1/2 inhibitor or low levels of the Tgfβ1 ligand. Recent studies by others suggested that quiescent stem cells are intermediaries between proliferative Lgr5+ stem cells and Paneth cells and that Tgfβ signaling is important in the formation of quiescent stem cells in other tissues. Therefore, we are currently asking whether Tgfβ signaling is important in the formation of quiescent stem cells in the intestine. Citation Format: Jared M. Fischer, Ashleigh Miller, Peter P. Calabrese, Darryl Shibata, R Michael Liskay. Transforming growth factor beta signaling regulates the fate of intestinal stem cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A14.

The adult intestinal epithelium of the gastrointestinal tract has a well-defined organizational structure. The epithelium can be divided into two regions, a functional region that houses differentiated cells (villi) and a proliferative region (crypts of Lieberkühn) that represents the epithelial stem cell niche. In the small intestine, finger-like projections protrude from the floor of the intestine, forming villi. Multiple proliferative crypts surround each villus base. All epithelial cells that populate the villus originate from these crypts. Multipotent epithelial stem cells reside in the crypts and give rise to four principal epithelial lineages: absorptive enterocytes, mucin secreting-goblet cells, peptide hormone secreting enteroendocrine cells, and Paneth cells. Three of these cell lineages differentiate as they migrate up and out of the crypts onto the adjacent villus. The fourth lineage, Paneth cells, differentiates with a downward migration to reside at the base of the crypt. It is believed that these different cell types are generated by asymmetric cell divisions of stem/progenitor cells. However, the molecular mechanisms of how stem cells differentiate into four different cell types are only partially understood. Aim: in the current study, we evaluated the role of β2SP (β2 spectrin) in the differentiation and asymmetric division of stem/progenitor cells in normal intestine epithelium. Results: Our preliminary data showed that the number of secreting-goblet cells and peptide hormone secreting enteroendocrine cells was reduced in β2SP+/− mice compared to the wt mice. The number of cells undergo asymmetric division was also 3 folds lower in β2SP+/− mice than in wt mice. In in vitro studies, polarization of Par3 and PKCζ was impaired in β2SP−/− MEF cells. The coimmunoprecipitation assay demonstrated that β2SP directly bound to Par3 and actin in normal intestine epithelial cells, indicating that β2SP was involved in the spindle orientation and positioning during the asymmetric division of intestine epithelial cells. Conclusion: β2SP interacts with Par3 and regulates spindle orientation and polarization in the process of cell division, thereby contributing to the differentiation and asymmetric division of intestinal stem/progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4258.

Although oral dosing is the most convenient route of drug administration, most anti-neoplastic drugs are administered intravenously due to poor bioavailability because of the epithelial barriers. A major organ involved in oral drug absorption is the small intestine where enterocytes are linked together by tight junctions (TJs). TJs regulate paracellular transport by creating a selectively-permeable barrier to small molecules, ions and water, thereby restricting the uptake of drugs. Transient modulation of the TJs is considered a potential strategy to improve drug delivery. Human small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. However, their geometry prevents access to the apical compartment of the epithelium (the outer, or luminal, surface being located within the organoid with the basal surface on the outside), making them unsuitable for the study of orally-administered drugs. Here we describe methods to generate transwell cell monolayers and inverted or ‘apical-out’ organoids, both derived from normal human small intestine, that allow direct access to the apical surface. We demonstrate that both formats provide an in vivo-like cell composition, comprising of all the intestinal cellular types, including stem cells, Paneth cells, goblet cells, enteroendocrine cells and enterocytes, and respond to treatment with ‘tool drug’ TJ modulators such as sodium caprate, chitosan, EGTA and various cytokines. Briefly, for the generation of transwell-grown cell monolayers, SI organoids were dissociated into single cells and plated in matrix-coated transwell inserts. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements. Treatments with TJ modulators were applied after TEER reached a plateau and effects on TJs were evaluated by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. For the generation of apical-out organoids, SI organoids were mechanically broken into fragments and seeded in low adhesion plates until the organoids switched polarity. Treatments with TJ modulators were applied and effects on TJs were evaluated by immunofluorescence. In conclusion, our models provide an innovative in vitro solution to study the effect of TJ modulators on increasing paracellular transport of orally-administered, anti-neoplastic drugs. Furthermore, as anti-neoplastic drugs can also have toxic effects on the intestinal epithelium by affecting TJs, thereby creating a leaky barrier, the models are useful to predict such effects and stratify drug candidates prior to in vivo studies. Citation Format: Valentina Ubertini, Stephanie French, James Wilson, Frida Ponthan. Small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 374.

The p53-inducible miR-34a and miR-34b/c genes are frequently silenced in colorectal cancer. To address the in vivo relevance of miR-34a/b/c function for suppression of intestinal tumor formation, we generated ApcMin/+ mice with deletions of the miR-34a and/or miR-34b/c genes separately or in combination. Combined deletion of miR-34a/b/c increased the number of intestinal stem cells as well as Paneth and Goblet cells, resulting in enlarged intestinal crypts. miR-34a/b/c-deficient ApcMin/+ mice displayed an increased tumor burden and grade and decreased survival. miR-34a/b/c-deficient adenomas showed elevated proliferation and decreased apoptosis and displayed pronounced bacterial infiltration, which may be due to an observed decrease in infiltrating immune cells and downregulation of barrier proteins. mRNA induction in miR-34a/b/c-deficient tumors was enriched for miR-34a/b/c seed-matching sites and for mRNAs encoding proteins related to epithelial–mesenchymal transition, stemness, and Wnt signaling. Accordingly, cells explanted from miR-34a/b/c-deficient adenomas formed tumor organoids at an increased rate. Several upregulated miR-34 targets displayed elevated expression in primary human colorectal cancers that was associated with lymph-node metastases (INHBB, AXL, FGFR1, and PDFGRB) and upregulation of INHBB and AXL in primary colorectal cancer was associated with poor patient survival. In conclusion, our results show that miR-34a/b/c suppress tumor formation caused by loss of Apc and control intestinal stem cell and secretory cell homeostasis by downregulation of multiple target mRNAs. Cancer Res; 77(10); 2746–58. ©2017 AACR.

