Tumor Biology and Metastasis

The Cell Cycle

The cell cycle comprises four phases (M phase, S phase, G1, and G2) (see Figure 2-1). Nonproliferating cells are usually arrested between the M (mitosis) and S (DNA synthesis) phases and are referred to as G0 cells. The majority of cells in normal tissues are in G0. Cells are stimulated to enter the cell cycle in response to external factors including growth factors and cell adhesion. During the G1 phase of the cell cycle, cells are responsive to mitogenic signals. Once the cell cycle has traversed the restriction point (R) in the G1 phase, cell cycle transitions become autonomous.

Progression through the cell cycle is mediated by the sequential activation and inactivation of a class of proteins called cyclin-dependent kinases (CDKs).^11,12 CDKs consist of an inactive conserved catalytic core and are regulated at the following three levels:

The first class of proteins induced in G1 following mitogenic stimulation of the cell cycle is the class D cyclins, which in turn activate CDK4 and CDK6. Cyclin D-CDK complexes cause phosphorylation of the substrate retinoblastoma protein (Rb), which results in dissociation of the transcription factor E2F from the Rb protein. The phosphorylation status of Rb plays a critical role in regulating G1 progression, and Rb is the molecular device that serves as the R point switch. Phosphorylated Rb releases E2F enabling transcription of numerous E2 responsive genes involved in DNA synthesis, and unphosphorylated RB remains associated with E2F, thereby inhibiting cell cycle progression. During G1 progression, activation of E2F also leads to induction of cyclin E. Cyclin E associates with CDK2 and the cyclin E-CDK2 complex maintains Rb in the phosphorylated state and is essential for cells to enter the S phase of cell cycle. At the G1/S phase transition, E2F induces cyclin A. During early S phase, cyclin D and E are degraded and cyclin A associates with CDK2 and CDK1; this kinase activity is essential for entry into S phase, completion of S phase, and entry into M phase. Mitosis is regulated by CDK1 in association with cyclins A and B causing phosphorylation of cytoskeletal proteins, including histones and lamins. This tight regulation on cell cycle progression prevents uncontrolled passage of normal cells through the cell cycle. The corollary of this is that loss of these control mechanisms can be a driving event in the development of cancer.

p53 Functions as a Genomic Guardian

The p53 response to stress may be mediated by DNA-dependent protein kinase (DNA-PK) or by the ATM kinase and leads to phosphorylation of the N terminus of p53. In normal cells, p53 is short lived; however, phosphorylated p53 is stabilized and can then function as a transcriptional regulator binding to sequences and transactivating a number of genes, including p21.^13,14 p21 has a high affinity for G1 CDK/cyclin complexes and acts as a CDKI inhibiting kinase activity, thereby arresting cells in G1. By holding cells in G1, the replication of damaged DNA is prevented, and the cell’s own DNA repair machinery has the opportunity to repair damage prior to reentering the active growth cycle (Figure 2-2).

The cellular levels of p53 protein are regulated by the product of another gene MDM2 (mouse double minute 2 oncogene).¹⁵ The principal role of MDM2 is to act as a negative regulator of p53 function. One mechanism for MDM2 to downregulate p53 is to target p53 for degradation. The p53 protein is maintained in normal cells as an unstable protein, and its interaction with MDM2 can target p53 for degradation via a ubiquitin proteosome pathway. MDM2 can also control p53 function by suppressing p53 transcriptional activity. MDM2 is a transcriptional target of p53, and expression is induced by the binding of p53 to an internal promoter within the mdm2 gene. MDM2 can in turn bind to a domain within the amino terminus of p53, thereby inhibiting the transcriptional activity and G₁ arrest function of p53 by masking access to the transcriptional machinery (see Figure 2-2).¹⁶

Cell Death

In contrast to necrosis, apoptosis is a distinct type of cell death most often characterized as the “programmed” self-destruction of cells that occurs in disease states, as well as part of the normal physiologic cell turnover. Whereas necrosis is characterized by swelling of the cell and lysis, in apoptosis there is cellular and nuclear shrinkage followed by fragmentation and subsequent phagocytosis. These morphologic features of apoptosis result from a number of apoptosis effectors (i.e., caspases) and regulators (particularly the Bcl-2 protein family). The molecular mechanisms involved in apoptosis are shown in Figure 2-3.^17,18 Apoptosis provides a controlled mechanism for eliminating cells that are irreversibly damaged and involves an adenosine triphosphate (ATP)-dependent activation of cellular pathways, which move calcium from the endoplasmic reticulum to the cytoplasm and activation of endonucleases. As noted previously, some of these pathways are mediated through caspases. However, a wide variety of signals can initiate an apoptotic response, including Fas ligand (CD95 or FasL) and its interaction with the Fas receptor, tumor necrosis factor (TNF) and its receptor interaction, and certain oncogenes. The Fas and TNF receptors are members of the death receptor family. These transmembrane proteins with cysteine rich extracellular domains and intracellular regions share a common structure termed the “death domain.” The proapoptotic ligands for these receptors are homotrimeric peptides that are either soluble or expressed at the surface of the adjacent cell. Ligand-induced receptor clustering promotes the binding of a soluble cytosolic adapter protein called Fas-associated death domain (FADD), which itself contains a death domain as well as a caspase binding site, to the clustered death domains of the receptors. This leads to activation of caspase 8 and downstream activation of effector caspases for apoptosis.

Where cells are damaged and are unable to repair DNA, p53 expression can upregulate p21 and cause cell cycle arrest or can aid in directing the cell into programmed death or apoptosis through upregulation and expression of Bax (a proapoptotic Bcl-2 family protein) and also through priming of caspases. In this, the expression of p53 can also downregulate expression of the Bcl2 gene itself (a prosurvival, negative regulator of apoptosis).

Summary

The cell cycle ensures a regulated process so that each cell can complete DNA replication before cell division occurs. The cell responds to growth and environmental signals through the cell cycle. Components of the cell cycle that have a stimulatory effect include the cyclins and the CDKs. The negative influences come from a series of checkpoints that respond to external stimuli. These include tumor suppressor genes such as p53 and Rb and also genes involved in DNA repair. The process of DNA replication is also subject to the introduction of errors, and this is closely monitored by a class of enzymes called DNA repair enzymes. Consequently, there are a number of safeguards within the cell cycle to ensure that normal cells are produced during division and that the DNA is accurately replicated. The next section will describe how these systems are overcome to produce a malignant cancer cell.

