-
[show abstract]
[hide abstract]
ABSTRACT: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities in vitro. Ectopic Klf4 expression impaired subcutaneous tumor growth and lung colonization in vivo. By contrast, Klf4 knockdown enhanced HCC cell migration. Interestingly, ectopic expression of Klf4 changed the morphology of murine HCC cells to a more epithelial phenotype. Associated with this, we found that expression of Slug, a critical epithelial mesenchymal transition (EMT)-related transcription factor, was significantly down-regulated in Klf4-expressing cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays showed that Klf4 is able to bind and repress the activity of the Slug promoter. Furthermore, ectopic Slug expression partially reverts the Klf4-mediated phenotypes. Consistent with a role as a tumor suppressor in HCC, analysis of the public microarray databases from Oncomine revealed reduced KLF4 expression in human HCC tissues in comparison with normal liver tissues in 3 out of 4 data sets. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found reduced KLF4 mRNA in 50% of HCC tissues. Importantly, an inverse correlation between the expression of KLF4 and SLUG was found in HCC tissues. Our data suggest that KLF4 acts as a tumor suppressor in HCC cells, in part by suppressing SLUG transcription.
PLoS ONE 01/2012; 7(8):e43593. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Intrahepatic and extrahepatic metastases are common findings in hepatocellular carcinoma (HCC). Insulin-like growth factor 2 (IGF2) expression is frequently induced in HCC, and serum IGF2 levels correlate with the presence of extrahepatic metastases. Yet, the role of IGF-induced signaling in the dissemination of HCC remains unclear. We have previously observed elevated IGF2 levels in tumors with metastatic potential in an HCC mouse model. Here, we demonstrate that inhibition of IGF2, or its receptor IGF1R, impairs the migration and invasion activities of murine HCC cells. Furthermore, inhibition of IGF1R also impairs the ability of HCC cells to colonize the lungs after introduction into the circulation through the tail vein but does not impair subcutaneous tumor growth. Collectively, these findings suggest that IGF1R-mediated signaling plays a causative role in tumor dissemination but is not required for tumor growth per se. Although previous studies indicate that IGF ligands can signal through IGF1R/insulin receptor (IR) heterodimers, and IR-A homodimers, we demonstrate that the IR is not required for invasion and metastasis by HCC cells. Finally, we identify matrix metalloproteinase 2 as a mediator of the invasive phenotype downstream of IGF1R-induced signaling. Thus, our studies demonstrate the importance of IGF2-induced signaling in the dissemination of HCC cells.
Neoplasia (New York, N.Y.) 10/2009; 11(9):835-45. · 5.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The presence of distant metastases is a common finding on diagnosis of pancreatic cancer; however, the mechanisms underlying the dissemination of this tumor type remain poorly understood. Loss of the p53 tumor suppressor protein has been associated with tumor progression and metastasis in several tumor types including pancreatic ductal adenocarcinoma. Here, we describe the generation of a progressive and metastatic pancreatic cancer mouse model after the somatic and sporadic delivery of avian retroviruses encoding the mouse polyoma virus middle T antigen to elastase-tv-a transgenic mice with a pancreas-specific deletion of the Trp53 tumor suppressor locus. In this model, the tumors metastasize most frequently to the liver, consistent with human pancreatic carcinomas. Analysis of metastatic lesions demonstrated that concomitant loss of the Ink4a/Arf locus was not required for metastasis; however, pancreas-specific deletion of a single Ink4a/Arf allele cooperated with Trp53 deletion in a haploinsufficient manner to accelerate tumor development. Thus, our findings illustrate the potential role of p53 loss of function in pancreatic tumor progression, demonstrate the feasibility of modeling pancreatic cancer metastasis after somatic and sporadic oncogene activation, and indicate that our model may provide a useful experimental system for investigation of the molecular mechanisms underlying pancreatic cancer progression and metastasis.
American Journal Of Pathology 05/2008; 172(4):1081-7. · 4.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The INK4A/ARF tumor suppressor locus is frequently inactivated in hepatocellular carcinoma (HCC), yet the consequences of this remain unknown. We recently described a HCC mouse model in which loss of the Ink4a/Arf locus accelerates the development of metastasis and enhances tumor cell migration and invasion in cell culture assays. We show here that knockdown of p19Arf in an HCC cell line increases invasion in cell culture assays. Furthermore, reintroduction of p19(Arf) into HCC cell lines lacking Ink4a/Arf inhibits tumor cell invasion, without affecting cell proliferation, or cell transformation as measured by soft agar colony formation. Inhibition of cell invasion by p19(Arf) was dependent on its C-terminal binding protein (CtBP) interaction domain but independent of Mdm2 binding and nucleolar localization. Indeed, RNA interference-mediated knockdown of CtBP1 or CtBP2 decreased cell invasion, and ectopic expression of CtBP2 enhanced tumor cell migration and invasion. Thus, our data indicate a novel role for the Arf tumor suppressor protein in regulating phenotypes associated with tumor progression and metastasis in HCC cells.