Introduction: Adenoma formation data in diabetics is inconclusive. Alcohol intake (AI), associated with colorectal neoplasia (CRN) has not been systematically studied in diabetes. We investigated effects of diabetes and AI in a prospective patient cohort at increased CRN risk. Methods: Diabetes diagnoses based on patients' problem list, corroborated by HbA1c and glucose levels. Self-reported AI was quantified as more than occasional/social. Cumulative adenoma number were computed using available records. In a subset of patients, CRN-related Paneth cell marker p87 was used to semi-quantify immunohistochemically the colonic regions binding this label. Results: 680 patients (348 diabetics 332 non-diabetic) were ascertained with 12.4-year median follow-up. Diabetics had more adenomas than non-diabetics (2.3±3.54 versus 1.81±2.91, p=0.05) and p87 levels were significantly higher in the descending colon (p Conclusion: Diabetics accumulate more adenomas than controls which is reflected in the increase of left-sided p87 expression. A combination of increased AI and diabetes particularly increases adenoma formation seemingly independent of factors such as age and smoking. Citation Format: Martin Tobi, Fadi Antaki, Stephanie Judd, Paula Sochacki, MaryAnn Rambus, Harvinder Talwar, Michael Lawson, Edi Levi, Benita McVicker. Does a combination of type 2 diabetes mellitus and increased alcohol intake affect prospective total adenoma accumulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2365.

Intestinal tumorigenesis driven by mutations in adenomatous polyposis coli gene requires the mammalian target of rapamycin complex 1 (mTORC1) activity, whereas the physiological roles of mTORC1 in the homeostasis of intestinal epithelial cells (IECs) remain virtually unknown. We here generated mice, in which tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, was specifically ablated in IECs (Tsc2 CKO mice). Tsc2 CKO mice manifested the enhanced proliferative activity of IECs in intestinal crypts as well as the promoted migration of these cells along the crypt-villus axis. In vitro study showed that conditional ablation of Tsc2 in IECs promoted development of intestinal organoids, while mTORC1 inhibitor, rapamycin, diminished this phenotype. Tsc2 CKO mice also manifested the increased apoptotic rate of IECs as well as the increased ectopic Paneth cells, which are one of the major differentiated IECs. In addition, such conditional ablation of Tsc2 resulted in a suppressed expression of Wnt target genes in the colonic epithelium. Finally, we found that Tsc2 CKO mice manifested the increased susceptibility for dextran sulfate sodium (DSS)-induced colitis. Although mTORC1 and Wnt signaling pathways are thought to cooperated to promote oncogenesis in the intestinal epithelium, our results thus suggest that the mTORC1 signaling unexpectedly counter-regulates the Wnt signaling with the maintenance of intestinal homeostasis. Citation Format: Takenori Kotani, Jajar Setiawan, Yoji Murata, Yasuyuki Saito, Takashi Matozaki. Role of Tsc2-mTORC1 signaling in homeostasis of intestinal epithelium and its relation to inflammation and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1771.

Background: In this study we evaluated the anti-cancer effects and gut modulating properties of the two Chinese medicinal formulas, known as HuaiHuaJinYinJiu (SL) and Floris Sophorae Powder (FSP). SL is comprised of Sophorae Flos (HuaiHua) and Lonicerae Japonicae Flos (JinYinHua) and has been traditionally prescribed for carbuncle treatment. The FSP is constituted of Sophorae Flos and Gardenia Jasminoides Ellis (ZhiZi) and has been used for the cure of hemafecia and abdominal pain. Methodology: A total of 27 ApcMin/+ mice were equally divided into three groups and each group were continuously gavage for 28 days with daily dosage of SL (1g/kg), FSP (1g/kg), and water respectively. At the end of the treatments, fecal samples were collected; mice were terminated and sampled for tissues for later investigations. Results: We observed that SL and FSP substantially reduced polyps by 32% and 37% in ApcMin/+ mice, respectively. Additionally, both formulas greatly strengthened the intestinal barrier of the ApcMin/+ mice by enhancing the population of Paneth and goblet cells, up-regulating E-cadherin, and down-regulating N-cadherin expressions. Meanwhile, SL and FSP remodeled the gut microbial composition by facilitating the growth of bacteria belonged to phyla Bacteroidetes and Verrucomicrobia, whereas suppressed the abundance of phyla Fusobacteria and Deferribacteres which are generally known for their pathogenesis. The growth of sulfate-reducing bacteria and bacteria in genus Helicobacter were inhibited in SL and FSP treated mice. We also detected the downregulation of the CagA - which is a cytotoxin-associated gene expressed highly in Helicobacter pylori and has been implicated with CRC development. SL and FSP, on the other hand, facilitated the growth of short-chain fatty (SCFA) producing bacteria that was positively correlated with upregulated expressions of the SCFAs-sensing receptors GPR41, GPR43, and GPR109a. We further found the downregulation of p-AKT, p-ERK, p-STAT3, and beta-catenin in the formula-treated mice. Inflammatory milieu of the SL and FSP treated mice were greatly improved by the polarization of M1 to M2 macrophages and modified with upregulation of anti-inflammatory cytokines (IL-4, IL-10, IL-12) and suppression of pro-inflammatory cytokines (iNOS, IL-1β, IL-17, IL-18, IL-23). Conclusion: Collectively, this study presents strong evidences that the modulation of gut microbiota and the associated metabolites as well as the reversal of the inflammatory gut epithelial barrier might account for the anti-cancer effects of SL and FSP TCM formulas in the ApcMin/+ mice. [This study is supported by Macau FDCT 103/2016/A3 to WLW Hsiao] Citation Format: Wenrui Xia, Imran Khan, Xiaoang Li, Guoxin Huang, Wai Kit Leong, Wei lin Liao, W.L. Wendy Hsiao. Two classic TCM formulas suppressed polyp counts in concurrence with improved gut microenvironment, and remodeled gut microbial composition of the ApcMin+ mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2825.