• Benign tumors: Broadly speaking, these tumors arise in any of the tissues of the body and grow locally. Their clinical significance is the ability to cause local pressure, cause obstruction, or form a space-occupying lesion, such as a benign brain tumor. Benign tumors do not metastasize.

• In-situ tumors: These are often small tumors that arise in the epithelium. Histologically, the lesion appears to contain cancer cells, but the tumor remains in the epithelial layer and does not invade the basement membrane or the supporting mesenchyme. A typical example of this is preinvasive squamous cell carcinoma (SCC) affecting the skin of cats, which is often referred to as Bowen’s disease.

• Cancer: This refers to a malignant tumor that has the capacity for both local invasion and distant spread by the process of metastasis.

Multistep Carcinogenesis

Cancer is the phenotypic end result of a whole series of changes that may have taken a long period of time to develop. Indeed, recent studies that have sequenced the genome of pancreatic and brain tumors have identified 63 and 60 genetic alterations on average in each cancer, respectively. From this large list of genetic alterations, there are a small number of commonly mutated genes that are “drivers” of the cancer phenotype.^19,20

The application of a cancer-producing agent (carcinogen) to tissues does not lead to the immediate production of a cancer cell. After the initiation step produced by the agent, there follows a period of tumor promotion. This promotion may be caused by the same initiating agent or by other substances, such as normal growth promoters or hormones. The initiating step is a rapid step and affects the genetic material of the cell. If the cell does not repair this damage, then promoting factors may progress the cell toward a malignant phenotype. In contrast to initiation, progression may be a very slow process and may not even manifest in the lifetime of the animal. Each stage of multistep carcinogenesis reflects genetic changes in the cell with a selection advantage that drives the progression toward a highly malignant cell. The age-dependent incidence of cancer suggests a requirement for between four and seven rate-limiting, stochastic events to produce the malignant phenotype.²¹

These sequential events in tumor formation are a consequence of changes at the genetic level. Over the past 25 years, cancer research has generated a rich and complex body of information revealing that cancer is a disease involving dynamic changes in the genome. Seminal to our understanding of cancer biology has been the discovery of the so-called cancer genes or oncogenes and tumor suppressor genes. Mutations that produce oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function have been identified through their alteration in human and animal cancer cells and by their elicitation of cancer phenotypes in experimental models.

Oncogenes

The RNA tumor viruses (retroviruses) provided the first evidence that genetic factors play a role in the development of cancer. The initial observation came in 1910 when Rous demonstrated that a filterable agent (later classified as a retrovirus and termed avian leukosis virus) was capable of producing lymphoid tumors in chickens.²² Retroviruses have three core genes (gag, pol, and env) and an additional gene that gives the virus the ability to transform cells. Retroviral sequences that are responsible for transforming properties are called viral oncogenes (v-onc). The names of these genes are derived from the tumors in which they were first described (e.g., v-ras from rat sarcoma virus).

Viral oncogenes were subsequently shown to have cellular homologues called cellular oncogenes (c-onc). Later, the term proto-oncogene was used to describe cellular oncogenes that do not have transforming potential to form tumors in their native state but can be altered to lead to malignancy.²³ Most proto-oncogenes are key genes involved in the control of cell growth and proliferation and their roles are complex. For simplicity, their sites and modes of action in the normal cell can be divided as follows (Table 2-1):

Growth Factors

Growth factors are molecules that act on the cell via cell surface receptors. Their contribution to carcinogenesis may be through excessive production of the growth factor or where a growth factor is expressed in a cell but does not normally function in that cell.

Growth Factor Receptors

Several proto-oncogene–derived proteins form a part of cell surface receptors for growth factors. The binding of ligand to receptor is the initial stage of delivery of mitogenic signals to cells. Their role in carcinogenesis may be through structural alterations in these proteins.

Protein Kinases

Protein kinases are associated with the inner surface of the plasma membrane and are involved in signal transduction following ligand-receptor binding. Structural changes in these genes and proteins lead to increased kinase activity that can have profound effects on signal transduction pathways.

Nuclear Proteins and Transcription Factors

Nuclear proteins and transcription factors encode proteins that control gene expression. These proto-oncogenes may have a role in cellular proliferation. Not surprisingly, changes in transcription factor activity may contribute to the development of the malignant genotype.

Tumor Suppressor Genes

Changes in genes can lead to either stimulatory or inhibitory effects on cell growth and proliferation. The stimulatory effects are provided by the proto-oncogenes as described. Mutations or translocations of these genes produce positive signals leading to uncontrolled growth. In contrast, tumor formation can result from a loss of inhibitory functions associated with another class of cellular genes called the tumor suppressor genes. The discovery of these genes began by observations of inherited cancer syndromes in children, in particular studies of retinoblastoma. In the early 1970s, epidemiologic studies of both retinoblastoma and Wilms’ tumor led Knudson to propose his “two hit” theory of tumorigenesis.

Cancer Arises through Multiple Molecular Mechanisms

The advances in our understanding of normal cell biology and the processes that lead to malignancy have increased dramatically over the past 30 years. The last decade has shown us that transformation of a normal cell into a malignant cell requires very few molecular, biochemical, and cellular changes that can be considered as acquired capabilities.^42,43 Further, despite the wide diversity of cancer types, these acquired capabilities appear to be common to all types of cancer. An optimistic view of increasing simplicity in cancer biology is further endorsed by the fact that all normal cells, irrespective of origin and phenotype, carry similar molecular machineries that regulate cell proliferation, differentiation, aging, and cell death.

We have discussed that tumorigenesis is a multistep process and that these steps reflect genetic alterations that drive the progression of a normal cell into a highly malignant cancer cell. This is supported by the finding that genomes of tumor cells are invariably altered at multiple sites. The spectrum of changes range from subtle point mutations in growth regulatory genes to obvious changes in chromosomal complement.