Cancer Research 02/2008; 68(2):476-82. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tumors develop through multiple stages, implicating multiple effectors, but the tools to assess how candidate genes contribute to stepwise tumor progression have been limited. We have developed a novel system in which progression of phenotypes in a mouse model of pancreatic islet cell tumorigenesis can be used to measure the effects of genes introduced by cell-type-specific infection with retroviral vectors. In this system, bitransgenic mice, in which the rat insulin promoter (RIP) drives expression of both the SV40 T antigen (RIP-Tag) and the receptor for subgroup A avian leukosis virus (RIP-tva), are infected with avian viral vectors carrying cDNAs encoding candidate progression factors. Like RIP-Tag mice, RIP-Tag; RIP-tva bitransgenic mice develop isolated carcinomas by approximately 14 wk of age, after progression through well-defined stages that are similar to aspects of human tumor progression, including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. When avian retroviral vectors carrying a green fluorescent protein marker were introduced into RIP-Tag; RIP-tva mice by intra-cardiac injection at the hyperplastic or early dysplastic stage of tumorigenesis, approximately 20% of the TVA-positive cells were infected and expressed green fluorescent proteins as measured by flow cytometry. Similar infection with vectors carrying cDNA encoding either of two progression factors, a dominant-negative version of cadherin 1 (dnE-cad) or Bcl-xL, accelerated the formation of islet tumors with invasive properties and pancreatic lymph node metastasis. To begin studying the mechanism by which Bcl-xL, an anti-apoptotic protein, promotes invasion and metastasis, RIP-Tag; RIP-tva pancreatic islet tumor cells were infected in vitro with RCASBP-Bcl-xL. Although no changes were observed in rates of proliferation or apoptosis, Bcl-xL altered cell morphology, remodeled the actin cytoskeleton, and down-regulated cadherin 1; it also induced cell migration and invasion, as evaluated using two-chamber transwell assays. In addition, myosin Va was identified as a novel Bcl-xL-interacting protein that might mediate the effects of Bcl-xL on tumor cell migration and invasion.
PLoS Biology 11/2007; 5(10):e276. · 11.45 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent activities critical to the prevention of cancer in mice and humans. Recent evidence from mouse models suggests that when p53 is absent, further loss of ARF can widen the tumor spectrum, and potentiate invasion and metastasis. A major target of the p53-independent activity of ARF is the COOH-terminal binding protein (CtBP) family of metabolically regulated transcriptional corepressors, which are degraded upon acute exposure to the ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, to repress epithelial and proapoptotic genes, and can mediate hypoxia-induced migration of cancer cells. The possibility that ARF could suppress tumor cell migration as part of its p53-independent activities was thus explored. Small-interfering RNA (siRNA)-mediated knockdown of ARF in human lung carcinoma cells led to increased cell migration, especially during hypoxia, and this effect was blocked by concomitant treatment with CtBP2 siRNA. Introduction of ARF into p53 and ARF-null human colon cancer cells inhibited hypoxia-induced migration. Furthermore, overexpression of CtBP2 in ARF-expressing cells enhanced cell migration, and an ARF mutant defective in CtBP-family binding was impaired in its ability to inhibit cell migration induced by CtBP2. ARF depletion or CtBP2 overexpression was associated with decreased PTEN expression and activation of the phosphatidylinositol 3-kinase pathway, and a phosphatidylinositol 3-kinase inhibitor blocked CtBP2-mediated cell migration. Thus, ARF can suppress cell migration by antagonizing CtBP2 and the phosphatidylinositol 3-kinase pathway, and these data may explain the increased aggressiveness of ARF-null tumors in mouse models.
Cancer Research 11/2007; 67(19):9322-9. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. HCC patients frequently present with disease that has metastasized to other regions of the liver, the portal vein, lymph nodes, or lungs, leading to poor prognoses. Therefore, model systems that allow exploration of the molecular mechanisms underlying metastasis in this disease are greatly needed. We describe here a metastatic HCC model generated after the somatic introduction of the mouse polyoma virus middle T antigen to mice with liver-specific deletion of the Trp53 tumor suppressor locus and show the cell autonomous effect of p53 loss of function on HCC metastasis. We additionally find that cholangiocarcinoma also develops in these mice, and some tumors display features of both HCC and cholangiocarcinoma, suggestive of origin from liver progenitor cells. Concomitant loss of the Ink4a/Arf tumor suppressor locus accelerates tumor formation and metastasis, suggesting potential roles for the p16 and p19 tumor suppressors in this process. Significantly, tumor cell lines isolated from tumors lacking both Trp53 and Ink4a/Arf display enhanced invasion activity in vitro relative to those lacking Trp53 alone. Thus, our data illustrate a new model system amenable for the analysis of HCC metastasis.