Prebiotics are considered as non-digestible food ingredients that stimulate the growth of beneficial gut bacteria, thus contribute to the health of the host. Recent evidences have suggested that prebiotics treatment may become one of the key therapeutic strategies for many diseases, especially for cancer. In our previous studies, both Gynostemma pentaphyllum saponin (GpS) and Ganoderma lucidum polysaccharides (GlP) demonstrated significant prebiotic effects in mouse models. In addition, GpS alone exerted significant anti-tumor effect by restoring the dysbiosis of the gut microbiota and microenvironment of the ApcMin/+mice. Thus, we hypothesized that supplement with GlP might enhance the anti-cancer effect of GpS through the modulation of the gut microbiota. To test the hypothesis, we treated ApcMin/+mice with four different treatments, i.e. GlP (750mg/kg), GpS (300mg/kg), GlP (750mg/kg) + GpS (300mg/kg) and the drinking water control. The results showed that GlP+GpS was more effective in suppressing the polyp growth compared to the GpS or GlP groups. GlP+GpS also improved the health of gut barriers by inducing Paneth cells and goblet cells, and reinstating the E-cadherin and N-cadherin expressions in a greater extend compared to the other groups. All treatments facilitated the polarization of M1 macrophage to M2 macrophage, and increased the anti-inflammatory cytokines (IL-4 and IL-10). Interestingly, expressions the SCFA receptors, GPR 43, 49 and 119 were markedly elevated in all treatment groups, with the most prominent expression observed in the GlP+GpS group. 16S rRNA sequencing data were analyzed by using alpha diversity analysis and weighted UniFrac distance analysis. Differential alterations of the composition of gut microbiota were observed in all treatment groups. Certain bacteria (Anoerodtipe, Bacteroides, Clostridium, Citribacter, Subdoligranulum, C. leptum) associated colitis and colorectal carcinoma were significant decreased, while some SCFA-producing bacteria (Blautia sp., Eubacterium saphenum, Clostridium aminobutyricum) were enhanced in the treated groups. We concluded that the anti-tumor effects of GpS and GlP might be in part through the modulation of the gut microbiota and the improvement of epithelial microenvironment, and the stronger effects were produced by the GpS+ GlP combined treatment. [This study was supported by Macau FDCT 015/2014/A1 and 103/2016/A3] Citation Format: Xiao-ang Li, Guoxin Huang Huang, Imran Khan, Wen-rui Xia, Wai kit Leong, W.L. Wendy Hsiao. Cooperative effects of mushroom polysaccharides and herbal saponins on tumor growth and gut microenvironment in ApcMin/+mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5144.

Cyclooxygenase-2 (COX-2) inhibition prevents adenoma formation in humans and mouse models of colon cancer. The selective COX-2 inhibitor celecoxib reduces COX-2 and prostaglandin E2 (PGE2) expression and adenomas in the intestine of Min/+ mice after treatment for several weeks, but prolonged treatment increases PGE2 production, resulting in drug-resistant tumor formation and transforming growth factor β (TGFβ)–dependent intestinal fibrosis. In this study, we examined pathways that regulate COX-2 expression and suppress chronic intestinal inflammation. We show that NF-κB signaling was inhibited in the ileum of Min/+ mice receiving long-term treatment with celecoxib. This effect was associated with inhibition of TGFβ-associated kinase-1 and IκB kinase α/β activities and reduced expression of the Toll-like receptor (TLR) 2 and TLR4 that enhance colonic barrier function. Additionally, we observed reduced activities of protein kinases c-Jun NH2-terminal kinase 1 and protein kinase A and transcription factor cyclic AMP–responsive element binding protein, regulators of COX-2 expression, which cross-talk with NF-κB. In ileum subjected to long-term celecoxib treatment, we noted relatively higher expression of COX-2, vascular endothelial growth factor, and interleukin-1β in Paneth cells, whereas NF-κB and COX-2 were more strongly expressed by an expanded population of stromal myofibroblasts. Our findings argue that celecoxib resistance is an acquired adaptation to changes in the crypt microenvironment that is associated with chronic intestinal inflammation and impaired acute wound-healing responsiveness. Cancer Res; 70(11); 4433–42. ©2010 AACR.

Intestinal metaplasia is often associated with human gastric carcinoma. Intestinalization seems to be a typical example of abnormal differentiation and is possibly a precancerous state. For investigation of intestinal metaplasia, a method for visualizing disaccharidases using Tes-Tape was developed; this method was applied to many specimens of stomach surgically removed for the treatment of gastric carcinoma. More than 130 specimens of human stomach were investigated. Intestinalization was classified into types I and II intestinal metaplasia. In type I intestinal metaplasia, sucrase, maltase, trehalase, alkaline phosphatase, goblet cells, and Paneth cells were present; while the type II intestinal metaplasia, sucrase and maltase were present but alkaline phosphatase and trehalase were absent. In type II, goblet cells were present but not Paneth cells. The histochemical technique for sucrase was newly devised. Some of the villi with goblet cells in the area of intestinalization in the stomach were not stained by sucrase activity, although most of the villi were stained. The presence of a third type of metaplasia was suggested. Purified sucrases obtained from the intestine and one case of type I intestinal metaplasia showed blood group reactivity due to the oligosaccharide side chain. However, purified sucrases obtained from two cases of type II intestinal metaplasia were negative in blood group reactivity. A close relation between distribution of alpha-fetoprotein and carcinoembryonic antigen in gastric carcinoma and that in surrounding intestinal metaplasia is discussed.

Molecular classification of colorectal cancer (CRC) based on gene expression profiling of tumor samples is known to be heavily affected by transcripts of stromal origin. As a consequence, current CRC transcriptional subtypes reflect an admixture of cancer cell-intrinsic traits and tumor microenvironment features. Whether selective analysis of the cancer cell transcriptome could improve CRC subtyping, remains an open issue. In patient-derived xenografts (PDXs), human transcripts only originate from cancer cells, because stromal transcripts are of mouse origin. We therefore assessed cancer-cell intrinsic transcriptional features of CRC by generating human-specific expression profiles of 515 PDXs from 244 CRC patients, and performing unsupervised class discovery. We identified five “CRC intrinsic subtypes” (CRIS A-E) only partially overlapping with the current ones, and robustly enriched for distinct molecular, functional and phenotypic traits: (i) CRIS-A: mucinous, glycolytic, CIMP, and enriched for microsatellite instability or mutations in KRAS; (ii) CRIS-B: marked TGF-β pathway activity, epithelial-mesenchymal transition and poor prognosis; (iii) CRIS-C: MSS, elevated EGFR signaling and sensitivity to EGFR-targeted treatments; (iv) CRIS-D: MSS, WNT activation, and IGF2 overexpression and amplification; (v) CRIS-E: MSS, Paneth cell-like phenotype and higher frequency of TP53 mutation. CRIS subtypes successfully categorized independent sets of primary and metastatic CRCs and cell lines, for a total of over 3000 samples profiled by microarrays and RNAseq. The new subtypes displayed unprecedented predictive and prognostic performances, indipendent from known markers including stromal signatures, whose integration with CRIS further enhanced prognostic significance. Citation Format: Claudio Isella, Sara E. Bellomo, Francesco Brundu, Francesco Galimi, Elisa Ficarra, Luigi Marchionni, Livio Trusolino, Andrea Bertotti, Enzo Medico. Cancer cell-selective transcriptome analysis reveals new colorectal cancer molecular subtypes with improved biological resolution and superior predictive and prognostic performance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1742. doi:10.1158/1538-7445.AM2017-1742

