Cancer Invasion and Metastasis: Molecular and Cellular Perspective

Cancer Invasion and Metastasis: Molecular and Cellular Perspective

Metastasis is the leading reason for the resultant mortality of patients with cancer. The past few decades have witnessed remarkable progress in understanding the molecular and cellular basis of this lethal process in cancer. The current article summarizes some of the key progress in this area and discusses the role of cell junctions, cell adhesions, epithelial-mesenchymal transition, angio and lymphangiogenesis and organ specific metastasis.

Introduction

Of primary importance in the prognosis of cancer patients is the sequence of events leading to the development of tumor cell invasion and metastasis. The course of tumor metastasis entails a series of stages that lead to the formation of secondary tumors in distant organs and is, largely, responsible for the mortality and morbidity of cancer.
Once tumor cells acquire the ability to penetrate the surrounding tissues, the process of invasion is instigated as these motile cells pass through the basement membrane and extracellular matrix, progressing to intravasation as they penetrate the lymphatic or vascular circulation. The metastatic cells then journey through the circulatory system invading the vascular basement membrane and extracellular matrix in the process of extravasation. Ultimately, these cells will attach at a new location and proliferate to produce the secondary tumor. Concentrating research efforts on identifying and understanding the mechanisms concerned in tumor cell invasion may lead to limiting tumor progression and, as a result, to a reduction in mortality for many cancer patients. In the following, we have summarized some of the recent progress in the area of cell adhesion, epithelial to mesenchymal transition, angiogenesis, lymphangiogenesis and organ specific metastasis in cancer.

Cancer Invasion and Metastasis: The Role of Cell Adhesion Molecules

Cancer metastasis is the spread of cancer cells to tissues and organs beyond where the tumor originated and the formation of new tumors (secondary and tertiary foci) is the single event that results in the death of most patients with cancer. At the time of cancer diagnosis, at least half of the patients already present clinically detectable metastatic disease.1 A higher number of patients will also have micrometastases that would be beyond conventional detection techniques. Thus, metastasis is the most life threatening event in patients with cancer. The process is composed of a number of sequential events which must be completed in order for the tumor cell to successfully metastasize, the so called metastatic cascade. This process contributes to the complexity of cancer as a multiplex disease. During the metastatic cascade, changes in cell-cell and cell-matrix adhesion are of paramount importance.2
The metastatic cascade can be broadly separated into three main processes: invasion, intravasation and extravasation. The loss of cell-cell adhesion capacity allows malignant tumor cells to dissociate from the primary tumor mass and changes in cell-matrix interaction enable the cells to invade the surrounding stroma; the process of invasion. This involves the secretion of substances to degrade the basement membrane and extracellular matrix and also the expression/ suppression of proteins involved in the control of motility and migration. The tumor must also initialize angiogenesis, without which the tumor would fail to develop, as local diffusion for transport of nutrients to and removal of waste products from the tumor site would suffice for tumors up to 2 mm in diameter.3The blood vessel within the tumor's vicinity can then provide a route for the detached cells to enter the circulatory system and metastasize to distant sites; the process of intravasation.4,5 Interaction between the tumor cell and the surrounding stroma is extremely important in the development of tumor angiogenesis.6 Once the tumor cell has arrived at a likely point of intravasation, it interacts with the endothelial cells by undergoing biochemical interactions (mediated by carbohydratecarbohydrate locking reactions, which occur weakly but quickly) develops adhesion to the endothelial cells to form stronger bonds, and thus penetrates the endothelium and the basement membrane; the process of extravasation. The new tumor can then proliferate at this secondary focus.
The metastatic cascade is therefore dependent on the loss of adhesion between cells, which results in the dissociation of the cell from the primary tumor, and subsequently the ability of the cell to attain a motile phenotype via changes in cell to matrix interaction.

Cellular Junctions

Epithelial cells are characterized by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. The cell to cell adhesion complex runs from the apical to the basal membranes and is composed of Tight Junctions (TJ), Adherens Junctions (AJ), Gap Junctions (GJ), Desmosomes and integrins (Fig. 1).

Figure 1.

Schematics showing the arrangement of cell-cell junctions and cell-matrix interactions.

