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10.1245/ASO.2003.02.021
Annals of Surgical Oncology 10:839-851 (2003)
© 2003 Society of Surgical Oncology
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EDUCATIONAL REVIEW

Recent Insights Into Angiogenesis, Apoptosis, Invasion, and Metastasis in Colorectal Carcinoma

William M. Boedefeld, II, MD, Kirby I. Bland, MD and Martin J. Heslin, MD

From the Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama.

Correspondence: Address correspondence and reprint requests to: Martin J. Heslin, MD, Department of Surgery, University of Alabama at Birmingham, 1922 Seventh Ave. South, KB 321, Birmingham, AL 35243; Fax: 205-975-5971; E-mail: marty.heslin{at}ccc.uab.edu

ABSTRACT

The numerous studies profiling mechanisms in colorectal carcinoma have implicated multiple pathways in the malignant progression of a colorectal epithelial cell. Such pathways as aberrations in the cell cycle, deviation from apoptosis, neovascularization of tumors, and invasion and metastasis of malignant epithelial cells have been shown to occur in the progression of a normal epithelial cell to an adenoma and carcinoma. Today, we continue to search for communications or connections between these pathways as we try to get a more global picture of the events responsible for the adenoma-carcinoma sequence. This review focuses on the latest developments of three well-characterized pathways implicated in colorectal carcinoma: angiogenesis, apoptosis, and invasion and metastasis. We will attempt to highlight clinical correlates, when available, with some of the more interesting molecules.

Key Words: Angiogenesis • Apoptosis • Invasion • Colorectal carcinoma

The purpose of this review is to summarize specific areas of molecular alterations in colorectal cancer. Because it is not possible to present an exhaustive review of every potential molecular alteration in colorectal cancer, we have chosen to focus on the evidence supporting the changes in the normal control mechanisms of angiogenesis, apoptosis, and invasion and metastasis.

ANGIOGENESIS

The concept of tumor angiogenesis as a mechanism for cancer progression was popularized in the 1970s by Folkman (Table 1).1 Angiogenesis, or new blood vessel growth from existing vessels, is regulated by a flux of positive and negative factors within a tumor. To create a proangiogenic environment within a tumor, there must be a concurrent increase in proangiogenic factors and a decrease in antiangiogenic factors. This environmental change results in the neovascularization of a tumor. Many potential angiogenic factors have been characterized, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), angiopoietins, thrombospondins (TSP), and ephrins.2


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  		TABLE 1. The history of angiogenesis
 
Vascular Endothelial Growth Factor
VEGF was one of the first proteins found to stimulate angiogenesis within a tumor. Although it was initially thought to work by an alternate mechanism, further studies supported the theory of direct stimulation of blood-vessel growth. In 1983, Senger et al.3 partially purified a 34- to 42-kDa protein from the hepatoma of a guinea pig and found that this protein induced microvascular permeability. He thus named this protein vascular permeability factor. In 1989, Ferrara and Henzel4 characterized a protein found to stimulate mitosis in endothelial cells and called this protein VEGF. Further cloning and expression studies of these proteins revealed that they were the same molecule.5,6 Since the initial characterization of VEGF, other proteins have been identified that have similar structures and functions as VEGF. These proteins are categorized as members of the VEGF family.7–10 Currently, this family includes six members: VEGF, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. At least five different isoforms of VEGF have been characterized.11 These different isoforms result from alternative splicing of the RNA and are named according to the number of amino acids in its primary structure: 121, 145, 165, 189, and 206. VEGF165 is believed to be the predominant isoform in most cells and will be the main one referred to in this review.12

To demonstrate the importance of VEGF and its isoforms in tumor angiogenesis, we must first characterize the receptors for these proteins. Currently, three receptors have been identified that act as targets for members of the VEGF family (Fig. 1). These receptors include VEGFR-1 (VEGF receptor-1), also known as fetal liver tyrosine kinase-1; VEGFR-2, or kinase domain region/fetal liver kinase-1; and VEGFR-3, or fetal liver tyrosine kinase-4.11,13 Studies examining the localization, structure, and function of these receptors demonstrated that they are relatively specific for endothelial cells and contain an intracellular tyrosine-kinase–signaling domain. VEGFR-1 and VEGFR-2 are predominantly expressed in vascular endothelium, whereas VEGFR-3 localizes mainly to lymphatic endothelium.14 Most VEGF activity occurs with VEGFR-1 and VEGFR-2, and VEGFR-3 seems to predominantly act as a receptor for VEGF-C and VEGF-D.15–17



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  		FIG. 1. The interactions of the six members of the vascular endothelial growth factor (VEGF) family, with their corresponding receptors. PlGF, placenta growth factor; VEGFR, VEGF receptor; Flt, fetal liver tyrosine kinase; Flk-1/KDR, fetal liver kinase-1/kinase domain region.