Objective: Poor tumor differentiation status is associated with worse patient outcome in colorectal cancer. However, the molecular basis for loss of differentiation in colon cancer is not well understood. We have found that the transcription factor Ets homologous factor (EHF) is highly expressed in the normal human and mouse colonic epithelium and is down-regulated in poorly-differentiated colorectal cancer cell lines. The aim of this study was to investigate the role of EHF in regulating normal colonic epithelial cell differentiation in vivo. Methods: A novel mouse model was generated in which the Ets DNA binding domain (exon 8) of EHF was flanked by loxP sites (EHFlox/lox). To inactive EHF in the intestinal epithelium, EHFlox/lox mice were crossed to Villin-CreERT2 mice and recombination induced by tamoxifen treatment. Tamoxifen and vehicle treated mice were monitored weekly for up to one year. At each endpoint, tissue was collected and the effect of EHF deletion on intestinal cell proliferation and differentiation assessed by qRT-PCR and immunohistochemistry. Results: Targeted inactivation of EHF in the small intestinal and colonic epithelium following tamoxifen treatment was confirmed at the DNA and RNA level. EHF inactivation in the intestinal epithelium had minimal effect on survival, weight gain and overall health of the animals. Furthermore, loss of EHF did not markedly affect cell proliferation or the overall architecture of the intestinal epithelium. Finally, EHF inactivation also had minimal effect on markers of absorptive, enteroendocrine or Paneth cell differentiation but did reduce the number of goblet cells in the colonic epithelium. Conclusion: EHF does not play a major role in regulating cell proliferation in the mouse intestine but may play a role in regulating goblet cell differentiation. The role of EHF in intestinal tumourigenesis and response to stress are currently being investigated. Citation Format: Camilla M. Reehorst, Ian Y. Luk, Rebecca Nightingale, Mercedes Davalos-Salas, Amardeep S. Dhillon, John M. Mariadason. Investigating the in vivo role of the EHF transcription factor in intestinal epithelial differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2017-3529

Lgr5 Stem Cells in self-renewal and cancer Hans Clevers Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. Current models state that 4-6 crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined (1). Lgr5/Gpr49 was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cell (CBC) generated all epithelial lineages over a 14 month period, implying that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers (2). We have established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ve stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche (3). Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in in Lgr5+ve stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fueling a growing microadenoma. These microadenomas display unimpeded growth and develop into macroscopic adenomas within 4-6 weeks. When APC is deleted in short-lived Transit Amplifying (TA) cells using a different Cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30 weeks, large adenomas are very rare in these mice. We conclude that stem cell-specific loss of APC results in progressively growing neoplasia. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept (4). In the stomach, Lgr5 expression is confined to a small population of cells at the gland base in the pyloric region, the gastroesophageal junction and the boundary between the corpus and squamous forestomach. This expression pattern mirrors that of the endogenous Wnt target gene Axin2. In-vivo lineage tracing reveals that the pyloric Lgr5+ve cells generate entire gastric units within 7-10 days and maintain this multipotent stem cell activity over at least 20 months. We conclude that Lgr5 is marking an active stem cell population at the base of the pyloric glands contributing to the long-term renewal of the gastric epithelium. Mutational activation of the Wnt pathway by APC deletion in these Lgr5+ve stem cells rapidly initiates tumor formation, implying a central role for Wnt signaling in self-renewal and cancer of the distal stomach (5). 1) Barker, N., van de Wetering, M., Clevers, H. The intestinal stem cell. Review Genes Dev. 22: 1856-1864. (2008) 2) Barker, N., Van Es, J.H., Kuipers, J., Kujala, P., Van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., Clevers, H. Identification of stem cells in small intestine and colon by the marker gene LGR5 Nature 449: 1003-1007 (2007) 3) Sato, T., Vries, R., Snippert, H., van de Wetering, M., Barker, N., Stange, D., van Es, J., Abo, A., Kujala, P., Peters, P., and Clevers, H. Single lgr5 gut stem cells build crypt-villus structures in vitro without a stromal niche. Nature 459 :262-265 (2009) 4) Barker, N., Ridgway, R.A., van Es, J.H.,van de Wetering, M., Begthel, H., van den Born, M., Danenberg, E., Clarke, A.R., Sansom, O.J., Clevers, H. Crypt Stem Cells as the Cells-of-Origin of Intestinal Cancer Nature 457: 608-611 (2009) 5) Barker, N., Huch et al. and H. Clevers Lgr5 Marks stomach stem cells Cell Stem Cell, in press Citation Format: Hans Clevers. Wnt, Lgr5 stem cells, and cancer [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY12-04

Herein, we report a model for how BRAF mutations, in contrast to KRAS mutations, specifically associate with and induce, in mouse colon organoids without a microenvironment, the phenotype of human sporadic right-sided colon adenocarcinomas (COAD). The human BRAF mutant COAD are particularly distinguished by having an increased incidence of promoter CpG island methylation, termed CpG island methylator phenotype (CIMP)-High. This latter phenotype contrasts with the majority of the COAD which are classified in the CIMP-low to intermediate groups and have mostly KRAS mutations. To understand how these mutations influence tumor evolution and the methylation landscape, we modeled the early carcinogenesis of colorectal cancer by inducing BRAFV600E and KRASG12D mutations individually in 3D organoids prepared from mouse proximal colon. The induction of BRAFV600E mutation, but not KRASG12D mutation, showed various features of progressive transformation. In vitro induction of BRAFV600E drove the organoids to derive cystic changes in morphology and promoted adoption of a stem cell niche independency characterized by growth in the absence of added stem cell niche factors including Wnt3a, R-Spondin, and Noggin. This independency is not due to increased secretion of niche factors by the Paneth-like cells in BRAFV600E mutant organoids. In addition, BRAFV600E organoids showed dysplastic changes such as high nuclear to cytoplasmic ratio and abnormal budding. The most exciting finding is that induction of BRAFV600E mutation, but not KRASG12D mutation, induced complete transformation of the organoids forming xenograft tumors in NOD/SCID mice. The tumors exhibit histological characteristics of human mucinous adenocarcinoma, which is the tumor type highly associated with BRAFV600E mutation in human COAD. Gene expression analyses revealed up-regulation of intestinal stem cell signature genes and down-regulation of genes related to intestinal differentiation in BRAFV600E organoids. In addition, BRAFV600E organoids showed increased expression levels of Wnt pathway target genes indicating an enhanced and sustained Wnt-signaling. Analyses of CpG-island methylation in a panel of genes showed increased CpG-island methylation only in the BRAFV600E mutant organoids. In conclusion, in mouse colon organoids, BRAFV600E drives a human right-sided COAD phenotype with adoption of a stem cell niche independency, activation of the Wnt pathway, and induction of CpG island methylation. Citation Format: Byunghak Kang, Julie In, Nicholas Zachos, David Huso, Shinji Maegawa, Jean-Pierre Issa, Hariharan Easwaran, Stephen B. Baylin. Oncogenic BRAFV600E drives stem cell niche factors-independent growth and tumorigenic transformation in colon organoids. [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 4273.