Tight Junctions (TJ)

The permeability of epithelial and endothelial cells is governed by the TJ and they are located at the apical membrane of the cell,7-9 (Fig. 1). The TJ is a region where the plasma membrane of adjacent cells forms a series of contacts that appear to completely occlude the extracellular space thus creating an intercellular barrier and intramembrane diffusion fence.10 In epithelial cells the TJ functions in an adhesive manner and can prevent cell dissociation.11 TJ in endothelial cells function as a barrier through which molecules and inflammatory cells can pass. Interaction with and penetration of the vascular endothelium by dissociated cancer cells is an important step in the formation of cancer metastases. TJ are the first barrier that cancer cells must overcome in order to metastasize. We have previously demonstrated that TJ of vascular endothelium in vivo function as a barrier between blood and tissues against metastatic cancer cells.12 Early studies demonstrated a correlation between the reduction of TJ and tumor differentiation and experimental evidence has emerged to place TJ in the frontline as the structure that cancer cells must overcome in order to metastasize.12-15 Although a considerable body of work exists on TJ and their role in a number of diseases, following the early work of Martinez-Paloma16 and others,17,18 it is only in recent years that there has been an upsurge in studies investigating their possible role in tumorigenesis and metastasis.

There have now been numerous studies on colorectal cancer,19-21 pancreatic cancers22-24 and an increasing number of studies performed on breast cancer.25-27 Changes in both tumor and endothelial cells are necessary for successful growth and spread of cancer cells and these changes are somewhat similar. A change in cancer cells by upregulation or downregulation of relevant TJ proteins results in loss of cellcell association, cell contact inhibition, leading to uncontrolled growth, loss of adhesion to and degradation of the basement. These must be a concurrent loss of cellcell association in the endothelium and modulation of TJ proteins involved in facilitating the passage of the cancer cells through this barrier.

HGF/SF (hepatocyte growth factor), a cytokine secreted by stromal cells and key to the development and progression of cancer, particularly during metastasis has been shown to be capable of modulating expression and function of TJ molecules in human breast cancer cell lines.28HGF decreased trans-epithelial resistance and increased paracellular permeability of human breast cancer cell lines, MDA-MB-231 and MCF-7. Q-PCR showed that HGF modulated the levels of several TJ molecule (occludin, claudin-1 and -5, JAM-1 and -2) mRNA transcripts in MDA-MB-231 and MCF-7 cells. Such data shows that HGF disrupts TJ function in human breast cancer cells by effecting changes in the expression of TJ molecules at both the mRNA and protein levels and that regulation of TJ could be of fundamental importance in the prevention of metastasis of breast cancer cells. Regulation of vascular permeability is one of the most important functions of endothelial cells, and endothelial cells from different organ sites show different degrees of permeability.29 Tumor blood vessels are more permeable on macro-molecular diffusion than normal tissue vessels. However, the cause and mechanism of hyperpermeability of human vessels had not been clear. Tumor cells release a number of factors that can assist their transmigration through the endothelium after treating endothelial cells with conditioned media from a highly invasive and metastatic melanoma cell line,29 with TJ being irreversibly damaged (as assessed using TER-trans-epithelial resistance). In fact, HGF has been shown to decrease TER and increase PCP (paracellular permeability) in human endothelial cells.8

An increasing number of studies have shown that numerous TJ components are directly or indirectly involved in cancer progression including ZO-1, ZO-2, claudin-7, claudin-1 and occludin.25 When human tissues and breast cancer cell lines were amplified for functional regions of occludin, tumor tissues showed truncated and/or variant signals. There was also considerable variation in the expression of occludin in the 10 human breast cancer cell lines investigated. Western blotting demonstrated that variants in the MDA-MB-231 and MCF-7 human breast cancer cell lines did not fit the expected occludin signals for changes in phosphorylation status. Immunostaining showed similarly disparate levels of expression. Ribozyme knockdown resulted in increased invasion, reduced adhesion and significantly reduced TJ functions. Q-RT-PCR analysis of 124 tumor and 33 background human breast tissues showed occludin to be significantly decreased in patients with metastatic disease. Immunohistochemical staining showed a decreased expression of occludin in the tumor sections. This study demonstrated for the first time that occludin is differentially expressed in human breast tumor tissues and cell lines. This loss of or aberrant expression has clear repercussions as to the importance of occludin in maintaining TJ integrity in breast tissues,25 (Fig. 2). Highly differentiated adenocarcinomas with well developed TJ provide an important insight into the usefulness of TJ molecules and are possible prognostic indicators and future targets for therapy. In breast cancer, ZO-1 has been demonstrated to be decreased in poorly differentiated tumors and correlated with increasing Grade and TNM (tumor-nodal) status.30 There are a respectable number of reports describing the dysregulation of transmembrane proteins in human cancers and in cell lines. This dysregulation can be the result of both upregulation and downregulation of expression, epigenetic changes and changes in activation and location of the proteins

Figure 2.

