Chapter IV: An ECM protease/inhibitor network regulates cell outgrowth
4.2 Introduction
Metastasis is the major cause of death from cancer (WHO Cancer, 2014). Determining the underlying mechanisms that tumors use to invade other tissues can provide valuable information in stopping metastasis. Specifically, targeted drugs can be developed against molecular factors that are responsible for metastatic behaviors. For instance, autocrine motility factor (AMF) induces tumorigenicity, and causes cell detachment from the primary tumor site through promoting cell motility (Iiizumi et al., 2008; Liotta et al., 1986). This leads to an increase in metastasis in colorectal, lung, kidney, breast, and gastrointestinal carcinomas (Baumann et al., 1990; Filella et al., 1991; Patel et al., 1995; Dobashi et al., 2006; Tsutsumi et al., 2002; Yanagawa et al., 2004;
Funasaka et al., 2007). Treatments have been developed against AMF, including carbohydrate phosphate inhibitors such as E4P, D- mannose-6-phosphate, and 5-phospho-D-arabinonate (5PAA), which block both AMF enzymatic activity and AMF-induced cell motility (Tanaka et al., 2002; Sun at al., 1999). Another treatment involves the use of antibodies against AMF (Talukder et al., 2000), which partially block HRG-induced invasiveness of human breast cancer MCF-7 cells. Therefore, identifying characteristics of metastatic cancers can yield effective treatments against this disease.
Metastasis occurs in several different stages, which include local invasion, intravasation (the entry of tumor cells into the bloodstream), circulation through the bloodstream, extravasation (the exit of tumor cells from capillary beds into the parenchyma of an organ), and colonization (Nguyen et al., 2009). Tumors initially begin by initiating genetic mutations that provide unlimited proliferative potential. These mutant cells tolerate cell division defects and an unstable genome, maintain progenitor-like phenotypes, and support other cell-autonomous functions that generate oncogenically transformed cells (Hanahan and Weinberg, 2000). However, in order to become metastatic, these tumors need to take on the additional ability to spread and proliferate in other tissues. This involves entering circulation, exiting circulation, and then infiltrating other organs. A subset of genes, known as metastasis initiation genes, promotes these invasive activities by inducing cell motility, epithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) degradation (Chiang and Massagué, 2008).
Genes that encode proteases are a major type of metastasis initiation gene. In metastasis, proteases degrade components of the ECM so that tumor cells can breach barriers that exist between tissues.
Several types of proteases are involved in degrading the ECM, including cathepsins, trypsins, threonine proteases, and matrix metalloproteases (Rakashanda et al., 2012). We are interested in studying the role of one family of zinc metalloproteases: the meprins. Meprins are a class of metalloproteases that are exclusively expressed in vertebrates and exist as two subunits: meprin α and meprin β (Sterchi et al., 2008). Meprins contribute to metastatic activity (Matters and Bond, 1999; Bond et al., 2005; Dietrich et al., 1996; Minder et al., 2012). Meprin β is upregulated in breast, pancreatic, and colon carcinoma cell lines (Matters and Bond, 1999; Bond et al., 2005;
Dietrich et al., 1996) and meprin α is expressed in three fold higher levels in metastatic colorectal cancer cells versus nonmetastatic cells (Minder et al., 2012). In vitro studies have shown that meprins cleave components of the ECM including laminin-1, laminin-5 (Köhler et al., 2000), collagen IV, fibronectin, and nidogen (Kruse et al., 2004). All of this work has been done using in vitro cell culture, and though important insights regarding meprin function have been determined, studying meprins in vivo can provide many new insights. By studying meprins within an organism, we wish to shed light on the mechanisms meprins use to degrade components of the ECM, identify upstream and downstream regulators of meprins and determine how meprins, affect cell shape change.
Developing cells exhibit behaviors similar to that of metastatic cancers, and model organisms are valuable tools for studying genes involved in metastasis. Cell motility and shape change are a key aspects of organism development. The caudal visceral mesoderm (CVM) cells migrate anteriorly in the Drosophila embryo to eventually form muscles of the gut (Kadam et al., 2012). Vertebrate neural crest cells undergo epithelial to mesenchymal transition followed by migration to give rise to critical components of the craniofacial skeleton, such as the jaws and skull, as well as melanocytes and ganglia of the peripheral nervous system (Bronner and Le Dourian, 2012). And in C. elegans, the anchor cell degrades its underlying basement membrane to form protrusions, which allows the anchor cell to induce vulval fates (Sherwood et al., 2005). Each of these systems has not only elucidated aspects of cell behavior, but also clarified mechanisms involved in metastasis. For instance, the FGF receptor heartless is involved in regulating CVM migration (Kadam et al., 2012), and blockage of FGFR can reduce metastasis from prostate cancer cells into bones (Wan et al., 2014). Both migrating neural crest cells as well as colon carcinoma cells lose cadherin expression at their leading edge (Nakagawa and Takeichi 1998; Prall 2007). Netrin/UNC-6 and its receptor, UNC-40, guide protrusion formation during AC invasion (Hagedorn et al., 2013); similarly, netrins are known to promote liver, colorectal, and cervical cancer cell metastasis (Yan et al., 2014; Ko et al., 2013; Zhang et al., 2013).
We wish to study meprin activity using a C. elegans cell: the uterine seam cell (utse). The C.
elegans uterine seam cell attaches the uterus to the body wall and undergoes cell outgrowth during development (Ghosh and Sternberg, 2014). During the L4 larval stage, the utse begins as an ellipsoidal cell, and then elongates outwards in a bidirectional manner along the anterior-posterior axis. Several genes involved in utse development have been implicated in cancer progression including Trio/unc-73, which is involved in the migration and invasiveness of gliblastoma cells (Fortin et al., 2012), NAV3/unc-53, which is found in colorectal cells (Carlsson et al., 2011), and several Rab GTPases (rab-1, rab-5, rab-6.2, rab-10, rab-11.1), which are upregulated in breast and ovarian cancers (Cheng et al., 2005). Since utse changes its shape and uses similar molecular inputs as metastatic cancer cells, we believe that it is an ideal model to study genes involved in cancer progression.
Here we focus on three C. elegans genes, nas-21, nas-22, and nas-26/toh-1, to study meprin activity. These three genes are members of a class of proteases known as nematode astacins (nas) (Möhrlen et al., 2003). The astacin family of proteases was isolated from the crayfish Astacus
astacus and encompass a family of than 120 reported sequences, detected in a wide range of organisms including bacteria, hexapodes, nematodes, molluscs, insects, and mammals (Gomis-Rüth 2003). Meprins belong to the astacin family (Sterchi et al., 2008) and therefore we believe that studying nas-21, nas-22, and toh-1 will increase our understanding of meprin function and regulation. In this work we use the utse to identify upstream and downstream regulators of these three genes, and show that nas-21, nas-22, and toh-1 are necessary for cell shape change, and characterize the effects of nas-21, nas-22, and toh-1 on the extracellular matrix.