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 AS POTENTIAL TARGETS AGAINST CANCER...... 40 4.2.1 Selective protease inhibitors as therapeutic agents ...... 41

4.3 DEMETHYLATION OF SPINT1 AND -2 AS THERAPEUTIC TARGETS IN CANCER ...... 42 4.3.1 DAC and AZA block methylation of SPINT2 by inhibition of DNMTs ...... 43 4.3.2 DAC and AZA as cancer treatments cause side effects ...... 44

4.4 INJECTION OF HAI-1 AND -2 AS THERAPEUTIC TREATMENT ...... 46

4.5 FURTHER PERSPECTIVES ...... 47

5 CONCLUSION ...... 48

REFERENCES ...... 49

APPENDIX 1 - CPG ISLANDS IN SPINT1 AND -2 ...... 57

APPENDIX 2 – ABBREVIATION LIST ...... 58

4/60 1 Introduction

Cancer is a well-known condition thought to arise from mutations and/or epigenetic changes of genes that influence the six hallmarks of cancer. These six hallmarks are characterized as sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis (Alberts et al., 2015; reviewed by Hanahan and Weinberg, 2011). Some of these hallmarks are associated with binding between the cellular mesenchymal-epithelial transition (c-MET) receptor and its specific ligand hepatocyte growth factor (HGF) (Figure 1A) (reviewed by Garajová et al., 2015). HGF is synthesized and secreted as the inactive precursor pro-HGF, which depends on site-specific cleavage to function as a ligand for the phosphorylation and activation of the c-MET-receptor (Figure 1A) (reviewed by Parr et al., 2010).

Figure 1. HGFA, matriptase and HAI-1 and -2 pathway. Part A illustrates cleavage of pro-HGFA and of a matriptase zymogen to their active forms, which can convert pro-HGF to HGF. HGF is then able to act as a ligand and bind to the c-MET receptor, leading to the activation of downstream signaling pathways involved in cancer progression. Both HAI- 1 and -2 are capable of inhibiting matriptase and HGFA, shown in part B. When inhibited, matriptase and HGFA are not capable of converting pro-HGF into its active form (Inspired by Parr et al., 2010).

5/60 A number of serine proteases, such as matriptase, hepatocyte growth factor activator (HGFA), and hepsin have been detected as potent converters of pro-HGF and are referred to as HGF activators (Figure 1A) (Chen et al., 2013; Miyazawa et al., 1993; Han et al., 2014). Increased activation of the HGF-cMET axis is thought to play a crucial role in the progression of several invasive and metastatic cancers and is suggested to enhance specific pro-oncogenic processes including cell migration, angiogenesis and invasion. The HGF-cMET axis has therefore been a target for treatment in most cancers, but is seen to develop resistance to several types of anticancer drugs and therapies (reviewed by Gherardi et al., 2012). HGFA and matriptase are up-regulated in many cancer tissues of epithelial origin (carcinomas) (Parr et al., 2004; Tanimoto et al., 2005; Yamauchi et al., 2004), which might contribute to the elevated activation of c-MET receptor. Active matriptase and HGFA are inhibited by the hepatocyte growth factor activator inhibitors (HAI-1 and -2) (Figure 1B) (Denda et al., 2002; Wang et al., 2009; Xu et al., 2012). These HAI proteins are seen down-regulated in some types of cancer (Yamauchi et al., 2004). The down-regulation of HAI-1 and -2 might play a critical role in the elevated activation of the HGF-cMET axis due to increased activation of HGFA and matriptase. It is plausible that up-regulation of these inhibitors could lead to less activation of the c-MET receptor due to less activated HGFA and matriptase. Because of the link between the ratio of activated matriptase/HGFA and HAI-1 and -2 and the activation of the HGF-cMET axis, experiments examining HAI-1 and -2 as possible therapeutic agents against cancer have been designed using gene- engineered mice. These studies suggest that when the ratio changes, favoring higher amounts of HAI- 1/HAI-2, the cancer either reaches a steady state with no further progression or the tumor size decreases (List et al., 2005; Sales et al., 2015).

1.1 Aim The aim of this project 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.

1.2 Reading guide This study is a literature review based on scientific reviews, primary articles and textbooks. The focus of this study is to elucidate how the ratio between matriptase/HGFA and HAI-1 and -2 can contribute to cancer progression, and how this may be prevented by down-regulating the level of active

6/60 matriptase and HGFA with use of HAI-1 and -2 and/or of synthetic constructed protease inhibitors as therapeutic agents. We have examined how the expression and activation of matriptase, HGFA, HAI-1 and -2 are regulated. Based on these findings, we have discussed how the ratio between matriptase/HGFA and HAI-1 and -2 can be changed, favoring more HAI-1 and -2. The subject requires background knowledge of cancer, therefore basic concepts of cancer progression are described. We have furthermore written a descriptive review section outlining current knowledge about HGFA, matriptase, HAI-1 and -2 regulation and their relation to cancer. These findings are then further discussed in relation to the potentials of HAI-1 and -2 as therapeutic targets for treatment with demethylating agents such as 5-Aza-2’-deoxycytidine (DAC) and 5-azacytidine (AZA). The alternative potential of directly injected HAI-1 and -2 as therapeutic agents against cancer is also discussed. An abbreviation list is shown in Appendix 2.

7/60 2 Background theory 2.1 Hallmarks of cancer A tumor is composed of a variety of cells and is malignant when the normal functions of the cells are changed and the tumor is able to spread to other tissues. This section describes the five of the six hallmarks of cancer which are influenced by activation of the HGF-cMET axis: Sustaining proliferative signaling, evading growth suppressors, resisting cell death, inducing angiogenesis, and activating invasion and metastasis. The sixth hallmark, enabling replicative immortality, is not linked to activation of c-MET, and is therefore not described. Instead, mutations and epigenetic changes affecting oncogenes and tumor suppressor genes will be described. DNA methylation is a substantial factor in tumor cells and its role in regulating tumor suppressor genes will therefore also be described in this section. Metastasis is an essential mechanism in the development of cancer and is influenced by epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET), and will further be outlined (Alberts et al., 2015; Griffiths et al., 2012; Nelson and Cox, 2013). At last the maintenance of cancer stem cells (CSCs) is also regulated by the HGF-cMET axis and is included in this section (Lim et al., 2014). The CSC theory contributes to the understanding of tumor development and resistance mechanisms towards chemotherapy.

2.1.1 Oncogenes Non-mutated oncogenes are called proto-oncogenes and code for different proteins, e.g. growth- factor receptors, signal-transduction proteins and transcriptional regulators that induce entry into the cell cycle or inhibit the apoptotic pathway, which normally destroys damaged cells. Thus, the proto- oncogenes stimulate cell growth and division. In order for these proteins to function, they need to be activated by regulatory signals. When proto-oncogenes are mutated they code for proteins called oncoproteins. The oncogene mutation is a gain-of-function dominant mutation, which means that the activity of the oncoproteins is enhanced in tumor cells, because they have been uncoupled from their normal regulatory pathway (Griffiths et al., 2012; Nelson and Cox, 2013; Reece et al., 2011). This enhanced activity of oncoproteins can either be caused by an overproduction or a modification of the protein. An overproduction of the oncoproteins is a result of regulatory mutations, gene amplification or chromosomal rearrangement. When a regulatory region of the oncogene is mutated, the gene expression is no longer regulated and therefore the oncoprotein is overproduced. In gene amplification, more copies of the oncogene are generated, leading to overproduction of the

8/60 oncoprotein. In chromosomal rearrangement, the DNA sequence of the regulatory region for the oncogene is changed, resulting in overproduction of the oncoprotein. The structurally modified oncoproteins with an enhanced activity are the result of a simple point mutation or a deletion that changes the DNA sequence of the oncoprotein-coding region. These modified oncoproteins cannot be inactivated and are therefore hyperactive (Alberts et al., 2015). Thus, they will constantly give signal for cell growth and division, which can result in formation of a tumor. One example of an oncogene mutation is in the rat sarcoma viral oncogene (ras) that codes for the RAS protein, which is a G protein (Griffiths et al., 2012; Nelson and Cox, 2013; Reece et al., 2011).

2.1.2 Tumor suppressor genes Contrary to the oncogenes, the non-mutated form of tumor suppressor genes code for regulatory proteins, which inhibit the cell cycle and thus reduce cell division. In addition other tumor suppressor genes code for proteins that activate apoptosis of damaged cells, or proteins important for repair of damaged DNA or for the control of cellular longevity (Griffiths et al., 2012; Nelson and Cox, 2013; Reece et al., 2011). In cancer development, these tumor suppressor genes can be mutated with loss- of-function recessive mutations or inactivated by epigenetic changes. When mutated in both alleles, the gene product loses much or all of its activity and when epigenetically changed there is a low level or no gene product (Alberts et al., 2015; Reece et al., 2011). When mutated or silenced the functions of the tumor suppressor proteins are lost and this will promote tumor growth, since cell division is no longer regulated (Griffiths et al., 2012; Nelson and Cox, 2013; Reece et al., 2011). Tumor cells generally have an atypical DNA methylation pattern in comparison with normal cells. In normal tissue, the CpG islands are normally unmethylated while many regions without CpG islands are methylated. However, these regions are demethylated in tumor cells. Furthermore, some CpG islands in cancer cells undergo de novo methylation, which is methylation by de novo methyltransferases (reviewed by Bergman and Cedar, 2013). The tumor suppressor genes are repressed by hypermethylation of the CpG islands placed in their promoter regions. When the promoter regions are hypermethylated the RNA polymerase cannot bind. Thus, the tumor suppressor genes are not transcribed and there is no gene product (Esteller, 2002). As mentioned, some of the proteins encoded by tumor suppressor genes are involved in repair of genetic defects. These genes are sometimes called caretaker genes, and mutations in these result in reduced DNA repair, which enhance the mutation rate in other genes. Among these unrepaired genes are the proto-oncogenes and tumor suppressor genes. Hence, mutations in caretaker genes are cancer promoting mutations since they might result

9/60 in tumor development (Griffiths et al., 2012; Nelson and Cox, 2013; Reece et al., 2011). One example of a tumor suppressor gene mutation is a mutation in the tumor protein p53 (TP53) gene, which codes for the protein p53 (NCBI, 2016). The p53 protein is a transcription factor, which promotes transcription of different genes that code for cell cycle inhibiting proteins. The mutated form of TP53 codes for an inactive p53 protein and if the TP53 gene is epigenetically silenced, there is no expression of the p53 protein. This means that the transcription is not activated and thus the cell cycle is not inhibited, leading to enhanced cell division when both alleles are mutated (Griffiths et al., 2012; Nelson and Cox, 2013; Reece et al., 2011).

2.1.3 Metastasis and angiogenesis When cancer is initiated the tumor usually develops in only one location and is referred to as a primary tumor (Martini et al., 2014). After a period of time, the cancer cells may start to spread toward locations distant from the primary tumor in a process called metastasis (Reece et al., 2011). An important part of metastasis is EMT, which is the mechanism where epithelial cells change from an epithelial phenotype to a mesenchymal phenotype. The mesenchymal phenotype is a less differentiated phenotype. EMT acts in different settings with different consequences. EMT also takes place during embryogenesis, during inflammation, and in wound healing. Metastasis is a process that takes place in a series of steps known as progression, intravasation, transport, extravasation, and colonization (Figure 2). EMT occurs between progression and intravasation (reviewed by Foroni et al., 2012).

