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Supp. Fig. 1 legend

Supplementary Figure 1. Dermo1Cre;Rosa-CAG-LSL-TdTomato mice.

FoxL1+-Telocytes (TCFoxL1+) are subepithelial cells that form a network underneath the epithelium. We have shown that without inflammatory stress, mice with loss of function in the BMP signalling pathway in TCFoxL1+ (BmpR1aΔFoxL1+) initiated colonic neoplasia. Although TCFoxL1+ are modulated in IBD patients, their specific role in this pathogenesis remains unclear. Thus, we investigated how the loss of BMP signalling in TCFoxL1+ influences the severity of inflammation and fosters epithelial recovery after inflammatory stress. BmpR1a was genetically ablated in mouse colonic TCFoxL1+. Experimental colitis was performed using a DSS challenge followed by recovery steps to assess wound healing. Physical barrier properties, including mucus composition and glycosylation, were assessed by alcian blue staining, immunofluorescences and RT-qPCR. We found that BmpR1aΔFoxL1+ mice had impaired mucus quality, and upon exposure to inflammatory challenges, they had increased susceptibility to experimental colitis and delayed healing. In addition, defective BMP signalling in TCFoxL1+ altered the functionality of goblet cells, thereby affecting mucosal structure and promoting bacterial invasion. Following inflammatory stress, TCFoxL1+ with impaired BMP signalling lose their homing signal for optimal distribution along the epithelium, which is critical in tissue regeneration after injury. Overall, our findings revealed key roles of BMP signalling in TCFoxL1+ in IBD pathogenesis.

Background & Aims Oropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo. We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas. Methods We generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo. Results Tumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients (P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment. Conclusions The single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine., Graphical abstract

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (

The homeostatic proliferation-differentiation gradient in the esophageal epithelium is perturbed under inflammatory disease conditions such as gastroesophageal reflux disease and eosinophilic esophagitis. Herein we describe the protocols for rapid generation (

Tumor-initiating cells and cancer stem cells refer to a subpopulation of self-renewing cells involved in tumor initiation and tumor maintenance, respectively. With this review, we aimed to define the functional and molecular differences between both cell types in the context of colon cancer. Recent evidence suggests that the two major stem cell populations in the normal intestinal crypt have tumor-initiating capacity and could be the cell-of-origin in colon cancer. Activation of the Wnt/β-catenin pathway (an early event in mouse intestinal carcinogenesis) in the crypt base columnar stem cells and reserve stem cells leads to adenoma formation, supporting a role in tumor initiation. On the other hand, colon cancer stem cells express several membrane markers facilitating their isolation by FACS and are associated with treatment resistance and higher metastatic potential. Tumor-initiating cells and cancer stem cells express distinct markers, display discrete biological functions, and can be studied using different molecular and cellular approaches. While cancer stem cells may be derived from tumor-initiating cells, these terms are not necessarily interchangeable and more likely reflect a specific cell state.

