Ben Rhoades

Genetic Modulation of PPARγ Phosphorylation Regulates Insulin Sensitivity

Abstract Obesity-associated diabetes is epidemic in industrialized societies. The nuclear receptor p eroxisome p roliferator- a ctivated r eceptor γ (PPARγ) is highly expressed in adipose tissue and the presumed molecular target for antidiabetic thiazolidinedione drugs that reverse insulin resistance but also promote weight gain. Phosphorylation reduces the activity of PPARγ in vitro, but physiological relevance has not been demonstrated. We have studied mice homozygous for a mutation (S112A) that prevents PPARγ phosphorylation. Surprisingly, the weights and adipose mass of PPARγ-S112A mice are not greater than wild-type. Remarkably, however, genetic prevention of PPARγ phosphorylation preserves insulin sensitivity in the setting of diet-induced obesity. Underlying this protection are smaller fat cells, elevated serum adiponectin, and reduced free fatty acid levels. Thus, the phosphorylation state of PPARγ modulates insulin sensitivity. Compounds that prevent PPARγ phosphorylation or ligands that induce the conformation of nonphosphorylated PPARγ may selectively enhance insulin sensitivity without increasing body weight.

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.

A mouse model of human oral-esophageal cancer

Squamous cancers of the oral cavity and esophagus are common worldwide, but no good genetically based animal model exists. A number of environmental factors as well as genetic alterations have been identified in these cancers, yet the specific combination of genetic events required for cancer progression remains unknown. The Epstein-Barr virus ED-L2 promoter (L2) can be used to target genes in a specific fashion to the oral-esophageal squamous epithelium. To that end, we generated L2-cyclin D1 (L2D1(+)) mice and crossbred these with p53-deficient mice. Whereas L2D1(+) mice exhibit a histologic phenotype of oral-esophageal dysplasia, the combination of cyclin D1 expression and p53 deficiency results in invasive oral-esophageal cancer. The development of the precancerous lesions was significantly reversed by the application of sulindac in the drinking water of the L2D1(+)/p53(+/-) mice. Furthermore, cell lines derived from oral epithelia of L2D1(+)/p53(+/-) and L2D1(+)/p53(-/-) mice, but not control mice, formed tumors in athymic nude mice. These data demonstrate that L2D1(+)/p53(+/-) mice provide a well-defined, novel, and faithful model of oral-esophageal cancer, which allows for the testing of novel chemopreventive, diagnostic, and therapeutic approaches.

Ha-Ras(G12V) induces senescence in primary and immortalized human esophageal keratinocytes with p53 dysfunction.

Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF–p53 pathway mediates Ha-RasG12V-induced senescence, and p19ARF−/− and p53−/− cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-RasG12V was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-RasG12V induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated β-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-RasG12V upregulated p16INK4a and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.

N-cadherin and keratinocyte growth factor receptor mediate the functional interplay between Ki-RASG12V and p53V143A in promoting pancreatic cell migration, invasion, and tissue architecture disruption.

ABSTRACT The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

IGFBP-3 regulates esophageal tumor growth through IGF-dependent and independent mechanisms

Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-RasV12-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-I from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.

Long-lived keratin 15+ esophageal progenitor cells contribute to homeostasis and regeneration

The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that Krt15+ basal cells are molecularly distinct from Krt15– basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, Krt15+ cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable Krt15+ progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium.

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

Mouse Intestinal Krt15+ Crypt Cells Are Radio-Resistant and Tumor Initiating

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.