 
VEGF expression is regulated by several factors: local tissue hypoxia, cytokines, oncogenes, tumor suppressor genes, and various growth factors.12 An environment of adequate tissue oxygenation is critical for cell survival. Tissue hypoxia has been associated with malignant progression, metastasis formation, and resistance to radiotherapy.18–20 Using in vitro studies, Minchenko et al.21 observed induction of the VEGF gene on incubating endothelial cells under hypoxic conditions, and subsequent experiments with an in vivo model of endothelial cells with local tissue hypoxia demonstrated similar findings. Other investigators have observed similar findings in different models, including Banai et al.,22 who, in a porcine model, clamped the left anterior descending artery for varying lengths of time to create localized areas of myocardial hypoxia. Hypoxia-inducible factor-1 (HIF-1) is believed to be the critical mediator that regulates VEGF expression under tissue hypoxia. This proangiogenic factor has been found to activate gene expression of various proteins under low oxygen tensions.23 HIF-1 acts as a transcriptional complex made up of two subunits—HIF-1{alpha} and HIF-1ß—and binds to a region known as the hypoxia-responsive enhancer region of select genes.24,25 Using tumor xenografts with hepatoma cells (Hepa-1c1c7), Maxwell et al.27 observed that hypoxic induction of HIF-1 stimulated the focal induction of VEGF. Further studies examining tissue hypoxia and VEGF regulation demonstrated that hypoxia not only increased the transcriptional activity of VEGF, but also increased the stability of its messenger RNA (mRNA).28 Examining cells under reduced oxygen tensions, Stein et al.29 demonstrated that although hypoxic conditions did upregulate transcription of VEGF, most of the increased levels resulted from the increase in the stability of its mRNA. Further regulatory mechanisms for VEGF have been described in association with other mediators, including transforming growth factor-ß, epidermal growth factor, interleukin-1{alpha}, interleukin-6, and prostaglandin E2.30–34

The biologic effects of VEGF not only include its presumed mitogenic activity on vascular endothelial cells and ability to induce angiogenesis, as described by Ferrara and Henzel,4 but also include the ability to inhibit apoptosis and act as a survival factor for endothelial cells.5 Gerber et al.35 demonstrated in human endothelial cells that the expression of the antiapoptotic proteins Bcl-2 and A1 was significantly induced on incubation with VEGF, and a significant correlation was found between the mRNA and protein levels for both. VEGF was also found to induce the expression of the matrix metalloproteinase (MMP) interstitial collagenase (MMP-1) in human umbilical vein endothelial cells both at the mRNA and protein levels, and it induced both the expression and activity of plasminogen activator, suggesting its role in extracellular proteolysis.36,37 VEGF also seems to contribute to tumor angiogenesis through the induction of microvascular permeability, with leakage of plasma proteins creating a proangiogenic fibrin-rich environment that stimulates the migration of fibroblast and endothelial cells.38