Summary Two principal stem cell pools orchestrate the rapid cell turnover in the intestinal epithelium. Rapidly cycling Lgr5+ stem cells are intercalated between the Paneth cells at the crypt base (CBCs) and injury-resistant reserve stem cells reside above the crypt base. The intermediate filament Keratin 15 (Krt15) marks either stem cells or long-lived progenitor cells that contribute to tissue repair in the hair follicle or the esophageal epithelium. Herein, we demonstrate that Krt15 labels long-lived and multipotent cells in the small intestinal crypt by lineage tracing. Krt15+ crypt cells display self-renewal potential in vivo and in 3D organoid cultures. Krt15+ crypt cells are resistant to high-dose radiation and contribute to epithelial regeneration following injury. Notably, loss of the tumor suppressor Apc in Krt15+ cells leads to adenoma and adenocarcinoma formation. These results indicate that Krt15 marks long-lived, multipotent, and injury-resistant crypt cells that may function as a cell of origin in intestinal cancer., Highlights • Krt15 marks multipotent and self-renewing crypt cells in the mouse small intestine • Krt15+ crypt cells are radio-resistant and contribute to regeneration following injury • Apc loss in Krt15+ cells leads to intestinal adenoma and adenocarcinoma formation • Krt15+ cells may function as a cell of origin in intestinal cancer, In this article, Rustgi and colleagues identify a new population of long-lived cells within the intestinal crypt, marked by the Krt15 promoter. Krt15+ cells are self-renewing, multipotent, and radio-resistant cells that facilitate recovery from radiation-induced injury. Furthermore, such cells, when targeted with Apc loss, undergo transformation to adenomas and adenocarcinomas.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell‐specific Imp1 deletion ( Imp1 Δ IEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn9s disease and ulcerative colitis. We demonstrate that Imp1 Δ IEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)‐mediated colonic injury. Imp1 Δ IEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B , ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1 Δ IEC mice revealed increased sensitivity of double‐mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.

Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer worldwide. The most commonly mutated gene in ESCC is TP53. Using a combinatorial genetic and carcinogenic approach, we generate a novel mouse model of ESCC expressing either mutant or null p53 and show that mutant p53 exhibits enhanced tumorigenic properties and displays a distinct genomic profile. Through RNA-seq analysis, we identify several endocytic recycling genes, including Rab Coupling Protein (Rab11-FIP1), which are significantly downregulated in mutant p53 tumor cells. In 3-dimensional (3D) organoid models, genetic knockdown of Rab11-FIP1 results in increased organoid size. Loss of Rab11-FIP1 increases tumor cell invasion in part through mutant p53 but also in an independent manner. Furthermore, loss of Rab11-FIP1 in human ESCC cell lines decreases E-cadherin expression and increases mesenchymal lineage-specific markers, suggesting induction of epithelial-mesenchymal transition (EMT). Rab11-FIP1 regulates EMT through direct inhibition of Zeb1, a key EMT transcriptional factor. Our novel findings reveal that Rab11-FIP1 regulates organoid formation, tumor cell invasion, and EMT.

The colon tumor microenvironment is becoming increasingly recognized as a complex but central player in the development of many cancers. Previously, we identified an oncogenic role for the mRNA-binding protein IMP1 (IGF2BP1) in the epithelium during colon tumorigenesis. In the current study, we reveal the contribution of stromal IMP1 in the context of colitis-associated colon tumorigenesis. Interestingly, stromal deletion of Imp1 (Dermo1Cre;Imp1LoxP/LoxP, or Imp1ΔMes) in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer resulted in increased tumor numbers of larger size and more advanced histologic grade than controls. In addition, Imp1ΔMes mice exhibited a global increase in protumorigenic microenvironment factors, including enhanced inflammation and stromal components. Evaluation of purified mesenchyme from AOM/DSS-treated Imp1ΔMes mice demonstrated an increase in hepatocyte growth factor (HGF), which has not been associated with regulation via IMP1. Genetic knockdown of Imp1 in human primary fibroblasts confirmed an increase in HGF with Imp1 loss, demonstrating a specific, cell-autonomous role for Imp1 loss to increase HGF expression. Taken together, these data demonstrate a novel tumor-suppressive role for IMP1 in colon stromal cells and underscore an exquisite, context-specific function for mRNA-binding proteins, such as IMP1, in disease states. Implications: The tumor-suppressive role of stromal IMP1 and its ability to modulate protumorigenic factors suggest that IMP1 status is important for the initiation and growth of epithelial tumors. Mol Cancer Res; 13(11); 1478–86. ©2015 AACR. See related article by Koltsova and Grivennikov, p. 1452

Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53(R175H)-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44(High)/CD24(Low) population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.