10/60 Figure 2. The different steps in the invasion-metastasis mechanism during cancer: Progression, EMT, intravasation, transport, extravasation, MET, and colonization. During progression, the cells break through the basement membrane into the stroma, referred to as reactive stroma due to influence of proteins in the stroma. The cells are influenced by the reactive stroma and EMT occurs in which the cells dedifferentiate into a mesenchymal phenotype. In intravasation these cells then enter the bloodstream and circulate until extravasation occurs, in which the cells break out of the bloodstream into new tissues. MET occurs after extravasation, where the cells with a mesenchymal phenotype differentiates back into cells with an epithelial phenotype. Colonization is then able to occur in the new tissue and a tumor can arise (Weinberg, 2013).

During progression, the tumor cells break through the basement membrane into the reactive stroma that initiates EMT. EMT can be induced by a series of different signals including the HGF-cMET axis, down-/up-regulation of different proteins or environmental conditions like hypoxia (Foroni et al., 2012; Toiyama et al., 2012). During EMT the cells are reprogrammed and are without the intercellular adhesions found in epithelial cells. Therefore, they act as individual cells unbound to each other and adapt a more invasive phenotype (Foroni et al., 2012; Toiyama et al., 2012). After EMT, the cells are found in the reactive stroma with access to circulation through the bloodstream (Figure 2). Intravasation is a process where the cells break through the membrane to the bloodstream, and afterwards are transported to a new location in the body. It is still not clear exactly what determines the location of the metastasis but the microenvironment is thought to play an important role (Foroni et al., 2012; reviewed by Sipos and Galamb, 2012). During extravasation, the cells break

11/60 out of the bloodstream into the stroma where MET occurs. MET is a process where the cells with a mesenchymal phenotype differentiates back into epithelial cells and regain a more adhesive and proliferative phenotype (Figure 2). After MET, the newly differentiated cells are located in another tissue and a new tumor can potentially arise (Foroni et al., 2012; Sipos and Galamb, 2012). The new tumor is referred to as a metastatic tumor.

Tumors have a high metabolic rate and the malignant tumors secrete signaling molecules that stimulate angiogenesis, which is the formation of new blood vessels from already existing ones. In tumor angiogenesis the new blood vessels grow into the area of the tumor, which means that the tumor cells again receive nutrients and oxygen. The result of tumor angiogenesis is further growth and metastasis of the malignant tumor (reviewed by Hanahan and Weinberg, 2011; Martini et al., 2014; Reece et al., 2011).

2.1.4 The cancer stem cell theory Another mechanism in cancer, influenced by the HGF-cMET axis, is CSC maintenance. The CSC theory states that CSCs constitute a subpopulation of the cells within a tumor and that only the CSCs are tumorigenic. This theory differs from the clonal theory, which states that all cells in a tumor are clones of one originator cell and that all of the cells are tumorigenic. CSCs are like normal stem cells able to self-renew and differentiate via asymmetric division, which means that one division produces two daughter cells. One of the daughter cells is a self-renewed undifferentiated tumorigenic CSC while the other is a non-tumorigenic transit amplifying cell, which is a rapidly dividing cell committed to differentiation. In this way, the CSCs create a heterogeneous offspring of cells that comprise a tumor and is thus responsible for tumor growth (reviewed by Deshpande and Rangarajan, 2015). In addition, CSCs can give rise to angiogenesis and are therefore important in tumor growth and metastasis (reviewed by Qiu et al., 2015). CSCs are also referred to as tumor initiating cells, because they are able to seed new tumors in mice (Deshpande and Rangarajan, 2015; Qiu et al., 2015). Furthermore, CSCs are thought to initiate EMT and are therefore important in invasion and metastasis. CSCs can survive treatments like chemotherapy, because they have an enhanced drug resistance compared to the rapidly dividing cancer cells. Due to this resistance the CSCs are responsible for recurrence of cancer. The drug resistance might be caused by an overexpression of specific ATP-binding cassette transporters in CSCs. The ATP-binding cassette transporters are efflux pumps that can pump chemotherapeutic substrates out of the cell. Thus, the substrates do not affect

12/60 the CSCs and they are able to survive. Furthermore, CSCs might be resistant to chemotherapy because they are slowly dividing cells and chemotherapeutic agents target the rapidly dividing cells (Deshpande and Rangarajan, 2015).

2.2 The HGF-cMET axis The interaction between HGF and its activators, e.g. HGFA and matriptase, is important for the understanding of the potential of matriptase, HGFA, and HAI-1 and -2 as cancer therapeutic targets. HGFA, matriptase, and HAI-1 and -2 will be elaborated in section 2.3, 2.4, and 2.5, respectively. In this chapter, the structure and function of HGF and c-MET will be described. Additionally, this section accounts for the molecular relation between dysregulated levels of active HGF and cancer hallmarks such as cell proliferation, angiogenesis, invasion, and metastasis. This section will furthermore describe the four different intracellular signaling pathways that are activated by the binding of HGF to its receptor c-MET: (1) the mitogen-activated protein kinase (MAPK) signaling cascade, (2) the PI3K-AKT-mTOR signal cascade, (3) the Janus kinase (JAK)/signal transducer activator of transcription (STAT) signal pathway, and (4) the nuclear factor-κB (NF-κB) signaling pathway (reviewed by Garajová et al., 2015).

2.2.1 Structure and activation of HGF HGF is a glycoprotein synthesized and secreted from cells with mesenchymal origin, i.e. embryonic mesenchymal cells and adult derivatives such as fibroblasts. HGF is secreted as an inactive single amino acid chain known as pro-HGF, which depends on a site-specific protease mediated cleavage in order to become a biologically active cytokine (reviewed by Parr et al., 2010). A number of different proteases have been shown to cleave pro-HGF, among these are the three serine proteases HGFA, matriptase, and hepsin (Han et al., 2014). Shimomura et al. (1992) were the first to purify the protease that proteolytically processed the single chain recombinant pro-HGF to the two-chain form recombinant HGF. A proteolytic processing of recombinant pro-HGF was achieved in serum-free cultures, in which protease inhibitors are often absent. The purified protease was later sequenced from cDNA and found to be the secreted serine protease HGFA (Miyazawa et al., 1993). The proteolytic processing of pro-HGF by matriptase has been shown by Chen et al. (2013). They found that active matriptase rapidly converts the pro-HGF single chain into the two-chain active form of HGF in a dose dependent manner. This was shown in vitro by immunoblotting using HGF

13/60 antibodies. The activated HGF is a heterodimer consisting of a heavy α-chain (54-65 kDa) and a light β-chain (31.5-34.5 kDa) held together by a disulfide bond (Figure 3). The α-chain holds an N-terminal hairpin loop domain and four Kringle domains (K1-K4). The β-chain holds a serine protease homology (SPH) domain, which is similar to the catalytic domain of serine proteases. However in HGF, the histidine and serine residues of the serine protease active site are replaced with glutamine and tyrosine residues, which leads to the absence of proteolytic activity (Naka et al., 1992). By functional analysis of HGF substitution and deletion variants, Lokker et al. (1992) found that the N- terminal hairpin loop and K1 domains are primarily responsible for receptor binding, whereas the SPH domain gives HGF its mitogenic activity when bound to c-MET. Furthermore, this study confirmed the necessity of the pro-HGF cleavage site in order for HGF to biologically function, but also found that pro-HGF showed receptor binding capacity, suggesting that activation of pro-HGF may occur while bound to c-MET on the cell surface.

Figure 3. Schematic representation of active HGF. The active form of HGF is a heterodimer consisting of an α- and a β-chain linked by a disulfide bond. The α-chain contains the N-terminal hairpin loop domain and K1-K4 domains. The β-chain contains a non-proteolytic SPH domain. The N-terminal hairpin loop domain and the K1 domain are primarily responsible for receptor binding, whereas the SPH domain gives HGF its mitogenic activity when bound to c-MET (Modified from Lokker et al., 1992).

2.2.2 Structure and Activation of c-MET Although HGF secretion is limited to cells of mesenchymal origin, it affects a variety of target cells, since the c-MET tyrosine kinase receptor is expressed on epithelial, endothelial, and hematopoietic progenitor cells. The c-MET receptor is a heterodimer consisting of the highly glycosylated and

14/60 entirely extracellular α-chain linked by a disulfide bond to the β-chain, which has a large extracellular segment, a transmembrane spanning segment, and an intracellular tyrosine kinase domain (Figure 4) (reviewed by Furge et al., 2000). The extracellular component of the receptor contains six domains: A semaphorin (SEMA) domain, a plexin, semaphorin, and integrin cysteine-rich (PSI) domain, and four immunoglobulin plexins transcription (IPT) domains (Figure 4). The intracellular component includes a juxtamembrane sequence, a catalytic region and a carboxy-terminal multifunctional docking site. The multifunctional docking site is responsible for the recruitment of many different transducers and adaptor proteins. HGF binds with high affinity to two c-MET molecules and causes a receptor dimerization. This conformational change induces autophosphorylation in the tyrosine residues Tyr1234 and Tyr1235 in the catalytic region, which regulates the kinase activity of the receptor. This is followed by a transphosphorylation of the Tyr1349 and Tyr1356 in the C-terminal region (Figure 4), which causes activation of the multiple docking site (Garajová et al., 2015).

Figure 4. Activation and structure of the c-MET receptor. The c-MET receptor consists of an α-chain linked by a disulfide bond to the β-chain. The β-chain has a large extracellular segment containing a SEMA domain, a plexin, semaphorin, and integrin cysteine-rich (PSI) domain, and four immunoglobulin plexins transcription (IPT) domains. The intracellular segment of the β-chain contains a juxtamembrane sequence, a catalytic region and a carboxy-terminal multifunctional docking site. When active, HGF binds to the receptor and causes autophosphorylation in the tyrosine residues Tyr1234 and Tyr1235 followed by a transphosphorylation of the Tyr1349 and Tyr1356 in the multifunctional docking site. This initiates recruitment of many different transducers and adaptor proteins (Modified from Garajová et al., 2015).

15/60 2.2.3 Functions of the HGF and c-MET Activation of the multiple docking site of c-MET, mediated by HGF binding, enables downstream signaling either through direct interaction with signaling molecules or through recruitment of adaptor proteins (Figure 5). Among many adaptor proteins, the growth factor receptor-bound protein 2 (Grb2) and Grb2-associated-binding protein 1 (Gab1) are recruited to the docking site, which both initiate the MAPK signaling pathway and the PI3K-AKT-mTOR signal cascade. The adaptor proteins are recruited along with several other signal transducers including PI3K, STAT3, and the RAS guanine nucleotide exchange factor son-of-sevenless (SOS) (Furge et al., 2000).

Figure 5. Binding of HGF to the c-MET receptor causes phosphorylation of the tyrosine residues Tyr1234 and Tyr1235 on the intracellular multiple docking site. This initiates recruitment of several adaptor proteins including Gab1 and Grb2, or direct interaction with signaling molecules such as STAT3. The different signaling pathways are outlined in the following four sections (Modified from Phan et al., 2015).