Intestinal crypts are maintained by long-lived intestinal stem cells (ISCs) which reside near the base of glands. Above the ISC pool, there are short-lived progenitors that can supply lineage-specific differentiated cell types into the villus. Notch and Wnt pathways play a key role for determining the stem/progenitor cell function and their cell fate, and regulating cancer development in the gastrointestine. Although it has been well established that ISCs are a major origin of intestinal cancer, it remains unknown whether intestinal progenitor population can give rise to cancer. We use Mist1-CreERT mice and induce Notch and Wnt activation by mating with LSL-Notch1-IC mice and/or Apc flox mice. We found that a bHLH transcriptional factor Mist1 is expressed in Paneth cells (Lysozyme+CD24high) as well as short-lived secretory progenitors (Lysozyme-CD24low) in the intestine. Mist1+ secretory progenitors are radio- and chemo-resistant, but do not show stem cell interconversion even after intestinal injury or Lgr5 ablation. However, aberrant Notch activation changes Mist1+ cells to Lgr5+ long-lived enterocyte progenitors, with loss of secretory lineage differentiation. Mist1+CD24low progenitor population can form intestinal organoids with Notch activation in vitro. Mist1+ secretory progenitors can give rise to intestinal cancer by simultaneous Notch and Wnt activation by loss of Apc gene, while loss of Apc in Mist1 lineage alone does not develop cancer. In the colon, Mist1 marks colonic secterory progenitors that become to lineage trace and can be a cancer-initiating cell after Notch activation or DSS-induced colonic injury. These results provide the clear evidence of cellular plasticity dependent on Notch signaling in the gut, and suggest short-lived progenitors as another cellular origin of cancer besides ISC pool. Citation Format: Yoku Hayakawa, Kosuke Sakitani, Woosook Kim, Yagnesh Tailor, Karan Nagar, Kazuhiko Koike, Samuel Asfaha, Timothy C. Wang. Mist1+ secretory progenitor cells can give rise to cancer in the intestine and colon. [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 912.

Recent work built on published transcriptional subtypes of colorectal cancer (CRC) has shown that tumor stromal content heavily impacts CRC classification, with clinical and biological implications. Lineage-dependent transcriptional components of stromal origin could therefore dominate over more subtle, yet relevant, gene expression traits inherent to cancer cells. Based on the notion that in patient-derived xenografts (PDXs) stromal cells of the human tumor are substituted by their murine counterparts, we assessed cancer-cell intrinsic transcriptional features by human-specific expression profiling on 515 PDXs from 244 CRC patients. We identified five CRC intrinsic subtypes (CRIS) endowed with molecular, functional and phenotypic peculiarities, many of which were not previously evidenced: CRIS-A was a MIN-like, KRAS-mutated, and secretory subtype with sustained glycolytic metabolism and inflammatory traits; CRIS-B grouped a subset of poorly differentiated tumors with active TGFβ signaling and EMT features; CRIS-C clustered KRAS wild-type CIN tumors exhibiting elevated EGFR pathway activity and MYC copy number gains; CRIS-D tumors were characterized by a typical stem phenotype with high WNT signaling, associated with strong enrichment in IGF2 amplification/overexpression and FGFR autocrine stimulation; CRIS-E again displayed WNT-related features, but associated with a Paneth-like phenotype and a TP53-mutated genotype. CRIS subtypes successfully categorized independent sets of primary and metastatic CRC tumors, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances. Integration of stroma-centered transcriptional profiles with cancer-cell intrinsic classification by CRIS further enhanced prognostic significance. Citation Format: Andrea Bertotti, Claudio Isella, Sara E. Bellomo, Francesco Brundu, Francesco Galimi, Elisa Ficarra, Livio Trusolino, Enzo Medico. Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value. [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 107.

Activation of the Wnt signaling is the initiating event in the majority of human colorectal cancers. In mouse models, disruption of the tumor suppressor gene, Apc, induces the adenomatous lesion. However, the analyses of initial events in animal models are hampered by difficulties to control the timing and the efficiency of introducing mutations. To overcome this problem, we used three dimensional organoid culture, which contain all epitherial components of intestinal cells with the defined organization of the crypt- and villus-like domains. In vitro 4-hydroxy-tamoxifen (4OHT) treatment of mouse organoids carrying Apc conditional mutation and CreERT2 allele led to the rapid and efficient gene aberration. Gene expression analysis revealed that the change in response to Apc inactivation was strikingly similar to those observed in the animal model. These observations demonstrated that the oragnoid culture is a useful tool to investigate the early cellular and molecular invents upon Apc inactivation. Recent analyses identified stem cells in normal intestine as well as cancer stem cells of human colorectal cancer. We found that Apc inactivation in the intestinal organoids led to the alteration in their expression profiles in a strikingly similar manner with those in animal models. The expression levels of tested stem cell markers were increased, except Aldh1. Immunofluorescence analysis demonstrated that their elevated epressions were caused by the increase of the stem cell population. Paneth cells, which provide the stem cell niche, consistently resided with the stem cells, indicating the accelerated growth of stem cells was accompanied by the differentiation process of the normal intestine. Notably, Aldh1 was exclusively expressed in the villus-like domains, which were decreased after 4OHT treatment. In the mouse intestine, it was expressed in the villi of normal intestine, and was not detected in the adenomatous lesion. These observations demonstrated that Apc inactivation led to the expansion of crypt-like domains by altering the proliferation and differentiation program of stem cells. Since the organoids share the features of the early response upon Apc inactivation with mouse models, they allowed detailed investigation of tumorigenesis. We have used the organoid culture system to elucidate the roles of oncogenic mutations as well as to evaluate the potential therapeutic targets. Citation Format: Ryoji Yao, Tetsuo Noda. The cellular hierarchy of intestinal tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4204. doi:10.1158/1538-7445.AM2015-4204