Metaplasia is the replacement of one differentiated somatic cell type with another differentiated somatic cell type in the same tissue. Typically, metaplasia is triggered by environmental stimuli, which may act in concert with the deleterious effects of microorganisms and inflammation. The cell of origin for intestinal metaplasia in the oesophagus and stomach and for pancreatic acinar-ductal metaplasia has been posited through genetic mouse models and lineage tracing but has not been identified in other types of metaplasia, such as squamous metaplasia. A hallmark of metaplasia is a change in cellular identity, and this process can be regulated by transcription factors that initiate and/or maintain cellular identity, perhaps in concert with epigenetic reprogramming. Universally, metaplasia is a precursor to low-grade dysplasia, which can culminate in high-grade dysplasia and carcinoma. Improved clinical screening for and surveillance of metaplasia might lead to better prevention or early detection of dysplasia and cancer.

Background & Aims Aberrations in the esophageal proliferation-differentiation gradient are histologic hallmarks in eosinophilic esophagitis (EoE) and gastroesophageal reflux disease. A reliable protocol to grow 3-dimensional (3D) esophageal organoids is needed to study esophageal epithelial homeostasis under physiological and pathologic conditions. Methods We modified keratinocyte-serum free medium to grow 3D organoids from endoscopic esophageal biopsies, immortalized human esophageal epithelial cells, and murine esophagi. Morphologic and functional characterization of 3D organoids was performed following genetic and pharmacologic modifications or exposure to EoE-relevant cytokines. The Notch pathway was evaluated by transfection assays and by gene expression analyses in vitro and in biopsies. Results Both murine and human esophageal 3D organoids displayed an explicit proliferation-differentiation gradient. Notch inhibition accumulated undifferentiated basal keratinocytes with deregulated squamous cell differentiation in organoids. EoE patient-derived 3D organoids displayed normal epithelial structure ex vivo in the absence of the EoE inflammatory milieu. Stimulation of esophageal 3D organoids with EoE-relevant cytokines resulted in a phenocopy of Notch inhibition in organoid 3D structures with recapitulation of reactive epithelial changes in EoE biopsies, where Notch3 expression was significantly decreased in EoE compared with control subjects. Conclusions Esophageal 3D organoids serve as a novel platform to investigate regulatory mechanisms in squamous epithelial homeostasis in the context of EoE and other diseases. Notch-mediated squamous cell differentiation is suppressed by cytokines known to be involved in EoE, suggesting that this may contribute to epithelial phenotypes associated with disease. Genetic and pharmacologic manipulations establish proof of concept for the utility of organoids for future studies and personalized medicine in EoE and other esophageal diseases.

Epidemiological and experimental evidence suggests that estrogen replacement therapy reduces the risk of colon cancer in postmenopausal women. Estrogen receptor beta (ERβ) is thought to be the principal mediator of the estrogen effect in the colon. Recent studies by our team suggested positive regulation of the transforming growth factor (TGF)β pathway by estrogen in mice colonocytes. We therefore wanted to investigate the effects of ERβ agonist treatment on intestinal tumorigenesis in ApcMin/+ mice. Weaned ApcMin/+ mice were injected subcutaneously three times a week for 12 wk with vehicle or ERβ-selective agonist, diarylpropionitrile (DPN, 5 mg/kg). DPN administration resulted in a significant reduction in small intestinal polyp multiplicity in both ApcMin/+ male and female mice. Furthermore, the mean diameter of small intestinal polyps was lower in DPN-treated than vehicle-treated males, along with lower BrdU incorporation indices in jejunal and colon epithelial cells of both sexes. DPN treatment also increased apoptosis in colon epithelium as measured by TUNEL assay and cleaved caspase 3 quantification. The effect of DPN on various components of the TGFβ pathway was also studied in colonocytes. DPN treatment increased expression of TGFβ1 and TGFβ3 transcripts, levels of nuclear and phosphorylated Smad2 as well as p27 cell-cycle inhibitor, a TGFβ pathway target gene. Our results demonstrate that DPN treatment reduces intestinal tumorigenesis in ApcMin/+ mice. Furthermore, we suggest that positive regulation of the TGFβ pathway by ERβ activation could contribute to the protective role of estrogen in intestinal tumor development. © 2010 Wiley-Liss, Inc.