16/60 2.2.3.1 The MAPK signaling cascade MAPKs are a group of serine or threonine protein kinases that, among other things, regulate transcription and thereby influence cell proliferation, cell survival, and differentiation. There are numerous MAPKs, the most investigated being the extracellular signal-regulated kinases (ERKs) (Figure 6). ERKs are indirectly activated by the RAS protein, which is stimulated by different tyrosine kinase receptors including c-MET. When HGF binds to c-MET, the adaptor protein Grb2 can form a complex with SOS, which then phosphorylates RAS (Garajová et al., 2015). This initiates a phosphorylation cascade, where RAS phosphorylates rapidly accelerated fibrosarcoma (RAF), which then again phosphorylates the MAPK/ERK kinases (MEKs), which finally phosphorylate ERKs (Roberts and Der, 2007). Moreover, the RAS-RAF-MEK-ERK pathway is initiated when sarcoma homology phosphatase 2 (SHP2) dephosphorylates Gab1. This too causes a GTP loading of RAS, which then commence the phosphorylation cascade (Figure 5) (Garajová et al., 2015).

Figure 6. The MAPK signaling cascade. When HGF binds to c-MET the Grb2-SOS complex is formed, and a phosphorylation cascade is initiated when the complex phosphorylates RAS. RAS phosphorylates RAF, which then phosphorylates MEK, which finally phosphorylates ERK. ERKs regulate transcription of numerous genes involved in cell proliferation, survival, and differentiation (Modified from Roberts and Der, 2007).

17/60 2.2.3.2 The PI3K-AKT-mTOR signal cascade Activation of the PI3K-AKT-mTOR signal cascade through the c-MET receptor occurs when the stimulated c-MET receptor recruits the adaptor proteins Grb2 and Gab1 (Figure 7). PI3Ks are activated by binding to Gab1 (Furge et al., 2000). The activated PI3Ks activate AKT by phosphorylation, which directly and indirectly can activate the mTOR complex 1 (mTORC1). AKT can increase the amount of active mTORC1 either by directly phosphorylating mTORC1 or indirectly by phosphorylating TSC2, which is one of the two members of the tuberous sclerosis complex that inhibits mTORC1. The phosphorylation of TSC2 destabilizes the tuberous sclerosis complex leading to increased mTORC1 activity. The activated mTORC1 leads to the release of transcription factors that trigger transcription of multiple oncogenes (Figure 7) (reviewed by Riquelme et al., 2016). This signal cascade plays a crucial role in regulation of gene expression involved in multiple cellular functions including cell growth, proliferation, metabolism, survival and angiogenesis (Furge et al., 2000; Riquelme et al., 2016). It has been demonstrated that there is an increased activity of the PI3K- AKT axis in cancer tissue (Riquelme et al., 2016).

Figure 7. The PI3K-AKT-mTOR signal cascade. The stimulated c-MET receptor recruits the adaptor proteins Grb2 and GAB1. PI3K then binds to Gab1 and gets activated. The activated PI3K phosphorylates and activates AKT, which increases the amount of active mTORC1 by directly phosphorylating the complex or indirectly by destabilizing the TSC1/TSC2 complex, leading to an increased amount of active mTORC1. The activation of mTORC1 leads to the release of transcription factors that regulate gene expression involved in cell growth, angiogenesis and proliferation (Modified from Furge et al., 2000; Riquelme et al., 2016).

18/60 2.2.3.3 The JAK/STAT signaling pathway The intracellular non-receptors, JAKs, and their downstream effectors, the STAT proteins, provide the cell with a direct communication from the transmembrane receptors to the nucleus, where the proteins regulate gene expression (Figure 8) (reviewed by O'Shea et al., 2015). This pathway is able to promote tumor cell proliferation, survival, and tumor invasion, which demonstrate the role it plays in cancer. It has been observed that STAT3 and STAT5 are the most important proteins for cancer progression (Garajová et al., 2015). The JAK/STAT signaling pathway is activated when the transmembrane receptor, e.g. c-MET, and the corresponding ligand, e.g. HGF, interact, leading to transactivation of the receptor-bound JAKs (Figure 8) (reviewed by Villarino et al., 2015). The activated JAKs are able to phosphorylate each other and the intracellular tail of the receptor, which creates a docking site for the STAT proteins. When the STAT proteins enter the docking site the JAKs mediate phosphorylation of the STATs, which will activate the STAT proteins and allow them to dimerize. The STAT-STAT dimer then translocates to the nucleus, where it directly binds to the DNA and regulates gene expression (O'Shea et al., 2015; Villarino et al., 2015).

Figure 8. The JAK/STAT signaling pathway. (1) The binding of HGF and c-MET activates the pathway by transactivating the receptor-bound JAKs. (2) The activated JAKs phosphorylate each other and the intracellular tail of c- MET, which creates a docking site for the STAT proteins. (3) The STAT proteins enter the docking site and the JAKs then mediate phosphorylation of the STATs, which activate them and allow them to dimerize. The STAT-STAT dimers then translocate to the nucleus where they regulate gene expression by binding to the DNA (Modified from O'Shea et al., 2015).

19/60 2.2.3.4 The NF-κB signaling pathway The NF-κB system consists of five proteins that are present in almost all cell types and tissues. The proteins of the NF-κB system form homo- and heterodimeric complexes that transcriptionally regulate target genes by binding to promoters or enhancer regions (Garajová et al., 2015). NF-κB regulates the genes that control cell proliferation, cell survival and apoptosis, stress responses and embryogenesis. The NF-κB system is inactive in the cytoplasm because the inhibitor κB (IκB) and the C-terminal half of p100 mask the nuclear localization signal (NLS) and thereby prevents the release of NF-κB proteins. The NF-κB system can be activated by two pathways: The canonical and the non-canonical pathway (Figure 9) (reviewed by Park and Hong, 2016). The canonical pathway is activated when c-MET is stimulated by binding of active HGF. This activates the IκB kinase (IKK) complex that phosphorylates two N-terminal serine residues of IκB. The phosphorylations then mediate degradation of IκB and activation of the NF-κB system. Activation of the non-canonical pathway occurs when IKKα is phosphorylated by NF-κB inducing kinase (NIK). This results in phosphorylation of p100. The phosphorylation mediates partial degradation and processing of p100 into mature p52 subunits. The newly generated p52 interacts with NF-κB proteins. In both pathways their respective inhibitors, IκB and the C-terminal half of p100 are destroyed and this unmasks the NLS, which allows the release of NF-κB proteins. The NF-κB proteins interact and translocate to the nucleus where they transactivate target genes by binding to gene promoters or enhancer regions (Figure 9) (Garajová et al., 2015; Park and Hong, 2016). Studies have shown that many different types of tumors have a constitutively active NF-κB proteins. Active NF-κB proteins turn on the expression of genes that keeps the cell proliferating and protects the cell from conditions that would have caused apoptosis (Park and Hong, 2016).

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Figure 9. The NF-κB signaling pathway. The canonical pathway is activated when HGF binds to c-MET. The stimulated c-MET activates the IKK complex, which phosphorylates IκB. This phosphorylation mediates degradation of IκB. The activation of the non-canonical pathway occurs when c-MET is stimulated by binding of HGF, which results in phosphorylation of IKKα by NIK. This leads to phosphorylation of p100. The phosphorylation of p100 mediates partial degradation and processing of p100 into mature p52 subunits. After IκB and partial p100 are destroyed the NLS is unmasked and this allows the release of NF-κB proteins that can interact with each other and translocate to the nucleus where they regulate genes by binding to promoters or enhancer regions (Inspired by Park and Hong, 2016).

2.3 HGFA structure and function Due to the increased level of active HGFA in cancer, the structure and the function of HGFA will be described.

HGFA is a serine protease that is synthesized in hepatocytes as the inactive pro-HGFA, and circulates in the blood (Miyazawa et al., 1993). The gene HGFAC encodes pro-HGFA (NCBI, 2015). The activation of pro-HGFA normally occurs in response to tissue injury and blood clotting (Miyazawa et al., 1993). Pro-HGFA consists of 655 amino acids and is cleaved to its active form, primarily by thrombin, between Arg407-Ile408 and Arg372-Val373 (Miyazawa et al., 1993; Shimomura et al., 1993). The active HGFA is a heterodimer, consisting of a 35 amino acid short chain and a 248 amino

21/60 acid long chain linked together by a disulfide bond (Shimomura et al., 1993). Two other enzymes are reported as possible activators of pro-HGFA in the cancer cell microenvironment, the human kallikrein-related peptidases (KLKs) 4 and 5. The efficiency of KLK5 is comparable to thrombin, while KLK4 activity is one-fifth of KLK5 and thrombin (reviewed by Kawaguchi and Kataoka, 2014). An important part of the HGFA structure is the oxyanion hole, which is formed by Ser195 and Gly193. It contains four substrate-binding subsites (S1-S4), which when active interact with P1-P4 residues of pro-HGF. HGFA also contains a 99-loop and a 220-loop known as the substrate specificity-determining loops (Figure 10). The 99-loop is important for the interaction with the inhibitors, HAI-1 and -2, due to its contribution to the formation of S2 and S4. Additionally the 220- , 180-, 140-loops are together with the N-terminal referred to as the activation domain (reviewed by Eigenbrot et al., 2010).

Figure 10. 3D-structure of HGFA. A model of the entire HGFA with certain loops pointed out: 38-, 60-, 99-, 140-, 170- , 180-, 220-loops. Ser195, Cys283, Cys259, Arg258 and, Arg260 are specific amino acids locations. The dotted green line marks the binding region and active site on HGFA (also shown in Figure 12) (Eigenbrot et al., 2010).

22/60 2.4 The structure and activation of matriptase In this section, the structure and the activation of matriptase will be described. The main focus will be matriptase activation due to the level of active matriptase that has been observed to be dysregulated during cancer. The matriptase activation is essential because active matriptase converts pro-HGF to the active form HGF. The knowledge about the regulation of the matriptase-coding gene ST14 (Kauppinen et al., 2010) is limited and therefore it will not be further described.

The gene ST14 encodes matriptase, which is a type II transmembrane serine protease that is expressed in e.g. different epithelial tissues (Kauppinen et al., 2010; reviewed by List, 2009; reviewed by Miller and List, 2013). Matriptase has a cytoplasmic N-terminus and an extracellular C-terminus (Figure 11). The extracellular region of the protease consists of one sperm protein, enterokinase, and agrin (SEA) domain, two C1r/s, Uegf, and bone morphogenic protein-1 (CUB) domains, four low density lipoprotein (LDL) receptor class A domains, and a C-terminal serine protease domain (reviewed by List et al., 2006; Miller and List, 2013).

Figure 11. Schematic representation of matriptase. Matriptase is a transmembrane protease with a cytoplasmic N- terminus and an extracellular C-terminus. It consists of one SEA domain, two CUB domains, four LDL domains and one serine protease domain. The SEA domain cleavage site is found within the SEA domain at Gly149, and the activation cleavage site is found within the serine protease domain at Arg614. Furthermore, matriptase contains the glycosylation sites Asn302 and Asn772, which may be important for activation (Inspired by List et al., 2006).