The aberrant expression of Occludin in human breast cancer. A shows the missing full length transcript of occludin in human breast cancer cell lines. B shows the erroneous expression of Occludin regions in MDA-MB-231 and MCF-7 cells. Successful knockdown of occludin by RT-PCR and western blotting (C top and bottom respectively) and loss of occludin from the cell membrane (D). Occludin knockdown reduced the transepithelial resistance and increased the paracellular permeability of human breast cancer cells (E and F). Loss of occludin observed in patient breast cancer tissues (G). In tissue samples, occludin was significantly reduced in patients with metastatic breast cancer (H).

Adherens Junctions (AJ)

AJ are cellcell microdomains that provide adherent strength and localize to the basal side of the TJ31 (Fig. 1). The integral membrane proteins of the AJ are of the cadherin family, with E-cadherin being most abundant in epithelia and VE-cadherin in endothelia (Fig. 1). Nectins are also found in AJ of epithelia. In polarized epithelia of vertebrates, the AJ is part of the tripartite junctional complex localized at the juxtaluminal region, which comprises the TJ, AJ, and desmosome aligned in this order from the apical end of the junction.32 In this type of epithelia, the AJ is specifically termed the zonula adherens or adhesion belt, as it completely encloses the cells along with the F-actin lining, called the circumferential actin belt.33 The AJs in other cell types assume different morphologies with the AJ in fibroblastic cells being spotty and discontinuous34 while those in neurons are organized into tiny puncta as a constituent of the synaptic junctions.35 A major function of AJs is to maintain the physical association between cells, as disruption of them causes loosening of cellcell contacts, leading to disorganization of tissue architecture.33

Classical or type I cadherins mediate adhesion at the adherens, cellcell or cellmatrix adhesive junctions that are linked to microfilaments. Type I classical cadherins are composed of five tandem extracellular cadherin domains (EC1-EC5), a single segment transmembrane domain and a distinct, highly conserved cytoplasmic tail that specifically binds catenins.36 In addition to cadherin homophilic binding, it has been reported that cadherin is also capable of heterophilic interactions with numerous extracellular and intracellular proteins. The key to their adhesive activity is the interaction between the catenin-binding sequence and submembrane plaque proteins β-catenin or plakoglobin (γ-catenin), which form the link to the actin cytoskeleton. α-catenin binds to a short region close to the N terminus of β-catenin forming a stable bond between the complex and the actin cytoskeleton.36 In addition to α-, β-, and γ-catenin, a fourth catenin-like protein capable of binding cadherin, p120ctn, has emerged as a key regulator of cadherin function.37 p120ctn was originally identified as a substrate for receptor tyrosine kinases and like the other catenin molecules, binds directly to the cytoplasmic domain of cadherin.37

Nectins are transmembrane proteins that are found in both TJ and AJ. In AJ, during the process of early cellcell contacts, nectins first accumulate at the contacts, and then cadherins follow them, suggesting that the former may guide the latter in their junctional localization. Nectin interaction serves for recruiting cadherins to heterotypic cellcell borders, which are otherwise distributed throughout cellcell borders.33 Thus, nectins recruit cadherins to the synaptic contacts formed between two distinct domains of hippocampal neurons, i.e., axons and dendrites, which express nectin-1 and nectin-3, respectively.38 Thus, nectins show important cooperation with classic cadherins in generating heterotypic cellcell contacts.33

Evidence has long accumulated to point toward a pivitol role for E-cadherin and the catenin complex in the control of cancer cell dissociation and spread. Tumor invasion and metastasis, both hallmarks of tumor malignancy, frequently coincide with the loss of E-cadherin-mediated cell-cell adhesion. Expression of E-cadherin, the most abundant adhesion molecule in adherens junctions of epithelia, is downregulated in most, if not all, epithelial cancers.39 Several studies have shown that reconstitution of a functional E-cadherin adhesion complex suppresses the invasive phenotype of many different tumor cell types.40-42 In the context of cancer, E-cadherin has been categorized as a tumor suppressor, given its essential role in the formation of proper intercellular junctions, and its downregulation in the process of epithelial-mesenchymal transition (EMT) in epithelial tumor progression.