Clinical evidence suggests that estradiol replacement therapy reduces colon cancer risk in ‘post’menopausal women. In colon epithelial cells, the estrogen receptor β (ERβ) is the predominant ER subtype and is thought to mediate the genomic effect of estrogens. The first aim of this study was to investigate the consequence of ERβ deficiency on intestinal tumorigenesis in the ApcMin/+ mouse model. Furthermore, to explore the biological mechanisms by which estrogens may influence the pathogenesis of colorectal cancer, we performed gene expression profiles in colonocytes from ovariectomized wild-type (WT) vs. ERβ−/− mice, treated with estradiol (E2) or vehicle. Specifically in female, ERβ deficiency was found to be associated with higher adenoma multiplicity in the small intestine, but not in the colon. Furthermore, tumors from ERβ−/−ApcMin/+ female mice were on average significantly larger than those from control ApcMin/+ mice. Higher steady-state proliferation in epithelial cells of the jejunum and colon from ERβ−/−ApcMin/+vs.ApcMin/+ female mice was confirmed by BrdU incorporation assay. Interestingly, functional categorization of microarray results revealed the TGFβ signaling pathway to be modulated in colonocytes, especially for the WT + E2vs. WT + Vehicle and the ERβ−/− + E2vs. WT + E2 comparisons. Using quantitative PCR analysis, we observed transcripts from ligands of the TGFβ pathway to be upregulated in colonocytes from E2-treated WT and ERβ−/− mice and downregulated in ERβ-deficient mice, mostly in an E2-independent manner. Therefore, our results demonstrate that ERβ deficiency enhances small intestinal tumorigenesis and suggest that modulation of the TGFβ signaling pathway could contribute to the protective role of estrogens on intestinal tumorigenesis. © 2008 Wiley-Liss, Inc.

Objective The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu. Design Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry. Results EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies. Conclusions Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.

Background: Intestinal stem cells regulate the emergence of different cell lineages across the crypt-villus axis and orchestrate cellular renewal and replenishment following injury (e.g. radiation). Two major ISC have been identified to date: a rapidly cycling crypt base columnar (CBC) stem cell and a more quiescent reserve stem cell located at the +4 position above the crypt base. CBCs marked by the Wnt target gene Lgr5 are dispensable for homeostasis and are quantitatively ablated by radiation injury. In contrast, quiescent ISCs are indispensable for homeostasis, regenerate CBCs, and are radioresistant. Keratins (Krt) are intermediate filaments required for architectural and mechanical support, but also contribute to cell migration and intracellular signaling. Interestingly, Krt15 has been characterized as a stem cell marker in the hair follicle. Here we described a radio-resistant Krt15+ stem cell population in the small intestine and demonstrate that cells within this population are capable of tumor initiation. Methods: Krt15-CrePR1 mice were crossed with Rosa26 reporter mice to perform lineage-tracing experiments in the mouse small intestine. High-dose radiation was used to investigate the role of Krt15 in tissue regeneration. Finally, Krt15-CrePR1 mice were crossed with Apcfl/fl mice to characterize the tumor-initiating properties of Krt15+ cells. Results: We observed that Krt15 marks a subpopulation of long-lived small intestinal crypt cells that are able to give rise to all differentiated lineages supporting their self-renewing and multipotent capacities. Interestingly, we observed that Krt15+ cells are able to form 3D organoids when seeded in Matrigel as a single cell suspension. Irradiation of Krt15-CrePR1;R26mTomato/mGFP mice with 12 Gy revealed that Krt15+ cells are radio-resistant and participate in tissue regeneration. Interestingly, Krt15 deficiency impairs tissue regeneration following high-dose radiation. Indeed, irradiated Krt15-/- mice display reduced crypt regeneration, crypt length, microcolonies and BrdU incorporation when compared to irradiated Krt15+/+ mice. Finally, we induced Apc loss in Krt15+ cells and observed formation of adenomas as well as adenocarcinomas in all Krt15-CrePR1;Apcfl/fl mice. Metastases were detected in some Krt15-CrePR1;Apcfl/fl mice. Conclusion: We have identified a long-lived Krt15+ subpopulation of intestinal crypt cells displaying stem cell-like properties that are radio-resistant and tumor initiating. This new stem cell population may open new avenues for therapeutic targeting in colon cancer. Citation Format: Veronique Giroux, Julien Stephan, Kathryn E. Hamilton, Priya Chatterjee, Sarah Andres, Andres J. Klein-Szanto, Christopher J. Lengner, Anil K. Rustgi. Keratin15 (Krt15) + are radio resistant and tumor-initiating cells in the mouse small intestine [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 5022. doi:10.1158/1538-7445.AM2017-5022

