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    Identification and Characteristics of Factors Regulating Hepatocellular Carcinoma Progression and Metastasis: A Dissertation

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    Authors
    Ahronian, Leanne G.
    Faculty Advisor
    Brian C. Lewis, PhD
    Academic Program
    Cancer Biology
    UMass Chan Affiliations
    Molecular, Cell and Cancer Biology Department
    Document Type
    Doctoral Dissertation
    Publication Date
    2014-03-28
    Keywords
    Dissertations, UMMS
    Carcinoma, Hepatocellular
    Gene Expression Profiling
    Genes, p53
    Kruppel-Like Transcription Factors
    Liver Neoplasms
    Point Mutation
    rho GTP-Binding Proteins
    Transcription Factors
    Tumor Suppressor Protein p53
    Hepatocellular Carcinoma
    Gene Expression Profiling
    p53 Genes
    Kruppel-Like Transcription Factors
    Liver Neoplasms
    Point Mutation
    rho GTP-Binding Proteins
    Transcription Factors
    Tumor Suppressor Protein p53
    Cancer Biology
    Digestive System Diseases
    Molecular Genetics
    Neoplasms
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    Abstract
    Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.
    DOI
    10.13028/M2002W
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/32063
    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/M2002W
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