Cancer Research 09/2007; 67(16):7589-96. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hedgehog signaling has been implicated in the development of several human cancers, including small cell lung carcinomas, medulloblastomas, basal cell carcinomas, and digestive tract tumors. Elevated levels of pathway components are observed in pancreatic ductal adenocarcinoma (PDAC) precursor lesions, and these levels increase further as lesions progress to more advanced stages. Yet the mechanisms by which hedgehog signaling contributes to pancreatic tumorigenesis were poorly understood. We recently published results showing that activated hedgehog signaling enhances the proliferation and survival of pancreatic duct epithelial cells, the presumptive target cells for PDAC development. We also demonstrated that sonic hedgehog (Shh) expression, in cooperation with loss of the Trp53 and Ink4a/Arf tumor suppressor loci, was sufficient to initiate the formation of early pancreatic lesions. Furthermore, Shh signaling enhanced K-Ras-mediated pancreatic tumorigenesis and reduced the dependence of tumor cells on the sustained activation of Ras-stimulated signaling pathways. Here we discuss the significance of these findings and the implications for therapy.
Cell cycle (Georgetown, Tex.) 08/2007; 6(13):1553-7. · 5.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of sonic hedgehog (Shh) signaling occurs in the majority of pancreatic ductal adenocarcinomas. Here we investigate the mechanisms by which Shh contributes to pancreatic tumorigenesis. We find that Shh expression enhances proliferation of pancreatic duct epithelial cells, potentially through the transcriptional regulation of the cell cycle regulators cyclin D1 and p21. We further show that Shh protects pancreatic duct epithelial cells from apoptosis through the activation of phosphatidylinositol 3-kinase signaling and the stabilization of Bcl-2 and Bcl-X(L). Significantly, Shh also cooperates with activated K-Ras to promote pancreatic tumor development. Finally, Shh signaling enhances K-Ras-induced pancreatic tumorigenesis by reducing the dependence of tumor cells on the sustained activation of the MAPK and phosphatidylinositol 3-kinase/Akt/mTOR signaling pathways. Thus, our data suggest that Shh signaling contributes to tumor initiation in the pancreas through at least two mechanisms and additionally enhances tumor cell resistance to therapeutic intervention. Collectively, our findings demonstrate crucial roles for Shh signaling in multiple stages of pancreatic carcinogenesis.
Proceedings of the National Academy of Sciences 04/2007; 104(12):5103-8. · 9.68 Impact Factor
-
Brian C Lewis
[show abstract]
[hide abstract]
ABSTRACT: The pancreas is specified during embryonic development from the gut endoderm. Among the signaling pathways required for the proper development of the organ are the notch and hedgehog signaling pathways. Both of these pathways are reactivated in pancreatic cancers, and sustained hedgehog signaling is required for the viability of most pancreatic cancer cell lines. Further, mouse models of the disease show activation of these pathways, and expression of pancreas progenitor markers. These findings indicate that developmentally regulated gene expression programs are important in the pathogenesis of pancreatic cancer.
Endocrinology & Metabolism Clinics of North America 07/2006; 35(2):397-404, xi. · 3.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have generated a mouse model for hepatocellular carcinoma using somatic delivery of oncogene-bearing avian retroviral vectors to the liver cells of mice expressing the viral receptor TVA under the control of the albumin gene promoter (Alb-TVA mice). Viruses encoding mouse polyoma virus middle T antigen (PyMT) induced tumors, which can be visualized with magnetic resonance imaging, in 65% of TVA-positive animals. While these tumors can exceed 10 mm in diameter, they do not invade locally or metastasize to the lungs. Delivery of PyMT-expressing viruses to Alb-TVA mice lacking an intact p53 gene does not increase tumor incidence. However, the resulting tumors are poorly differentiated, invasive, and metastatic to the lungs. Gene expression microarrays identified over 100 genes that are differentially expressed between tumors found in p53 wild-type and p53 null mice. Some of these genes, such as cathepsin E and Igf2, have been previously implicated in tumor cell migration and invasion. Tumors induced in p53 null, TVA transgenic mice by PyMT mutants with changes in specific tyrosine residues fail to form metastases, indicating that metastasis is dependent on both the oncogene and the absence of p53.
Molecular and Cellular Biology 03/2005; 25(4):1228-37. · 5.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have generated a mouse model for pancreatic cancer through the somatic delivery of oncogene-bearing avian retroviruses to mice that express TVA, the receptor for avian leukosis sarcoma virus subgroup A (ALSV-A), under the control of the elastase promoter. Delivery of ALSV-A-based RCAS vectors encoding either mouse polyoma virus middle T antigen (PyMT) or c-Myc to elastase-tv-a transgenic, Ink4a/Arf null mice induced the formation of pancreatic tumors. RCAS-PyMT induced pancreatic tumors with the histologic features of acinar or ductal carcinomas. The induced pancreatic lesions express Pdx1, a marker for pancreas progenitor cells, and many tumors express markers for both exocrine and endocrine cell lineages, suggesting that the tumors may be derived from progenitor cells. In contrast, RCAS-c-myc induced endocrine tumors exclusively, as determined by histology and detection of differentiation markers. Thus, specific oncogenes can induce the formation of different pancreatic tumor types in a single transgenic line, most likely from one or more types of multipotential progenitor cells. Our model appears to be useful for elucidating the genetic alterations, target cells, and signaling pathways that are important in the genesis of different types of pancreatic cancer.
Genes & Development 01/2004; 17(24):3127-38. · 11.66 Impact Factor