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer characterized by a high rate of metastasis, limited therapeutic options, and a poor prognosis. However, there is limited information regarding the molecular mechanisms underlying the metastatic properties of ESCC. p53 is one of the most commonly mutated genes in ESCC, and our group has shown that esophageal cells lines expressing a mutation in human p53 shows signs of malignancy and increased invasion in 3D organotypic culture. A mouse model of mutant p53 (R172H) in ESCC is lacking in the field. To elucidate the role of mutant p53 in ESCC we developed a novel mouse model utilizing a genetic and carcinogenic approach. L2cre;p53-/- and p53R172H/- mice were generated and treated with 4NQO in their drinking water for 16 weeks, which resulted in the development of ESCC. Compared to wildtype mice, p53R172H/- mice and p53-/- mice exhibited a decreased tumor latency time, increased tumor frequency and a more severe tumor diagnosis. However, p53R172H/- mice and p53-/- mice displayed similar tumorigenic properties. RNA-seq was performed on cell lines established from wildtype and p53 mouse models and reveled different gene expression profiles between wildtype, p53R172H/-, and p53-/- cells. p53R172H/- cells displayed an increase in mesenchymal and decrease in epithelial marker expression, supporting the idea that they are undergoing EMT. In addition, several endocytic recycling related genes, including Rab11-fip1, Rab25, and Myo5b were downregulated in mutant and null p53 compared to wildtype cells. Further examination of the differing genetic profiles in our mouse models can provide novel insight into the role of mutant p53 in ESCC tumorigenesis and lead to the identification of new therapeutic targets. Citation Format: Ashley Lento, Apple Long, Veronique Giroux, Qiaosi Tang, Morgan Sammons, Andres Klein-Szanto, Shelly Berger, Anil Rustgi. Investigating the role of mutant p53 in esophageal squamous cell carcinoma [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 2578. doi:10.1158/1538-7445.AM2017-2578

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

The estrogen-related receptor alpha (ERRα) is a nuclear receptor that acts primarily as a regulator of metabolic processes, particularly in tissues subjected to high-energy demand. In addition to its control of energy metabolism and mitochondrial biogenesis, ERRα has recently been associated with cancer progression. Notably, increased expression of ERRα has been shown in several cancerous tissues, including breast, ovary and colon. However, additional studies are required to gain insight into the action of ERRα in cancer biology, particularly in non-endocrine-related cancers. Therefore, using a short hairpin RNA-mediated approach, we investigated whether ERRα is required for the rapid growth of colon cancer cells and to maintain their neoplastic metabolic state. Results show that silencing ERRα significantly impaired colon cancer cell proliferation and colony formation in vitro as well as their in vivo tumorigenic capacity. A pronounced delay in G1-to-S cell cycle phase transition was observed in ERRα-depleted cells in association with reduced cyclin-dependent kinase 2 activity and hyperphosphorylated state of the retinoblastoma protein along with disturbed expression of several cell cycle regulators, including p15 and p27. Interestingly, ERRα-depleted HCT116 cells also displayed significant reduction in expression of a large set of key genes to glycolysis, tricarboxylic acid cycle and lipid synthesis. Furthermore, using (14)C isotope tracer analysis, ERRα depletion in colon cancer cells resulted in reduced glucose incorporation and glucose-mediated lipogenesis in these cells. These findings suggest that ERRα coordinates colon cancer cell proliferation and tumorigenic capacity with energy metabolism. Thus, ERRα could represent a promising therapeutic target in colon cancer.

