10.5.1 Protease Activity at the Invasive Front
Proteolysis must function at the tumor cell surface to facilitate invasion and degradation of the basement membrane. Extracellular proteinases, transmembrane proteinases, cell-surface molecules, and intracellular factors all contribute to generating pericellular zones of proteolysis. Mechanisms known to underlie the proteolytic activation at the cell membrane include the activation of plasmin by uPA and its receptor uPAR, and the activation of pro-MMP-2 within a trimolecular complex generated by MT1-MMP, MMP-2, and TIMP-2 (Hernandez-Barrantes et al, 2000; Overall et al, 2000). MT1-MMP activity associated with tumor progression has been observed at the leading edge of migrating cells. For the cell surface activation of pro-MMP-2 in the trimolecular complex, MT1-MMP must itself be activated by a proprotein convertase called furin. Mature furin can cycle between the Golgi and the cell surface, and activate MT1-MMP at both locations. The association of pro-MMP-2 with α2β1 integrin-bound collagen was found to provide a reserve of the enzyme for subsequent activation of the trimolecular complex. By comprehensively comparing multiple MMPs in experimental systems that utilize 3D matrix composites, Weiss and colleagues found that MT1-, MT2-, or MT3-MMPs constitute the minimal requirement for basement membrane transmigration, and that MT1-MMP is the dominant protease mobilized for cancer cell trafficking through 3D interstitial ECM barriers (Rowe and Weiss, 2009). Several secreted MMPs, especially MMP-2 and MMP-9, have also been extensively studied in human cancer and experimental systems and have been shown to facilitate tumor cell invasion and motility, although their primary function in vivo may be to facilitate bulk ECM turnover during tissue remodeling (Kessenbrock et al, 2010).
The integrin αvβ3 may also localize active MMP-2 to the cell surface. Their colocalization was observed on newly developing blood vessels and on the tumor invasive front, and inhibition of their binding reduced tumor growth and angiogenesis (Silletti et al, 2001). Recently, it was shown that MT1-MMP catalyzes shedding of the α3 integrin ectodomain in ovarian carcinoma cells, and this associates with formation of multicellular aggregates, an important step in ovarian cancer metastasis (Moss et al, 2009). Shedding of MMP-9–dependent E-cadherin may also play a role in the dissemination of ovarian cancer cells. CD44 provides a means of anchoring active MMP-9 to the cell surface of invadopodia in breast cancer and melanoma cells, and was found to be critical for MMP-9-mediated cell migration (Dufour et al, 2010). The association of CD44 with hyaluronic acid also has been shown to increase MMP-2 secretion and CD44 has been found to recruit MMP-7 and direct localization of MT1-MMP to the cell membrane. These findings highlight the complex spatial coordination between adhesion molecules and enzymatic activity, which bring about controlled activation of metalloproteinases and, ultimately, the digestion of ECM at the leading edge of invasive tumor cells.
10.5.2 Protease Activity in the Tumor Microenvironment
Beyond ECM degradation, studies using transgenic and knockout mouse models of MMPs and TIMPs have documented that proteolysis affects the early, as well as the late, stages of cancer progression. Protease activity impacts directly on cell growth, cell survival, angiogenesis, and inflammation (Fig. 10–9). Metalloproteinases and TIMPs alter the release of potent growth factors such as VEGF, TGF-β, and IGF-II, which are either sequestered in the ECM or exist in complexes with their binding proteins. Proteolytic cleavage of already synthesized factors results in altered bioavailability of growth signals to cancer cells and impact the process of cell proliferation. Activation of EGFR (see Chap. 8, Sec. 8.2), which is overexpressed in many human cancers, follows ADAM-mediated release of members of the EGF family of growth factors, including amphiregulin, TGF-α, and HB-EGF. Similarly, processing of Notch (see Chap. 8, Sec. 8.2.4), a master regulator of cell differentiation, requires a 3-step proteolytic activation with ADAMs performing the second cleavage of the receptor. MMPs and TIMPs also influence apoptosis signals, such as those feeding into Fas-mediated death receptor signaling (see Chap. 9, Sec. 9.4.4). In addition, proteolytic activity regulates capillary ingrowth, vascular stability and access of tumor cells to vascular and lymphatic networks. Overall, manipulation of the expression of TIMPs reveals that they universally inhibit angiogenesis, invasion, and metastasis, but their effects on cell proliferation and apoptosis are both tissue-specific and context-dependent (Cruz-Munoz and Khokha, 2008).
