BREAST CANCER METASTATIC DORMANCY AND EMERGENCE, A ROLE FOR ADJUVANT STATIN THERAPY by Colin Henry Beckwitt Bachelor of Science in Biological Engineering, Massachusetts Institute of Technology, 2013 Submitted to the Graduate Faculty of The School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2018 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Colin Henry Beckwitt It was defended on May 22, 2018 and approved by Chairperson: Donna Beer Stolz, PhD, Associate Professor, Department of Cell Biology Zoltán N. Oltvai, MD, Associate Professor, Department of Pathology Partha Roy, PhD, Associate Professor, Departments of Bioengineering and Pathology Kari N. Nejak-Bowen, MBA, PhD, Assistant Professor, Department of Pathology Linda G. Griffith, PhD, School of Engineering Teaching Innovation Professor, Departments of Biological and Mechanical Engineering Dissertation Advisor: Alan Wells, MD, DMSc, Thomas J. Gill III Professor, Department of Pathology ii Copyright © by Colin Henry Beckwitt 2018 iii BREAST CANCER METASTATIC DORMANCY AND EMERGENCE, A ROLE FOR ADJUVANT STATIN THERAPY Colin Henry Beckwitt University of Pittsburgh, 2018 Breast cancer is responsible for the most new cancer cases and is the second highest cause of cancer related deaths among women. Localized breast cancer is effectively treated surgically. In contrast, metastatic cancers often remain undetected as dormant micrometastases for years to decades after primary surgery. Emergence of micrometastases to form clinically evident metastases complicates therapeutic intervention, making survival rates poor. The often long lag time between primary tumor diagnosis and emergence of metastatic disease motivates the development or repurposing of agents to act as safe, long term adjuvants to prevent disease progression. The statin drugs have been FDA approved for the treatment of hypercholesterolemia for three decades. Despite their well-studied effects on lipid homeostasis, statins feature beneficial pleiotropic effects on other organ systems and pathologies. Several epidemiological studies have demonstrated statins do not affect cancer incidence but do reduce breast cancer mortality. Previous in vitro work has demonstrated growth-suppressive and apoptosis-inducing effects of statins in tumor cell lines. However, the mechanisms and markers that contribute to statin’s suppressive effects on tumor cells remain unclear. We hypothesize that statins benefit cancer mortality by directly suppressing the outgrowth of dormant metastatic disease. The data presented herein demonstrate that statins reduce tumor cell growth and survival, in part due to suppression of Ras and Akt signaling. Importantly, these effects are limited to the membrane-permeant lipophilic statins. In ex vivo and in vivo models of metastatic breast cancer, iv statins reduce the proliferation at the site of metastasis but not the primary tumor. Tumor cells expressing membrane E-cadherin are relatively more resistant to statin therapy, which implies that the clinical cancer mortality benefit observed is due to decreased outgrowth of dormant epithelial micrometastases into clinically evident mesenchymal macrometastases. These data motivate the further study of statins as safe, long term adjuvants to prevent emergent breast cancer. Moreover, these data recommend a therapeutic switch to lipophilic statins in breast cancer patients taking statins for other diseases. That statins selectively suppress metastatic but not primary tumor growth is promising for their use as safe, long term adjuvants to reduce recurrence and mortality from metastatic breast cancer. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIX 1.0 BREAST CANCER PATHOPHYSIOLOGY AND THERAPY ............................. 1 1.1 INTRODUCTION ............................................................................................... 1 1.2 CLINICAL EPIDEMIOLOGY .......................................................................... 1 1.3 DEVELOPMENT, ANATOMY, AND PHYSIOLOGY .................................. 7 1.4 DISEASE PROGRESSION ................................................................................ 9 1.5 CLINICAL AND MOLECULAR PATHOLOGY ......................................... 14 1.5.1 Histologic Classification of Breast Cancer .................................................. 14 1.5.2 Clinical Classification of Breast Cancer ...................................................... 16 1.5.3 Molecular Classification of Breast Cancer .................................................. 19 1.6 CELLULAR AND MOLECULAR PATHOLOGY ....................................... 22 1.6.1 The Estrogen Receptor .................................................................................. 22 1.6.1.1 History .................................................................................................. 22 1.6.1.2 Molecular Biology ............................................................................... 24 1.6.2 The Progesterone Receptor........................................................................... 26 1.6.3 The ErbB Receptor Family ........................................................................... 28 1.6.3.1 The Epidermal Growth Factor Receptor (EGFR) ........................... 28 1.6.3.2 The Human Epidermal Growth Factor Receptor 2 (HER2) .......... 30 1.6.3.3 The Human Epidermal Growth Factor Receptor 3 (HER3) .......... 32 1.6.3.4 The Human Epidermal Growth Factor Receptor 4 (HER4) .......... 33 1.6.3.5 ErbB Family Summary ...................................................................... 33 vi 1.6.4 Ras – MAPK Signaling.................................................................................. 34 1.6.5 PI3K – Akt Signaling ..................................................................................... 38 1.6.6 Ras – MAPK and PI3K – Akt Crosstalk ..................................................... 40 1.6.7 Tumor Heterogeneity .................................................................................... 41 1.7 SURGERY .......................................................................................................... 42 1.8 RADIATION THERAPY ................................................................................. 45 1.9 CHEMOTHERAPY .......................................................................................... 49 1.9.1 Anthracyclines ............................................................................................... 49 1.9.2 Alkylating Agents .......................................................................................... 52 1.9.2.1 Cyclophosphamide .............................................................................. 53 1.9.2.2 Platinum-containing drugs ................................................................. 54 1.9.3 Mitotic Inhibitors ........................................................................................... 55 1.9.3.1 Taxanes ................................................................................................ 56 1.9.3.2 Other Microtubule Inhibitors ............................................................ 58 1.9.4 Antimetabolite Drugs .................................................................................... 60 1.9.4.1 5-Fluorouracil and Capecitabine ....................................................... 60 1.9.4.2 Gemcitabine ......................................................................................... 62 1.10 BIOLOGICAL AGENTS .................................................................................. 63 1.10.1 Anti-Estrogen Therapies .............................................................................. 64 1.10.1.1 Selective Estrogen Receptor Modulators (SERMs) ....................... 64 1.10.1.2 Selective Estrogen Receptor Degraders (SERDs) .......................... 67 1.10.1.3 Aromatase Inhibitors ........................................................................ 68 1.10.1.4 Luteinizing Hormone Releasing Hormone (LHRH) Analogues ... 71 vii 1.10.2 HER2 Targeted Therapies ........................................................................... 71 1.10.2.1 Trastuzumab and T-DM1 ................................................................ 72 1.10.2.2 Pertuzumab........................................................................................ 74 1.10.2.3 Lapatinib ............................................................................................ 74 1.10.3 EGFR Targeted Therapies .......................................................................... 75 1.10.4 CDK4/6 Targeted Therapies ....................................................................... 76 1.10.5 PARP Targeted Therapies ........................................................................... 77 1.10.6 PD-1 and PD-L1 Targeted Therapies ......................................................... 78 1.11 COMMON SYSTEMIC THERAPY REGIMENS ......................................... 79 1.12 CLINICAL NEED ............................................................................................. 80 2.0 STATIN PHARMACOLOGY .................................................................................. 82 2.1 DISCOVERY ..................................................................................................... 82 2.2 PHARMACODYNAMICS ..............................................................................