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.³ The 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 cancers^22-24 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.²⁸ HGF 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 (more...)

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.⁴³ Patients 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 filaments^31,45 (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.⁵¹ reported 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

Epithelial-Mesenchymal Transition

Mechanisms of EMT

There is growing acknowledgment that the detachment and escape of cells from the primary tumor mimics the developmental process known as epithelialmesenchymal transition (EMT) (Fig. 3), a dynamic process permitting polarized epithelial cells to go through multiple biochemical and morphological changes enabling them to assume a mesenchymal phenotype with enhanced migratory and invasive capabilities.^77-80

Figure 3.

Schematic description of EMT/MET showing effectors of these processes; dissociation/ association of cell to cell adhesions together with characteristic markers of either epithelial or mesenchymal cells.

Initiation of the process of EMT entails the loss of cell-cell adhesions; activation of transcription factors; alterations in expression of specific cell-surface proteins; reorganization and expression of cytoskeletal proteins; and production of ECM degrading enzymes. Consequently, the course of EMT involves a shift in the characteristic morphology and gene expression pattern of epithelial cells resulting in the acquisition of a characteristic mesenchymal, migratory phenotype.^81,82

EMT Biomarkers

A number of biomarkers have been found to be useful indicators for EMT (Table 1.).

Table 1.

Biomarkers of EMT .

E-Cadherin

It is essential that weakening of cell-cell adhesion occurs to allow cells to become motile and metastasise and a modification in the adhesive properties of cells is a necessary element of the metastatic process. Cell adhesion molecules (CAMs) regulate cell-cell and cell-matrix adhesion and are implicated in almost all stages of metastasis, therefore alterations in normal levels of CAMs such as E-cadherin will be significant in tumor progression. E-cadherin is a member of a family of Ca²+ dependent CAMs made up of intracellular, extracellular and transmembrane domains. These domains play vital roles in cellular recognition during morphogenesis and development and are responsible for cell-cell adhesion⁸⁷ thus holding a central role in the maintenance of tissue integrity. E-cadherin and its adhesion complex play an essential function in the adhesion of breast cancer cells, being involved in the control of tumor progression and metastasis. Members of the complex, such as β-catenin, act as regulators of cell adhesion, and also of cell signaling and transcription regulation.⁸⁸ Studies exploring the expression of E-cadherin and α-catenin in tumor tissues have shown that loss of both molecules is linked to an increased invasiveness of tumor cells.⁸⁹ Evidence for this comes from in vitro and in vivo studies which demonstrate that E-cadherin expression is inversely correlated with the motile and invasive behavior of tumor cells and also with metastasis in cancer patients.⁹⁰ Further studies have revealed that the relocalization of β-catenin to the nucleus correlates with the acquisition of the mesenchymal phenotype,^91,92 and is associated with the loss of E-cadherin. This reduction of cell surface E-cadherin causes the cells to be receptive to initiation of EMT.⁹³ Numerous reports have indicated that E-cadherin plays a role in meningiomas, tumors of the central nervous system; with upregulation and nuclear localization of β-catenin in 60% of anaplastic memingiomas.⁹⁴

Transcription Factors in EMT

Important transcription factors shown to be significant in EMT, as they affect the regulation of E-cadherin expression, are Slug and Snail (SNAI1),⁹⁵ Zeb-1⁸⁵ and Twist.^96,97 Importantly, Snail has been identified as having a significant role in the differentiation of epithelial cells into mesenchymal cells during embryonic development^98,99 with Slug and Snail effecting the downregulation of E-cadherin expression by binding directly to two proximal E2-boxes of the E-cadherin promoter.^84,100 It has been shown that Snail and E-cadherin expression are inversely correlated in squamous cell carcinoma¹⁰¹ and cancer of the breast.¹⁰² Snail also represses expression of genes encoding tight junction components, such as claudins and occludins.¹⁰³

The basic helix-loop-helix protein Twist is also a key transcription factor in EMT and is known to trigger EMT mechanisms possibly by the regulation of the E-cadherin to N-cadherin switch. It is not known if E-cadherin expression can be repressed directly by Twist however, forced N-cadherin expression exerts a dominant effect over E-cadherin in breast cancer cells.^104,105 Similarly, expression of N-cadherin in normal epithelial cells results in downregulation of E-cadherin expression.¹⁰⁴ Work on glioblastoma (GBM) by Mikheeva et al.¹⁰⁶ has shown that TWIST1 promotes GBM invasion through instigation of mesenchymal molecular and cellular changes. This study showed, however, that this effect was not reliant on a cadherin switch as a reduction in levels of E-cadherin and consequent increase in N-cadherin did not occur with TWIST1 overexpression.

