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    COMBATING β-CATENIN DRIVEN HEPATOCELLULAR CARCINOMA

    by

    Evan R. Delgado

    B.S. in Biochemistry and Molecular Biology, State University of New York at Albany, 2009

    Submitted to the Graduate Faculty of

    School of Medicine in partial fulfillment

    of the requirements for the degree of

    Doctor of Philosophy

    University of Pittsburgh

    2014

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    UNIVERSITY OF PITTSBURGH

    SCHOOL OF MEDICINE

    This dissertation was presented

    by

    Evan R. Delgado

    It was defended on

    January 30, 2014

    and approved by

    Dr. George Michalopoulos, MD. PhD. Maud L. Menten Professor, Department of Pathology

    Dr. Aaron Barchowsky, PhD., Professor, Department of Environmental and Occupational Health

    Dr. Michael Tsang, PhD., Associate Professor, Department of Developmental Biology

    Dr. Shanmugam Nagarajan, PhD., Associate Professor, Department of Pathology

    Dissertation Advisor: Dr. Satdarshan P. S. Monga, MD., Professor, Department of Pathology

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    Copyright © by Evan R. Delgado

    2014

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    Hepatocellular Carcinoma (HCC) is the most common primary liver tumor and is a

    major cause of cancer related death worldwide with few avenues of treatment that benefit

    patients. There are many causes of HCC making the cancer difficult to treat as a homogenous

    disease. Many molecular pathways are deregulated during the onset of hepatocarcinogenesis, and

    one commonly activated signaling cascade in HCC is the Wnt/β-catenin pathway. β-Catenin

    plays multiple roles in cellular processes from maintenance of cellular adhesions to regulating

    regenerative signals required for the liver to grow. In cancer, β-catenin signaling is aberrantly

    regulated and has now been shown to play roles in tumor cell proliferation, survival and

    metabolism contributing to disease progression. Knowing the prevalence and importance of β-

    catenin in HCC, it is critical to develop targeted, personalized therapeutics which may impact

    multiple aspects of tumor biology. Here, we have identified several different avenues that target

    active Wnt signaling. We demonstrate the importance of computational biology to identify novel

    small molecules (SMs) to target β-catenin signaling. SM treatment results in decreased β-catenin

    signaling leading to decreases in downstream targets affecting HCC growth and survival. We

    also utilized antisense treatments which target β-catenin at the genetic level decreasing β-catenin

    protein expression and leading to subsequent cell death and decrease in tumor burden.

    Additionally, we identified angiogenesis as a notable event regulated by Wnt/β-catenin signaling

    COMBATING BETA-CATENIN DRIVEN HEPATOCELLULAR CARCINOMA

    Evan R. Delgado, B.S.

    University of Pittsburgh, 2014

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    In fact, inhibiting Wnt signaling in HCC cells led to reduced production of pro-angiogenic

    secreted factors which in turn inhibited angiogenic characteristics in tumor associated endothelial

    cells. Our studies validate the significance of targeting β-catenin in HCC that should lead to

    notable effects on tumor growth and development. With the current studies providing the final

    proof-of-concept, we now believe that β-catenin directed therapies may in fact be plausible, and

    have potential clinical implications.

    PUBLICATIONS

    1) Evan Delgado, Hiro Okabe, Jing Yang, Yixian Zhang, Lee M. Greenberger, Satdarshan P. S. Monga. Combatting β-catenin driven hepatocellular carcinoma by utilization of Locked Nucleic Acids. (In progress) 2) Evan Delgado, Jing Yang, Juhoon So, Stephanie Leimgruber, Michael Kahn, Tohru Ishitani, Donghun Shin, Gabriella Mustata, Satdarshan P. S. Monga. Identification and characterization of a novel small molecule inhibitor of beta-catenin signaling in hepatocellular carcinoma. American Journal of Pathology. (submitted). 3) Evan Delgado, Raman Bahal, Jing Yang, Jung Min Lee, Danith H Ly, Satdarshan P. S. Monga. β-catenin Knockdown by Cell Permeable Gamma Guanidine-Based Peptide Nucleic Acid Reveals its New Roles in Hepatocellular Cancer. Current Cancer Drug Targets. (Jul 1, 2013).