Matriptase is synthesized in the rough endoplasmic reticulum (ER) as an inactive single-chain zymogen, i.e. a proenzyme. Matriptase can be activated by two endoproteolytic cleavages. First, the SEA domain at the N-terminal end of the inactive matriptase is cleaved at Gly149 (Figure 11) in the domain’s Gly-Ser-Val-Ile-Ala motif (List et al., 2006; Wang et al., 2009; Xu et al., 2012). The N- terminal portion with the SEA domain is still associated with the C-terminal portion containing the

23/60 other matriptase domains. This maintained association might be due to a non-covalent interaction and was shown in an in vitro experiment with the mouse homologue of matriptase, epithin (Cho et al., 2001). The cleavage process is suggested to take place in the ER or the Golgi apparatus. In polarized epithelial cells, such as epithelial ductal cells, the inactive matriptase is then moved to the basolateral plasma membrane (Figure 12) (List et al., 2006; Wang et al., 2009; Xu et al., 2012). The second cleavage is at Arg614 (Figure 11) within the cleavage site Arg-Val-Val-Gly-Gly of the serine protease domain at the surface of the cell, which leads to the conversion of matriptase into its active form (reviewed by Kawaguchi and Kataoka, 2014; List et al., 2006). Glycosylation might also influence the activation of matriptase. The first CUB domain and the catalytic domain of matriptase contain the N-glycosylation sites Asn302 and Asn772, respectively (Figure 11). In a study by Oberst et al. (2003) it was shown in cultured breast cancer cells that the first CUB domain and the serine protease domain of matriptase have to be glycosylated in order to induce the activation of the enzyme (Oberst et al., 2003). When matriptase is active, it can activate its substrates, which have been identified to be e.g. HGF. Matriptase is only active in a short period, due to the binding of HAI-1, which rapidly inactivates matriptase. In epithelial ductal cells matriptase and HAI-1 forms a 120 kDa complex, which is shed from the basolateral plasma membrane as a 95 kDa or a 110 kDa complex (Figure 12) (List et al., 2006; Wang et al., 2009; Xu et al., 2012).

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Figure 12. Synthesis, activation, and inhibition of matriptase. (1) The inactive form of matriptase is synthesized in the rough ER. (2) It has been suggested that the first cleavage of the matriptase zymogen in the SEA domain takes place in the ER or the Golgi apparatus. After cleavage the N-terminal SEA domain is still associated with the C-terminal portion of matriptase. (3) Then the inactive matriptase is targeted to the basolateral plasma membrane of the cell, (4) where the second cleavage in the serine protease domain of matriptase takes place, leading to activation of matriptase, which is rapidly inhibited by HAI-1 at the cell surface. (5) A 120 kDa complex of matriptase and HAI-1 is formed, and this complex is shed from the membrane as a 95 kDa or a 110 kDa matriptase-HAI-1 complex (Modified from Wang et al., 2009).

2.5 HAI-1 and -2 In this section, the structure and function of HAI-1 and -2 as inhibitors of HGFA and matriptase will be described. HAI-1 and -2 are expressed in different kinds of epithelial tissues, such as in the intestines, tubules, placenta, and prostate. They are expressed in different levels in various cell lines, and play crucial roles in preventing tumorigenesis (Kataoka et al., 2000; Parr et al., 2004; Yamauchi et al., 2002). This section will therefore also elaborate the different interactions between HGFA/matriptase, and HAI-1/-2.

25/60 2.5.1 The structure and function of HAI-1 and -2 The SPINT1 and -2 genes encode HAI-1 and -2, respectively (Itoh et al., 2000). Both HAI-1 and -2 contain two Kunitz domains (KDs), and these domains inhibit the serine proteases HGFA, matriptase, and hepsin (Denda et al., 2002; Herter et al., 2005). The HAI proteins are type 1 transmembrane proteins composed of an intracellular C-terminus, an extracellular N-terminus, and a transmembrane domain. In addition, HAI-1 contains a LDL-receptor like domain (Figure 13) (Denda et al., 2002; Kataoka et al., 2003). Denda et al. (2002) suggest that the N-terminal KD1 of HAI-1 is responsible for the strong inhibition of HGFA and matriptase, hence it prevents conversion of pro-HGF to active HGF. The C-terminal KD2 of HAI-1 shows weak inhibition against HGFA and matriptase. Furthermore, HAI-1 contains another domain called motif at N-terminus with seven cysteines (MANSC), which Guo et al. (2004) suggest might play a role in the binding to HGFA and/or matriptase. Guo et al. (2004) suggest that the trypsin-like serine protease domains of HGFA and matriptase could be the potential binding regions for MANSC. However, the domain has shown to have a weak inhibitory effect and they therefore suggest that MANSC could have other roles.

Figure 13. Schematic representation of HAI-1 and-2. HAI-1 and -2 consist of an intracellular C-terminus, a transmembrane domain, an extracellular N-terminus, two kunitz domains, and in addition HAI-1 contains a LDL receptor- like domain and a MANSC domain (Inspired by List et al., 2006; Parr et al., 2010).

The transmembrane HAI proteins can undergo ectodomain shedding, where the extracellular part, including the domains, is released from the cell surface by a proteolytic cleavage (reviewed by Hayashida et al., 2010; reviewed by Parr et al., 2010). Shedding regulates the protein expression on

26/60 the cell surface, and thus allows the proteins to interact with other factors that could influence the cellular level, such as the interactions between HAI-1/-2 and HGFA/matriptase. The ectodomain shedding can be regulated by different mechanisms, such as phosphorylation, polarized secretion, intracellular trafficking, activation of the enzyme sheddase, and protein-protein interactions (Hayashida et al., 2010; Parr et al., 2010). HAI-1 and -2 are suggested to prevent HGFA and matriptase from attaching and cleaving the pro-HGF to its active form, HGF (Hayashida et al., 2010; Parr et al., 2010).

2.5.2 HAI’s expression and regulation of SPINT1 and -2 A recent study by Sechler et al. (2015) showed a difference in HAI-1 expression based on the γ- catenin-expressing human lung cancer cell lines, H157 and H1299, respectively. γ-catenins are proteins that serve to anchor adjacent cells, and are often absent or down-regulated in human lung cancer patients (Winn et al., 2002). By RT-qPCR and immunoblotting with the use of specific antibodies, the study showed that H157 cells express HAI-1 mRNA and p53, while the H1299 cells does not. The expression of a wild-type p53 cDNA induced the expression of HAI-1 in H1299 cells, suggesting that the expression of HAI-1 mRNA is γ-catenin dependent through a p53 mechanism, indicating that these two factors might have an important role in preventing cancer formation. Sechler et al. (2015) also revealed the findings of a p53 binding site at the SPINT1 promoter.

By immunohistochemistry staining with antibodies, Yamauchi et al. (2002) found that HAI-2 was expressed in the primary spermatocytes where HAI-1 was barely expressed. HAI-2 may therefore have an influence in the development of spermatogenesis. The study suggests that SPINT2, in testis, has two different transcription start sites (~30 and ~360 nucleotides upstream the translation initiation site ATG) but it is still poorly understood. However, other investigations have showed that only the ~30 site is used to express HAI-2 in the testis. The study suggest that some of the transcription factors, such as GATA-1 and NF-κB, that are present close to the ~360 non-functional site, are involved in a transcriptional regulation, and hence might act by blocking or suppressing transcription of SPINT2 (Yamauchi et al., 2002).

In summary, γ-catenin and p53 are important for the expression of HAI-1 and might have an important role in cancer prevention. Further, transcription of SPINT2 is regulated by different transcription factors, such as NF-κB.

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2.5.3 HAI-1 and -2 inhibit HGFA through different interactions Transmembrane HAI-1 is capable of binding to and inhibiting HGFA, whereas the transmembrane HAI-2 does not have the ability to inhibit HGFA (Kataoka et al., 2000; Parr et al., 2010). Kataoka et al. (2000) outlined two possible reasons for the lack of inhibitory effect toward HGFA by the transmembrane HAI-2. They suggest that the LDL receptor like domain, which is not present in HAI- 2, could be involved in the interaction between HGFA and the transmembrane HAI-1. Another possibility is that the HAI-2 KDs are exposed to the intracellularly environment and thus not accessible to extracellular HGFA (Kataoka et al., 2000). Although it has been shown that HAI-2 does not inhibit HGFA in its transmembrane form, a study by Kawaguchi et al. (1997) showed that HAI- 2 interacts with and inhibits HGFA when secreted. In agreement with Kawaguchi et al. (1997), a study by Qin et al. (1998) also showed that HAI-2 inhibits HGFA when secreted. Furthermore, they showed that the KD1 of HAI-2 is mainly responsible for this inhibition. Shimomura et al. (1999) and Denda et al. (2002) used immunoblotting to measure the level of HAI-1, and suggested that HAI-1 exists in a transmembrane integrated form of 66 kDa, and after the ectodomain shedding exists in two forms of approximately 39-40 kDa and 58 kDa, respectively. It has been shown that the 39-40 kDa form has the strongest inhibitory effect against HGFA compared to the 58 kDa form. Inhibitors like HAI-1 are capable of inhibiting HGFA due to specific interactions with the active site of HGFA (reviewed by Eigenbrot et al., 2010). Only the KD1 of HAI-1 is known to be responsible for inhibition of HGFA. HAI-1 inhibits HGFA by forming a tight interaction between KD1 and all substrate- inhibitor specificity-determining loops of HGFA, including the 99-loop and the 220-loop (Figure 14). Furthermore, the KD1 also makes two main-chain-main-chain hydrogen bonds with HGFA, similar to the binding with pro-HGF (Eigenbrot et al., 2010).

In summary, only HAI-1 has the ability to inhibit HGFA both when in its transmembrane form and in its shedded form. HAI-2 is only able to inhibit HGFA when secreted from the membrane. This inhibition is primarily due to the interaction between KD1 of the HAI proteins and the substrate- inhibitor specificity-determining loops of HGFA. Further, HAI-1 is shedded in two forms where the smallest form have the strongest inhibitory effect.

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Figure 14. 3D-structure of HGFA and the KD1-domain of HAI. The left 3D-structure shows HGFA with its active site and the binding domain are shown in green. The purple and pink structure shows the 3D-structure of the KD1-domain found in HAI-1 and HAI-2, which acts as a pseudo-substrate that binds to the active site on HGFA (Modified from Eigenbrot et al., 2010).

2.5.4 The interactions between HAI-1/ -2 and matriptase The activation of matriptase depends on different factors, e.g. HAI-1. A model suggested by Oberst et al. (2003) described that HAI-1 may influence matriptase activation in the human breast cancer cell line BT549. HAI-1 and matriptase form an activation complex via their LDL domains, which can initiate a process in which a matriptase zymogen activates another matriptase zymogen. In contrast Friis et al. (2014) have outlined a theory regarding HAI-1, showing that the absence of endogenous HAI-1 might not influence expression of matriptase, or matriptase activation and localization within the cell. Thus, the primary function of HAI-1 is instead to inhibit the active matriptase. Friis et al. (2014) used intestinal epithelial cells to study matriptase and HAI-1, and the results are only validated in this cell type. Studies by List et al. (2005) and Sales et al. (2015) indicate the inhibiting effects of HAI-1 and -2 on matriptase in mice. In the study by Sales et al. (2015), matriptase was overexpressed in basal keratinocytes from mice, which lead to the development of tumors. A simultaneous overexpression of HAI-2 resulted in a significant decrease in rapid tumor formation. The study by List et al. (2005) showed that overexpression of matriptase in the skin of mice lead to tumor formation, and this effect of overexpressed matriptase was decreased by overexpression of HAI-1 in the skin of mice.