Nevertheless many of the genes regulated by TWIST1 in GBM cell lines mirror those which it regulates in cancer metastasis which suggests some overlap with that of TWIST1-mediated EMT in carcinomas.¹⁰⁶ In work on medulloblastoma, evidence for a significant role for EMT has been seen with intermittent hypoxic conditions in the tumor microenvironment.¹⁰⁷ Hypoxia is recognized as a factor involved in overexpression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) with overexpression promoting uPAR-mediated survival signaling in various cancers.¹⁰⁸ Likewise, hypoxia/overexpression of uPAR in cancer cells promotes EMT and thus invasiveness and metastasis. The study by Gupta also showed that when medulloblastoma cells are exposed to intermittent hypoxia this initiates various molecular and phenotypic changes consistent with EMT, as the cell signaling molecules vimentin, N-cadherin, Snail are overexpressed in these medulloblastoma cells with a reduction in the epithelial markers ZO-1 and E-cadherin.

Angiogenesis and Lymphangiogenesis in Cancer Metastasis

Angiogenic and Lymphangiogenic Cascade

The angiogenic process is made up of a complex multi-step cascade, which is tightly regulated through the balance of a number of pro- and anti- angiogenic factors. Tumor cells frequently tip this balance in favor of blood vessel production through the secretion of pro-angiogenic factors as summarized in Fig. 4. The production of angiogenic factors from a source tissue or tumor bind to and activate endothelial cells of a neighboring blood vessel. Following activation, the endothelial cells begin to produce enzymes that break down the basement membrane of the blood vessel creating tiny pores. Endothelial cells then proliferate and migrate through these pores, toward the angiogenic source, a mechanism that involves a variety of adhesion molecules to aid movement of the new blood vessel toward the source and also the production of various enzymes, such as matrix metalloproteinases, at the sprouting tip, to facilitate this movement through the extrα-cellular matrix. Endothelial cells of the new vessel then undergo a tubule formation phase, where these cells roll to form a tube like structure before establishment of a blood vessel loop between the source and the existing vessel. Finally, structural stabilization of this loop is obtained through recruitment of additional cell types, such as smooth muscle cells, providing support to the vessel and allowing blood flow to the angiogenic source.¹²⁵

Figure 4.

Summary of key steps involved in the angiogenic cascade

While the vasculature system and lymphatic system are structurally different, the process of lymphangiogenesis shares similarities with the angiogenesis process. New lymphatic vessel growth can be stimulated by a variety of factors such as members of the vascular endothelial growth factor (VEGF) family (e.g., VEGF-C and VEGF-D), which induce sprouting of new vessels and proliferation of lymphatic endothelial cells (LEC),^126,127 a process which, similar to angiogenesis, is utilized by metastasising tumor cells. Key angiogenic and lymphangiogenic factors are summaried in Table 2.

Table 2.

Key angiogenic and lymphangiogenic factors .