Cancer cells have defects in regulatory circuits that govern cellular proliferation and homeostasis and survival. A model has been proposed that suggests that the vast array of genetic abnormalities associated with cancer are a manifestation of eight alterations in cellular physiology that collectively contribute to malignant growth. First proposed in 2000 and updated in 2011, these “hallmarks” of cancer constitute an organizing principle for rationalizing the complexities of cancer and are underpinned by two overarching themes: genome instability and chronic inflammation.^42,43 The eight acquired characteristics can be summarized under the following headings (see Figure 1-3):

In addition, cancer exhibits another dimension of complexity in that they contain an array of “normal” cells that contribute to the acquisition and maintenance of the cancer hallmarks by creating the tumor microenvironment. The next section is an overview of these traits and the strategies by which they are acquired in cancer cells. The process of metastasis requires angiogenesis and invasion, as such the traits of sustained angiogenesis, and tissue invasion and metastasis will be collectively reviewed under a final section on metastasis.

Self-Sufficiency in Growth Signals

Normal cells require mitogenic stimuli for growth and proliferation. These signals are transmitted to the nucleus by the binding of signaling molecules to specific receptors, the diffusion of growth factors into the cell, extracellular matrix components, or cell-to-cell adhesions or interactions. As previously discussed, many oncogenes act by mimicking normal growth signals. Tumor cells are not dependent on external mitogenic stimuli for proliferation and sustained growth but are self-sufficient. The liberation from dependency on exogenous signals severely disrupts normal cellular homeostasis. Arguably, the most fundamental trait of cancer cells is their ability to sustain chronic proliferation. By deregulating these signals, cancer cells become masters of their own destinies, promoting signaling (typically through intracellular kinase domains) to promote progression through the cell cycle, increases in cell size, increases in cell survival, and changes in energy metabolism.

The cancer cell can acquire this capability in the following ways:

Although the acquisition of growth signaling autonomy by cancer cells is conceptually satisfying, it is in fact too simplistic. One of the major problems of cancer research is to focus on the cancer cell in isolation. It is now apparent that we must also consider the contribution of the tumor microenvironment to the survival of cancer cells. Within normal tissues, paracrine and endocrine signals contribute greatly to growth and proliferation. Cell-to cell growth signaling is also likely to operate in cancer cells and may be as important as some of the autonomous mechanisms of tumor growth. It has recently been suggested that growth signals for the proliferation of carcinoma cells are derived from the tumor stromal elements (e.g., cancer or carcinoma-associated fibroblasts [CAFs]). It is therefore possible that the survival of tumor cells not only relies on the acquisition of growth signal autonomy but may also require the recruitment or modulation of stromal cells to provide these growth signals.

Insensitivity to Antigrowth Signals or Evading Growth Suppressors

Within the normal cell, multiple antiproliferative signals operate to maintain cellular quiescence and homeostasis. These signals include soluble growth inhibitors that act via cell surface receptors and immobilized inhibitors that are embedded in the extracellular matrix and on the surface of nearby cells. The signals operate to push the cell either into G0 or into a postmitotic state (usually associated with the acquisition of specific differentiation–associated characteristics) and are thus intimately associated with cell cycle control mechanisms. Cells monitor their external environment during the progression through G1 and, on the basis of external stimuli, decide whether to proliferate, become quiescent, or enter into a postmitotic state.

Most cellular programs that negatively regulate cell growth and proliferation depend on the actions of tumor suppressor genes. At the basic level, most of the antiproliferative signals are funneled through the Rb protein and its close relatives. Disruption of Rb allows cell proliferation and renders the cell insensitive to antiproliferative signals such as that provided by the well-characterized transforming growth factor-β (TGF-β).³² The Rb protein integrates signals from diverse extracellular and intracellular sources and can control cell cycle progression. The other major tumor suppressor is p53, which integrates intracellular signals and can promote either cell cycle arrest or apoptosis (depending on the degree of cellular stress or damage). However, the effects of p53 expression are highly context dependent. Loss of Rb or p53 is associated with the malignant phenotype through the cell’s ability to evade antigrowth signals.

In addition to tumor suppressor gene loss, cells can also evade antigrowth signals by the following alternative cellular programs:

Evading Cell Death: The Roles of Apoptosis, Autophagy, and Necrosis

The growth of any tumor depends not only on the rate of cell division but also the rate of cellular attrition (mainly provided by apoptotic mechanisms). Basic molecular and pathologic studies of tumors have confirmed that acquired resistance toward apoptosis and other types of death is a hallmark of all types of cancer.

Cancer cells, through a variety of strategies, can acquire resistance to cell death and apoptosis. One of the most common ways is through loss of function of the tumor suppressor protein p53. Loss of p53 protein function occurs most often through mutation but can also be via sequestration or inactivation of the protein by viral proteins or by amplification of other oncogenes such as MDM2. Removal of normal p53 function leads to failure of the cells’ sensor mechanism for DNA damage. When the cell suffers an insult such as UV radiation, hypoxia, or exposure to DNA-damaging agents, signals are funneled through p53 to cause either cell cycle arrest or apoptosis. Failure of this mechanism can contribute to the progression of the cell toward malignancy and promoting the accumulation of additional genetic defects that are not corrected at defined checkpoints in cell cycle progression.³⁵

The mechanisms involved in apoptosis are now well established, as are the strategies by which cancer cells evade its actions. However, recently there have been conceptual advances involving other forms of “programmed cell death” as a barrier to cancer development. A notable example is the emerging role that autophagy plays in cancer development. Autophagy is a normal cellular response that operates at low basal levels in cells but can be induced in states of cellular stress such as nutrient deficiency. In autophagy, there is controlled breakdown of cellular organelles that yields energy and cellular substrates that can be used for a variety of cellular functions. Recent evidence suggests that autophagy may be involved in both tumor cell survival and, paradoxically, tumor cell death, depending on the cellular state. The link between apoptosis and autophagy suggests that autophagy may represent another barrier for cells to overcome before they can attain malignancy. In contrast, it has also been shown that irradiation or cytotoxic drug treatment in late-stage tumors may promote autophagy, leading to cells attaining a state of reversible dormancy. The situation seems to suggest that autophagy is a barrier to tumor development in early disease but, in late stage disease, may allow cancer cells to survive severe cellular stress.⁴⁴

In both autophagy and apoptosis, the process does not lead to the release of any “proinflammatory” signals. In contrast, the process of necrosis, observed in larger tumors, causes release of signals that support an influx of inflammatory cells. For many years, this has been considered to be a positive event, helping to expose the immune system to tumor antigens and promote immune destruction. However, recent evidence suggests that some phenotypes of inflammatory macrophages can actually support tumor growth through fostering of angiogenesis, cancer cell proliferation, and invasion. Consequently, our understanding of macrophage phenotypes in cancer progression is an area of active research.