29/60 A study by Larsen et al. (2013) indicates that HAI-2 prevents shedding of matriptase from the basolateral plasma membrane, thus HAI-2 is important for matriptase accumulation in the membrane. Matriptase and HAI-2 were expressed together in Madin-Darby Canine Kidney epithelial cells, resulting in matriptase localizing to the membrane. KD1 of HAI-2 seems to be important for this accumulation of membrane-bound matriptase. Furthermore, Larsen et al. (2013) did not observe any HAI-2 at the basolateral plasma membrane in the cell line, Caco-2, which are human colon epithelial cells. The expressed HAI-2 was primarily observed intracellularly, suggesting that HAI-2 and matriptase interact at an intracellular location. This location is suggested to be the ER.

In summary, HAI-1 and -2 both have an inhibitory effect on matriptase. HAI-1 might have an influence on both the activation and the inhibition of matriptase, although some studies have showed that HAI-1 does not have any influence on matriptase activation. The interaction of matriptase and HAI-2 seems to take place intracellularly, and further HAI-2 may prevent shedding of matriptase, leading to matriptase accumulation in the membrane.

2.5.5 HAI-2 regulates matriptase activation through prostasin HAI-2 might regulate activation and shedding of matriptase through the protease prostasin, which may influence the matriptase activation (Friis et al., 2014). A study by Friis et al.(2013) showed that matriptase zymogen and prostasin zymogen interact when they are co-expressed in epithelial cells, and they create a zymogen activation complex together. The two proteases then activate each other. The results of Friis et al. (2013) indicate that the activation by prostasin of matriptase does not depend on a catalytic activity from prostasin or on the conversion of the prostasin zymogen. Friis et al. (2013) suggest that the activation of matriptase is due to an allosteric interaction with prostasin. The serine protease domain of matriptase may undergo a conformational change, leading to autoactivation of matriptase. In this process, prostasin functions as a non-catalytic co-factor. The results of Friis et al. (2014) indicate that HAI-2 and prostasin interact in intestinal epithelial cells, and that HAI-2 inhibits matriptase activation by regulating the activity of prostasin. By regulating prostasin, HAI-2 controls the activation and shedding of matriptase, and furthermore the formation of the complex with matriptase and HAI-1. In addition, another study by Bergum et al. (2012) examined the correlation between the expressions of matriptase and prostasin in regards to cancer. The results of the study showed a significant correlation between the expressions of prostasin and matriptase in breast cancer cell lines and breast cancer tissue. 21 different breast cancer cell lines were used, and in 17 of the cell

30/60 lines co-expression of matriptase and prostasin was observed. Neither matriptase nor prostasin were expressed in the remaining four cell lines. Co-expression of matriptase and prostasin protein in the breast cancer tissue was detected in 43 out of 71 samples. Furthermore, in 12 of the samples either matriptase or prostasin were expressed (Bergum et al., 2012).

In summary, matriptase is activated by an interaction with prostasin. HAI-2 is able to decrease the activity of prostasin and thereby inhibit matriptase, controlling the activation and shedding of matriptase.

31/60 3 Review

This chapter provides an overview of current knowledge about the expression and regulation of HGFA, matriptase, c-MET and HAI-1 and -2 in relation to cancer. In addition, studies showing a disrupted ratio between HAI/HGFA and HAI/matriptase in cancer are described. The knowledge from this section will be further used in chapter 4 to discuss how matriptase, HGFA, HAI-1 and -2, and the microenvironmental factors should be regulated in proportion to each other regarding prevention of cancer progression and metastasis.

3.1 Expression of HAI-1 and -2 is decreased in cancer This section focuses on studies examining the expression of HAI proteins in cancer tissues. This is followed by a description of studies suggesting that the down-regulation of HAI-2 is due to hypermethylation around the SPINT2 starting site. In relation to the regulation of HAI, one study suggesting post-transcriptional modification of HAI-2 by N-glycosylation resulting in two species of HAI-2 is presented.

3.1.1 HAI-1 and -2 are inversely correlated with tumor progression A study by Yamauchi et al. (2004) compared the mRNA levels of HAI-1 and -2 in normal renal tissue to the expression levels in renal cell carcinoma (RCC) tissue using RT-qPCR. They observed significantly decreased levels of HAI-1 and -2 mRNA in RCC tissue. Significantly decreased HAI-2 protein levels were also observed from immunohistochemical staining of prostate cancer samples compared to HAI-2 levels in normal benign prostate hyperplasia. Furthermore, the HAI-2 mRNA levels were lowest in poorly differentiated tumors, which are often considered aggressive and high grade tumors (Bergum and List, 2010). Similar results of significantly decreased HAI-2 mRNA levels were obtained from aggressive malignant glioma tissue compared to a low-grade glioma cell line. Again, the lowest HAI-2 levels were observed in the most aggressive tumors (Hamasuna et al., 2001). Unexpectedly, Parr et al. (2004) found an increased expression of HAI-1 and -2 mRNA in breast cancer samples compared to control samples of normal breast tissue. Parr et al. (2004) explain these inconsistent findings based on another study reporting lower interaction between HAI-1 and its antigen (lower immunoreactivity) in cancer cells adjacent to the normal tissue, especially at the invasion front. Hence, the higher mRNA levels measured by Parr et al. (2004) could be from cells at the invasion front, whereas cells inside the tumor are likely to have a decreased HAI 1- and -2 mRNA

32/60 expression. This is in accordance with the immunohistochemical staining of both HAI proteins, which showed a lower staining in cancer cells than in normal mammary epithelial cells. Further investigation by Parr et al. (2004) revealed that the levels of HAI-1 and -2 mRNA were significantly reduced in grade 3 tumors compared with the amount in grade 1 tumors. In addition, the level of HAI-2 mRNA was also significantly decreased in grade 3 tumors in comparison with the level in grade 2 tumors. These findings support the statement of HAI-1 and -2 being inversely correlated with tumor progression.

In summary HAI-1 and -2 are decreased in cancer tissue compared to normal tissue. In addition, the levels of HAI-1 and -2 decrease as the tumor grade increases.

3.1.2 Decreased levels of HAI-2 mRNA might be caused by hypermethylation The SPINT2 gene encoding HAI-2 has a CpG island around the starting site, and has been examined by Fukai et al. (2003) in relation to human hepatocellular carcinoma (HCC). Their results indicated that when the promoter of SPINT2 was hypermethylated, the gene was silenced, and in primary HCC tumors from HCC patients, a hypermethylated SPINT2 promoter was frequently observed. 21 of 26 HCC tumors showed hypermethylation of the 5’ CpG island, and in the nontumorous tissue only in 7 of 26 cases hypermethylation were observed. Tung et al. (2009) also showed this correlation between hypermethylation and silenced SPINT2 in HCC. Hypermethylation of the SPINT2 promoter was observed in 71 % of the cases with decreased levels of HAI-2 mRNA in HCC cell lines. In addition, the same correlation between hypermethylated SPINT2 promoter and expression of HAI-2 was observed in other types of cancer such as gastric cancer and RCC (Dong et al., 2010; Morris et al., 2005). Currently there are no studies regarding hypermethylation of the SPINT1 promoter. Pereira et al. (2016) investigated the promoter methylation and expression level of SPINT2 in prostate cancer, by using immunohistochemistry and methylation specific PCR. The study clarified whether it is methylation of the promoter or post-translational alteration that is responsible for the down-regulation of HAI-2. The results indicate that methylation on the CpG sites on the SPINT2 promoter region does not have an impact on the expression of HAI-2 mRNA. In addition, the study showed, by in silico analysis, that the measured HAI-2 mRNA level does not correlate with the level of the HAI-2 protein. Thus, Pereira et al. (2016) suggest that down-regulation of the HAI-2 protein can be due to a post- translational glycosylation, and thereby a possibly misfolding of the HAI-2 protein. Another

33/60 mechanism causing decreased expression of HAI-2 could be other molecules, e.g. GATA-1 and NF- κB, blocking the starting site as found in testis by Yamauchi et al. (2002).

In summary, the CpG island of the SPINT2 promoter is hypermethylated in some types of cancer resulting in decreased expression of HAI-2, although this is not the case in all types of cancer.

3.1.3 Post-transcriptional modification of HAI-2 indicates two species of HAI-2 A study by Lai et al. (2015) shows that the HAI-2 protein is expressed as two species of different size. One is suggested to be an immature form of HAI-2 of 25-kDa, which remains in the ER, and the other mature form is found in two sizes on 30-40 kDa, which is located in granules/vesicles. The two forms of HAI-2 are due to different N-glycans on Asn-57. Both HAI-2 species undergo N- glycosylation by addition of a 14-sugar glycan to Asn-57 but only the mature form has N- acetylglucoamine branching. The same study examined their respective ability to inhibit matriptase in breast cancer cells. The data suggested that the immature HAI-2 is not capable of inhibiting matriptase and the mature HAI-2 only inhibits matriptase to a minor extent. 50 % of the molecular mass of the naturally occurring HAI-2 is contributed by N-glycans attachment to HAI-2. The unmodified HAI-2 polypeptide has a size of 14 kDa, determined by western blot. Addition of N- glycans to HAI-2 increases the molecular weight with 9-17 kDa. Using two HAI-2 monoclonal antibodies, a survey with a variety of different cell cultures was performed, from which Lai et al. (2015) conclude that human epithelial and carcinoma cells express multiple forms of HAI-2.

From immunoblots, three bands were detected with apparent sizes of 25, 30 and 40 kDa, respectively. Further binding experiments suggested that the N-glycan on the 25 kDa HAI-2 form is an oligo- mannose type without N-glycan branching and the N-glycan on the 30 and 40 kDa form is a complex type with extensive N-glycan branching by addition of N-acetylglucoamine. Blocking of N-glycan branching by inhibition of α-mannosidase decreased the level of the 30- and 40 kDa HAI-2 form but increased the amount of the 25 kDa HAI-2 form. This suggests that the 25 kDa HAI-2 form might be a precursor for the 30- and 40 kDa HAI-2 form. This hypothesis is supported by the location of the 25 kDa HAI-2 form in the ER and the other form in vesicles/granules that have exited the Golgi apparatus, which might be where the completion of the N-glycan branching occurs. The relative expression of the two HAI-2 forms was analyzed in a variety of human epithelial and carcinoma cells

34/60 and revealed that the two HAI-2 species were co-expressed in a cell-specific manner, but always in an equal ratio (Lai et al., 2015).. In summary, there are two species of HAI-2 due to different N-glycosylations, which result in different sizes. The immature 25 kDa HAI-2 form is not capable of inhibiting matriptase, whereas the mature 30- and 40 kDa HAI-2 form is able to inhibit matriptase.

3.2 Altered levels of active HGFA in cancer This section focuses on studies describing the altered levels of HGFA in cancer and how hypoxia inducible factor (HIF) -1α stimulates the expression of HGFA under hypoxic conditions.