Metalloproteinases (MMPs) play multiple roles in tumor progression and metastasis. These enzymes are contributed by both stromal cells and tumor cells. Different MMPs are implicated in the various processes. Specific MMPs that promote each of the listed processes during metastatic dissemination are shown as examples. The figure shows vertically an expansion of intravasation and extravasation events in the vasculature of the tumor and metastatic organ, respectively. TAM, Tumor-associated macrophage. CAF, Cancer-associated fibroblast. (Adapted from Nguyen et al., 2009).
Several nonneoplastic host cells, including fibroblasts, endothelial cells, leukocytes, and bone marrow-derived cell populations, are recruited during tumor development, and the composite of these stromal and cancer cells creates a complex microenvironment (Fig. 10–10). Metalloproteinase activity contributes to generating this microenvironment and can further facilitate tumor progression. For example, metalloproteinase activity, as regulated by the TIMP3-ADAM17 interactions, is important for the systemic release of molecules such as cell-surface-bound TNF-α. This pleiotropic cytokine is situated at the apex of cytokine cascades and NF-κB signaling that underlies immune cell crosstalk with cancer cells. Chemokines that influence immune cell motility are similarly processed by proteases. MMP cleavage of members of the monocyte chemoattractant protein (MCP) family of chemokines renders them receptor antagonists with inflammation-dampening effects (McQuibban et al, 2002). MMP-1 and MMP-3 process CCL8/MCP-2, which has antitumor activity in a melanoma model. MMP-8 is mainly produced by neutrophils, and was shown to exert a tumor-suppressive effect in a model of carcinogen-induced skin cancer (Balbin et al, 2003). Other chemokines, including CXCL1/KC (neutrophil-attracting) and CXCL11 (Th1-lymphocyte-attracting), are also substrates of MMPs and thus, proteolysis can affect neutrophil content or T-cell response (see Chap. 21, Sec. 21.4). Metalloproteinases can also cleave RANKL, which can subsequently promote metastasis to bone.
Mutual interactions between tumor cells and the microenvironment promote progression to metastasis. Tumor cells can express some of the factors listed above, such as Snail or Slug, to promote progression. Futhermore, tumor cells can influence their microenvionment and induce the production of tumor-promoting factors from neighboring or incorporated "normal" cells (CAFs and TAMs). Many of the critical factors for tumor survival and motility, such as TNF-α and TGF-β, are provided by the tumor microenvironment. CAF, Cancer-associatrd fibroblast; TAM, tumor-associated macrophage.
10.5.3 Ameboid Movement and Cell Motility
The idea that cell penetration of ECM-imposed structural barriers depends exclusively on proteolysis has been challenged by studies in collagen gels showing that tumor cells can acquire rounded, amoeboid-like shape and perform mechanical displacement of intact ECM fibrils by relying on increased cell deformation and reduced cell-ECM adhesion. This protease-independent amoeboid tumor cell migration is likened to that of leukocytes (Croft and Olson, 2008). The importance of this alternate mechanism of migration in the metastatic process is currently uncertain, partially because of concerns about whether these gels truly mimic the 3D ECM barriers found in vivo (Sabeh et al, 2009).