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    TABLE OF CONTENTS

    PREFACE .................................................................................................................................... 15

    1.0 INTRODUCTION ...................................................................................................... 17

    1.1 CANCER EPIDEMIOLOGY ........................................................................... 18

    1.1.1 Worldwide cancer burden ............................................................................ 18

    1.1.2 Cancer related deaths worldwide ................................................................. 19

    1.1.3 Four most common cancers: Breast, Colorectal, Lung, and Liver ........... 19

    1.2 HEPATOCELLULAR CARCINOMA ........................................................... 21

    1.2.1 Major Global Risk Factors for HCC ........................................................... 22

    1.2.2 HCC in the United States .............................................................................. 25

    1.3 HEPATOCARCINOGENESIS ........................................................................ 26

    1.3.1 Stepwise process to hepatocellular oncogenesis .......................................... 27

    1.3.2 Biochemical Pathways known to be involved in HCC ............................... 30

    1.4 WNT/β-CATENIN SIGNALING ..................................................................... 34

    1.4.1 Wnt/β-catenin signaling in HCC .................................................................. 35

    1.5 CURRENT THERAPEUTIC STRATEGIES FOR HCC ............................. 37

    1.5.1 Sorafenib and TACE in HCC ....................................................................... 38

    1.5.2 Current clinical trial developments ............................................................. 39

    1.5.3 Preclinical Developments .............................................................................. 43

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    1.6 CURRENT CONTRIBUTIONS OF BETA-CATENIN TARGETED

    THERAPEUTICS IN HCC ............................................................................................... 46

    2.0 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL SMALL

    MOLECULE INHIBITOR OF BETA-CATENIN SIGNALING IN HEPATOCELLULAR

    CANCER CELLS ....................................................................................................................... 48

    2.1 IDENTIFICATION OF SMALL MOLECULES TARGETING

    WNT/BETA-CATENIN SIGNALING ............................................................................. 49

    2.1.1 Structural similarity search reveals ICG-001 analogs ............................... 49

    2.1.2 In silico toxicity prediction reveals promising small molecules ................ 50

    2.2 BIOLOGICAL VALIDATION OF LEAD SMALL MOLECULES ............ 51

    2.2.1 Identifying small molecules capable of inhibiting Wnt/β-catenin signaling

    in vitro 51

    2.2.2 Toxicity studies reveal PMED-1 to be top candidate ................................. 52

    2.3 INVESTIGATING TIME AND DOSE DEPENDENCY OF PMED-1 ........ 52

    2.3.1 Wnt/β-catenin activity assay studies PMED-1 efficacy .............................. 54

    2.3.2 Inhibiting cell cycle progression and growth supports activity assay ....... 54

    2.4 HCC CELL LINE SPECIFICITY OF PMED-1 EXAMINED ..................... 56

    2.4.1 Multiple HCC cell lines are susceptible to PMED-1 via TOPFlash assay 56

    2.4.2 PMED-1 causes consistent inhibition of Wnt target gene expression in

    multiple HCC cell lines .............................................................................................. 57

    2.5 ACTIVITY SPECIFICITY OF PMED-1 MIMICS ICG-001 ....................... 59

    2.5.1 Co-immunoprecipitation of CBP/β-catenin shows PMED-1 activity ....... 59

    2.5.2 PMED-1 does not show off target kinase inhibition ................................... 60

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    2.6 POSSIBLE IN VIVO APPLICATION FOR PMED-1 ................................... 62

    2.6.1 Addressing stability of PMED-1 in vitro ...................................................... 62

    2.6.2 Inhibition of Wnt signaling in Zebrafish with PMED-1 ............................ 63

    2.6.3 Characterizing duration of Wnt signaling inhibition in vivo ..................... 64

    2.7 DISCUSSION OF APPLICABILITY OF PMED-1 ....................................... 66

    3.0 β-CATENIN KNOCKDOWN BY CELL PERMEABLE GAMMA GUANIDINE-

    BASED PEPTIDE NUCLEIC ACID REVEALS ITS NEW ROLES IN

    HEPATOCELLULAR CANCER ............................................................................................. 70

    3.1 DEVELOPING AN ANTISENSE AGAINST BETA-CATENIN ................. 71

    3.1.1 Design and synthesis of Peptide Nucleic acids ............................................ 71

    3.1.2 Uptake of Peptide Nucleic Acids Leads to Inhibited Wnt/β-catenin

    Signaling in HCC cells ...........................................................