Maria E. Vega

Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene.

p120-catenin (p120ctn) interacts with E-cadherin, but to our knowledge, no formal proof that p120ctn functions as a bona fide tumor suppressor gene has emerged to date. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop preneoplastic and neoplastic lesions in the oral cavity, esophagus, and squamous forestomach. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.

Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors.

Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.

Differential ability of Tribbles family members to promote degradation of C/EBPα and induce acute myelogenous leukemia

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPalpha), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha, which account for their differential ability to induce leukemia.

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.

Transformation by Tribbles homolog 2 (Trib2) requires both the Trib2 kinase domain and COP1 binding

Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)-binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for Trib2-induced AML. Deletion or mutation of the COP1-binding site abrogated the ability of Trib2 to degrade CCAAT/enhancer-binding protein-α (C/EBP-α), block granulocytic differentiation, and to induce AML in vivo. Furthermore, COP1 knockdown inhibited the ability of Trib2 to degrade C/EBP-α, showing that it is important for mediating Trib2 activity. We also show that the Trib2 kinase domain is essential for its function. Trib2 contains variant catalytic loop sequences, compared with conventional kinases, that we show are necessary for Trib2 activity. The kinase domain mutants bind, but cannot efficiently degrade, C/EBP-α. Together, our data demonstrate that Trib2 can bind both COP1 and C/EBP-α, leading to degradation of C/EBP-α. Identification of the functional regions of Trib2 that are essential to its oncogenic role provides the basis for developing inhibitors that will block Trib functions in cancer.

A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human cancer with poor prognosis due to late diagnosis and metastasis. Common genomic alterations in ESCC include p53 mutation, p120ctn inactivation, and overexpression of oncogenes such as cyclin D1, EGFR, and c-Met. Using esophageal epithelial cells transformed by the overexpression of EGFR and p53R175H, we find novel evidence of a functional link between p53R175H and the c-Met receptor tyrosine kinase to mediate tumor cell invasion. Increased c-Met receptor activation was observed upon p53R175H expression and enhanced further upon subsequent EGFR overexpression. We inhibited c-Met phosphorylation, resulting in diminished invasion of the genetically transformed primary esophageal epithelial cells (EPC-hTERT-EGFR-p53R175H), suggesting that the mechanism of increased invasiveness upon EGFR and p53R175H expression may be the result of increased c-Met activation. These results suggest that the use of therapeutics directed at c-Met in ESCC and other squamous cell cancers.

Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4

Barretts 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.

Abstract 5194: Notch regulates squamous differentiation, cell plasticity and tumor heterogeneity in esophageal carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: The Notch receptor family regulates cell fates and may act as a tumor suppressor in the squamous epithelia. While squamous differentiation is maintained in esophageal squamous cell carcinoma (ESCC) and its precursor lesions, the roles of Notch in esophageal tumor biology remain elusive. Methods: Transformed human esophageal cells expressing EGFR, p53R175H and cyclin D1 (EPC2-T) and ESCC cell lines were stably transduced with ICN1, an active form of NOTCH1 (N1) in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or gamma-secretase inhibitors (GSI). 8xCSL-luciferase reporter was transiently transfected to assess Notch activity. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2. Squamous epithelium was reconstituted in organotypic (3D) culture, a form of tissue engineering. In addition, cell growth was assessed in soft agar and immunodeficient mice. Quantitative RT-PCR, Western blotting, immunohistochemistry (IHC) and flow cytometry were done to determine gene expression. Primary ESCC tissues (n=20) were analyzed by IHC. Results: In primary ESCC, N1 was localized to well-differentiated tumor cell nests expressing involucrin (IVL), a squamous differentiation marker, while downregulated in poorly-differentiated cells lacking IVL within a single tumor tissue, implying N1 in tumor heterogeneity. In cell lines, ICN1 activated Notch-dependent transcription and squamous differentiation in the absence of DNMAML1. In 3D culture, DNMAML1 and GSI not only blocked squamous differentiation but induced massive invasion and epithelial-to-mesenchymal transition (EMT) with Wnt activation as suggested by loss of E-cadherin and nuclear localization of β-catenin. In agreement, DNMAML1 upregulated LEF1, Zinc finger E-box binding proteins ZEB1 and ZEB2, transcription factors all essential in EMT and maintenance of cancer stem cells, facilitating transforming growth factor (TGF)-β1-induced EMT in EPC2-T cells. Moreover, DNMAML1 enriched a unique subset of CD44-bright and CD24-dim cells and augmented colony formation, tumorigenicity as well as chemotherapeutic drug resistance against Cisplatin. Importantly, Cre-mediated removal of chromosomally integrated floxed DNMAML1 reactivated Notch to allow terminal differentiation with IVL induction. Moreover, ZEB knockdown greatly impaired TGF-β-mediated EMT while restoring chemotherapeutic drug sensitivity. Conclusions: These data indicate that Notch activation promotes squamous differentiation whereas Notch inhibition enriches poorly differentiated tumor cells with cancer stem cell potential with drug resistance, which involves ZEB and Wnt activation, thus providing a novel mechanistic insight into how Notch signaling may contribute to tumor heterogeneity in ESCC. 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 5194. doi:10.1158/1538-7445.AM2011-5194

252 CD44 Defines Highly Plastic Esophageal Squamous Cancer Cells With Tumor Initiating Capabilities

Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barretts esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription and decreased expression of its own targets P21, P27 and E-Cadherin. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling.

Abstract CN04-03: Esophageal cancer and the tumor microenvironment

Squamous cell cancers are the most common type of epithelially derived human cancers and arise from a number of diverse sites. They share a number of common genetic alterations and environmental exposures. Employing oral and esophageal cancers as prototypes, we have developed three‐dimensional (3D) or organoptypic models that represent a form of human tissue engineering (Genes and Development 2007; Journal of Clinical Investigation 2008). These approaches underscore the combinatorial roles of EGFR signaling and p53 mutation in fostering tumor cell migration and invasion in the microenvironment. Microarray analyses reveal specific effectors in the adhesion family of genes that might be exploited as biomarkers for detection and targets for new therapeutics. We have also generated genetically engineered mouse models that reveal the effects of cyclin D1 overexpression in concert with p53 loss, as well as the role of p120‐catenin loss in the initiation and progression of cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):CN04-03.