Limitless Replicative Capacity

Over 30 years ago the pioneering observations of Hayflick established that when normal human or animal cells are grown in culture, they demonstrate a finite replicative lifespan. That is, they are capable of a finite number of cell divisions, after which they undergo what has been termed replicative senescence and are incapable of any further cell division. The mechanism underlying the replicative clock that monitors this process has been the subject of intense research. This process has further evoked considerable interest as it is also one of the mechanisms that must be overcome to establish the immortal phenotype that is characteristic of the cancer cell.^45-47

In mammalian cells, the DNA is organized into chromosomes within the nucleus and these are capped by specialized DNA-protein structures known as telomeres. The major function of these structures is protection, but they are progressively eroded at each cell division because of the inability of DNA to completely replicate itself. The result is that there is progressive telomeric attrition as cell populations double. After an estimated 50 cell divisions, cells enter an irreversible (and prolonged) state of cellular senescence (sometimes referred to as mortality stage 1 [M1]). This period is characterized by arrest of proliferation without loss of biochemical function or viability. At the end of this period, cells exhibit altered morphology and chromosomal instability, a state often referred to as crisis (mortality stage 2 [M2]) (Figures 2-5 and 2-6). Thus telomeric attrition is intimately involved with the aging of cells. Cancer cells must overcome replicative senescence and take on an immortal phenotype.

It has now been demonstrated in human tumors and, more recently, tumors of the dog, that telomere maintenance is a feature of virtually all cancer types.^46-53 Tumor cells succeed in telomeric maintenance by the expression of the enzyme telomerase. From studies on cellular senescence, expression of the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of cancer cells and has thus become the most common marker of malignant cells. Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, at the telomeres. In humans, telomerase expression is repressed in most somatic tissues, and telomeres shorten with each progressive cell division. In contrast, telomerase activity is a common finding in many human malignancies, resulting in stabilized telomere length. The telomerase complex consists of an RNA subunit that contains a domain complementary to the telomeric repeat sequence TTAGGG and a catalytic protein component. The catalytic protein component acts as a reverse transcriptase and can catalyze the addition of telomeric repeats onto the ends of chromosomes, using the RNA subunit as a template. It is now well documented that the level of telomerase in malignant tissue compared to normal tissue is much higher, and this differential is greater than that for classic enzymatic targets such as thymidylate synthase, dihydrofolate reductase, or topoisomerase II.

Telomerase biology is complex, and the mechanisms by which telomerase becomes reactivated in tumor cells is the subject of intense research. However, this represents an exciting opportunity for further understanding the complex biology of cancer and also the identification of completely novel targets for therapy.

Reprogramming Energy Metabolism

The sustained growth and proliferation of a cancer requires a corresponding adjustment of energy metabolism to ensure this growth can be fueled. Under normal conditions, cells respire aerobically in that they metabolize glucose to pyruvate with a net gain in energy as ATP.⁴³ Cancer cells can undergo a “metabolic switch” so that glucose is metabolized to lactate, in the presence or absence of oxygen, causing a net energy deficit. There is a corresponding upregulation of glucose transporters (e.g., GLUT-1), which increases uptake of glucose into the cytoplasm. This process is exploited in positron-emission tomography (PET) imaging as tumors will preferentially uptake a radiolabeled analog of glucose (¹⁸F-fluorodeoxyglucose [FDG]). This metabolic switch is sometimes referred to as the Warburg effect (Figure 2-7).⁵⁴

It is difficult to appreciate the survival advantage of this mechanism. One hypothesis is that the switch allows the diversion of glycolytic intermediates into other biosynthetic pathways that support the production of new cells. This is supported by the observation that the Warburg effect can also be detected in growing cells in the embryo. An expansion of the theory of reprogramming of energy metabolism in cancer suggests that cancer cells are advantaged by a flexibility in their ability to derive ATP. This may come from a number of metabolic pathways that derive ATP from glucose metabolism under aerobic and anaerobic conditions but may also include efficiencies in metabolizing amino acids and lipids toward ATP and other biomolecule synthesis. Such metabolic flexibility may be necessary during primary tumor development but even more so during metastatic progression (see next section).

Metastasis-Associated Genes and Metastasis Suppressor Genes

Cancer cells are not unique in their ability to complete the individual steps required for metastasis (Figure 2-8). For example, leukocytes and neuronal cells have the ability to invade tissue planes and cross vascular barriers. Several types of leukocytes demonstrate the phenotype of intermittent adherence to vascular endothelium and are able to resist anoikis. It is also true that stem cells of various phases of differentiation are able to perform many of these steps during development and in the adult.⁵⁶ What is unique about metastatic cells is that each must be able to perform all the steps required for successful metastasis. An extension of this argument is that the genetic changes that permit the metastatic process are not unique to a metastatic cancer cell; however, the metastatic cancer cell must have the appropriate set of genetic changes available to complete all the steps of the metastatic cascade.

Literally hundreds of genes and their resultant proteins have been suggested to contribute to the development of cancers and to their eventual ability to metastasize. It is possible for a single genetic change in cancer to contribute many of the metastasis-associated processes or for several genes to work together toward a single metastasis-associated process. Metastatic cancers may achieve the metastatic phenotype through distinct constellations of genetic and “epigenetic” events that in their respective sums complete the list of necessary metastasis-associated processes needed for successful metastasis. Two classes of genes have been broadly defined as contributing to the metastatic phenotype. These include metastasis promoting genes^57,58 and metastasis suppressors.^59,60 These genes have functions in normal development and physiology (i.e., cell migration, tissue invasion, and angiogenesis discussed earlier) that are subverted by the cancer cell in the acquisition of the metastatic phenotype.