3.2.1 Up-regulated levels of HGFA in cancer tissue In the study by Yamauchi et al. (2004), the expression of both HAI-1 and -2 and HGFA mRNA was measured. Contrary to the level of HAI-1 and -2, the expression level of HGFA mRNA was detectable in RCC whereas it was hardly detectable in normal renal tissue. It was observed that the ratio between both HAI-1 and -2 mRNA and HGFA mRNA in normal renal tissue was altered in RCC, leading to an enhanced level of active HGF. The study by Parr et al. (2004) showed that the mRNA expression of HGFA was significantly higher in human breast cancer tissue compared to normal breast tissue. Another study measured the amount of active HGFA in serum by an enzyme-linked immunosorbent assay (ELISA). The results showed that the amount of active HGFA was significantly higher in serum of patients with multiple myeloma, which is cancer in plasma cells, compared to healthy patients (Wader et al., 2008). Overall, the studies showed that HGFA is up-regulated in cancer compared to normal tissue.

3.2.2 Regulation of HGFA by HIF-1α By using RT-qPCR and western blotting, a study showed that both HGFA mRNA and HGFA protein were up-regulated by HIF-1α under hypoxic conditions in the pancreatic cancer cells, PK8 (Kitajima et al., 2008). Hypoxia is a known condition in most human tumors. HIF-1α is a transcription factor known only to be present in hypoxic tissues, due to its degradation by prolyl hydroxylase when oxygen is present under normoxic conditions. HIF-1α binds to specific gene sequences known as hypoxia-responsive elements (HREs), which are found in promoter regions of e.g. HGFAC. When HIF-1α is bound to the HREs, transcription of the target genes is stimulated. It has been observed that

35/60 HIF-1α is associated with poor prognosis and resistance to therapy in different types of cancer (Brown, 2007; reviewed by Kitajima and Miyazaki, 2013). A study by Kitajima et al. (2008) found that the HGFA promoter is activated by HIF-1α under hypoxia, possibly by binding to the HRE element of the HGFA promoter. This leads to an overexpression of HGFA and thereby an increase in the conversion of pro-HGF to active HGF. This increased amount of HGF activates the HGF-cMET pathway in PK8 cells, showing that HIF-1α induces invasion and metastasis through this pathway (Kitajima et al., 2008). HIF-1α is not only present under hypoxic conditions, but has also been shown to be stabilized by oncogenic mutations and radical oxygen species (ROS) that block the activity of prolyl hydroxylase. Further, HIF-1α does not only regulate gene expression of HGFAC, but also the expression of genes important in angiogenesis, chemoresistance, invasion, metastasis and anti- apoptosis in gastric cancer (Kitajima and Miyazaki, 2013).

3.3 Active matriptase in relation to cancer progression This section focuses on studies describing the expression level of matriptase in cancer, and how matriptase protein levels and activity levels can be altered by regulation on three different levels: (1) Altered matriptase expression, (2) decreased interaction with HAI-1 and -2, and (3) induced matriptase zymogen activation by environmental changes such as acidic microenvironment and oxidative stress.

3.3.1 Up-regulated matriptase in cancer tissue Tanimoto et al. (2005) investigated the expression level of matriptase in ovarian cancer, by the use of epithelial cells from patients, and control epithelial cells. The expression of matriptase was found in all the epithelial cells from cancer patients, and in some of the cells derived from normal ovaries. In addition, matriptase was found both in and on the cell surface of the ovary epithelial cancer cells. Based on these findings, Tanimoto et al. (2005) investigated the ratio between the matriptase expression found in the cancer cells and the cells derived from normal ovaries. The data indicated a significantly higher matriptase expression in the cancer cells. Zoratti et al. (2015) also reported increased expression of matriptase in cancer compared to normal tissue. A specific model with transgenic mice predisposed for developing mammary carcinoma was used to mimic cancer progression in human mammary carcinoma. Matriptase expression in human mammary carcinoma was measured as a control for the mouse model. The results showed low levels of matriptase protein

36/60 in normal mammary cells and increased expression in the mammary carcinoma cells. This indicates that matriptase is up-regulated in cancer cells. Zoratti et al. (2015) also found that matriptase was expressed in its proteolytically active form in normal mammary epithelial cells. The active form of matriptase was significantly up-regulated in the mammary carcinoma cells determined by gelatin zymography, which is used to analyze the proteolytic activity.

3.3.2 Dysregulation of the ratio between HAI-1 and matriptase promotes cancer progression A study by Vogel et al. (2006) examined the expression of matriptase and HAI-1, and the matriptase/HAI-1 ratio in colorectal carcinogenesis. The mRNA level of matriptase in carcinomas was significantly decreased compared to the level in healthy tissue, and furthermore the tumor grade was increased when the level of matriptase mRNA was down-regulated. The mRNA level of HAI-1 was also significantly down-regulated in patients with cancer compared to healthy patients, and the tumor grade was increased as the HAI-1 level decreased. In carcinomas and in tissue with severe dysplasia and mild or moderate dysplasia, a significantly higher ratio between the levels of matriptase mRNA and HAI-1 mRNA was observed. This was shown in comparison to tissue from healthy patients. The dysregulation of the ratio in the colorectal carcinogenesis might occur at an early stage due to the observations of a disrupted ratio in the mild or moderate dysplasia tissue. A changed matriptase/HAI-1 ratio in cancer can be due to a decreased level of HAI-1, an increased level of matriptase or it can be a combination of down-/up-regulation of HAI-1 and matriptase (Vogel et al., 2006).

3.3.3 Regulation of active matriptase through control of matriptase zymogen activation The activation of matriptase can be regulated by inducing the matriptase zymogen activation by an acidic microenvironment and oxidative stress.

3.3.3.1 Acidic microenvironment induces matriptase zymogen activation In general, acidic conditions has been shown to influence tumor cells and maintain tumor growth (Tannock and Rotin, 1989). A study by Tseng et al. (2010) investigated the influence of acidic microenvironment on matriptase zymogen activation in the non-tumorigenic human mammalian epithelial cell-line 184 A1N4. They found that cells exposed to citric acid buffer at pH 6.0 showed an increased matriptase activation compared to cells at physiological pH (7.4), which suggests that matriptase activation depends on pH rather than upstream signaling. The correlation between pH and

37/60 the rate of matriptase activation has also been examined in vitro by Wang et al. (2014). They found that inactive matriptase undergoes activation spontaneously at physiological pH, but at a slow rate. When the pH in the cell was lowered to a more acidic level, the spontaneous activation of matriptase accelerated. This was shown in both intact 184 A1N4 cells and in cell-free systems with an isolated cell membrane. More specifically, the matriptase activation rate was determined by measuring matriptase-HAI-1 complex formation, corresponding to the rate of matriptase activation, at different pH values. At physiological pH, matriptase activation was induced after 40-60 minutes, and the activation reached a maximal limit after approximately 80 minutes. About 10 % of the matriptase zymogens were activated at the maximal limit. When pH was decreased to 6.5, matriptase activation occurred after less than 10 minutes, reaching a maximal level after 20 minutes. At this maximal level, around 80% of the matriptase zymogens were activated (Wang et al., 2014). Matriptase zymogen activation might therefore be dependent on the pH rather than upstreaming signaling.

3.3.3.2 Oxidative stress induces matriptase zymogen activation Inflammation is a regulated response that causes recruitment of activated inflammatory cells, which in turn change the stromal microenvironment including making it more acidic or more oxidizing (Wang et al., 2014). Several normal cell lines have shown induced matriptase zymogen activation when exposed to oxidative stress. Wang et al. (2014) demonstrated this by exposing 184 A1N4 cells to cobalt chloride and cadmium chloride, which is known to induce ROS production. Both metal ions caused induced matriptase activity in a dose dependent manner, cadmium chloride being more efficient than cobalt chloride. Another study by Chen et al. (2011) also found a regulatory effect of oxidative stress on matriptase zymogen activation in cultured human keratinocytes (HaCaT) cells exposed to hydrogen peroxide, which also causes oxidative stress. Additionally, they examined the matriptase/HAI-1 ratio in 23 distinct inflammatory skin diseases, and it was demonstrated that the ratios were the same as in normal cells, even though induced matriptase activity was found in 16 of the diseases. Furthermore, the cells with increased levels of cleaved/active matriptase were mostly surrounded by inflammatory cells, suggesting that active matriptase might be part of the keratinocyte response to inflammation.

In summary, matriptase zymogen activation can be induced by oxidative stress. A study showed that it is possible to observe the same ratio between matriptase and HAI-1 in normal and inflammatory skin tissue, although the amount of active matriptase is higher in inflammatory skin tissue compared to normal skin tissue.

38/60 3.4 Overexpression of c-MET in cancer tissue Altered c-MET stimulation might also be caused by an overexpression of the c-MET receptor, which is found in many types of cancer.

A study by Elenitoba-Johnson et al. (2003) found higher levels of c-MET transcripts in diffuse large B-cell lymphoma (DLBCL) compared with follicular lymphoma from the same patient. Another study by Tjin et al. (2006) also investigated the expression of c-MET in B-cell malignancies by immunohistochemistry and immunoblotting. In agreement with Elenitoba-Johnson et al. (2003) their results showed very strong staining of c-MET, indicating an overexpression of c-MET in DLBCL. This is in alignment with findings of changes in c-MET expression in solid tumors, due to mutation/amplification of the receptor, and/or changes in kinase activity in a wide palette of different types of cancer such as colon, lung, gastric and pancreatic cancer (reviewed by Salgia, 2009). Overexpression of c-MET has also been detected in breast cancer (Lengyel et al., 2005). An increased amount of c-MET is associated with poor prognosis in cancer (Baschnagel et al., 2014).

39/60 4. Discussion 4.1 Importance of the ratio between HGFA/matriptase and HAI-1/ -2 The studies, mentioned in section 3, indicate that HAI-1 and -2 are down-regulated in cancer tissue, while matriptase and HGFA are up-regulated, explaining the enhanced activation of the HGF-cMET axis in cancer tissue. A study by Vogel et al. (2006) found an unexpected decrease in matriptase mRNA in cancer cells compared to normal hyperplasia, but with a corresponding decrease in HAI-1. The down-regulation in both mRNA levels, favored the level of matriptase in the ratio between the two. Yamauchi et al. (2004) have observed the same dysregulated ratio between HGFA mRNA and the HAI-1/-2 mRNA, favoring more HGFA mRNA. As previously mentioned, cancer progression and metastasis are caused by a dysregulated ratio between the HAI-1/-2 and matriptase/HGFA, which favors more matriptase/HGFA. In addition, many studies examine the mRNA expression levels of matriptase, HGFA, HAI-1 and -2. The expression levels of matriptase, HGFA, and HAI mRNA are not necessarily a measure of their active form. Chen et al. (2011) showed that the ratio between matriptase mRNA and HAI-1 mRNA can be similar in normal skin tissue and inflammatory skin tissue, despite increased levels of active matriptase in the inflammatory skin tissue. This indicates that it is important to measure the level of active protein in addition to the level of mRNA in order to perform the most comprehensive analysis. As mentioned in section 3.1.3, there are two different species of HAI-2, which differ in their glycosylation, one is active, whereas the other is not able to inhibit matriptase (Lai et al., 2015). This may give rise to a higher level of HAI-2 mRNA than what will become the active HAI-2 protein, which is able to inhibit active matriptase. Active matriptase and HGFA activate pro-HGF, which binds to the c-MET receptor initiating the HGF-cMET axis. Thus, increased levels of active matriptase and HGFA lead to increased activity of the HGF-cMET axis, which promotes cancer progression and metastasis. Increased levels of active HGFA and matriptase are caused by enhanced gene expression, decrease in HAI-1 and -2 levels, and/or change in environmental conditions. Therefore, regulation of HGFAC, ST14, SPINT1 and -2 should be considered as therapeutic possibilities for prevention of cancer progression and metastasis.