Wild-type TP53 , a potent tumor suppressor gene, is responsible for initiating appropriate responses to a variety of cellular stresses and is the most commonly mutated gene in human cancers. Strikingly, most TP53 mutations are missense mutations within one of six hotspot codons in the DNA-binding domain (DBD). Recent evidence suggests that the …

unstable as evidenced by the presence of high level of DNA damage, centrosome amplification, and aneuploidy. AIM: To investigate role and possible mechanisms of Klf4 in preserving genetic stability. METHODS: To determine the ability of Klf4 restoration in correcting the observed genetic instability in MEFS null for Klf4 (Klf4-/-), we overexpressed Klf4 in Klf4-/MEFs and compared the results to wild type (Klf4+/+) and non-transfected Klf4-/MEFs. Cell cycle profiles and proliferation curve was performed to determine growth characteristics. Quantification of chromosome number and immunohistochemical staining of centrosomes were performed to assess chromosome integrity. To determine the role of Klf4 in correcting DNA damage, we overexpressed or not Klf4 in Klf4-/MEFs followed by γ-irradiation or not in parallel with Klf4+/+ and Klf4-/MEFs. Using immunohistochemistry, γH2AX and p53BP1 foci were counted at 0, 1h, 4h and 24h after γ-irradiation. The levels of p53, p21, cyclin E, Cdk2 and γH2AX in Klf4+/+ and Klf4-/MEFs, with and without Klf4 overexpression, were determined by Western blotting. RESULTS: Overexpression of Klf4 in Klf4-/MEFs suppressed cell proliferation compared to untransfected Klf4+/+ and untransfected Klf4-/MEFs. Centrosome staining showed a 10-fold decrease in the percent of cells with ≥3 centrosomes after overexpressing Klf4 in Klf4-/MEFs compared to untransfected Klf4-/MEFs. A higher percentage of Klf4-/MEFs cells were detected with increased and sustained ≥5 γH2AX and p53BP1 foci following γ-irradiation compared to irradiated Klf4+/+ MEFs. Overexpression of Klf4 in Klf4-/MEFs reduced the percentage of cells with ≥5 γH2AX foci to a level similar to that in Klf4+/+ MEFs, while the percent of cells with ≥5 p53BP1 foci were reduced to the same level as irradiated Klf4+/+ but at a faster rate. Karyotype analysis showed that Klf4-transfected Klf4-/MEFs had a 25% decrease in number of cells exhibiting aneuploidy in contrast to untransfected Klf4-/MEFs. Western blot analysis showed that p53, cyclin E, Cdk2 and γH2AX levels were reduced and p21 was elevated following Klf4 overexpression in Klf4-/MEFs. CONCLUSION: Results of this study demonstrate that overexpression of Klf4 in MEFs null for the Klf4 alleles display evidence of improved genetic stability as illustrated by reduced DNA damage, aneuploidy, and centrosome amplification. These results indicate that KLF4 plays a crucial role in preserving genetic stability.