10.5.4 Cancer-Associated Fibroblasts
Mesenchymal cell types, including cancer-associated fibroblasts (CAFs) and pericytes, coevolve with cancer cells during tumor progression and become an integral part of the paracrine communication (see Fig. 10–10). CAFs were thought initially to arise from local fibroblasts by acquiring a modified "activated" phenotype. However, later studies linked their origin to bone marrow-derived cells or transdifferentiation from epithelial or even endothelial cells. TGF-β is considered a critical activating factor for CAFs, which are typically identified through the expression of a set of markers (ie, α-smooth muscle actin [α-SMA]; fibroblast specific protein-1 [FSP-1]/S100A4; FAP; neuron-glial antigen-2 [NG-2]; platelet-derived growth factor-β receptor [PDGFR-β]), whereas pericytes are more loosely defined but are thought to depend on PDGF and TGF-β signaling. These stromal cell types can contribute to cancer promotion by the delivery of key growth signals (HGF, FGF) and survival signals (insulin-like growth factors [IGFs]) that can counter death signals and activate downstream oncogenic signaling. They inherently provide ECM components for interaction with integrins resulting in the activation of specific signal transduction pathways. CAFs also overexpress metalloproteinases, chemokines (SDF-1 or CXCL12, IL-6, CXCL8) and angiogenic factors (VEGFs, FGFs) that can lead to the generation of a proangiogenic and proinflammatory microenvironment. Consistent with this, when they are coinjected with tumor cells CAFs promote xenograft growth. They have been observed at the sites of metastases, and SDF-1 is known to promote the recruitment of endothelial progenitors, whereas activated pericytes can affect vessel permeability, both of which are critical mechanisms in angiogenesis. The gene expression profiles of CAFs have demonstrated their heterogeneity in individual tumors and resulted in identification of CAF subsets, which may have prognostic value. The importance of understanding these cells, originally considered to be bystanders, is emphasized by the observation that mice deficient in specific CAF markers show decreased metastasis, and that CAFs may alter the drug-sensitivity of cancer cells (Ostman and Augsten, 2009).
10.5.5 Tumor-Associated Macrophages
Tumor-associated macrophages (TAMs) are important players in promoting cancer progression (see Fig. 10–10) and high TAM content in the tumor mass correlates with poor prognosis of patients (Qian and Pollard, 2010). In the local microenvironment, tumor cells can direct the differentiation of TAMs, which exhibit several protumorigenic and prometastatic functions, including induction of inflammation, secretion of growth factors and MMPs, promotion of angiogenesis, and suppression of cytotoxic effects (Sica et al, 2008). The inflammatory state of TAMs is controlled by the transcription factor NF-κB, which, in turn, is activated through toll-like receptors (TLRs; see Chap. 21, Sec. 21.2.2). TAMs exert most of their inflammatory effects through cytokines. Cytokines such as IL-6 cause the endothelial lining of the tumor vessels to become leaky, resulting in the recruitment of more inflammatory cells and providing escape routes into the bloodstream for tumor cells. TNF-α produced by TAMs can activate NF-κB and AP-1 family of transcription factors in the tumor cells, stimulating their cell proliferation and survival. Specific inhibition of NF-κB activity in myeloid cells through ablation of IkB kinase (IKK)-α resulted in a reduction of inflammation and inhibition of tumor progression. In contrast, inactivation of STAT3 (a transcription factor that functions to suppress inflammation—see Chap. 8, Sec. 8.3.1) in myeloid cells is associated with abundant expression of inflammatory cytokines such as TNF-α and IL-6 that have been shown to promote chronic colitis and invasive colorectal cancer in animal models (Grivennikov et al, 2009). Another crucial cytokine produced by TAMs is IL-23. IL-23 acts by enhancing the activity of Th17 cells and inhibiting the activity of T-regulatory cells. The Th17 cell is a T-helper cell subclass with strong inflammatory effects, and they are generally associated with tumor progression.
10.5.6 Epithelial-to-Mesenchymal Transition
Epithelium is a highly polarized structure composed of polygonal-shaped cells with abundant cell–cell tight junctions. It lines the outer surface, as well as the inner organ surface, of the body. Epithelial cells make contact with a basal membrane, and both cell–cell and cell–matrix adhesions are necessary for their survival. Different epithelial cell populations are heterogeneous, depending on the tissue/organ involved. In contrast, mesenchymal cells are spindle-shaped and highly migratory, which is essential for their role in supporting tissue/organ development. EMT (Fig. 10–11) is a process during which an epithelial cell loses its apical–basal polarity and becomes a mesenchymal-like cell with increased migratory ability, resistance to apoptosis, and increased production of ECM components (Kalluri and Neilson, 2003). During this process, epithelial cells often lose the expression of cell-surface and cytoskeletal proteins mediating adhesion, such as E-cadherin, cytokeratin, zona occludens 1 (ZO-1), and laminin. Instead, they gain proteins that are often seen on mesenchymal cell surface such as N-cadherin, vimentin, fibronectin, and α-SMA. The resulting mesenchymal-like cell eventually detaches from the basal membrane and migrates away from the epithelial layer. This process occurs extensively in the embryo at different stages of maturation and development of organs, and in wound healing (Hay, 1995). In tumors it is hypothesized that this embryonic program may be reactivated to drive an initial important step in metastasis, but may then be reversed mesenchymal to epithelial transition (MET) when the cell establishes a new growth (Kalluri and Weinberg, 2009). This latter (MET) step may help to explain why it has been difficult to observe evidence of the EMT process in human tumor specimens (Tarin et al, 2005).