The use of high through-put and genome-wide investigations has uncovered many putative metastasis-associated genes in cancer. It should be noted that many metastasis-associated genes have functions that also contribute to tumor formation and progression. Several of these metastasis-associated genes have been validated in canine and feline cancers.^61,62 For example, the metastasis-associated gene ezrin was identified using genomic approaches in murine and human studies of metastasis.⁶³ Ezrin is a membrane-cytoskeleton linker protein that functionally and physically connects the actin cytoskeleton to the cell membrane. The degree of ezrin expression in the primary tumor of dogs with osteosarcoma was shown to predict a more aggressive course of disease, defined by metastasis to the lung. Furthermore, recent studies have confirmed the connection between ezrin and protein kinase C (PKC) signaling in murine, canine, and human osteosarcoma cells.^64,65

Metastasis suppressor genes have been identified in several human cancers. These genes are thought to also have normal functions in the regulation of motility, invasion, and angiogenesis. The loss of these genes is not thought to be associated with the formation of tumors; however, their loss is thought to contribute to specific steps in the metastatic cascade. The characterization, biology, and clinical impact of metastasis suppressor genes have been recently reviewed elsewhere.⁶⁶ The loss of reduced expression of metastasis suppressors has not been documented in canine or feline cancers at this time.

The following sections provide descriptions of the critical metastasis-associated processes (see Figure 2-8). Examples of genetic changes or resulting protein changes that contribute to each process is highlighted in each section, with particular emphasis on those with demonstrated associations with veterinary malignancies.

Intravasation

Following the successful growth of the primary tumor, intravasation of a cancer cell into the vascular or lymphatic circulation is the first step required during the metastatic cascade. The process of intravasation requires that a tumor cell be motile and able to digest, modulate, or escape the extracellular matrix.^67-69 The specific mechanisms used by cancer cells to invade and intravasate include the classical model of enzymatic degradation of the extracellular matrix referred to as mesenchymal invasion (see later discussion on epithelial-mesenchymal transition). Unlike mesenchymal invasion, tumor cells may also develop so-called amoeboid invasion, in which they individually slip between fibers of the extracellular matrix without evidence or a need for enzymatic degradation.⁷⁰ Finally, a distinct method termed collective invasion refers to the en masse regional extension of a tumor into surrounding tissues.⁷¹ Such collective invasion is observed clinically in dogs with oral SCC and biologically high-grade/histologically low-grade fibrosarcoma. The low rate of distant metastasis associated with collective invasion suggests a lack of functional attributes necessary for true distant metastasis. It is likely that individual metastatic tumors may utilize distinct invasion programs at distinct points during metastatic progression. Not surprisingly, studies with intravital imaging (single cell imaging of cancer cells in animals) have demonstrated that only a minority of the cells in the primary tumor develop any of these forms of invasion.^72,73 Efforts are underway to define the genetic features of this minority population.^74,75

In the classic mesenchymal form of invasion, matrix proteases and metalloproteases (MMPs) are believed to be necessary for the invasive phenotype. Expression of members of this enzyme family have been found in most human and several canine and feline malignancies, including canine osteosarcoma.^76-79 The activity of MMPs in osteosarcoma and mast cell cancers has been correlated with grade and metastatic propensity.^79,80 Similar correlative studies have been undertaken in human patients.⁸¹ The importance of MMP activity during this early step in metastasis prompted the development of pharmacological inhibitors of MMPs. Anticancer activity of these agents in preclinical animal models and in sporadic human patients was evident; however, in randomized trials, the activity of MMPs was not observed.^82,83 The failure of these clinical trials may be explained at many levels and does not refute the importance of invasion as a critical step for a metastatic cell. Rather, these results suggest potential redundancy in the types of invasion (mesenchymal versus amoeboid) that may exist within a given cancer.^84,85 Recent evidence also suggests that the expression of matrix-degrading enzymes and other growth factors may not be necessary in the tumor cells themselves but may be provided by inflammatory cells (i.e., macrophages) recruited by the growing tumor.⁸⁶

Survival in the Circulation (Resisting Anoikis)

Frisch and Francis reported the induction of apoptosis after disruption of the interaction between epithelial cells and the extracellular matrix.⁹³ This phenomenon was termed anoikis, from the Greek word for homelessness. In normal tissues, anoikis is a mechanism for maintaining tissue homeostasis and integrity.⁹⁴ For the metastatic cancer cell, survival during dissemination requires resistance to anoikis. In normal tissues, anoikis is prevented by two systems: cell-matrix anchorage and cell-cell interactions.⁹⁵ Anchorage of cells to the extracellular matrix is mediated primarily by transmembrane receptors referred to as integrins. Formation of active heterodimers trigger an intracellular cascade resulting in activation of effectors of growth and survival.⁹⁶ Integrin family members have been identified in canine sarcomas and lymphomas.^97-102 Cell-cell anchorage in many epithelial tissues is mediated by cadherins, a family of calcium-binding glycoproteins. Intracellularly, cadherins form complexes with members of the catenin protein family that link them to the actin cytoskeleton, as well as survival-promoting signal transduction cascades.¹⁰³ Loss of either cell-cell or cell-matrix interaction in normal cells triggers the activation of the caspase proteases, the hallmark of apoptotic cell death. Metastatic cancer must resist this contact-dependent death in order to be successful and do so through two nonmutually exclusive mechanisms. The first is by maintaining cell-cell contacts with other tumor cells (homotypic interactions) or with host cells such as platelets and inflammatory cells (heterotypic interactions) during metastatic progression. Both homotypic and heterotypic interactions generate intracellular signals that prevent the initiation of anoikis. Additionally, cancer cells may overexpress proteins that directly inhibit anoikis. For example, the integrin pair α_vβ₃ is frequently overexpressed in malignancy, including prostate cancer and melanoma.^104,105 This overexpression subverts the need for ligand binding and results in the generation of survival signals.¹⁰⁶ Proteins reported to be involved in resistance to anoikis include Trk B, focal adhesion kinase (FAK, the immediate effector of integrin signaling), galectin-3, and TGF-β, among others. Certainly more molecular mediators of anoikis resistance have been identified and reviewed elsewhere.¹⁰⁷ These molecules may be valuable antimetastasis targets for novel therapy.