Colorectal cancer (CRC) is the 2nd leading cause of cancer death in U.S. With lifetime incidence rates as high as 1 in 20, it is critical to better define the molecular events controlling intestinal homeostasis and tumor initiation/progression. In normal intestinal homeostasis, the Notch pathway plays a fundamental role in controlling epithelial cell fate through juxtacrine signaling between membrane-bound ligands to the Notch receptor present on contacting cells. This complex promotes intestinal epithelial cell fate through transcription factors Hes1 and Atoh1/Math1, allowing for differentiation of absorptive enterocyte lineage by Hes1 or secretory (goblet, enteroendocrine and Paneth) cell lineage controlled by Atoh1. In CRC however, constitutive Notch activation is observed allowing for enhanced Hes1 expression associated with tumor progression and self-renewal of tumor-initiating cells. Yet, a clear mechanism for the constitutive activation of this pathway in CRC is not known. Here, we investigated the regulation of Notch signaling by the mRNA decay factor Tristetraprolin (TTP) in CRC and TTP knockout mice. TTP is a member of the TIS11 family of RNA-binding proteins, and the major factor promoting decay of mRNAs bearing AU-rich element (ARE) motifs in their 3′UTR. TTP plays an essential role in controlling pro-inflammatory and oncogenic gene expression in the GI tract, however during CRC progression, TTP expression is silenced in tumors. In this study, TTP-inducible CRC cells were used to show that expression of TTP suppresses proliferation and anchorage-independent growth, impacts cell cycle, and reduces xenograft tumor growth. Interestingly, TTP expression altered CRC cells such that cells now displayed a secretory cell-like morphology, consistent with studies showing effects of Notch signaling attenuation. Alcian blue staining of acidic mucins was significantly increased in TTP-expressing CRC cells and in xenograft tumors, suggesting a role for TTP in directing secretory cell differentiation. Furthermore, gene expression arrays and reporter gene assays showed that levels of Notch signaling genes were significantly impacted by TTP and TTP preferentially promoted a 3-fold downregulation of Notch1 receptor as compared to Notch2-4. The ability of TTP to target Notch1 mRNA for rapid decay occurred through its ARE-containing 3′UTR as observed using 3′UTR reporter and RNA-binding assays. To determine if these effects were specific to tumor cells, intestinal cell lineage was examined in TTP knockout mice with a remarkable reduction in goblet cell number observed as compared to wild-type mice. These findings demonstrate a novel role for TTP in controlling intestinal cell differentiation via post-transcriptional regulation of Notch1 and provide mechanistic insights into constitutive Notch activation observed in CRC occurring through loss of TTP expression. Citation Format: Shufei Zhuang, Liangyan Hu, Sandhya Sanduja, Shahid Umar, Shrikant Anant, Dan A. Dixon. The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2130. doi:10.1158/1538-7445.AM2015-2130

Colorectal tumors are hierarchically organized in which cancer stem cells (CSCs) are at the top of the hierarchy and give rise to non-tumorigenic differentiated tumor cells (DTC). The maintenance of normal intestinal stem cells requires ‘niche’ signals that are provided, at least in the small intestine, by neighboring differentiated Paneth cells. CSC maintenance may also depend on niche signals although little is known about the nature of such signals and their potential source. For the present study we tested the hypothesis that DTCs could provide CSC-maintaining signals. We used CSC-enriched colonosphere cultures that were established from human colorectal tumors and CSC-derived stably differentiated offspring that was derived from them. We found that DTCs established from multiple colorectal tumors greatly increased the clone-forming capacity of CSCs. This effect was reproduced by DTC-conditioned medium, suggesting the involvement of a secreted factor. By using a cytokine array we identified stem cell factor (SCF) as a major DTC-secreted factor. While SCF was mainly produced by DTCs, its receptor, c-Kit, was mainly expressed by CSCs. Indeed, recombinant SCF enhanced clone-forming capacity to a similar extent as DTC-conditioned medium. Furthermore, basal and DTC-stimulated clone-forming capacity was strongly reduced in the presence of an SCF-neutralizing antibody or in the presence of the cKit inhibitors Imatinib or ISCK03. qPCR and western blot analysis showed that inhibition of cKit caused a strong reduction in the expression of stem cell genes, including LGR5, CD133, OLMF4 and SOX2. Furthermore, DTCs induce the E-to N-cadherin switch in CSCs and to a strong increase in the expression of EMT markers such as vimentin, ZEB1, ZEB2, SNAIL1 and fibronectin. cKIT inhibitors prevented EMT. Our results identify a critical role for non-tumorigenic DTCs in the support of colorectal CSCs. DTCs induce EMT and EMT-associated stem cell characteristics at least in part via paracrine SCF-cKit signaling. We conclude that cKit inhibitors that are widely used in the clinic to treat gastrointestinal stromal tumors (GIST) and acute myeloid leukemia (AML), may have therapeutic value in the treatment of (a subgroup of) colorectal tumors. Citation Format: Szabolcs Fatrai, Susanne J. van Schelven, Inne H.M. Borel Rinkes, Onno Kranenburg. Differentiated tumor cells secrete stem cell factor (SCF) to promote maintenance of cancer stem cells and induce EMT in colorectal tumors. [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 1045. doi:10.1158/1538-7445.AM2014-1045

Colorectal cancer (CRC) is one of the leading causes of cancer mortality in the Western countries. In most of the CRC patients, an initial mutation occurs in the APC or CTNNB1 genes, leading to the ligand-independent activation of the canonical Wnt pathway. Besides having a central role in the development of CRC, the Wnt pathway plays a critical role also in the maintenance of the normal intestine. Attempts at therapeutic inhibition of this pathway could thus lead to serious side effects in CRC patients. We have previously shown that the Prox1 transcription factor is induced in the intestinal epithelium by mutations activating the Wnt pathway and it critically contributes to CRC progression via an unknown mechanism. Here we provide evidence that Prox1 expression is induced in the Lgr5+ adenoma stem cells early after Apc deletion. Our in vivo models and ex vivo organoid experiments suggest that Prox1 silencing or deletion restricts the expansion of the Lgr5+ adenoma stem cell population both in humans and in mice. Interestingly, silencing the phospholipid binding protein Annexin A1 (Anxa1), a gene suppressed by Prox1, is responsible for several of the effects of Prox1 on adenoma cells, such as the re-organization of the actin cytoskeleton, enhanced stem cell activity and tumor growth. Furthermore, Prox1 deletion abnormally increases the mTORC1 pathway activity, which results in decreased survival of the adenoma stem cells. Since Prox1 is expressed at low level only in rare neuroendocrine cells and in some Paneth cells in the wild type intestinal epithelium, furthermore, its genetic deletion in the adult gut does not lead to obvious phenotypes, Prox1 may serve as an attractive therapeutic target for restricting the progression of early intestinal adenomas. Citation Format: Zoltan Wiener, Ville Hyvönen, Jenny Högström, Tanja Holopainen, Arja Band, Pauliina Kallio, Olli Dufva, Caj Haglund, Olli Kruuna, Guillermo Oliver, Yinon Ben-Neriah, Kari Alitalo. The Wnt-target Prox1 promotes colorectal cancer stem cell survival to fuel tumor growth. [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 1908. doi:10.1158/1538-7445.AM2014-1908