During tumor progression, epithelial tumor cells often undergo EMT to gain anchorage-independent survival and motility. Epithelial cells usually express markers such as E-cadherin, ZO-1, laminin, and desmoplakin. Through the aberrant expression of transcription factors such as Snail and Slug, these epithelial cells lose their epithelial markers and start to express mesenchymal markers, such as N-cadherin, vimentin, β-catenin, and α-SMA. Many of these markers are also functionally associated with the epithelial or mesenchymal characteristics. For example, E-cadherin and N-cadherin activate different intracellular programs that give rise to the epithelial-like or mesenchymal-like properties.
Various studies link loss of E-cadherin to EMT. Treatment with a monoclonal anti–E-cadherin antibody disrupted cell–cell junctions in MDCK cells, activation of a fusion protein that abolishes E-cadherin expression induced EMT in mouse mammary epithelial cells, while expression of E-cadherin induced a MET-like process with reestablishment of cell junctions and decreased proliferation in cells that had already undergone EMT. Cancer cell lines with loss of E-cadherin expression show higher tumorigenicity as xenografts in nude mice, and, in some cancers, E-cadherin levels relate inversely to prognosis. Mutations in the E-cadherin gene causing either loss or truncation of the protein have been identified in human breast and gastric cancers, possibly rendering these tumors more prone to EMT and metastasis. Similarly, epigenetic mechanisms such as transcriptional repression and promoter silencing by hypermethylation also contribute to E-cadherin downregulation in various carcinomas. Transcription factors that play roles in EMT such as TWIST, Snail (Snai1), and Slug (Snai2), also repress E-cadherin expression (Medici et al, 2008).
TGF-β, normally a negative regulator of epithelial cell growth, is another key player in EMT. In various in vitro studies, TGF-β has been shown to induce EMT-like changes in epithelial cell lines. Two pathways downstream of TGF-β are at least partially responsible for its transforming effect, Smad and p38/RhoA (see Chap. 8, Sec. 8.4.4). Studies using in vitro cell lines suggest that activation of p38 is indispensable. Other signals that induce EMT include HGF, which activates the receptor tyrosine kinase c-Met, as well as EGF and PDGF, activating their respective receptors. Multiple recent studies have linked noncoding microRNAs (see Chap. 2, Sec. 2.4.3) as regulators of EMT, both positively and negatively. MicroRNA family members, miR-200 and miR-205, prevent EMT by inhibiting Zeb-1 and Zeb-2, known repressors of E-cadherin expression. In contrast, miR-21 expression is elevated in many carcinomas and supports TGF-β-dependent EMT (Shi et al, 2010).
Hypoxia drives the expression of a large number of metastasis-related genes through the specific HIF1/2 transcription factors and has been found to relate to metastatic disease in many experimental models and in some clinical studies (Finger and Giaccia, 2010; Lunt et al, 2009). As discussed in Chapter 12, Section 12.2, cells in tumors may be exposed to hypoxia as a consequence of diffusion limitations (prolonged or chronic hypoxia) or of perfusion limitations (acute or cyclic hypoxia). Both types of exposure are reported to modify gene expression, and in experimental studies in vitro, both were found to affect metastatic properties of cells. In animal models or patients, it is currently not possible to determine directly whether cells which form metastases derive from areas of chronic or acute hypoxia, but studies that have deliberately induced increased levels of acute (cyclic) hypoxia in tumors in animal models have demonstrated increase development of metastases, suggesting that exposure to acute (cyclic) hypoxia can play an important role in the metastatic process (Lunt et al, 2009). Many specific mechanisms of metastasis are reported to be affected by hypoxia, as discussed below, but the specific genes involved may be different in different cell types.