Because of the persuasive data demonstrating the importance of VEGF in tumor angiogenesis, an abundance of studies have examined the prognostic value of VEGF expression, tumor vessel count, and expression of VEGFRs in sporadic colon cancer. Using immunohistochemical staining of human colon carcinomas, Takahashi et al.39 observed a significant correlation between the intensity of staining for VEGF and metastatic disease. The same group also noted a significant correlation between kinase domain region positive–staining endothelial cells and metastatic disease, further supporting the role of VEGF in colorectal cancer progression. A separate study evaluated factor VIII, VEGF, basic fibroblast growth factor, proliferating cell nuclear antigen, and the presence of vascular, lymphatic, and perineural invasion in human colon cancer patients with node-negative disease.40 Multivariate analysis identified vessel count as a significant correlate with time to recurrence. Vessel count also correlated significantly with expression of VEGF, further implicating these parameters with prognosis. VEGF-D, whose activity has been suggested to play a role in the development of lymphatic vessels, was examined in 69 patients with colorectal cancer at Nottingham City Hospital in Helsinki, Finland.41 VEGF-D was found to be an independent prognostic factor of both overall and disease-free survival. It is interesting to note, though, that the VEGF-D receptor, VEGFR-3, was not found to be a significant prognostic parameter in these patients. In a separate investigation that used Northern blots to evaluate mRNA levels of VEGF, Ishigami et al.42 observed an association between overexpression of VEGF and mortality, further implicating the importance of VEGF in cancer progression. Taken together, these studies support the theory of VEGF induction as an indicator of poor prognosis. This concept has resulted in a number of clinical trials attempting to evaluate antiangiogenic therapy in colorectal cancer; however, currently no antiangiogenic drugs are approved by the Food and Drug Administration for adjuvant therapy in colorectal cancer (Figs. 1–5GoGoGo).



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  		FIG. 2. Death-receptor path-way of apoptosis. FADD, Fas-associated death domain–containing protein; Apaf, apoptotic protease-activating factor-1.

 


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  		FIG. 3. Bcl-2 family categorized into subfamilies demonstrating which members promote and which ones inhibit apoptosis. CED, cell death defective; BH, Bcl-2 homology.

 


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  		FIG. 4. Matrix metalloproteinase (MMP) overexpression and progression of colorectal normal mucosa to adenoma and carcinoma.

 
Platelet-Derived Endothelial Cell Growth Factor
PD-ECGF, also known as thymidine phosphorylase, is believed to have two functions in colorectal cancer. First, this molecule has been implicated as an angiogenic stimulator. Takahashi et al.43 performed immunostaining for PD-ECGF on 96 colon cancer specimens and observed that PD-ECGF was higher in specimens with high vessel counts and low VEGF expression than in cancer specimens with high vessel counts and high VEGF expression. This implicated PD-ECGF as an important angiogenic factor for tumors with low levels of VEGF expression. Second, PD-ECGF catalyzes the reversible phosphorolysis of pyrimidines such as thymidine, a role important for normal metabolism. This mechanism of action of PD-ECGF in colorectal neoplasia is thought to enhance the activation of fluoropyrimidines, thereby increasing the sensitivity of colorectal carcinoma to 5-fluorouracil treatment. Evidence supporting this theory was illustrated from transfection assays in which PD-ECGF complementary DNA was placed into multiple cell lines, including the HT29 human colorectal cell line, and demonstrated an increased sensitivity to 5-fluorouracil.44–46 As a prognostic parameter, the data for PD-ECGF are conflicting. Saito et al.47 examined 86 colorectal cancer specimens that were immunostained for PD-ECGF and observed that patients with high PD-ECGF expression had a lower incidence of local and distant metastasis. In a similar experiment, Takebayashi et al.48 immunostained 163 colon cancer specimens for PD-ECGF and found that cancer specimens staining for PD-ECGF significantly correlated with tumor size, extent of invasion, lymph node metastasis, and lymphatic and venous invasion.33 Thus, further studies are needed to determine the prognostic value of PD-ECGF expression.

Thrombospondins
The TSP family consists of five macromolecules that act at both the cell surface and extracellular matrix, regulating cellular interactions associated with angiogenesis.49 These five macromolecules are further divided into subfamilies based on the three-dimensional structure. TSP-1 and -2 are trimeric proteins and are grouped in subfamily A, whereas TSP-3, TSP-4, and cartilage oligomeric matrix protein are pentameric proteins that form subfamily B.49 TSP-1 and -2 have been more extensively studied and are believed to have significant roles in tumor angiogenesis. TSP-1 is released from the {alpha} granules of platelets, as well as macrophages, endothelial cells, and fibroblasts, and is upregulated in healing wounds.50–54 TSP-1–null mice have normal developmental angiogenesis; however, there are pronounced alterations in wound-repair angiogenesis in mice deficient in TSP-1.55 Overexpression of TSP-1 resulted in delayed wound healing and impaired granulation tissue, decreased proliferation of endothelial cells, reduction of fibroblast migration into the wound, and a 30% reduction in the blood vessel density in granulation tissue.56 TSP-2 is mainly released from fibroblasts in healing skin wounds; TSP-2–null mice not only demonstrate increased vascularity in wounds, but also display faster wound healing.57 Both TSP-1 and -2 have been implicated as inhibitors of tumor growth and angiogenesis in cell-line studies; however, data supporting their roles in prognosis remain controversial.58–62 The antiangiogenic effect of these two molecules has been attributed to a reduction in both the number and size of the blood vessels, whereas its anti–tumor-growth effect has been theorized to be a barrier effect that is created around the tumor and that prevents outward growth.60 To demonstrate the clinical importance of these molecules, Maeda et al.63 examined 100 colorectal specimens for TSP-1 expression and found that TSP-1 expression inversely correlated with prognosis: TSP-1–negative tumors had a significantly worse prognosis. Tokunaga et al.64 evaluated 61 patients with colon carcinomas by using reverse transcriptase–polymerase chain reaction methods for TSP-2 and VEGF, and patients with TSP-2–positive/VEGF-negative tumors had a significantly better prognosis. These studies support the theory that a positive balance of antiangiogenic factors in colorectal tumors portends a better prognosis.