Recent studies in triple-negative breast cancer (TNBC), which is characterized by negativity for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2), have shown there is a high risk breast cancer that lacks specific targets for treatment selection. Chemotherapy is, therefore, the primary systemic modality used in the treatment of this disease, but reliable parameters to predict the chemosensitivity of TNBC have not been clinically available.43Patients with E-cadherin-negative and Ki67-positive expression showed significantly worse overall survival time than those with either E-cadherin-positive or Ki67-negative expression. Multivariate analysis showed that the combination of E-cadherin-negative and Ki67-positive expression was strongly predictive of poor overall survival in TNBC patients receiving adjuvant chemotherapy. The authors demonstrated that adjuvant therapy is beneficial for Stage II TNBC patients and that the combination of E-cadherin and Ki67 status might be a useful prognostic marker indicating the need for adjuvant chemotherapy in Stage II TNBC patients.43

E-cadherin inactivation with loss of cell adhesion is the hallmark of lesions of the lobular phenotype and E-cadherin is typically absent, as seen by immunohistochemistry in both lobular carcinoma in situ and invasive lobular lesions, suggesting it occurs early in the neoplastic process. In invasive lobular lesions, the cadherin-catenin complex was examined; complete complex dissociation was defined as negative membranous E-cadherin, α- and β-catenin expression.44 E-cadherin was found to be absent in all lesions and positive in all normal tissues. Membranous a and β-catenin expressions decreased with the transition from lobular lesions to invasive lesions, while TWIST expression increased. Gene expression paralleled IHC-staining patterns with a stepwise downregulation of E-cadherin, α and β-catenins from normal to lobular to invasive lesions, and increasing expression of TWIST from normal to lobular to invasive lesions. The decreasing membranous catenin expression in tandem with increasing levels of TWIST across the spectrum of lobular lesions suggests that cadherin-catenin complex dissociation is a progressive process in human breast cancer.44

Desmosomes

In cell-cell junctions, desmosomes form adherent points in the form of a continuum of cells within tissues by linkage of their integral membrane proteins (desmocollin and desmoglein) via desmoplakins (plakophilin and plakoglobin) to intermediate filaments31,45 (Fig. 1). Desmosomes are crucial for tissue integrity by their very strong adherence that resists calcium-depletion in developed tissue, but can be regulated by protein kinase C when dynamic remodelling of cellcell adhesion is required.45 Desmosomes not only provide mechanical stability but also facilitate cellcell communication through signal transmission.46 The desmosome is divided into three parallel identifiable zones, arranged symmetrically on the cytoplasmic faces of the plasma membranes of bordering cells and separated by the extracellular domain, which in mature desmosomes is bisected by a dense midline. Each desmosomal plaque consists of a thick outer dense plaque and a translucent inner dense plaque. The five major desmosomal components are the desmosomal cadherins, represented by desmogleins (14) and desmocollins (13), the armadillo family members, plakoglobin and the plakophilins (13), and the plakin linker protein desmoplakin, which anchors the intermediate keratin filaments.46

Recent studies using mouse genetic approaches have uncovered a role for desmosomes in tumor suppression, demonstrating that desmosome downregulation occurs before that of adherens junctions to drive tumor development and early invasion, suggesting a two-step model of adhesion dysfunction in cancer progression.47 Studies have shown that an increased expression of desmosome proteins, such as Desmoglein 2 and 3 and PKP3, can be observed in certain cancers of the skin, head and neck, prostate and lung compared with normal tissue, and that this overexpression is associated with enhanced tumor progression.46,48-50