Evasion of the Immune System

At all stages of metastatic progression, metastatic tumor cells must evade detection and destruction by the immune system. The ability of the host immune system to recognize and destroy tumor cells (immunosurveillance) was first proposed by Paul Ehrlich in 1909. Molecular support of the theory of immunosurveillance has come with studies of mice deficient in immunomodulatory and proinflammatory molecules such as interferon-γ (IFN-γ), interleukin-12 (IL-12), and perforin. Mice deficient in these molecules are known to develop tumors more readily than wild-type mice. Clinical evidence for immunosurveillance against cancer was first reported by Coley over 100 years ago. Through the administration of bacteria, Coley’s toxin, Coley was able to induce fever and tumor regression in patients with cancer. Evidence in dogs with osteosarcoma further supports the potential value of cancer immunotherapy.¹⁰⁸ Indeed, survival times in dogs who develop bacterial infection at the site of a limb salvage surgery are significantly longer than in dogs who do not develop infection. Interestingly similar parallels may be seen in human osteosarcoma patients. In immunocompetent hosts, it is believed that immunosurveillance removes a large number of cancer cells from the primary tumor, from the circulation, and at distant metastatic sites. Cancer cells employ a wide variety of mechanisms to effect this evasion. The mechanisms for immunosurveillance and evasion from immunosurveillance by cancer are reviewed elsewhere and are summarized in Chapter 13 of this text.¹⁰⁹

Modification of the immune system to treat cancer continues to be an attractive therapeutic strategy.^110-112 Clinical trials based on this concept have been reported throughout the veterinary literature in dogs with melanoma, soft tissue sarcoma, hemangiosarcoma, osteosarcoma, and others, using a variety of immune-based therapies.¹¹³ Indeed, this principle has been the basis for the development and approval of a therapeutic vaccine directed against a melanoma antigen in dogs with melanoma.¹¹⁴

Arrest in Target Tissues

The arrest of circulating tumor cells at distant sites is thought to occur by two distinct but potentially overlapping mechanisms. These include size-dependent “trapping” of tumor cells within the lumen of small vessels (capillaries and veins) in the target organ, and/or receptor mediated interaction involving the tumor cell and the host vasculature.¹¹⁵ Data supporting the “trapping” phenomenon comes from single-cell imaging studies in which metastatic cancer cells were observed to primarily arrest at distant vascular beds as a result of size-dependent restrictions of large tumor cells in small blood vessels.^116,117 This work was primarily conducted for metastasis to the liver and more recently metastasis to the brain.¹¹⁸ This trapping phenomenon suggests that the site for metastasis from a primary tumor is largely guided by the location of the first (small vessel) vascular bed encountered by a tumor cell. Alternatively, several groups have suggested the role of specific adhesion molecules as being necessary for initial tumor arrest in the microenvironment of the secondary metastatic sites.^119-121 It is likely that both of the mechanisms play a role in the initial seeding of distant metastatic target organs. The dominant mechanism of arrest may be primarily defined by tumor type or target organ.

Early Survival at Distant Sites

Once the cancer cell or cancer embolus has arrested at its distant sites, it may immediately move out of the circulation into the target organ or may stay within the circulation.¹²² In either location, the cancer cell must survive in a new microenvironment. Early survival of metastatic cells at secondary sites is a significant hurdle for the cancer cell. Several studies have shown the ability of cancer cells to arrest at multiple organs in the body. Within hours, the number of remaining cells is dramatically reduced and within days, the number of viable cells may be as low as 0.1% of the original number of cells, even for the most aggressive cancer models.^63,123 The organ selectivity for cancers is to a large extent defined by the organs in which a cancer cell can survive following initial arrest. The “seed and soil” hypothesis first articulated by Paget and then more recently by Fidler et al suggests that the success of a cancer, or metastasis, is defined by interactions between the seed (tumor cell) and the soil (the tumor microenvironment). Arrest of tumor cells in target organs may or may not be receptor mediated (discussed earlier); however, adhesion requires receptor-ligand interactions. Contributors to these steps include the cell adhesion molecules (CAMs). Multiple CAMs have been identified and are named based on their cell specificity (e.g., N-CAM for neural and L-CAM for liver). CD44 is a specific adhesion molecule (H-CAM for homing), initially identified as the receptor for the matrix component hyaluronic acid on hematopoietic cells. Expression of splice variants of CD44 on tumor cells has been demonstrated to correlate with poor prognosis in a wide variety of human tumors from acute myelogenous leukemia (AML) and Hodgkin’s disease to breast and colon cancer and osteosarcoma.¹²⁴ Chemokines are a class of chemotactic cytokines that function in leukocyte trafficking and function. In metastasis, chemokines may also contribute to the metastatic process through enhanced metastatic cell survival.¹²⁵ Chemokines may be active in the recruitment of a leukocytic infiltrate to intravasated, circulating tumor cells, therein creating an embolus that can better resist shearing stress associated with circulation or through the generation of matrix degrading proteins at the distant metastatic site.

The Premetastatic Niche and Modulation of the Microenvironment

It is increasingly clear that the sites (microenvironments) of successful secondary metastasis are modulated by the presence of a primary tumor and then by the arrival of metastatic cells at the secondary site. The concept of the premetastatic niche suggests that a primary tumor modulates the microenvironment of a secondary site before the arrival of most metastatic cells.¹²⁶ The modulation of the premetastatic niche appears to be accomplished by primary tumor-induced mobilization and recruitment of specific primarily bone marrow–derived cells (bone marrow niche) to the secondary microenvironment.¹²⁷ These bone marrow–derived cells are myeloid in origin and express the vascular endothelial growth factor receptor (VEGFR). Interestingly, the sites of metastatic tumor arrest appear to preferentially include sites in which these myeloid-derived cells were first recruited. Furthermore, targeting these VEGFR-positive cells using pharmacologic and genetic tools has effectively prevented metastatic development in murine models.¹²⁷ Ongoing studies will likely uncover greater complexity of the populations of cells recruited to the premetastatic niche and potentially therapeutic targets for antimetastatic therapy (see Figure 1-5).