Angiopoietins
Angiopoietins are a family of growth factors that serve as ligands for the endothelium-specific tyrosine kinase receptor TIE-2.65 Currently, there are four known angiopoietins, but only angiopoietins-1 and -2 have been implicated in colorectal tumorigenesis. Angiopoietin-1 is believed to have a stabilizing effect on blood vessels through its agonistic effect on TIE-2 receptors, promoting the interaction between endothelial cells and the surrounding extracellular matrix.66 Angiopoietin-2 is theorized to have antagonistic effects on the TIE-2 receptor, causing destabilization of the surrounding vasculature.67 Using immunohistochemical staining for CD31, Ahmad et al.68 used HT29 colon cancer cells transfected with angpoietin-1 and -2 complementary DNA and injected them subcutaneously into nude mice to evaluate tumor vessel counts. This group demonstrated that angiopoietin-1–transfected tumors had significantly lower blood vessel densities, whereas angiopoietin-2–transfected tumors had significantly higher vessel counts, as well as increased tumor growth. Although the exact mechanism is still unknown, current data suggest that angiopoietin-2 in the presence of VEGF induces angiogenesis, whereas angiopoietin-1 overexpression in the presence of VEGF is antiangiogenic.

Ephrins
The erythropoietin-producing hepatoma amplified sequence family is a relatively new family of tyrosine kinase receptors that are believed to function in tumor angiogenesis. Currently this family consists of 14 receptors and 8 ligands and has been shown to be critical for normal embryonic vasculature development.69 Numerous studies have demonstrated upregulation or overexpression of these receptors in different human tumors, including glioblastoma, melanoma, and breast, lung, and colorectal carcinoma.70,71 The ligands for these receptors, termed ephrins, reside within or are anchored to the plasma membrane. These receptors and ligands are subclassified into an A and B class on the basis of structure, sequence homology, and binding affinity. Because of their relatively recent implications in tumor angiogenesis, only a few groups have specifically examined their role in colorectal carcinomas. Using immunohistochemical staining for the ephrin family members EphB4 and ephrin-B2 in human colon cancer specimens, Liu et al.72 observed overexpression of both ephrin-B2 and EphB4 in all tumor specimens evaluated compared with adjacent normal mucosa; this suggests a role in colon cancer progression.

In summary, angiogenesis remains a complex process in which we continue to find new roles for existing molecules and find new molecules that contribute to already-established mechanisms. Numerous factors contribute to tumor angiogenesis, and neovascularization within a tumor seems to depend on the relative balance between pro- and antiangiogenic factors. Although the role of VEGF in tumor angiogenesis is substantial, several other factors are implicated in this dynamic process.

APOPTOSIS

An essential aspect for human life is the ability of a cell to reproduce and create an identical copy of itself. Aberrations of such processes can result in instability of the genome and, unless monitored at checkpoints, can lead to tumorigenesis. Apoptosis provides a pathway for abnormal cells to be removed before additional genetic mutations accumulate, resulting in a malignant cell. Although apoptosis, or programmed cell death, plays an essential role in the development and functioning of multicellular organisms, the exact mechanisms controlling this process remain elusive. Much has been written on hypothesized pathways; however, many of these pathways are contradictory, making new insights confusing rather than enlightening. Important regulators that have been well characterized in apoptosis include caspases, the tumor necrosis factor (TNF) receptor family, adapter proteins, and the Bcl-2 family (Fig. 2). Because of the abundance of hypothesized molecules in this process, but limited space in this review, we focus only on the well-characterized members of apoptosis.

