Roskilde University – Bachelor Project in Natural Sciences 6th semester, Spring 2016, house 14.2. Down-regulation of active HGFA and matriptase as therapeutic targets against cancer Gloire Irakunda, Ida Axholm, Karoline Knudsen List, Kiwi Kjøller, Rikke Svendsen & Simone Radziejewska Andreasen Supervisor: Cathy Mitchelmore Preface This study is a 6th semester bachelor project in medical and molecular biology on the Natural Science Bachelor at Roskilde University. The project is written in the spring semester of 2016 by Gloire Irakunda, Ida Axholm, Karoline Knudsen List, Kiwi Kjøller, Rikke Svendsen, and Simone Radziejewska Andreasen. The group would particularly like to thank our supervisor Cathy Mitchelmore, associate professor at the Institute of Science and Environment at RUC, for her guidance and supervision. In addition, we would like to thank Karin List, associate professor at Barbara Ann Karmanos Cancer Institute at Wayne State University School of Medicine, for feedback and answering clarifying questions. Furthermore, we would like to thank group 6 and their supervisor Michelle Vang for constructive criticisms. 1/60 Abstract The cellular mesenchymal-epithelial transition (c-MET) receptor is activated by the hepatocyte growth factor (HGF). The proform of HGF (pro-HGF) is cleaved and activated by the serine proteases matriptase and hepatocyte growth factor activator (HGFA). Only the active form of HGF is able to activate the c-MET receptor. Matriptase and HGFA are inhibited by the HGF activator inhibitors (HAI-1 and -2), which are encoded by the serine protease inhibitor kunitz type 1 and -2 (SPINT1 and -2) genes. An enhanced activation of the c-MET receptor is associated with increased cell proliferation, angiogenesis, invasion, and metastasis in cancer. The aim of this literature review is to examine how the ratio between active matriptase/HGFA and HAI-1 and -2 is disrupted in cancer, and what the therapeutic potentials of matriptase, HGFA, HAI-1 and -2 are. Based on in vitro studies we hypothesize that one mechanism leading to this disruption is due to change in pH and the oxidative conditions, which influence the activation of HGFA and matriptase. However, it might be difficult to control these environmental factors and the potential of selective protease inhibitors is therefore also discussed. Further, the disruption is likely to be caused by hypermethylation of SPINT1 and -2. As treatment against cancer, it might be possible to demethylate the SPINT genes or to directly inject HAI proteins and/or synthetic constructed protease inhibitors. Further investigations are needed before it is possible to conclude whether matriptase, HGFA, HAI-1 and -2 are efficient as therapeutic targets in cancer. 2/60 Table of content PREFACE ............................................................................................................................................................................................. 1 ABSTRACT .......................................................................................................................................................................................... 2 1 INTRODUCTION ............................................................................................................................................................................ 5 1.1 AIM ................................................................................................................................................................................................................... 6 1.2 READING GUIDE .............................................................................................................................................................................................. 6 2 BACKGROUND THEORY ............................................................................................................................................................. 8 2.1 HALLMARKS OF CANCER ............................................................................................................................................................................... 8 2.1.1 Oncogenes ............................................................................................................................................................................................8 2.1.2 Tumor suppressor genes.................................................................................................................................................................9 2.1.3 Metastasis and angiogenesis ..................................................................................................................................................... 10 2.1.4 The cancer stem cell theory........................................................................................................................................................ 12 2.2 THE HGF-CMET AXIS................................................................................................................................................................................. 13 2.2.1 Structure and activation of HGF .............................................................................................................................................. 13 2.2.2 Structure and Activation of c-MET .......................................................................................................................................... 14 2.2.3 Functions of the HGF and c-MET .............................................................................................................................................. 16 2.3 HGFA STRUCTURE AND FUNCTION ........................................................................................................................................................... 21 2.4 THE STRUCTURE AND ACTIVATION OF MATRIPTASE .............................................................................................................................. 23 2.5 HAI-1 AND -2 .............................................................................................................................................................................................. 25 2.5.1 The structure and function of HAI-1 and -2 ......................................................................................................................... 26 2.5.2 HAI’s expression and regulation of SPINT1 and -2 ........................................................................................................... 27 2.5.3 HAI-1 and -2 inhibit HGFA through different interactions ............................................................................................ 28 2.5.4 The interactions between HAI-1/ -2 and matriptase ....................................................................................................... 29 2.5.5 HAI-2 regulates matriptase activation through prostasin ............................................................................................ 30 3 REVIEW ......................................................................................................................................................................................... 32 3.1 EXPRESSION OF HAI-1 AND -2 IS DECREASED IN CANCER .................................................................................................................... 32 3.1.1 HAI-1 and -2 are inversely correlated with tumor progression ................................................................................... 32 3.1.2 Decreased levels of HAI-2 mRNA might be caused by hypermethylation ................................................................ 33 3.1.3 Post-transcriptional modification of HAI-2 indicates two species of HAI-2 ............................................................ 34 3.2 ALTERED LEVELS OF ACTIVE HGFA IN CANCER ...................................................................................................................................... 35 3.2.1 Up-regulated levels of HGFA in cancer tissue ...................................................................................................................... 35 3.2.2 Regulation of HGFA by HIF-1α .................................................................................................................................................. 35 3.3 ACTIVE MATRIPTASE IN RELATION TO CANCER PROGRESSION ............................................................................................................. 36 3.3.1 Up-regulated matriptase in cancer tissue ............................................................................................................................ 36 3.3.2 Dysregulation of the ratio between HAI-1 and matriptase promotes cancer progression ............................... 37 3/60 3.3.3 Regulation of active matriptase through control of matriptase zymogen activation ........................................ 37 3.4 OVEREXPRESSION OF C-MET IN CANCER TISSUE.................................................................................................................................... 39 4. DISCUSSION ................................................................................................................................................................................ 40 4.1 IMPORTANCE OF THE RATIO BETWEEN HGFA/MATRIPTASE AND HAI-1/ -2 ................................................................................. 40 4.2 HGFA AND MATRIPTASE