EMT-promoting genes, such as TWIST and Snai1/2, can be upregulated by hypoxia in multiple cell lines, including ovarian, renal cell, pancreatic, and colon cancer lines, causing loss of E-cadherin. Other important adhesion molecules that can be mediated by hypoxia are the β1 integrins that have been found to correlate with invasive capacity in pancreatic cancer cell lines. Hypoxia also can induce the expression of uPA, uPAR, MMP-2, and MMP-9 in a variety of cell lines, leading to an increase in metastatic invasion both in vitro and in vivo. Blocking uPAR activity with a monoclonal antibody was reported to almost abolish metastatic disease in mice bearing human melanoma xenografts. Similarly, hypoxic exposure of a human breast carcinoma line in vitro resulted in the downregulation of TIMP and concomitant upregulation of MMP-9, causing increased invasive capacity, an effect that could be blocked by using an inhibitor of MMPs. Other studies demonstrated hypoxia-mediated upregulation of MMP-2 activity and a positive correlation with metastatic ability in lung and melanoma tumor models. Hypoxia has been shown to increase transcription of c-MET thereby sensitizing the cells to HGF, significantly increasing the invasive capacity of tumor cells.
The SDF-1/CXCR4 signaling complex plays a key role in tumor cell motility and homing. In vivo studies using a human breast cancer line showed that the formation of both spontaneous and experimental lung metastases could be significantly reduced using a monoclonal antibody against CXCR4, demonstrating its importance in the metastatic process. CXCR4 and SDF1 have been shown to be upregulated by tumor hypoxia, facilitating the development of metastatic disease. The presence of SDF-1 at secondary sites, such as lung, lymph nodes, and bone, concomitant with the expression of its receptor on CTCs may enable cell adhesion and extravasation at the secondary site. The ECM protein, lysyl oxidase (LOX), has also been identified as a hypoxia-regulated gene involved in metastatic disease (Erler et al, 2006). LOX was found to be positively correlated with tumor hypoxia in breast cancer patients, and there was a significant relationship between LOX expression and distant metastases. In vitro studies demonstrated a role for LOX in invasion and migration through regulation of FAK activity, suggesting multiple roles for hypoxia-induced LOX in the formation of metastatic disease. Another important ECM protein that is hypoxia-regulated is the secreted glycophosphoprotein osteopontin (OPN), which is expressed by multiple different cell types (osteoclasts, osteoblasts, epithelial cells, and endothelial cells). OPN has roles in cell adhesion, angiogenesis, prevention of apoptosis, and the anchorage-independent proliferation of tumor cells. Its expression has been found to correlate with increased metastatic potential in breast, prostate, colon, and head and neck cancers, and in soft-tissue sarcoma (Anborgh et al, 2010).
VEGF-A is a potent inducer of tumor angiogenesis (see Chap. 11, Sec. 11.4.1) that is upregulated in response to hypoxia by both HIF-1 dependent and independent mechanisms. Its receptors, VEGFR-1 and -2, are also induced under hypoxic conditions. The role of this protein in metastatic disease has been examined extensively, with some studies suggesting a link between VEGF-A expression and metastatic disease, and others not, consistent with the concept that angiogenesis is a result of multiple factors of varying importance in different tumor types. However, in driving the development of neovasculature, VEGF-A may provide a mechanism of transport for the tumor cells, as well as enhancing the intravasation and extravasation stages of the metastatic process because of increased vascular permeability. VEGF-A also plays a role in macrophage migration, and TAMs are reported to localize in hypoxic regions in tumors.
Using expression microarray, OPN was identified to be the most consistently upregulated gene in relation to tumor progression (Agrawal et al, 2002). High levels of OPN in the serum also have been reported to correlate with increased levels of hypoxia and poorer treatment outcome in head and neck and non–small cell lung tumors (Le et al, 2003; Mack et al, 2008).