Caspases
Caspases (cysteine aspartyl protease) consist of a family of 14 cysteine proteases that possess a unique structure motif of three domains: an amino terminal domain, a large subunit, and a small subunit.73–75 Initially, these proteins are synthesized as inactive zymogens, termed procaspases. On activation, each individual caspase has multiple and varying substrate-specificities determined by the size of its binding site.76 Caspases function in at least three separate but important processes of apoptosis: cytokine maturation, initiation of apoptosis, and coordination of the effector pathway for apoptosis. Caspase-1, initially called interleukin-1ß–converting enzyme, was the first caspase identified, and this enzyme, along with caspase-4 and caspase-5, is primarily involved in the proteolytic cleavage and activation of cytokines. Caspases-2, -8, and -9 initiate the caspase cascade and are thus termed initiator caspases. Caspases-3, -6, and -7 function in the proteolysis and activation of proteins necessary for completing the apoptotic process and are thus termed effector caspases. The specificity of the recognition motifs for these enzymes makes them unique; caspases require a four–amino acid sequence that differs among specific caspases and cleaves on the carboxyl side of an aspartate residue.77 Cheng et al.78 and Clem et al.79 examined the relationship between members of the caspase and Bcl-2 families. Cheng et al. found that caspase-3 cleaved both Bcl-2 and Bcl-xL, removed their antiapoptotic properties, and formed a C-terminal product with proapoptotic properties. Clem et al. discovered that caspase-8 cleaved the proapoptotic Bcl-2 family member Bid and resulted in a C-terminal cleavage product that activated the release of cytochrome c from mitochondria, another potential mechanism for regulating apoptosis.80,81 Thus, the major role of caspases appears in both the initiation and effector phases of the apoptotic process. More work is necessary to define specific roles for each of the molecules and their potential prognostic or therapeutic roles in colorectal cancer; however, some progress has been made. Adachi et al.82 examined the role of caspase activity in apoptosis in a colorectal cancer cell line and found that 5-fluorouracil–induced apoptotic cells had increased activities of caspases-3 and -8. Uchida et al.83 used another colon cancer cell line to transfect a caspase-8 vector in the presence of 5-fluorouracil and found a significant induction of apoptosis with both treatment modalities versus either one alone, suggesting a possible role for combination treatment in 5-fluorouracil–resistant tumors.

TNF Receptor
The TNF receptor family is a large group of transmembrane receptors that contain a characteristic extracellular cysteine-rich domain.84 A subgroup of receptors in this family has been implicated in apoptosis: these receptors include TNF receptor-1, CD95, death receptor (DR)3, DR4, DR5, and DR6. The TNF receptor family has a multitude of biologic effects, including cell proliferation, differentiation, apoptosis, and cell death, depending on the stimulus and the target cell. Using flow cytometry, Meterissian et al.85 demonstrated in multiple human colorectal cell lines that stimulation of the TNF receptor with a monoclonal antibody induced apoptosis. Unique to this group of receptors is a 65–amino acid sequence in the intracellular region, termed death domain, that facilitates intermolecular signaling between the receptor and effector molecules of apoptosis.86 The death domain serves as a cytoplasmic portion of the TNF receptor that facilitates interactions with adaptor proteins, allowing for caspases to aggregate and become activated. Adaptor proteins such as apoptotic protease-activating factor-1, Ced-4, and Fas-associated death domain–containing protein/mediator of receptor-induced toxicity are vital in the interaction between activated receptors and downstream target molecules.87–89 Important to these proteins are the well-characterized domains that allow for the molecular interactions between the TNF receptors, adapter proteins, and caspases. Three such domains that have been well characterized include the death domain, the death-effector domain, and the caspase-recruitment domain.90,91

Because of the intimate relationship in the apoptotic pathway of TNF receptors to the caspase cascade, these molecules have become potential targets for cancer chemotherapeutics. Further, with most colorectal cancer patients having a mutation of the tumor-suppressor gene p53, this pathway provides a p53-independent path to apoptosis. Although in theory this concept is very promising, the significant side effects of systemic TNF in the clinical setting have continued to challenge investigators.