Reduced expression of Desmocollin 2 has been reported in colorectal carcinomas, suggesting that it may play a role in the development and/or progression of colorectal cancer. Kolegraff et al.51reported that the loss of Desmocollin-2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/β-catenin signaling. Inhibition of Akt prevented the increase in β-catenin-dependent transcription and proliferation following Desmocollin-2 knockdown and attenuated the in vivo growth of Desmocollin-2 -deficient cells. This provides evidence that loss of Desmocollin-2 contributes to the growth of colorectal cancer cells and highlights a novel mechanism by which the desmosomal cadherins regulate β-catenin signaling.51

Oral squamous cell carcinomas and pre-malignant dysplasia can be suβ-classified according to their in vitro replicative lifespan, where the immortal dysplasia and carcinoma subsets have p16(ink4a) and p53 dysfunction, telomerase deregulation and genetic instability and the mortal subset do not. It has been demonstrated that desmosomal proteins exhibit a distinct expression pattern in oral mucosa when compared with epidermis in vivo. Microarray data from a large panel of lines shows that the transcript levels of Desmoglein 2 and Desmocollin2/3 are reduced in immortal dysplasia and carcinoma cells.52 Interestingly, Desmoglein 2 was upregulated. Reduction of Desmoglein 3 and upregulation of Desmoglein 2 were found in two independent microarray data sets. Significantly, we demonstrated that reduction of Desmoglein 3 and upregulation of Desmoglein 2 was reversible in vitro by using RNAi-mediated knockdown of Desmoglein 2 in carcinoma cells. The remaining desmosomal proteins were largely disrupted or internalized and associated with retraction of keratin intermediate filaments in oral squamous cell carcinomas lines. These findings suggest dysfunction and loss of desmosomal components are common events in the immortal class of oral squamous cell carcinomas and that these events may precede overt malignancy.52

There are numerous links between the desmosome and the adherens junction. A decrease in the levels of the desmosomal plaque protein, plakophilin3, leads to a decrease in desmosome size and cell-cell adhesion. Gosavi et al.53 investigated whether plakophilin3 is required for desmosome formation. Plakophilin3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared with vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that plakophilin3, plakoglobin and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either plakoglobin or E-cadherin led to a decrease in the levels of plakophilin3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that plakoglobin and E-cadherin recruit plakophilin 3 to the cell border to initiate desmosome formation.53

Gap Junctions (GJ)

GJ are unique cell-to-cell channels that allow diffusion of small metabolites, second messengers, ions and other molecules between neighboring cells31 (Fig. 1). GJ communication is essential for electrical transduction, signaling and nutrition. The channels can be open or closed, a highly dynamic process regulated at multiple levels, with the integral membrane proteins forming these channels in vertebrates being the connexins of which over 20 family members have now been identified in humans; connexin43 the most abundantly expressed connexin.31 ZO-1 acts as a scaffold in GJ and recruits signaling proteins. Connexins are also known to interact with Occludin and also form complexes with CAR and β-catenin.54

For decades, cancer was associated with GJ defects. However, more recently it appeared that connexins can be re-expressed and participate in cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer are known to be connexin deficient, Lamiche et al.55 investigated whether their bone-targeted metastatic behavior could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates in the differentiation of osteoblastic cells. It appeared that Cx43 behaved differently in those cell lines and induced different phenotypes. In LNCaP, connexin43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those connexin43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of connexin43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of connexin43 may govern the ability of prostate cancer cells to metastasize in bones.55

In colorectal tumors, loss of connexin43 expression is correlated with significantly shorter relapse-free and overall survival. Connexin43 was further found to negatively regulate growth of colon cancer cells, in part by enhancing apoptosis and was found to colocalize with β-catenin and reduce Wnt signaling.56 This study represents the first evidence that Cx43 acts as a colorectal cancer tumor suppressor and that loss of Cx43 expression during colorectal cancer development is associated with reduced patient survival. Connexin43 was downregulated or aberrantly localized in colon cancer cell lines and colorectal carcinomas, which is associated with loss of gap junction intercellular communication. Such data indicate that Cx43 is a colorectal cancer tumor suppressor protein that predicts clinical outcome.56

Integrins and Selectins

There is accumulating evidence for the role of integrins and selectins in cancer progression of various cancer types, including colon and lung carcinomas and melanomas.57 While selectin-mediated tumor cells arrest and adhesion contribute to metastasis, integrin-mediated interaction from both tumor cells and the surrounding environment further contribute to cancer progression.