10.5.8 Metastatic Niches and Microvesicles
A recent addition to the metastatic process is the concept that a "premetastatic niche" can be created in distant organs to which CTCs can "home" (Carlini et al, 2011). Although tumor cells are the driving force of metastasis, these new findings suggest that the host cells within the tumor microenvironment play a key role in influencing metastatic behavior. Specifically, bone marrow-derived hematopoietic progenitor cells expressing VEGFR-1 have been shown to precede the arrival of even single metastatic cells at distant sites. They are postulated to build a microenvironment suitable for tumor cell growth, although blockade of VEGFR-1 was insufficient to prevent metastasis in the widely used B16 melanoma and Lewis lung tumor metastasis models (Dawson et al, 2009). Molecular processes that have been identified in niche creation include the upregulation of specific integrins and their ECM ligands or increased expression of inflammatory chemoattractants. For example, α4β1 integrin expression on progenitor cells can allow their interaction with fibronectin-expressing metastatic cells. Secreted soluble factors are key players in bone marrow cell mobilization during metastasis, and S100A8 or S100A9 expressed in the niche can attract macrophages. Metalloproteinases are also likely candidates for promoting the formation of a metastatic niche, as this involves altered matrix proteins, VEGF, TGF-β, and TNF-α bioavailability, chemokine activity, and immune cell interaction. In addition hypoxia-induced LOX was recently associated with the development of metastatic niches (Erler et al, 2009). Mechanisms for how such niches are initiated, the extent of the role they play in the overall metastatic process, and whether factors secreted from the primary tumor are required for their initiation are currently unclear and require further investigation.
Membrane vesicles (exosomes) derived from both tumor and host cells have also been recognized to promote tumor growth and metastasis, possible through promotion of metastatic niches (Peinado et al, 2011). Microvesicles are generated by the outward budding and fission of membrane vesicles from the cell surface. They are released from cells upon activation, malignant transformation, stress, or death, and can be found in various biological fluids, including blood and urine. These structures contain "cargo" including proteins (receptors, antigens), lipids, and nucleic acids (DNA, mRNA, microRNA), and can be endocytosed by other cells or interact with their cell surface receptors through fusion. This process occurs frequently in platelets and tumor cells, and such exosomes can provide a means for horizontal transfer of bioactive molecules to stimulate tumor progression, immune response, invasion, angiogenesis, and metastasis. For example, microvesicles from platelets were found to induce angiogenesis and metastasis in lung and breast cancers, and those released from tumor cells contained tetraspanins that could recruit endothelial cells. Molecular information harbored in the circulating microvesicles is being explored for its prognostic and predictive significance (Pap, 2011).
10.5.9 Metastasis-Suppressor Genes
A small number of genes are reported as metastasis-suppressor genes. These genes are defined strictly as those whose products reduce the metastatic behavior of tumor cells without affecting their tumorigenic capacity. The detailed mechanisms by which these genes act are diverse and involve every step in the metastatic cascade. However, only a few genes have been investigated in enough detail to be certain about their mechanism(s) of action or even that they truly conform to the second part of the definition. The nm-23 (NME-1) gene was the first metastasis-suppressor gene isolated and it has several isoforms (Lee et al, 2009). Inverse correlations between expression levels and metastatic potential have been observed in a number of different cancers including breast cancer and melanoma. NME-1 has nucleoside diphosphate kinase (NDPK) activity as well as exonuclease and histidine kinase activity. It also appears to play a role in maintenance of genomic stability. Mutations in both NDPK and exonuclease activity still allow suppression of metastasis and the activities associated with them vary by cell type. It is unclear which of the various activities plays the critical role in suppressing cancer metastasis. The KAI-1/ CD82 gene is a member of the Tetraspanin (TM4SP) family of adhesion molecules that play a role in lymphocyte differentiation and function. It has been reported to have a p53 binding site in its promotor, and the loss of KAI-1 correlated with loss of p53. Loss of expression is implicated in metastases in cancers of the prostate, breast and colon and in melanoma. KiSS-1 appears to be involved in cell signaling, as a posttranslationally-modified version of this protein, called metastatin, has been reported to bind to a G-protein–coupled receptor (Axor12) and preliminary evidence suggests that activation of this receptor can alter signaling through FAK. MKK4 is also a molecule associated with signaling, through the stress-activated protein kinase (SAPK) pathway (see Chap. 8, Sec. 8.2.4), and both these molecules may act to increase the likelihood that a tumor cell will be able to initiate growth at a new (metastatic) site. Breast metastasis suppressor-1 (BrMS-1), which is frequently altered in late-stage breast cancers, is a transcriptional repressor and causes downregulation of phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2). In vivo experiments have demonstrated that BrMS-1 inhibits several steps of metastasis, including the ultimate step, colonization at the secondary site. To date, however, whether gene regulation effects are direct versus indirect has not been clearly demonstrated. Hurst and Welch (2011) have reviewed clinical and experimental information about these and other possible metastasis-suppressor genes.