Bcl-2 Family
Bcl-2 is the prototypical antiapoptotic protein that has been well studied in multiple human cancers, including colorectal carcinoma.92 This is just one member of a large class of molecules classified in the Bcl-2 family (Fig. 3). The members of this family share at least one conserved domain (if not all four) termed Bcl-2 homology regions, or BH-1, -2, -3, and -4.93 These domains are functionally important to this family, because studies suggest that they dictate the effect the Bcl-2 family member will have on the cell. Many of the prosurvival Bcl-2 members contain conserved BH-1 and -2 domains. The BH-3 domain seems to be an important apoptotic factor, and there exists a subset of Bcl-2 family members (the BH-3 subfamily) that contain only this domain and that are very effective proapoptotic proteins.94 Currently, the Bcl-2 family is subdivided into three subfamilies: the prosurvival Bcl-2 subfamily, the proapoptotic Bax subfamily, and the proapoptotic BH-3 subfamily. The exact mechanism by which these proteins cause or inhibit apoptosis remains elusive; however, there are several theories that attempt to explain these mechanisms. One intriguing aspect of these proteins is their ability to heterodimerize with one another.95 This inherent binding ability of these proteins forms the basis of the model involving the prototypical antiapoptotic factor Bcl-2 and the prototypical proapoptotic member Bax.96 Under this model, the propensity of a cell to undergo apoptosis depends on the relative amounts of Bcl-2/Bax heterodimers, Bax/Bax homodimers, and Bcl-2/Bcl-2 homodimers. An excess of the proapoptotic homodimers (Bax) will result in apoptosis, whereas an excess of the Bcl-2 homodimers results in cell survival. Another theory related to the inherent ability of these family members to interact relates to the creation of a conglomerate of molecules, including cytochrome c, caspases, and adapter proteins.87 Other hypotheses relating to Bcl-2 functions include creation of membrane channels, regulation of caspase activity, and inhibition of cytochrome c export out of the mitochondria.97,98 The idea that Bcl-2 members form channels within membranes originated from studies that examined the structure of Bcl-xL. These studies demonstrated similarities between the membrane-inserting domains of certain bacterial toxins (i.e., diphtheria toxin) and Bcl-xL.

The expression of Bcl-2 has been evaluated in human colorectal adenoma and carcinoma samples in several studies, and the overall conclusion is that it tends to be overexpressed predominantly in adenomas compared with carcinomas, suggesting that it functions early in the adenoma-carcinoma sequence.92,99 Also, patients with colorectal carcinomas that had high levels of Bcl-2 expression had a better prognosis. Studies examining Bax in human colorectal carcinoma samples demonstrated that colorectal carcinomas with high levels of Bax tended to have a poor prognosis.33,48,100 These studies are somewhat perplexing in that high levels of an antiapoptotic factor indicated a better prognosis, whereas high levels of proapoptotic factors tended to indicate a poor prognosis.

Caenorhabditis elegans
The nematode Caenorhabditis elegans provides an excellent but simplistic model for the molecular events that occur in apoptosis. In this model, four proteins interact to promote apoptosis. These four proteins include cell death defective (CED)-3, -4, and -9 and EGL-1.101 This serves as a simplistic representation of the human model because there is no redundancy in this process. Only a single protein exists for each class of apoptotic regulators mentioned previously. CED-3 acts as the effector caspase, and CED-4 functions as the adapter protein whose role is to activate CED-3. CED-4 is the homolog of human apoptotic protease-activating factor-1, which functions in a cytochrome c–dependent mechanism, activating caspace-3 and leading to apoptosis in humans.87 CED-9 plays the role of the antiapoptotic human Bcl-2; in cells destined for survival, CED-9 binds to CED-4 and prevents it from activating CED-3. EGL-1 functions as the BH-3–only protein that initiates apoptosis. When cells are programmed for death, EGL-1 binds to and inactivates CED-9, thus allowing CED-4 to activate CED-3 and promoting apoptosis.