Integrins

Integrins are large and complex transmembrane glycoproteins that consist of two distinct chains, α and β-subunits, which form a non-covalent heterodimer and combine to form 24 unique canonical α/β receptors.57 Integrins mediate cell adhesion and directly bind components of the extracellular matrix, such as fibronectin, vitronectin, laminin, or collagen and provide anchorage for cell motility and invasion. Integrins mediate bidirectional signaling where intracellular signals induce alterations in the conformation.57 Integrins participate in multiple cellular processes, including cell adhesion, migration, proliferation, survival, and the activation of growth factor receptors. As many human tumors originate from epithelial cells, integrins expressed on epithelial cells are generally also present in tumor cells and therefore, integrins have become linked with patient survival and metastatic status. Recent studies have shown that expression of αv integrins is elevated in the prostate cancer stem/progenitor cell subpopulation compared with more differentiated, committed precursors. Van den Hoogen et al.58 examined the functional role of αv integrin receptor expression in the acquisition of a metastatic stem/ progenitor phenotype in human prostate cancer. Stable knockdown of αv integrin expression in PC-3M-Pro4 prostate cancer cells coincided with a significant decrease of prostate cancer stem/ progenitor cell characteristics (α2 integrin, CD44, and ALDH(hi)) and decreased expression of invasion-associated genes Snail, Snail2, and Twist. Consistent with these observations, αv-knockdown strongly inhibited the clonogenic and migratory potentials of human prostate cancer cells in vitro and significantly decreased tumorigenicity and metastatic ability in preclinical models of orthotopic growth and bone metastasis. This indicates that integrin αv expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human prostate cancer cells with metastasis-initiating capacity.58,59

Lu et al.59 investigated the expression of osteopontin and integrin αv (ITGAV, main receptor of the osteopontin) in laryngeal and hypopharyngeal squamous cell carcinoma and any correlation of the expression quantity with tumor biological behavior. The expression quantity of osteopontin and integrin αv in primary and metastatic carcinomas is significantly higher than in normal tissues. The expression of osteopontin and integrin αv in the well-differentiated group was significantly lower than in moderately and poorly differentiated groups; the expression quantity of osteopontin and integrin αv in groups with lymph node metastasis was significantly higher than in groups without lymph node metastasis. The authors conclude that the expression of osteopontin and integrin αv significantly influenced the differentiation and metastasis of the laryngeal and hypopharyngeal squamous cell carcinoma. Overexpression of both proteins may have contributed to invasion and metastasis of the laryngeal and hypopharyngeal squamous cell carcinoma, and therefore, they both may have value as a target for chemotherapy in laryngeal and hypopharyngeal squamous cell carcinoma treatment.59

Selectins

The selectins: E-selectin, P-selectin, and L-selectin are adhesion molecules that are crucial for binding of circulating leukocytes to vascular endothelium during the inflammatory response to injury or infection. Accumulated evidence indicates that selectins regulate adhesion of circulating cancer cells to the walls of blood vessels.60 Selectin ligands are transmembrane glycoproteins expressed on leukocytes and cancer cells that promote bond formations with selectins to mediate inflammatory processes and selectins and their ligands also participate in signal transduction to regulate diverse cellular functions.60

Haematogenous metastasis of small cell lung cancer is still a poorly understood process and represents the life threatening event in this malignancy.61 In particular, the rate-limiting step within the metastatic cascade is not yet clearly defined although, many findings indicate that extravasation of circulating tumor cells is crucially important as most tumor cells within the circulation undergo apoptosis. If extravasation of small cell lung cancer tumor cells mimics leukocyte-endothelial interactions, small cell lung cancer cells should adhere to E- and P-selectins expressed on the luminal surface of activated endothelium. The adhesion to E- and P-selectin under physiological shear stress with regard to adhesive events, rolling behavior and rolling velocity was determined in the human small cell lung cancer cell lines SW2, H69, H82, OH1 and OH3. OH1 SCLC cells adhered best to recombinant human (rh) E-selectin FC-chimeras and human lung endothelial cells (HPMEC), H82 small cell lung cancer cells adhered best to activated human umbilical vein endothelial cells (HUVEC) under physiological shear stress. As OH1 cells had also produced by far the highest number of spontaneous lung metastases when xenografted into pfp/rag2 mice in previous experiments the findings implicate that adhesion of small cell lung cancer cells to E-selectin is of paramount importance in small cell lung cancer metastasis formation.61

Cell-Matrix Interactions

Controlled interaction between the cells and the extracellular matrix is essential for many processes, including normal development, migration and proliferation.31 Interaction between the cell and the matrix can occur through a number of routes; cell adhesion molecules (CAM) including integrins, selectins, cadherins, the Ig superfamily, CD44 and focal adhesions.

