Various cell types use the cell invasion program during normal animal development

During blastocyst implantation, the embryo builds a physical connection between itself and the maternal endometrium. This is provided by the trophoblast cells, which are cells forming the surface of the blastocyst. First, the trophoblast cells adhere the blastocyst to the uterine cells by forming cellular protrusions that reach into the endometrial epithelia. The surface of the endometrium is formed by the decidual cells that are newly formed and removed every menstruation cycle. Afterwards, the trophoblast cells give rise to two different cell types that are both important to invade the endometrium. The syncytiotrophoblast (ST) cell is made by fusion of trophoblast cells and mediates fetal and maternal exchange of nutrients. In addition, the ST cell starts invasion by secreting lytic enzymes (Serine proteases, cathepsin, plasminogen activators and matrix metalloproteinase [MMP]), which digest the extracellular matrix surrounding the decidual cells. Then, decidual cells detach and the syncytiotrophoblast cell can reach and penetrate the underlying basal lamina. As a result the invasive protrusions reach the stroma and the embryo is firmly connected to the endometrium (www.embryology.ch). The trophoblast cells differentiate also into extravillous trophoblast cells (EVT). EVT cells are highly invasive cells that penetrate into the endometrial tissue, invade maternal arteries and thereby provide the embryo with blood [101]. During invasion, EVT cells produce pro-invasive proteins like MMPs and downregulate production of anti-invasive proteins like cell adhesion molecules (e.g. integrin and e-cadherin).

During neurulation, neural crest cells (NCC) are formed dorsal to the neural fold. Once the neural tube is formed NCCs undergo an epidermal to mesenchymal transition (EMT) and start to migrate towards their final location in the body. Finally, NCCs give rise to a high variety of cells, such as neurons and glia from the peripheral nervous system (PNS), skeletal elements, tendons and smooth muscles. Migration of neural crest cells depends on the extracellular matrix and interactions of integrins with laminin and fibronectin [102]. Moreover, migrating NCCs have been shown to secrete matrix modifying enzymes, such as plasminogen activators, thereby modulating the ECM [103]. A subpopulation of NCCs develop into melanocytes, the melanin-forming pigment cells. Once melanocytes reach the ectoderm they start to dissolve its basal lamina and invade the skin [104].

The ability of cells to cross basal laminae is also of great importance for the immune

General Introduction

and start to recruit leukocytes from nearby blood vessels. Chemokine signals from the macrophage strengthen the interaction between leukocytes and the endothelial cells of the blood vessel. Integrins presented on the surface of leukocytes allow tight binding to the blood vessel wall. Finally, the leukocytes start to express proteases that allow to cut the basal lamina in the blood vessel and to migrate into the affected organ. This step is called extravasation of leukocytes.

Since it is obvious that unwanted invasion of cells can cause serious damage to an organism, activation of invasion must be tightly regulated in space and time. A central part of controlling invasive behavior is regulation of matrix modifying enzymes, such as MMPs, serine proteases and cathepsin. MMPs are a family of homologous proteases that all depend on Zn-ions for their catalytic activity [105]. They are secreted as inactive proenzymes and activated by hydrolysis. For example, plasmin, a blood component that degrades blood plasma proteins, is also known to activate MMPs. Active MMPs can be inhibited by tissue inhibitors of matrix metalloproteins (TIMP). Thereby, MMPs are regulated at various levels.

MMPs are grouped based on their substrate specificity as gelatinases, collagenases and stromyelysins.

On the other hand also the ECM influences adhesion, migration and invasion of a cell by interacting with its integrin receptors that are present on the cell surface. Matrix components like laminin and fibronectin are known to mediate these interactions. There are several examples for matrix components that regulate MMP expression. It has been shown that certain matrix components promote MMP secretion.

Trophoblast implantation is an example for cellular invasion that has been extensively studied at the genetic level. There, invasive behavior is controlled by growth factors, their binding partners and matrix proteins. Invasion is promoted, for example, by the insulin-like growth factor II (IGFII) and inhibited by transforming growth factor-ß (TGF-ß) signaling. TGF-ß is thought to exert its effect as an anti-invasive signal in different ways. TGF-ß upregulates integrins, which might lead to stronger cell-matrix interaction and thus inhibit cell migration.

In addition, it regulates important matrix proteins. Tissue inhibitor of metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) are up- and urokinase-like plasminogen activator (uPA) is downregulated [106], which leads to lower MMP activity.

General Introduction

During C. elegans development, the AC, a specialized cell in the C. elegans gonad, acquires at a specific time point of development the ability to invade the neighboring vulval tissue. So far, investigators have found only few components that are necessary for the AC to acquire the invasive ability. The transcription factor fos-1, which is the C. elegans homologue of the Fos proto-oncogene is necessary in the AC for invasive behavior [100]. Further, fos-1 has been shown to positively regulate expression of three matrix related components in the AC (Fig.4). The cadherin homolog cdh-3 is involved in cellular adhesion and thereby affects hypodermal morphogenesis [107]. Zincmetallo protease-1 (zmp-1) is structurally similar to Mmp genes and may therefore act as a matrix degrading enzyme [108]. him-4 is homologous to the poorly characterized vertebrate matrix component hemicentin. In C. elegans, HIM-4 is a component of the ECM that organizes epithelial cell adhesions into fine line-shaped junctions [109].

C. elegans as a model to study cellular invasion has only recently been established [100]. The discovery of fos-1 as a regulator is interesting since the vertebrate homolog Fos is involved in invasive types of cancer. If rat fibroblasts are transformed with v-fos, the viral homolog of the cellular proto-oncogene c-fos, these cells become invasive [110] . Moreover, the

Fig.4: fos-1a regulates AC invasion in C. elegans.

The transcription factor fos-1a Fos is necessary for transcriptional activation of the AC invasion effectors, cdh-3, zmp-1 and him-4 [100]. But it is unknown whether fos-1a Fos directly regulates these target genes. Moreover it is unknown what factors regulate fos-1a Fos itself.

General Introduction

heterodimeric transcription factor activator protein-1 (AP1), which consists of fos and jun, positively regulates MMP-9 expression in cancer cells [105]. Since studies of C. elegans development are able to shed light on conserved signaling pathways (e.g. RTK/RAS/MAPK, Notch signaling), it is promising that by studying AC invasion in C. elegans, new insights can be gained to better understand the invasive behavior of cancer cells in vertebrates.

General Introduction