Genetic changes in intestinal epithelial stem cells are more important for oncogenesis of intestinal tumors than such changes in non-stem cells. A large-scale sequencing analysis of human colorectal carcinoma (CRC) revealed that Wnt/β-catenin signal activation is the most frequent genomic changes (92-97%) in human colorectal carcinoma (CRC). However, fundamental significance of its signal activation of intestinal stem cells is still remains unsolved. Kruppel-like factor 5 (KLF5) is one of KLF zinc finger transcription factor with diverse biological functions. KLF5 has stronger expression in the bottom of intestinal crypts, where the stem cells reside, and weaker expression in villi as the epithelial cells differentiate into more mature cells, which is complementary pattern of KLF4. KLF5 is implicated in the renewal and maintenance of ES cells, and has the potential to induce iPS cells, although weaker than KLF4. In this study, we found that inducible deletion of KLF5, specifically in Lgr5+ intestinal epithelial stem cells, suppressed the proliferation and nuclear localization of β-catenin, and generated some apoptotic cells in the intestinal crypts of mice. Furthermore, we demonstrated that inducible deletion of KLF5 in all intestinal epithelia caused mislocalization of Paneth cells, which constitute the stem cell niche. These data indicate that KLF5, rather than KLF4, is required for the maintenance and differentiation of Lgr5+ intestinal stem cells. Moreover, we detected frequent genome amplification at the KLF5 locus in human colorectal cancers (CRCs), and enhanced expression of KLF5 in CRC samples. In mice, Lgr5+ stem cell-specific induction of an active β-catenin mutant generated severe lethal adenomas and carcinomas. By contrast, when the induction of active β-catenin was performed simultaneously with deletion of KLF5 in stem cells, oncogenesis was almost completely suppressed. The simultaneous deletion of KLF5 under the induction of the active mutant of β-catenin in the stem cells suppressed the transcription of HDAC1and Sphk1, which promotes CRCs, and upregulated the transcription of Aim2 and Foxo3, tumor suppressors in CRCs, in both the FACS sorted stem cells and the transit amplifying cells. These results indicate that oncogenesis of intestinal tumors induced by Wnt/β-catenin signal activation (92-97% in CRCs) in stem cells requires KLF5, as in the case of Myc, and that KLF5 is also an essential controlling factor for homeostatic turnover and maintenance, niche formation, and differentiation of the intestinal epithelial stem cells. These suggest that KLF5 is a promising target oncogene for CRC therapy directed at colon cancer stem cells. Citation Format: Takeo Nakaya, Masahiko Kuroda, Ryozo Nagai. KLF5 is essential for oncogenesis of intestinal tumors and control of intestinal stem cells. [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 2293. doi:10.1158/1538-7445.AM2013-2293

Colorectal tumors are hierarchically organized: A small cancer stem cell (CSC) pool drives the formation of proliferating and differentiating tumor cells that make up the bulk of the tumor but lack tumor-initiating capacity. CSCs are thought to be derived from normal intestinal stem cells (ISCs) through the acquisition of multiple pro-tumorigenic genetic alterations. The maintenance of ISCs requires ‘niche’ signals that are provided, at least in the small intestine, by neighboring Paneth cells. CSC maintenance may also depend on niche signals although little is known about the nature of such signals and their potential source. Here we tested the hypothesis that differentiated tumor cells (DTC) could provide CSC-maintaining signals by making use of CSC-enriched colonosphere cultures that were freshly established from human colorectal tumors. We found that DTCs established from multiple colorectal tumors greatly increased the clone-forming capacity of CSCs from their own and from other tumors. This effect was reproduced by DTC-conditioned medium, suggesting the involvement of a secreted factor. By using a cytokine array and three CSC-supporting DTC cultures we identified 24 factors that were secreted by all three DTCs. Analysis of the gene expression profiles of two independent cohorts of primary colorectal tumors showed that only one of these secreted factors, stem cell factor (SCF), was significantly correlated with metastasis formation and poor overall survival. In human colonospheres SCF was mainly produced by DTCs, while the receptor for SCF, c-Kit, was expressed by CSCs. Indeed, recombinant SCF enhanced clone-forming capacity to a similar extent as DTC-conditioned medium. Furthermore, basal and DTC-stimulated clone-forming capacity was strongly reduced in the presence of an SCF-neutralizing antibody or in the presence of the cKit inhibitors Imatinib or ISCK03. qPCR analysis showed that inhibition of cKit caused a strong reduction in the expression of stem cell genes, including LGR5, CD133, OLMF4 and SOX2. In addition, both purified SCF and DTC-conditioned medium induced cKit-dependent expression of EMT and mesenchymal genes, including ZEB1, SNAIL1, fibronectin and vimentin. Our results identify a critical role for non-tumorigenic DTCs in the support of colorectal CSCs via paracrine SCF-cKit signaling. We conclude that cKit inhibitors that are widely used in the clinic to treat gastrointestinal stromal tumors (GIST) and acute myeloid leukemia (AML), may also have therapeutic value in the treatment of (a subgroup of) colorectal tumors. Citation Format: Szabolcs Fatrai, Susanne J. van Schelven, Inne H.M. Borel Rinkes, Onno Kranenburg. Stem Cell Factor (SCF) secreted by differentiated tumor cells helps maintaining the cancer stem cell phenotype in colorectal tumors. [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 269. doi:10.1158/1538-7445.AM2013-269