4.2 HGFA and matriptase as potential targets against cancer HGFA and matriptase are regulated by other factors than HAI-1 and -2, such as environmental factors like pH or oxidative stress found in malignant tumors. Matriptase activation depends on prostasin activity in certain tissues, and Bergum et al. (2012) have showed that matriptase and prostasin are co-

40/60 expressed in breast cancer. Thus, another potential target could be inhibition of the prostasin activity in certain tissues. However, this might be less efficient than using HAI-1 and -2 as therapeutic agents, since prostasin is only involved in matriptase activation, which it is only partly responsible for matriptase activation is also enhanced by decreased pH and increased oxidative stress, and thus these factors should be decreased in regards to lower the amount of active matriptase to prevent further cancer progression and metastasis. In addition, the expression of HGFAC is enhanced by HIF-1α, which is highly expressed in cancer either due to mutations, hypoxia or ROS. However, it may not be possible to control pH and the oxidative conditions in the cell microenvironment. Furthermore, the activity of HGFA is dependent on activation by numerous proteins such as thrombin, KLK4 and -5, which could be potential therapeutic targets. However, direct inhibition of active matriptase and HGFA might be more efficient compared to controlling pH, oxidative conditions and/or several proteins such as thrombin, KLK4 and -5.

4.2.1 Selective protease inhibitors as therapeutic agents The regulation of ST14 expression is not well investigated, but maybe other environmental factors affect ST14 and thus it might be difficult to regulate the gene expression of matriptase. However, gene engineered mice with a 99% reduction of epidermal matriptase mRNA expression levels (ST14 hypomorphic mice) are shown to have normal survival and capability of producing rearing offspring. These findings are unlike the ST14 null mice, which suffer from neonatal lethality. Furthermore, the ST14 hypomorphic mice were suffering from rather mild symptoms similar to those observed in a human disorder ARIH, which is an inherited disorder linked to homozygosity for a point mutation in ST14. Among others, the symptoms in ST14 hypomorphic mice were expressed as wrinkled and scaly skin, patchy and sparse fur, and conical or notched teeth. These findings of rather mild side effects of radical matriptase inhibition, indicating that selective matriptase inhibitors might be suitable as therapeutic agents (List et al., 2007).

One suggested selective inhibitor of matriptase is the small-molecule selective matriptase inhibitor, IN-1, which is shown to reduce active matriptase levels. IN-1 is also shown to efficiently abrogate c- MET activation and block breast cancer cell proliferation (Zoratti et al., 2015). Another selective inhibitor of matriptase is CVS-3983, which has been examined in vivo by Galkin et al. (2004). CVS- 3983 was investigated in mice using xenograft human prostate cancer, which is surgical human graft inserted in mice. The grafts were derived from two different human prostate cancer cell lines

41/60 (CWR22R and CWRSA6). When treated with CVS-3983, the volumes of the tumors developed from CWR22R and CWRSA6 were significantly decreased by 65.5% and 56.3%, respectively. The only observed difference between CVS-3983-treated mice and control mice was the tumor sizes. Galkin et al. (2004) suggest that CVS-3983 inhibits tumor growth without causing cell death. CVS-3983 is rather selective towards matriptase, but might be capable of inhibiting other proteases.

Selective inhibitors of HGFA have also been tested. Han et al. (2014) synthesized various tetrapeptide ketothiazoles (KTs), which were tested as inhibitors for e.g. HGFA. The KTs were constructed as analogues that resembled the substrate cleavage site of pro-HGF in the P4-P1 portion. Some of these KTs blocked the activation of pro-HGF in an HGFA enzymatic assay. Furthermore, the inhibitors were examined in the invasive breast cancer cell line, MDA-MB-231. These cells expressed a high level of c-MET and pro-HGFA, and they were further treated with pro-HGF. Many of the inhibitors caused an efficient decrease in the c-MET signaling of the cells. Another study by Tjin et al. (2006) also showed inhibition of active HGFA using anti-HGFA monoclonal P1-4 in DLBCL. Thus, inhibitors, which contain a portion similar to the substrate cleavage site of HGF, can impair conversion of pro-HGF by blocking HGFA activation, and may further inhibit cancer progression and metastasis.

Thus, the designed selective protease inhibitors are potent therapeutic agents against cancer progression. Selective protease inhibitors might have a greater specificity in inhibiting proteases than HAI-1 and -2, which have other functions than regulating proteases. High selectivity and specificity is crucial in terms of side effects. Moreover, specific inhibitors of matriptase might only cause mild side effects such as abnormal skin and hair, which are relatively harmless compared to the side effects of chemotherapy. Similar studies regarding HGFA are limited, but this should be investigated in order to consider possible side effects of HGFA inhibition.

4.3 Demethylation of SPINT1 and -2 as therapeutic targets in cancer A study with specific intestinal epithelial cells, in mice, with deletion of the SPINT1 gene, tested the tumorigenesis and activation of pro-HGF. The results showed enhanced tumor formation in the small intestines in mice (15 weeks old) lacking the SPINT1 gene. Furthermore, the activation of pro-HGF in the same mouse models were examined, and already 10 weeks after birth, the intestinal tissue showed tumor progression, enhancement of HGF, and phosphorylated c-MET. This suggests that lack

42/60 of the SPINT1 gene can be crucial to cancer formation in intestinal epithelial cells (reviewed by Kawaguchi and Kataoka, 2014). Furthermore, List et al. (2005) showed that when the SPINT1 gene is overexpressed, the tumor formation is decreased. Thus, SPINT1 is a potential target in regards to decreasing cancer progression and metastasis. As mentioned in section 3 the decrease in both mRNA expression and protein levels of HAI-1 and -2, in cancer, might be caused by hypermethylation of the starting site. Hypermethylation of the CpG island in the SPINT2 promoter is correlated with decreased expressions of HAI-2 and is observed in several types of cancer (Dong et al., 2010; Fukai et al., 2003; Morris et al., 2005; Tung et al., 2009). The SPINT1 gene encoding HAI-1 has currently not been investigated regarding hypermethylation, even though SPINT1 has a CpG island around the starting site consisting of more CpG sites than SPINT2 (Mitchelmore, personal communication 2016) (Appendix 1). It is therefore reasonable to suggest that decreased expression levels of HAI-1 and -2 in cancer are caused by hypermethylation of the starting site. Since several studies show that hypermethylation of the SPINT2 promoter cause down-regulation of HAI-2 during cancer progression, demethylation of the SPINT2 promoter might restore the level of HAI-2. Potential therapeutic targets could be DNA methyltransferases (DNMTs), which methylate DNA. Epigenetic therapy of cancer could be the inhibition of DNMTs, which might lead to hypomethylation. The U.S. food and drug administration has approved the two demethylating agents AZA and DAC for clinical testing (reviewed by Duenas-Gonzalez et al., 2016).

4.3.1 DAC and AZA block methylation of SPINT2 by inhibition of DNMTs A potent inhibitor of DNMTs is the S-phase specific demethylating agent DAC that induces cell death and differentiation. When DAC enters the cell, it is phosphorylated by deoxcytidine kinase and then further phosphorylated into the active form Aza-dCTP. Aza-dCTP is incorporated into the DNA, where the 5-azacytosine ring of Aza-dCTP bind covalently to the enzyme DNMT1, a type of DNMT that methylates the DNA. This binding inactivates the enzyme, which results in hypomethylation (Dong et al., 2010; reviewed by Momparler, 2013; reviewed by Wongtrakoongate, 2015). The other demethylating agent AZA can also incorporate into the DNA and thereby inhibit DNMTs by blocking DNA methylation. However, AZA is able to incorporate into RNA, where it disrupts normal RNA function (Wongtrakoongate, 2015). The hypomethylation will in both cases lead to reactivation of tumor suppressor genes that were silenced by hypermethylation (Momparler, 2013; Wongtrakoongate, 2015). The study by Dong et al. (2010) showed that the hypermethylated SPINT2 promoter is demethylated in gastric cancer cell lines treated with DAC. Increased levels of HAI-2

43/60 mRNA indicate that DAC reactivates SPINT2. Tung et al. (2009) demonstrated that the hypermethylated SPINT2 in HCC is demethylated when the cells are treated with DAC and this treatment increases the level of HAI-2 mRNA. A study by Morris et al. (2005) also observed that the hypermethylated SPINT2 in RCC is demethylated after treatment with AZA, resulting in up-regulated expression of HAI-2. This suggests that DAC and AZA are agents that potentially can restore the level of HAI-2 in cancer where the ratio between HAI-2 and matriptase or HAI-2 and HGFA is dysregulated.

4.3.2 DAC and AZA as cancer treatments cause side effects Even though DAC and AZA increase the HAI-2 mRNA level by demethylation of the SPINT2 promoter, it is important to remember that both agents are not gene specific and could therefore cause demethylation of gene regions that are normally methylated, which might cause side effects. A potential side effect could be an up-regulation of matriptase, HGFA or hepsin if the genes encoding these proteins are also hypermethylated in cancer. However, most studies show HGFA and matriptase to be up-regulated in cancer, and it is therefore unlikely that these genes are hypermethylated. Furthermore, DAC and AZA are given as intravenous infusion (reviewed by Momparler et al., 2014) and the two agents might therefore affect both cancer and normal cells. Momparler (2013) and Momparler et al. (2014) have observed that a side effect of DAC treatment is myelosuppression, a condition where the bone marrow activity is decreased resulting in fewer red blood cells, white blood cells and platelets. Another side effect of DAC is granulocytopenia, a condition where the number of granulocytes is decreased (Momparler, 2013). These side effects highlight the toxicity of DAC. However, DAC and AZA have been recognized as having lower toxicity than traditionally chemotherapy (Wongtrakoongate, 2015), and therefore therapy with these two agents might provide a safer treatment. Furthermore, Momparler et al. (2014) observed that DAC can induce complete remission of patients with acute myeloid leukemia, but most of the patients experienced relapse. They suggest that the treatment with DAC require a combination with other chemotherapeutic agents to increase the effectiveness of the therapy. These other agents probably have side effects, which should also be taken into account regarding cancer treatment with DAC or AZA.