In conclusion, the mechanisms responsible for facilitating apoptosis continue to challenge the basic scientist. We have made great strides in our knowledge of caspases, TNF receptors, adapter proteins, and the Bcl-2 family, and, through work on the roundworm C. elegans, we have produced an excellent model of the events that occur in apoptosis. However, there are still many aspects of apoptosis that need to be more clearly defined, including the multitude of pathways for activating caspases, the expanding role of mitochondria in apoptosis, and the underlying mechanisms by which the Bcl-2 family regulates apoptosis.

INVASION AND METASTASIS

One important mechanism for the development and progression of colorectal cancer is the ability of transformed cells to invade and traverse the basement membrane. This process facilitates the progression of adenomas to carcinomas and metastasis of carcinomas. A special class of enzyme implicated in this process is MMP. This family of enzymes includes enzymes that are not only part of the degradative process, but also inhibitors that limit their activity. In general, remodeling of the extracellular matrix is an integral part of normal tissue growth and differentiation. MMPs generally function to degrade proteoglycans and matrix glycoproteins. Important to cancer progression, loss of basement membrane integrity may correlate with an increased probability of distant metastasis and poor prognosis.102 Therefore, overexpression of MMPs may be one part of the multistep process by which the neoplastic cell can proliferate and metastasize. In this part of the review, we describe the major classes of MMPs and tissue inhibitors of MMPs (TIMPs) and then focus on the in vitro and in vivo animal data, as well as clinical associations in human correlative studies.

MMPs are a group of structurally related endopeptidases that are involved in the normal processes of tissue degradation and remodeling, such as angiogenesis or wound repair. MMPs are involved with many pathologic conditions, such as arthritis, and are implicated in tumor development.103 Each of the individual MMPs shares several similar protein domains, and they vary on the basis of structure, location relative to the cell membrane, substrate, and the ability to activate other MMPs, creating a cascade effect. The primary categories of the MMP family are collagenases, stromelysins, matrilysins, gelatinases, membrane-type MMPs, and TIMPs.104 Collagenases (MMP-1, -8, and -13) are primarily involved with the degradation of all types of collagens, with limited activation of other MMPs. Stromelysins (MMP-3, -10, -11, and -12) tend to degrade elastins, proteoglycans, laminin, and fibronectin and can activate collagenases. Matrilysins (primarily MMP-7) is the most basic MMP and has the broadest activity; it degrades a wide range of proteoglycans, laminins, fibronectins, gelatins, collagen IV, and elastins and is a primary activator of some collagenases and of gelatinase A (MMP-2). Gelatinases (MMP-2 and -9) degrade gelatins, collagen, and elastins; they have little documentation of a role in the activation of other MMPs. Membrane-type MMPs (1 through 6) are a class of MMPs that are anchored to the cell membrane and may participate in the degradation of extracellular matrix components, but they seem to have a major role in the activation of gelatinases.105,106 The TIMPs are proteins that inhibit the activated form of many members of the MMP family and may have activity in malignant tumors.107 In fact, clinical trials evaluating this class of proteins have been performed with a number of tumor types (pancreatic, gastric, and lung cancer) and have shown little efficacy for improved survival overall, except for, perhaps, stabilization of disease in a subset of gastric cancer patients.108–110 It is largely unclear whether this class of inhibitors will have an effect on cancer control in their present form.

To date, three MMPs have been most associated with colorectal adenomas and carcinoma. MMP-2 (gelatinase A) is mainly associated with the degradation of type IV collagen. Overexpression has been reported in gastric, pancreatic, and colorectal cancer.111,112 MMP-7 (matrilysin) seems to be expressed by neoplastic cells and may function in the early phases of neoplastic growth.113 This was demonstrated in colorectal adenomas compared with normal mucosa and in mouse models of intestinal neoplasia.113,114 Recent data have suggested that increased levels of MMP-9 (gelatinase B) mRNA in colorectal cancer, compared with normal mucosa, were associated with significantly shorter disease-free and overall survival.115

Heslin et al.116 demonstrated that MMP-7 overexpression was an early event in the carcinogenic cascade, because normal mucosa progresses to adenoma. As adenomas acquire the ability to become invasive adenocarcinomas, the data show that MMP-7 remains increased, and through either coactivation by MMP-7 or activation by other mediators not yet defined in these tissues (other MMPs, prostaglandins, growth factors, and so on), both MMP-2 and MMP-9 were increased in expression compared with normal mucosa. This may allow for tumor cells to invade or for new blood vessel growth to occur in previously normal tissues. MMP-2 and -9 seem to be co-regulated in invasive carcinomas, as demonstrated by a significant direct association between these factors (Fig. 4). This study also showed that there was no correlation of MMP-7 expression with increasing American Joint Committee on Cancer stage; however, long-term follow-up with correlation with recurrence and survival was not complete.