Background: AGR2 represents Anterior Gradient 2, initially discovered in Xenopus where its expression at the anterior pole of the developing embryo induces forebrain and cement gland formation. In salamanders, AGR2 plays a role in nerve-dependent limb regeneration. AGR2 induces foci formation and anchorage independent growth in vitro. Increased AGR2 is observed in adenocarcinoma of the prostate, breast, pancreas, esophagus and ovaries. AGR2 overexpression transforms NIH3T3 cells resulting into tumor xenografts in nude mice. AGR2 induces expression of amphiregullin, EGFR ligand by affecting Hippo signaling pathway. In normal tissues it is expressed in the secretory lineage, paneth, enteroendocrine and goblet cells of the small intestine. AGR2's expression and role in stomach development and disease has not been studied. Stomach consists of three main regions: the fundus, lined with squamous keratinized epithelium, the corpus and the pylorus (glandular stomach). The corpus glands consist of the parietal cells, chief cells, foveolar cells, enterochromaffin cells, neck cells and stem cells. Parietal cells secrete acid for digestion, chief cells make digestive enzyme, pepsinogen and the foveolar cells secrete mucus. Proposed functions of neck cells include a role in mucus secretion or as a progenitor for chief cells. AGR2 is expressed by neck cells in the normal stomach. Hypothesis and methods: AGR2 expression in neck cells is critical for normal development of the stomach. This hypothesis is tested by generating a conditional AGR2 knock out mouse in which the floxed exons are excised with CMV induced expression of Cre-recombinase. Immunohistochemisty with lineage specific antibodies was performed to analyze the phenotype. Results: AGR2 loss results in weight loss and enlarged stomach in the mice. Histological analysis of stomach from AGR2 knock out mice demonstrates that AGR2 expression in the mucus neck cells is dispensable to terminal differentiation of chief cells. Loss of AGR2 results in incomplete maturation of chief cells. AGR2 loss is accompanied with increased cell proliferation in the stomach compared to the litter mate controls. The dividing cells exclude the lineage markers: H-KATPase (parietal cells), Muc5AC (faveolar cells), and intrinsic factor (chief cells). A significant expansion of TFF2 positive cells is observed that is also referred to as Spasmolytic polypeptide-expressing metaplasia (SPEM). In humans, SPEM is associated with precancerous stomach and is observed upon parietal cell loss.TFF2 in the normal stomach is expressed in the neck cells. Previous studies show that loss of parietal cells results in SPEM in the stomach. Loss of AGR2 triggers expansion of TFF2 expressing cells/SPEM before the loss of parietal cells. Conclusions: • AGR2 expression in neck cells is critical for terminal differentiation of chief cells. • Loss of AGR2 results in hyperplasia and SPEM in the stomach 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 1325. doi:1538-7445.AM2012-1325

Intestinal homeostasis relies upon accurate reprogramming of highly proliferative, multipotent colon epithelial stem/progenitor cells into 3 specialized cell types (enterocytic, goblet, and enteroendocrine cells) that migrate along the crypt axis toward the lumen and another (Paneth cells) that partitions to the bottom of the crypt. This reprogramming is modeled in immortalized cell lines such as Caco-2 and HT29Cl.16E and Cl.19A that proliferate in an undifferentiated state in culture but acquire absorptive (Caco-2) or secretory (16E and 19A) phenotypes in response to contact inhibition of growth or treatment with the short chain fatty acid butyrate. These processes are dependent not only on tightly coordinated changes in gene expression but also posttranscriptional regulation. miRNA silencing is one mechanism of gene regulation by which 21bp oligonucleotides recruit a silencing complex to the 3’ untranslated regions (UTRs) of target genes. We have previously identified a functional miRNA target in the 3’ UTR of Mybl2, one of 14 genes downregulated during growth arrest- and butyrate- induced differentiation of Caco2, 16E, and 19A cells. We also demonstrated that overexpression of miR-365, a miRNA predicted to bind this target, downregulates Mybl2 protein in proliferating Caco2 cells. Although these cell lines are good models for colon epithelial cell maturation, demonstrating that these observations occur in vivo in differentiating cells along the crypt-luminal axis of the human colon is necessary to accurately define mechanisms that maintain intestinal homeostasis. Here we have developed a technique to mechanically isolate intact, unamplified RNA from serial 10μ sections along the crypt-luminal axis of the human colon in vivo, from which we have determined the expression of several miRNAs. Expression of a subset of miRNAs is similar in both differentiating Caco2 cells in vitro and cells isolated form the human crypt-luminal axis in vivo : miR-365, 324-5p, 588, let-7a1, 331-3p, and 34a are significantly increased in differentiated versus proliferating colon epithelial cells in vitro and in vivo, though for the latter two the increases in vivo are more modest than those in vitro. On the other hand, miR-145, significantly upregulated in maturing Caco2 cells in vitro, is dowregulated in differentiated cells in vivo. Importantly, these results demonstrate that miR-365 likely plays an important part in Mybl2 regulation in differentiating human colon epithelial cells in vivo as we have shown in vitro, and they establish a method to determine the mechanisms of expression of critical proliferation and differentiation mediators, such as Mybl2, at a 10μ resolution along the human crypt-luminal axis in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 138. doi:10.1158/1538-7445.AM2011-138

Short-term chronic exposures of rats to furan were recently found by us to preferentially induce a unique liver lobe pattern of development of small intestinal metaplasia and subsequent cholangiofibrosis, being essentially localized to the caudate and right liver lobes (L. W. Elmore, and A. E. Sirica, Cancer Res., 51: 5752-5759, 1991). We now demonstrate the preferential development of primary hepatic adenocarcinomas exhibiting small intestine mucosal cell differentiation, which have arisen at 70 to 90% incidences from the right/caudate liver lobes of Fischer 344 adult male rats by 16 months after their receiving furan by gavage at a daily dose of 30 mg/kg of body weight, five times a week, for 9, 12, and 13 weeks, respectively. In contrast, the incidences of primary hepatocellular carcinomas that developed in the furan-treated rats ranged from 0 to 20%, with the two hepatocellular carcinomas observed to be originating from the median/left liver lobes. Twenty-six of 27 hepatic adenocarcinomas analyzed exhibited glands containing on average 30.2% goblet cells, 2.1% Paneth cells, and 0.5% serotonin-positive neuroendocrine cells. Phenotypically, the glandular epithelial cells of the furan-induced intestinal-type adenocarcinomas were immunohistochemically positive for cytokeratin 19, but exhibited a heterogeneous pattern of immunohistochemical staining for gamma-glutamyl transpeptidase and showed no detectable immunostaining for transforming growth factor alpha. In addition, many of the glandular structures within these primary hepatic adenocarcinomas showed evidence of basement membrane disruption, as demonstrated by both electron microscopy and immunohistochemical staining for basement membrane laminin. While these intestinal-type adenocarcinomas appeared to have spread intrahepatically, none showed evidence of extrahepatic metastases. However, six of eight randomly selected adenocarcinomas grew progressively and retained their intestinal pattern of differentiation following serial transplantation into the fat pads of young adult Fischer 344 recipient rats. In this study, we also observed one primary hepatic cholangiocarcinoma that was characterized by a more native biliary rather than intestinal-type of differentiation. Interestingly, this was the only primary liver cancer observed by us to exhibit extrahepatic metastasis. In conclusion, our current findings clearly indicate that the small intestinal metaplasia and subsequent cholangiofibrosis developing early in the right/caudate liver lobes of furan-treated rats do not simply reflect reactive changes, but strongly correlate with the high incidences of intestinal-type of primary hepatic adenocarcinoma that occurs in the right/caudate liver lobes of rats after long-term exposures to furan.