The argument for the use of DAC and AZA is that they up-regulate the level of HAI-2 in cancer, and this might lead to less active matriptase and active HGFA, resulting in a decreased activation of the HGF-cMET axis. In addition, tumor suppressor genes have been shown to be methylated in cancer,

44/60 therefore treatment with DAC and AZA might result in up-regulation of these genes. It might not be enough to up-regulate HAI-2 in regards to decrease the level of active matriptase and HGFA. The efficiency of DAC and AZA as cancer treatment may also depend on HAI-1, which inhibits both matriptase and HGFA. As mentioned, SPINT1 contains more CpG sites in the CpG island around the starting sites than SPINT2. This could indicate that cancer cells with decreased level of HAI-1 might be caused by a hypermethylated SPINT1 promoter. DAC and AZA might be able to demethylate SPINT1 and thereby increase the level of HAI-1. If the down-regulation of HAI-1 is not due to hypermethylation of SPINT1, the level of HAI-1 will not be up-regulated by DAC and AZA. The activity of the HGF-cMET axis is further decreased if both HAI-1 and -2 are up-regulated in cancer tissue (Parr and Jiang, 2006). The study by Pereira et al. (2016) showed that the SPINT2 promoter region in PCa cell lines was not hypermethylated, and was therefore not the cause of the decreased level of HAI-2 mRNA. Thus, DAC and AZA are not able to increase the level of HAI-2 mRNA in PCa cell lines since the decreased level of HAI-2 mRNA must be caused by other regulatory mechanisms. Therefore, it should be taken into consideration that DAC or AZA might not work in all cancer types, because the down-regulated levels of HAI-1 and/or -2 might not always be caused by hypermethylations of the SPINT1 and/or -2 promoters. Furthermore, the ectodomain shedding of HAI-1 and -2 is, as mentioned, regulated by different mechanisms, such as phosphorylation, polarized secretion, intracellular trafficking, activation of the enzyme sheddase, and protein-protein interactions. These factors could affect the level of accessible HAI-1 and -2 that inhibit HGFA and matriptase. This might have an influence on the efficiency of DAC and AZA as cancer-treatment, since the shedding and activation of the expressed HAI proteins depend on several factors, and should therefore be taken into account. In addition, it is also important to consider if the side effects of the two agents are too severe before they are used as cancer treatment. If the side effects are not too severe and the effects of DAC and AZA restore the HAI-2 level in regards to the level of active matriptase and HGFA, these two chemotherapeutic agents could be combined with current chemotherapy.

45/60 4.4 Injection of HAI-1 and -2 as therapeutic treatment If the side effects of DAC and AZA are too severe or if the SPINT genes are not hypermethylated, an alternative approach could be to increase the protein levels of HAI-1 and -2 by directly injecting the proteins into the bloodstream or cancer tissue. A study by Parr and Jiang (2006) showed that when biologically active recombinant HAI (rHAI)-1 and/or -2 were added to human fibroblasts, the amount of active HGF was significantly reduced. This indicates that HAI-1 and -2 inhibit the activation of pro-HGF through either matriptase or HGFA. The data showed that addition of either rHAI-1 or -2 reduced the amount of active HGF with 50 %, and in combination reduced the amount of active HGF with 75 %. The study also showed evidence that addition of stromal fibroblasts with both rHAI-1 and rHAI-2 to breast cancer cells reduce invasion of breast cancer cells dramatically (Parr and Jiang, 2006). If this is valid for other cancer types and if it is possible to directly inject both rHAI-1 and rHAI-2 into the tumor, this treatment could possibly reduce tumor invasion. In addition, if it is possible to ensure that only the active HAI-1 and -2 are injected, it could be beneficial to inject HAI proteins directly into the tumor compared with regulation of SPINT1 and -2. If the HAI levels are regulated with DAC or AZA, both active and inactive HAI-1 and -2 will be produced. However, injection might not be a long-acting treatment since the natural expression of HAI-1 and -2 is still silenced or down-regulated. Eventually the injected proteins might be degraded, and thus the treatment may only have a temporary effect on cancer progression. However, it could be combined with other chemotherapeutic agents to create a more efficient treatment against cancer progression. Furthermore, it should be considered that the injected rHAI-1 and -2 could spread to the healthy tissue, and thus disrupt the HAI homeostasis in healthy tissue. One side effect of a disrupted HAI homeostasis could be a disruption in the maintenance of the skin caused by increased inhibition of matriptase. Chen et al. (2014) suggest that the primary physiological function of matriptase in human skin is to regulate proliferation and early differentiation of keratinocytes. Furthermore, it has been demonstrated that matriptase-prostasin-HAI-1 cell protease network appears to be an essential process for maintenance of skin barrier function (Chen et al., 2014). Therefore, an injection of rHAI- 1 and -2 could possibly lead to side effects in the maintenance of the skin barrier function. However, skin problems could be considered a mild side effect compared to side effects caused by general chemotherapy.

46/60 4.5 Further perspectives As mentioned the levels of active HGFA and matriptase are regulated by microenvironmental factors. Hence, the regulation of SPINT1 and -2 could also depend on several microenvironmental factors, and might influence the expression of HAI-1 and -2, which could be interesting to further investigate. In addition, it could be interesting to examine if the CpG island of SPINT1 is hypermethylated in cancer. This could be examined using the same methods as the ones used to measure hypermethylation of SPINT2. Inhibition of hepsin could also be a target in cancer treatment, as it is known to cleave pro-HGF to its active form and is like HGFA and matriptase, inhibited by HAI-1 and -2. Furthermore, it has been observed to be up-regulated in prostate and ovarian cancer, where it has an effect on progression and metastasis (Kirchhofer et al., 2005; Klezovitch et al., 2004). Down- regulation of hepsin may cause side effects due to its normal physiological functions, although this should be further examined. Another alternative approach that could prevent cancer progression and metastasis is to use the HGF antagonist, NK4, which is a HGF variant containing 4 kringle domains and a N-terminal hairpin domain. NK4 binds to c-MET and completely blocks HGF binding, resulting in suppressed tumor invasion and metastatic spread. An advantage of using NK4 is that it is not biologically active in human colorectal cancer cells (Parr et al., 2000), and thus it may not affect other biological processes. As previously mentioned, KD1 of HAI-1 and -2 is shown to have the strongest inhibitory effect on HGFA and matriptase, and an efficient synthetic inhibitor could therefore be an analog to KD1. Furthermore, the glycosylation of HAI-2 seem to have an impact on its inhibitory effect and should therefore be taken into consideration regarding construction of a synthetic inhibitor.

47/60 5 Conclusion

HGFA and matriptase are mostly up-regulated in different kinds of cancer, while HAI-1 and-2 are mostly down-regulated. It is important to consider the ratio between the active forms of the HAI proteins and HGFA or matriptase. This is due to increased cancer progression and metastasis when activation of the c-MET receptor is enhanced due to excess level of matriptase and HGFA compared the HAI level. Thus, HGFA, matriptase, HAI-1 and -2 could be potential targets in regards to down- regulation of active HGFA and matriptase levels, thereby decreasing cancer progression and metastasis. The levels of active HGFA and matriptase depend on gene expression, the amount of HAI-1 and -2 and/or change in environmental conditions. It might be complicated to control these environmental conditions, and thus other factors might be more efficient to regulate. A different approach of down-regulating the level of active HGFA and matriptase is to increase the level of HAI- 1 and -2 and/or design synthetic protease inhibitors, which mimic the inhibitory features of the HAI proteins. Studies have shown that overexpression of the two HAI-1 and -2 decrease tumor progression. The decreased level of HAI-2 in some types of cancer are caused by hypermethylation of SPINT2. Hypermethylation of SPINT1 has not been investigated however, the decreased level of HAI-1 might be affected by hypermethylation due to the CpG island found around the starting site of SPINT1. Hence, an up-regulation of the HAI proteins could be obtained by demethylating the SPINT genes by treatment with DAC or AZA. Another way to decrease the level of proteases might be to directly inject HAI proteins or synthetically designed protease inhibitors into the bloodstream or cancer tissue. However, the injection with HAI proteins might not be a long-acting treatment due to potential degradation and non-specific targeting against only matriptase and HGFA. The synthetically designed protease inhibitors might be more efficient therapeutic agents due to their specific and selective targeting against matriptase and HGFA. All the suggested treatments may have side effects, which should be further investigated before it can be concluded whether HAI, matriptase and HGFA are suitable targets in cancer, and which of the therapeutic targets is the most efficient for prevention of cancer progression and metastasis.

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56/60 Appendix 1 - CpG islands in SPINT1 and -2

Figure 1. SPINT1 from the UCSC genome browser on Human DNA. The green box indicates CpG island and is located near the promoter region on the SPINT1.

Figure 2. SPINT2 from the UCSC genome browser on Human DNA. The green box indicates CpG island and is located near the promoter region on the SPINT2.

57/60 Appendix 2 – Abbreviation list

AKT: Protein kinase B, also referred to as AKT AZA: 5-azacytidine c-MET: Cellular Mesenchymal Epithelial Transition CpG: 5'—C—phosphate—G—3' CSC: Cancer stem cell CUB: C1r/s, Uegf, and bone morphogenic protein-1 domains DAC: 5-Aza-2’-deoxycytidine DLBCL: Diffuse large B-cell lymphomas DNMTs: DNA methyltransferases ELISA: Enzyme-linked immunosorbent assay ER: Endoplasmic reticulum ERK: Extracellular signal-regulated kinase EMT: Epithelial mesenchymal transition Gab1: Grb2-associated-binding protein 1 GATA-1: Globin transcription factor 1 GDP: Guanosine diphosphat Grb2: Growth factor receptor-bound protein 2 GTP: Guanosine triphosphate HaCat: Cultured human keratinocyte cells HAI: Hepatocyte growth factor activation inhibitor HCC: Human hepatocellular carcinoma HGF: Hepatocyte growth factor HGFA: Hepatocyte growth factor activator HIF-1α: Hypoxia-inducible factor 1α HREs: Hypoxia-responsive elements IKK: IκB kinase IPT: Immunoglobulin plexins transcription JAK: Janus kinase KD: Kunitz domain KLKs 4 and 5: Kallikrein-related peptidases 4 and 5 K1-K4: Kringle domains 1-4

58/60 LDL: Low density lipoprotein receptor MANSC: Motif at N-terminus with seven cysteines MAPK: Mitogen-activated protein kinase MEK: Mitogen-activated protein kinase kinase MET: Mesenchymal epithelial transition mTOR: Mammalian target of rapamycin mTORC1: Mammalian target of rapamycin complex 1 NF-κB: Nuclear factor-κB NIK: NF-κB inducing kinase NK4: N-terminal hairpin domain and 4 kringle domains NLS: Nuclear localization signals p53: Tumor protein 53 PI3K: Phosphoinositide 3-kinase B PK8 cells: Pancreatic cancer cells pro-HGF: Preform-hepatocyte growth factor activator PSI: Plexin, semaphorin, and integrin (cysteine-rich domains) RAF: V-raf murine sarcoma (viral oncogene homolog B1) ras: Rat sarcoma viral oncogene RAS: Rat sarcoma viral oncogene homolog RCC: Renal cell carcinoma rHAI: Recombinant HAI ROS: Radical oxygen species RT-qPCR: Reverse transcriptase quantitative polymerase chain reaction SEA: Sperm protein, enterokinase, and agrin domain sema: Semaphorin domain SHP2: Sarcoma homology phosphatase SOS: Son-of-sevenless (ras guanine nucleotide exchange factor) SPH: Serine protease homology SPINT1: Serine protease inhibitor, Kunitz type 1 / serine peptidase inhibitor, Kunitz type 1 SPINT2: Serine protease inhibitor, Kunitz type 2 / serine peptidase inhibitor, Kunitz type 2 ST14: Suppressor of tumorigenicity 14 STAT: Signal transducer activator of transcription

59/60 STAT3: Signal transducer and activator of transcription-3 STAT5: Signal transducer and activator of transcription-5 TP53: Tumor protein 52 gene TSC2: Tuberous sclerosis complex 2 TSC1/TSC2: Tuberous sclerosis complex 1 and 2

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