Others have shown MMP-7 to be primarily expressed on the tumor cell surface, and, therefore, this is likely a direct effect of disordered signaling pathways.113 This early change may allow abnormal cells the ability to overcome normal cell-cell growth inhibition. Similarly, this may be the first step in the local proteolysis of the basement membrane to allow for invasion. MMP-2 and MMP-9 tend to be overexpressed by the stroma surrounding the tumor, which may be a reaction to MMP-7 or other mediators known to induce MMP expression.117 The response of stromal cells that produce MMPs may also be due to angiogenesis induced by the tumor.

In vitro data suggest that MMP-7 expression is related to the invasiveness of the primary tumor cells.118 This study demonstrated that MMP-7 levels in the tumor cells varied directly with the tumorigenicity. MMP-7 has been previously demonstrated in one study to be focally overexpressed in approximately 50% of benign adenomas and, in a separate study, to be overexpressed in the more dysplastic and invasive portions of tumors by immunostaining.113,119 Others have demonstrated that, in patients with familial adenomatous polyposis, MMP-7 is constitutively overexpressed in all polyps regardless of size or dysplasia, whereas in sporadic adenomatous polyps, MMP-7 expression was correlated with both size and dysplasia.120 Swallow et al.,121 in an elegant in vitro study, evaluated colorectal cancer cell lines with and without metastatic potential for the ability to induce monocytes to produce MMP-2 and MMP-9. Each of the previously described cell lines was cocultured with monocytes but separated by a membrane. MMP-2 and MMP-9 activity was increased by the monocytes cocultured with cancer cells of high metastatic potential. Neither MMP-2 nor MMP-9 was produced by the colorectal cancer cells; this demonstrates that colorectal cancer cell lines with metastatic potential have the ability to induce MMP-2 and MMP-9 activity in the monocytes through a soluble stimulus. For these particular MMPs, these in vitro data support the notion that cancer cells induce stromal cells to degrade the extracellular matrix and basement membrane through a paracrine-type effect. Separately, Zeng et al.115 evaluated the ratio of MMP-9 expression by Northern blot analysis in normal mucosa and carcinomas and correlated this ratio with recurrence and survival. An increased ratio was significantly related to Dukes’ stage, the presence of distant metastases at the time of presentation, and an independent prognostic factor associated with a decreased disease-free survival.

In conclusion, MMP-7 seems to be an early event in the colorectal adenoma-to-carcinoma progression, with subsequent increases of MMP-2 and MMP-9 in colorectal carcinomas. Evaluation of other compounds thought to be upstream regulators of MMP expression (prostaglandins, growth factors, and so on), as well as potential downstream effects on apoptosis or angiogenesis, remains to be completed. Also, correlation of MMP expression with recurrence and tumor mortality would be important with long-term follow-up. The clinical application of MMP inhibitors for cancer control has yet to be demonstrated to be clearly beneficial.

There is a tremendous amount of ongoing investigation into the many pathways implicated in colorectal carcinogenesis. Although it is important to have an in-depth knowledge of specific pathways involved in carcinogenesis, determining the interactions among these pathways will further help us gain an understanding of colorectal cancer. In this article, we attempted to provide core information on the molecular events of three specific pathways of colorectal carcinoma. When available, clinical studies were added to supplement the molecular data. Also, we attempted to provide information on studies that have examined mediators across these three pathways, such as studies examining the biologic effects of VEGF on antiapoptotic proteins and MMPs. Although the three pathways of angiogenesis, apoptosis, and invasion/metastasis have three very different mechanisms with different but important end points implicated in colorectal carcinoma, we believe that the interactions among such mechanisms are important as well.

FOOTNOTES

This is a review of three well-characterized pathways implicated in the malignant progression of a colorectal epithelial cell. We will provide data on the latest developments in angiogenesis, apoptosis, and invasion and metastasis as they relate to colorectal carcinoma.

Received for publication February 25, 2003. Accepted for publication June 23, 2003.

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