Following survival at the distant site, the tumor cell must proliferate and modulate its new environment. In most cases, it is believed that cancer cells extravasate from the circulation and then proliferate in the new organ. However, it is also possible for proliferation to occur within blood vessels, in a process referred to as intravascular metastases, and then expand out into the local tissue before further proliferation occurs.¹²⁸ In both situations, modulation of the new environment is necessary for appropriate growth and progression of the metastatic lesions. It is now recognized that part of this modulation is based on tumor-induced changes in the stroma.^129,130 These stromal changes may result in the production of growth factors or signals that are used by the tumor for further growth. These stromal cell–derived growth factors provide important signals for tumor cell proliferation and progression, including angiogenesis. The importance of tumor-stromal interaction has suggested that the “induced” tumor-stroma may be a credible target for novel cancer therapeutics.¹³¹

Angiogenesis

It is now well accepted that the development of new blood vessels from endothelial progenitors (vasculogenesis) or from existing blood vessels (angiogenesis) is required for cancer progression and metastasis.^132,133 Endothelial cells or endothelial progenitors are activated by tumor-derived growth factors and result in new capillaries at the tumor site.¹³⁴ In healthy tissue, endothelial cell proliferation is controlled by a balance between protein factors that activate endothelial cells and those that antagonize activation. Malignant tumors provide signals that result in endothelial cell survival, motility, invasion, differentiation, and organization. These steps are required to create a supportive vasculature for the tumor. In many ways, these required endothelial processes share parallel features with the processes required for the success of a metastatic cancer cell itself. The creation of new blood vessels requires the tumors to recruit circulating endothelial cells to their site, presumably through the release of growth factors such as vascular endothelial growth factor (VEGF). Circulating endothelial cells must survive at their new site with the help of survival signals (e.g., thrombospondin-I [TSP-I]) and form vascular tubes that then reorganize to sustain blood flow. The resulting vasculature of cancers is typically aberrant with often poorly organized and chaotic vascular structures that are leaky, with limited adventitial development and excessive branching.¹³⁵ Once developed, this angiogenic phenotype (the result of the angiogenic switch) is associated with a diverse pattern of ongoing angiogenesis and neovascularization. This ongoing process is likely complex and involves a wide variety of tumor and microenvironmental-derived growth factors and signaling molecules. Adding to this complexity, it is likely that certain phases of cancer progression are associated with and require periods of antiangiogenesis. Indeed, these hypovascularized states may directly contribute to the progression of certain cancers.¹³⁶

Many lines of evidence support the importance of angiogenesis in the biology of metastasis. The vascularity of a primary tumor (measured by microvessel density) has been correlated with metastatic behavior for most human and many veterinary tumors. The expression of angiogenesis-associated growth or survival factors and their receptors (i.e., VEGFR) in serum and in tumors, respectively, has also been correlated with outcome; more recently, functional imaging studies using magnetic resonance imaging (MRI) and other means has provided correlates of vascularity with poor outcome.^137,138 The strength of this biologic argument has supported the development of a number of novel therapeutic agents with antiangiogenic activities. These agents have moved through discovery and development and are now approved drugs for cancer (see Section C in Chapter 14).

Metastasis from Metastases

For most solid tumors the appearance of a single metastatic nodule is followed shortly by the development of additional metastases within the same target organ and tertiary sites. It is unlikely that these metastatic sites all emerge from distinct and unique clones within the primary tumor and nearly simultaneously progress through the metastatic cascade to yield synchronous metastases at the distant site. It is reasonable and perhaps more likely that metastases develop from other metastatic sites. In this hypothesis a small number of successful clones colonize distant sites. As these clones move through progression to become successful metastases, the process of metastasis continues, resulting in metastasis from metastases. Although data supporting this hypothesis are limited, the implication for the treatment and management of cancer patients is substantial. If true, the process of metastasis from metastases would suggest that all steps in the metastatic cascade occur continuously, both before and after detection of metastases in patients. As such, all of the steps in the metastatic cascade may be targets for future therapeutic intervention.

Ongoing Controversies and Areas of Research in the Field of Metastasis

Genome Instability

The majority of the acquired hallmarks of cancer require changes in the genome through mutation, amplification, or chromosomal translocation. However, the process of random mutation is inefficient because of the complex and fastidious maintenance mechanisms of the normal cell that monitor DNA damage and regulate repair enzymes. As such, it is actually difficult to explain why cancers arise in animals at all because the acquisition of all traits would seem an impossible task. To explain this, it may be argued that genomes must attain increased mutability or genome instability in order to overcome the redundant homeostatic mechanisms that ordinarily prevent the emergence of a cancer cell.^1,2

The following mechanisms have been suggested that may support the development of genomic instability⁴³:

Tumor-Promoting Inflammation

For decades it has been recognized that tumors contain inflammatory and immune cell infiltrates that have classically been considered to be an attempt by the immune system to eradicate the tumor. However, recent evidence suggests that tumor-associated inflammation may paradoxically have a tumor-promoting effect. Inflammation can contribute to neoplastic progression through one of the following various mechanisms.^43,152,153

Many of the cells that contribute to this are components of the innate immune system, particularly macrophages with a specific, cancer-promoting phenotype. Specifically, they form part of the tumor microenvironment that supports the maintenance of the cancer phenotype (see Figure 1-5).

The Pathway to Cancer

The eight acquired capabilities of cancer cells and the two overarching enabling characteristics of genome instability and tumor inflammation have been outlined. It is important to stress that the pathways by which cells become malignant are highly variable. Mutations in certain oncogenes can occur early in the progression of some tumors and late in others. As a consequence, the acquisition of the essential cancer characteristics may appear at different times in the progression of different cancers. Furthermore, in certain tumors, a specific genetic event may, on its own, contribute only partially to the acquisition of a single capability, while in others it may contribute to the simultaneous acquisition of multiple capabilities. However, irrespective of the path taken, the hallmark capabilities of cancer will remain common for multiple cancer types and will help clarify mechanisms, prognosis, and the development of new treatments.