Organ Specific Metastasis

Cancer metastases are responsible for the majority of cancer-related deaths. From a primary tumor to a distant site and eventually developing a secondary tumor, cancerous cells need to proceed along a series of interrelated and sequential steps, including invasion through extracellular matrix, intravasation, survival in the circulation, extravasation into a distant site, and progressive growth at that site. The metastatic procedure is an inefficient process whereby the vast majority of circulating tumor cells are not able to progressively grow at distant sites. A latent period may exist between infiltration of cancer cells at a distant site and colonization leading progressively to the growth of a secondary tumor. Such a period can be as long as a couple of years seen in some metastases of breast cancer after initial management, and it can also be as short as a few months in lung cancer which may develop a metastasis rapidly within a few months of diagnosis. The cellular origin, intrinsic properties of the tumor, tissue affinities and circulation patterns determine not only the sites of tumor spread, but also the temporal course and severity of metastasis to vital organs. In addition to the above aspects of metastases, certain metastatic cells exhibit tissue tropism, preferring to grow in certain organs (Table 3). In breast cancer, for example, metastasis affects the bone and the lung, and less frequently the liver, brain, and adrenal medulla. Although the genetic and epigenetic basis of these metastatic properties is yet to be fully established, acquisition of the ability to complete each step involved in metastasis is thought to be driven by the accumulation of genetic mutations and epigenetic events that may result in a cells acquisition of metastatic traits during the process of developing a secondary tumor.

Table 3.

Common metastatic sites of certain solid tumors .

The organs mostly assaulted by metastases are lung, liver, brain and bone¹⁴⁶ (Fig. 5). The lungs are the commonest site of metastases for many primary tumors. However, there is a great difference in propensity between the malignancies. It is just as high as 90% in melanomas at autopsy. The lungs serve as first filter for tumor cells spreading through blood circulation in malignancies whose venous drainage flows directly into the lungs. The tumors of testis, melanoma, osteosarcoma, and head and neck tumors have the highest incidence of pulmonary metastases.¹⁴⁶ The liver is one of the most common sites for metastatic disease, accounting for 25% of all metastases to solid organs.¹⁴⁷ In the United States and Europe, secondary liver neoplasms are far more common than primary hepatic neoplasms. In the adult oncology patient, most are metastatic carcinomas, of which adenocarcinomas are the predominant subtype, followed by squamous cell carcinomas and neuroendocrine carcinomas. Other tumor types that metastasize to the liver include melanomas, lymphomas, and rarely sarcomas. The most frequent metastasis to the brain occurs in patients with lung, breast, melanoma, renal, and colorectal tumors.¹⁴⁸ In 2700 cases from the Memorial Sloan-Kettering Cancer Center in New York, the distribution of primary cancers was as follows: 48% lung, 15% breast, 9% melanoma, 1% lymphoma (mainly non-Hodgkin), 3% GI (3% colon and 2% pancreatic), 11% genitourinary (21% kidney, 46% testes, 5% cervix, 5% ovary), 10% osteosarcoma, 5% neuroblastoma, and 6% head and neck tumor . Once metastasis to the brain is diagnosed, the median survival of untreated patients is 12 mo. Bone metastases are most commonly seen in prostate, breast and lung cancer, which are leading malignancies in female and/or male having the highest incidence and mortality rates.^149-151 Bone metastasis usually leads to severe morbidities, which always persist until the death of patients, including bone pain, hypercalcemia, pathological fracture, spinal cord compression and consequent paralysis. In the following part, we generally reviewed the process and molecular mechanisms of organ specific metastases with a focus on bone metastasis.

Figure 5.

Organ specific metastases from primary tumors.

Conclusion

Metastasis, the leading cause of mortality in patients with cancer, is receiving increasing attention in both scientfic and clinical research. Yet the mechanisms remain poorly understood and methods in combatting metastasis remain limited. It is however pleasing to observe some of the major progresses in this vital area of cancer research. With the increasing knowledge in gene expression, cellular behavior, biological events in the spread paths of cancer cells, there are now new prospects of taking some of the observations into the diagnosis, prognosis and treatment in the metastatic disease. For example, new knowledge on barrier function and paracelluar permeability may allow one to devise new direction in controlling the trepassing cancer cells and their entry into the destination tissues and organs. New biomarkers in areas such as epithelial to mesenchymal transtion offer new opportunities in predictive methods of metastatic potential of a primary tumor and new target for therapy. Angiogenesis has already been a fruitful area in new therapies and the organ specific spread of a solid tumor may allow new method of detection and a new way of targeting metastatic tumor cells. Although enormous challenges remain, it is anticipated that these lines of research will steadily find their into clinical practice.

Acknowledgment

The authors wish to thank Cancer Research Wales, the Albert Hung Foundation, the Breast Cancer Hope Foundation, and the Welsh Assembly Government for supporting their work.

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