The nuclear receptor ERRα acts principally in pair with its coactivator PGC-1α as a regulator of metabolic processes particularly in tissues subjected to high-energy demand. Besides its implication in energy metabolism and mitochondrial biogenesis, ERRα was recently associated with tumorigenesis. Increased expression of ERRα was noted in different cancerous tissues as breast, ovary and colon. Also, a new form of ERRα lacking the exon5 (ERRα Δ5) was recently identified and suggested to be a dominant negative of ERRα and interestingly to be differently expressed in normal and cancerous tissues. Since a protumorigenic role is emerging for this metabolic regulator, we investigated the role of ERRα and its spliced form ERRα β5 in colon carcinogenesis. METHODS: shRNA-mediated silencing of ERRα and overexpression of ERRα β5 were performed by lentivirus infection in DLD1 and HCT116 human colon cancer cells. The interaction between both proteins was investigated as well as their respective roles in various tumorigenic processes. RESULTS: Silencing of ERRα by shRNA reduced proliferation of DLD1 and HCT116 cells as measured by growth kinetics and growth assays in soft agar and reduced tumor formation in NUDE mice. Furthermore, FACS-scan analysis revealed that ERRα silencing delays G1-S phase transition in colon cancer cells. Differential expression of a splice variant of ERRα (ERRα Δ5) in cancer vs normal tissues would support the importance of ERRα in colorectal carcinogenesis. This is the case at least for colon tumors as cancerous samples display lower amount of ERRα β5 and higher amount of ERRα compared to adjacent normal tissues. Also, the ERRα β5 expression is surprisingly regulated by proteosomal degradation in normal and colon cancer cell lines and this could explain the low abundance of the ERRα spliced form. Interaction between both variants has been characterized by immunoprecipitation assays, confirming the conserved dimerization potential of ERRα β5 variant with ERRα. Furthermore, luciferase assays revealed that ERRα β5 is transcriptionaly inactive and its expression inhibits the transcriptional activity of ERRα on its target genes, suggesting that this variant acts as a dominant negative for ERRα. Interestingly, the reexpression of ERRα β5 in colon cancer cells reduces soft agar colony formation. CONCLUSION: Strong ERRα expression is associated with cancerous tissues and is required for the intense proliferation of colon cancer cells. Interestingly, the poorly active splice variant ERRα β5 is lost in cancerous colon tissues and has antiproliferative properties when reintroduced in colon cancer cells. Since this splice variant could interact with ERRα and inhibit its activity, ERRα β5 reduction of expression in cancerous tissue could allow ERRα to fully promote colon cancer cell proliferation. 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 1305. doi:1538-7445.AM2012-1305

Colon cancer is a major cause of death by cancer in North America. Recently, we found that both subunits of the integrin α6β4 are up-regulated in colon primary tumours. The α6 integrin subunit exists as two distinct splice isoforms, α6A and α6B. It is the α6A isoform that was shown to be associated with the proliferative cell population of the normal human colon and up-regulated in colorectal cancer. The aim of this study was to analyze the role of integrin α6Aβ4 in human colon cancer, more precisely its effect on cell proliferation and its mechanism of regulation. To investigate the function of integrin α6Aβ4 in colon cancer, expression of splice variant α6A was specifically abolished, using shRNA technology, in colon adenocarcinoma cell lines (Caco-2/15, T84, SW480, SW620, DLD-1 and HT29). The function of splice variant α6A of the α6β4 integrin was investigated in vitro and in tumour xenografts. In all colon cancer cell lines tested, knockdown of the integrin α6A subunit resulted in a decrease of up to 90% of the α6A variant without affecting the levels of the α6B variant. Furthermore, no compensatory expression of α6Bβ4 integrin was observed following the abolition of the α6A subunit. In the majority of colon cancer cell lines, the knockdown of α6A resulted in a significant decrease in proliferation as show by BrdU incorporation. Moreover, it was accompanied by reduced activity of the Wnt/β-catenin pathway as measured by a decrease of responsive β-catenin/TCF promoter activity and increased β-catenin phosphorylation at Ser37/Thr41 by GSK3β. Preliminary data confirmed that in these cells, the Wnt/β-catenin pathway can be stimulated by pharmacological inhibition of GSK3β (SB216763), as displayed by a decrease of β-catenin phosphorylation at Ser37/Thr41 and an increase in responsive β-catenin/TCF promoter activity. Analysis of splice variants of the α6 integrin subunit in human colorectal cancer revealed that α6A was increased in 80% of tumours as compared to their matched resection margins and a significant correlation was observed between expression of α6A and c-myc in the tumours. Furthermore, to determine the role of the integrin α6A subunit on colon cancer tumorigenesis in vivo, control and α6A knockdown T84, SW620, HT29 and DLD-1 cells were injected into nude mice. Results showed that abolition of the integrin α6A subunit led to a significant reduction in tumour growth for T84, SW620 and HT29 colon cell lines. Moreover, analysis of tumours showed an increase in β-catenin phosphorylation at Ser37/Thr41 in T84 and HT29. Our results show for the first time that splice variant A of the integrin α6 subunit regulates the Wnt/β-catenin pathway to promote cancer cell growth. Since 80% of colon cancers display an up-regulation of integrin α6A expression, targeting this integrin in colon cancers may offer new therapeutic value. 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 30. doi:1538-7445.AM2012-30