Integrins

Integrin-mediated adhesions to the extracellular matrix are among the first adhesion junctions where bidirectional signaling occurs.31 At the extracellular side integrins bind directly to the extracellular matrix which includes collagen, fibronectin and laminins etc. Cytoplasmic partners include talins, paxillin, focal adhesion kinase and linkage to α-actinin and actin-stress fibers. These focal adhesion complexes control a variety of signaling pathways regulated by the interplay with the extracellular partners. Substantial cross-talk between the diverse cellcell and cellextracellular matrix junctions has been found, and the architecture of the epithelial monolayer is highly regulated by their concerted actions.31

Cell Adhesion Molecules (CAM)

Cell adhesion molecules (CAM) facilitate cellular processes such as cell proliferation, migration, and differentiation and are essential during development and for maintaining the integrity of tissue architecture in adults.62 CAMs include cadherins, integrins, selectins, and the immunoglobulin superfamily (IgSF). In normal tissue, CAM expression is tightly regulated. However, aberrant expression of CAMs disrupts normal cell-cell and cell-matrix interactions and can facilitate tumor formation and metastasis. A number of IgSF members have been identified as biomarkers for cancer progression and have also been associated with metastatic progression in a range of huma tumors.62

CD44

CD44 is a multifunctional cell surface adhesion molecule that is involved in cell-cell and cell-matrix interaction and has been implicated in tumor cell invasion and metastasis. In humans, the CD44 family is encoded by a single gene located on chromosome 11p13 and comprises at least 20 exons. Exons 15, 1618 and 20, are spliced together to form a CD44 transcript that has become known as the standard isoform (CD44s). At least ten exons can be alternatively spliced and inserted into the standard isoform at an insertion site between exons 5 and 16 to give rise to variant isoforms of CD44. Thus, exons 615 are variant exons and are typically identified as v1v10.63 CD44 is the principal ligand for hyaluronic acid (HA), a major component of the extracellular matrix. However CD44 can also bind to other ECM components including collagen, fibronectin, laminin and non-ECM component such as osteopontin and serglycin. CD44 is expressed on a variety of cells and tissues including T- lymphocytes, B-cells, monocytes, granulocytes, erythrocytes, many epithelial cell types; Keratinocytes, chondrocytes, mesothelial and some endothelial cells. It is also expressed in many cancer cell types and their metastases in particular; high molecular weight forms of CD44 show restricted expression in tumors and may correlate with tumor development and metastasis and have potential diagnostic and prognostic value in some cancers. Additionally, it has been shown in experimental models that CD44 can inhibit tumor growth and metastatic spread. Further investigation is still needed but CD44 may yet prove to be a potential target for cancer therapy.63

The importance of non-coding RNA transcripts in regulating microRNA (miRNA) functions, especially the 3' untranslated region (UTR), has been revealed in recent years. Genes encoding the extracellular matrix normally produce large mRNA transcripts including the 3UTR. How these large transcripts affect miRNA functions and how miRNAs modulate the extracellular matrix protein expression are largely unknown. Jeyapalan and Yang64 demonstrated that the overexpression of the CD44 3UTR results in enhanced cell motility, invasion and cell adhesion in human breast carcinoma cell line MDA-MB-231. They also found that expression of the CD44 3UTR enhances metastasis in vivo. Computational analysis indicated that miRNAs that interact with the CD44 3UTR also have binding sites in other matrix encoding mRNA 3UTRs, including collagen type 1α1 (Col1α1) repressed by miR-328 and fibronectin type 1 (FN1) repressed by miR-5123p, miR-491 and miR-671. Protein analysis demonstrated that expression of CD44, Col1a1, and FN1 were synergistically upregulated in vitro and in vivo upon transfection of the CD44 3UTR. The non-coding 3UTR of CD44 interacts with multiple miRNAs that target extracellular matrix properties and thus can be used to antagonize miRNA activities.64