• Cancer cells and cancer stem cells (CSCs)

• Endothelial cells

• Pericytes

• Immune cells

• Tumor-associated fibroblasts

Cancer Cells and Cancer Stem Cells

The concept that a cancer can arise from any cell in the body, along with the stochastic model of tumorigenesis, has recently been challenged.^154,155 Although tumor heterogeneity is a well-established concept, a new dimension to heterogeneity has been established that suggests that a tumor may contain a hierarchical structure similar to many normal organ systems. Recent evidence suggests the existence of CSCs (sometimes termed tumor-initiating cells), which form the founder cell population for a tumor (see Figure 1-8). It was first extensively documented for leukemia and multiple myeloma that only a small subset of cancer cells are capable of extensive proliferation. For example, when mouse myeloma cells were obtained from mouse ascites, separated from normal hematopoietic cells, and put in clonal in vitro colony-forming assays, only 1 in 10,000 to 1 in 100 cancer cells were able to form colonies. Even when leukemic cells were transplanted in vivo, only 1% to 4% of cells could form spleen colonies.^156-161 Because the differences in clonogenicity among the leukemia cells mirrored the differences in clonogenicity among normal hematopoietic cells, the clonogenic leukemic cells were described as leukemic stem cells. It has also been shown for solid cancers that the cells are phenotypically heterogeneous and that only a small proportion of cells are clonogenic in culture and in vivo.^162-165 For example, only 1 in 1000 to 1 in 5000 lung cancer, ovarian cancer, or neuroblastoma cells were found to form colonies in soft agar. Just as in the context of leukemic stem cells, these observations led to the hypothesis that only a few cancer cells are actually tumorigenic and that these tumorigenic cells could be considered as CSCs. Although the field and concept of CSCs is a controversial one, CSCs may prove to be a common constituent of many, if not all, cancer types. Features of the CSC model also fit well with a view of metastasis in which only a small number of cells within a tumor have the ability (and plasticity) to endure the stresses of metastatic progression, survive during a dormant period, and then progress, proliferate, and differentiate into the complex heterogenous metastatic lesion.

If tumor growth and metastasis are driven by a small population of CSCs, this might explain the failure to develop therapies that are consistently able to eradicate solid tumors.^154,155 Although currently available drugs can shrink metastatic tumors, these effects are usually transient and often do not appreciably extend the life of patients. One reason for the failure of these treatments is the acquisition of drug resistance by the cancer cells as they evolve; another possibility is that existing therapies fail to kill CSCs effectively.

Stem cell populations in human cancers have been identified in breast, bone, brain, colon, pancreas, liver, ovary, and skin.^154,155 The actual origin of CSCs within solid tumors has not been clarified and may actually vary between tumor types. In some tumors, it may be that the resident adult or somatic stem cell may serve as the tumor-initiating cell, and in other tumors, the initiating cell may be the resident progenitor or transit-amplifying cell. A characteristic of the CSCs is that they undergo asymmetric division and self-renew, providing a continual resident population of highly resistant cancer cells.

Existing cancer therapies have been largely developed against the bulk population of tumor cells because they are often identified by their ability to shrink tumors. Because most cells with a cancer have limited proliferative potential, an ability to shrink a tumor mainly reflects an ability to kill these cells. It seems that normal stem cells from various tissues tend to be more resistant to chemotherapeutics than mature cell types from the same tissues. If the same is true of CSCs, then one would predict that these cells would be more resistant to chemotherapeutics than tumor cells with limited proliferative potential. Even therapies that cause complete regression of tumors might spare enough CSCs to allow regrowth of the tumors. Therapies that are more specifically directed against CSCs might result in much more durable responses and even cures of metastatic tumors. In veterinary oncology, putative stem cell populations have been identified for breast, bone, brain, and liver.^{154,155,166-168} Interestingly, stem cell populations appear to have altered DNA repair pathways, which may explain their resistance to conventional drugs. Identification of these populations, coupled with the availability of microarray and microRNA array technology, is allowing the identification of potential therapeutic targets.

Recent research has linked the acquisition of CSC characteristics with the EMT program, described previously in the section on metastasis.¹⁶⁸ Cells that undergo EMT also take on characteristics reminiscent of the CSC phenotype. For example, they have the ability to self-renew and may support the ability of cells to colonize outside of the primary tumor. One may speculate that the signaling processes that support EMT may also serve to maintain the CSC population within a tumor and may also suggest plasticity in CSC populations.

Endothelial Cells

Much of the heterogeneity in tumors is found in the stromal compartment, and many of these cells are endothelial cells, which form the tumor-associated vasculature. VEGF and fibroblast growth factor are prominent signaling pathways in formation of these vessels. More modern sequencing techniques have also identified new pathways that may represent important signaling systems for neoangiogenesis and may represent therapeutic targets (e.g., notch signaling).

Pericytes

Pericytes are mesenchymal cells that wrap around the endothelial tubing of the blood vessels. Pericytes are considered a major cell type supporting the tumor vasculature.

Immune Inflammatory Cells

As described previously, an environment of chronic inflammation is an important enabling characteristic that supports the acquisition of cancer-related traits. Cells of the innate immune system are particularly crucial for the maintenance of the tumor environment. In particular, tumor macrophages with a specific pro-tumor phenotype can enhance cellular proliferation, invasion, and neoangiogenesis.

Cancer-Associated Fibroblasts

Cancer-associated fibroblasts comprise the supporting structure for many tumors but can also promote invasion, cell proliferation, and neoangiogenesis.⁴³ The importance of the various cell types that make up the tumor microenvironment cannot be overstated. Although there is a complex signaling network between and within cancer cells that maintains the cancer phenotype, superimposed on top of this are the complex signaling networks between stromal components and stromal cells and cancer cells. Although we have considered earlier the potential evolution of cancer cells as they evolve from early disease to late-stage disease, it is quite possible that there is a similar evolution in the supporting structures that is dictated by the cancer itself. For example, incipient tumors may recruit stromal elements, which in turn reciprocate by promoting cell proliferation and angiogenesis. Evolution of the cancer population may then in turn feed back on the stromal population to further reprogram them to support the growing tumor. This of course could be extended to show the role of the stroma in promoting invasion and metastasis. These mechanisms underpin the complexity of understanding cancer pathogenesis as the process is highly dynamic and context dependent.

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