Besides its well-known function in energetic metabolism, the nuclear receptor Estrogen-Related Receptor Alpha (ERRα) has also been shown to be overexpressed in breast, ovarian, uterine, prostate and colon cancer and to be associated to a poor prognosis in some of these cancer types. Interestingly, an ERRα splice variant mainly expressed in non cancerous tissues has been reported. This variant missing the fifth exon (ERRα Δ5) is still expected to interact with DNA and form dimers but cannot recruit coactivators, therefore being devoid of any transcriptional activity. AIM: Determine the role of ERRα as well as ERRα Δ5 in intestinal tumorigenesis. METHODS: shRNA-mediated silencing of ERRα and overexpression of ERRα Δ5 were performed by lentivirus infection in DLD1 and HCT116 human colon cancer cells. The interaction between both proteins was investigated as well as their respective roles in various tumorigenic processes. RESULTS: Silencing of ERRα by shRNA reduced proliferation of DLD1 and HCT116 cells as measured by growth kinetics and anchorage-independent growth assays in soft agar. We also observed that ERRα knockdown cells grew into smaller tumors when subcutaneously implanted in NUDE mice. Furthermore, FACS-scan analysis revealed that ERRα silencing delays G1 to S phase transition in colon cancer cells. Differential expression of a splice variant of ERRα (ERRα Δ5) in cancer vs normal tissues would support the importance of ERRα role in colorectal carcinogenesis. This is the case at least for colon tumors as cancerous samples display lower amount of ERRα Δ5 and higher amount of ERRα compared to adjacent paired normal tissues, as measured by Western Blot analysis. Interaction between both variants has been characterized by immunoprecipitation assays, confirming the conserved dimerization potential of ERRα Δ5 variant with the full length ERRα. Furthermore, luciferase assays revealed that ERRα Δ5 is transcriptionaly inactive and its expression inhibits the transcriptional activity of ERRα on its target genes, suggesting that this variant acts as a dominant negative for ERRα. Interestingly, the reintroduction of ERRα Δ5 in human colon cancer cells leads to the reduction of cell growth and soft agar colony formation. CONCLUSION: Strong ERRα expression is associated with cancerous tissues and is required for the intense proliferation of colon cancer cells. Interestingly, the poorly active splice variant ERRα Δ5 is lost in cancerous colon tissues and has antiproliferative properties when reintroduced in colon cancer cells. Since this splice variant could interact with ERRα and inhibit its activity, ERRα Δ5 reduction of expression in cancerous tissue could allow ERRα to fully promote colon cancer cell proliferation. 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 2045. doi:10.1158/1538-7445.AM2011-2045