CD44 is also a causal factor for tumor invasion, metastasis and acquisition of resistance to apoptosis. CD44 knockdown using inducible short hairpin RNA (shRNA) significantly reduces cell growth and invasion. Short hairpin RNA against CD44 and pGFP-V-RS-vector was used for knockdown of CD44 expression in SW620 colon cancer cells. Short hairpin RNA against CD44 reduced the expression of CD44. Cell proliferation, migration and invasion were markedly inhibited and apoptosis was increased in shRNA CD44-transfected cells. Knockdown of CD44 decreased the phosphorylation of PDK1, Akt and GSK3β, and β-catenin levels. Decreased phosphorylated Akt led to an increase in phosphorylated FoxO1 and induced cell cycle arrest in the G0-G1 phase and a decrease in the S phase. The levels of Bcl-2 and Bcl-xL expression were downregulated, while the levels of BAX expression and cleaved caspase-3, -8 and -9 were increased. CD44 knockdown by way of shRNA inhibited cell proliferation and induced cell apoptosis which suggests that it could be used as a therapeutic intervention with the anti-survival/pro-apoptotic machinery in human colon cancer.65

Focal Adhesions

Focal adhesion kinase (FAK), a crucial mediator of integrin and growth factor signaling, is a novel and promising target in cancer therapy. FAK resides within focal adhesions which are contact points between extracellular matrix (ECM) and cytoskeleton, and increased expression of the kinase has been linked with cancer cell migration, proliferation and survival.66 Migration is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interplay with focal adhesions. At the leading edge of a migrating cell, it is the re-arrangement of actin and its attachment to focal adhesions that generates the driving force necessary for movement.67 Signaling by the FAK-Src complex plays a crucial role in regulating the formation of protein complexes at focal adhesions to which the actin filaments are attached. Cortactin, an F-actin associated protein and a substrate of Src kinase interacts with FAK through its SH3 domain and the C-terminal proline-rich regions of FAK. Wang et al.67 showed that the autophosphorylation of Tyr(397) in FAK, which is necessary for FAK activation, was not required for the interaction with cortactin, but was essential for the tyrosine phosphorylation of the associated cortactin. At focal adhesions, cortactin was phosphorylated at tyrosine residues known to be phosphorylated by Src. The tyrosine phosphorylation of cortactin and its ability to associate with the actin cytoskeleton were required in tandem for the regulation of cell motility. Cell motility could be inhibited by truncating the N-terminal F-actin binding domains of cortactin or by blocking tyrosine phosphorylation (Y421/466/475/482F mutation). In addition, the mutant cortactin phosphorylation mimic (Y421/466/475/482E) had a reduced ability to interact with FAK and promoted cell motility. The promotion of cell motility by the cortactin phosphorylation mimic could also be inhibited by truncating its N-terminal F-actin binding domains. This suggests that cortactin acts as a bridging molecule between actin filaments and focal adhesions. The cortactin N-terminus associates with F-actin, while its C-terminus interacts with focal adhesions. The tyrosine phosphorylation of cortactin by the FAK-Src complex modulates its interaction with FAK and increases its turnover at focal adhesions to promote cell motility.67

Clinical Considerations

A number of cell adhesion molecules have now become classed as clinical indicators and there is a clear trend toward using them for prognosis or diagnosis. The number of studies identifying these molecules as biomarkers are legion and cannot be thoroughly reviewed here. Some timely examples are as follows: The TJ transmembrane protein claudin-7 has achieved status as a prognostic indicator in invasive ductal carcinoma of the breast68 and is a candidate expression marker for distinguishing chromophobe renal cell carcinoma from other renal tumor subtypes, including the morphologically similar oncocytoma.69 Moreover, decreased claudin-7 correlated with high tumor grade in prostate cancer70 and is able to regulate the expression of prostate specific antigen.71 When considering potential targets for therapy, claudin-1 has been found to act as a cancer invasion/metastasis suppressor in addition to its use as a prognostic predictor and potential drug treatment target for patients with lung adenocarcinoma.72 E-Cadherin and vimentin have now been described predictive markers of outcome among patients with non-small cell lung cancer treated with erlotinib.