10.5.10 Genome-Wide Analyses and Metastatic Signatures
Recent advances in sequencing and microarray technologies (see Chap. 2) have enabled genome-wide analyses of primary tumors and researchers are increasingly using -omic approaches (eg, genome, transcriptome, proteome, or methylome) to study metastasis. One of the most common approaches is the gene expression profiling of tumors (transcriptome), and this approach has been applied to examining metastatic propensity of tumors. A seminal paper identified a group of 70 genes expressed in the primary tumors of breast cancer patients, which formed a profile that was capable of predicting survival and the likelihood of developing distant disease (van de Vijver et al, 2002). In a retrospective analysis, this profile was claimed to outperform the best predictions based on histological and clinical criteria, thus providing a capability to select early stage patients who needed adjuvant chemotherapy, and thereby avoiding exposing those who did not need such treatment to the toxicity involved (Knauer et al, 2010). Another study identified an expression profile involving 128 genes that distinguished between primary tumors and metastases of a range of different types of adenocarcinomas (Ramaswamy et al, 2003). This gene set was subsequently refined down to a group of 17 genes that retained a broad diagnostic ability to predict outcome in a range of tumor types (lung, breast and prostate adenocarcinomas and medulloblastoma but not lymphoma). Interestingly, predictive gene signatures from various studies often do not overlap with each other. This has been explained by the heterogeneity of the various patient pools from which the signatures are derived.
These technologies have been extensively applied to breast cancer, allowing specific subgroups with prognostic differences to be identified. Various tests using molecular profiling are currently commercially available to assist treatment decisions for patients with early stage breast cancer (Eroles et al, 2012). Clinical studies have demonstrated benefit in such tests for choice of drug treatment in patients with node-negative breast cancer, who may have undetected distant metastases, but there remain questions about which patient populations are most appropriate for these tests and whether they are applicable to other cancers (Oakman et al, 2010).
In addition to global RNA expression profiles, changes in genome-wide DNA copy number have been examined in cancers. Hu et al (2009) found consistent copy number gains on chromosome 8q22 in breast cancer patients with poor prognosis. Through computational analyses of this region, they were able to identify metadherin (MTDH) as a metastasis-promoter gene. Knocking down MTDH in several cell lines resulted in significant reduction of lung metastases in an experimental model of metastasis in mice. Hu et al (2009) also showed that gain of MTDH was associated with poor prognosis in an independent patient cohort. In another study on colorectal cancer, comparison of DNA copy number profiles from metastasis-free patients and patients harboring liver or peritoneal metastases identified copy number gains on chromosome 20q that preferentially associated with liver metastasis (Bruin et al, 2010).
Whole-genome sequencing is another powerful tool for understanding the metastatic process. In pancreatic cancer, comparison of mutations between primary malignancies and their corresponding metastases led to the identification of single-nucleotide changes that only existed in the metastases (Campbell et al, 2010). Furthermore, relying on extensive analyses of the sequencing data, an evolutionary path of the tumor cells was constructed and sequential mutations acquired by tumor cells that eventually led to metastases were identified (as illustrated by Fig. 10–2). Whole-genome methylation profiling is also being applied to metastasis research. Fang et al (2011) identified a methylation signature associated with low metastatic risk and high survival rate in breast cancer. The methylation status was able to account for many of the transcriptional differences between the poor- and good-prognosis patients, and was independent of other breast cancer markers. Its predictive potential is shared by other malignancies, such as glioma and colon cancer. As these new sequencing technologies become more affordable and more widely available, it is expected that they will lead to deeper insights in metastatic dissemination of each cancer type to specific distant organs.