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Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.

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Holland-Frei Cancer Medicine. 6th edition.

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What Makes a Cancer Cell a Cancer Cell?

, MD, PhD.

Phenotypic Alterations in Cancer Cells

Treatment of animals or cells in culture with carcinogenic agents is a means of studying discrete biochemical events that lead to malignant transformation. Studies of cell transformation in vitro, however, have many pitfalls. These “tissue-culture artifacts” include overgrowth of cells not characteristic of the original population of cultured cells (eg, overgrowth of fibroblasts in cultures that were originally primarily epithelial cells), selection for a small population of variant cells with continued passage in vitro, or appearance of cells with an abnormal chromosomal number or structure (karyotype). Such changes in the characteristics of cultured cell populations can lead to “spontaneous” transformation that mimics some of the changes seen in populations of cultured cells treated with oncogenic agents. Thus, it is often difficult to sort out the critical malignant events from the noncritical ones. Malignant transformation can also be induced in vivo, by treatment of susceptible experimental animals with carcinogenic chemicals or oncogenic viruses or by irradiation, but identification of critical biochemical changes in vivo is even more tenuous because it is difficult to discriminate toxic from malignant events and to determine what role a myriad factors, such as the nutritional state of the animal, hormone levels, or endogenous infections with microorganisms or parasites, might have on the in vivo carcinogenic events. Moreover, tissues in vivo are a mixture of cell types, and it is difficult to determine in which cells the critical transformation events are occurring and what role the microenvironment of the tissue plays. Most studies that are designed to identify discrete biochemical events occurring in cells during malignant transformation have therefore been done with cultured cells, because clones of relatively homogeneous cell populations can be studied and the cellular environment defined and manipulated. The ultimate criterion that establishes whether or not cells have been transformed, however, is their ability to form a tumor in an appropriate host animal. The recently developed ability to generate immortalized “normal” cell lines of a given differentiated phenotype from human embryonic stem cells has enhanced the ability to study cells of a normal genotype from a single source.27 Such cell lines may also be generated by transfection of the telomerase gene into cells to maintain chromosomal length.

Over the past 60 years, much scientific effort has gone into research aimed at identifying the phenotypic characteristics of in vitro-transformed cells that correlate with the growth of a cancer in vivo. This research has tremendously increased our knowledge of the biochemistry of cancer cells. However, many of the biochemical characteristics initially thought to be closely associated with the malignant phenotype of cells in culture were subsequently found to be dissociable from the ability of those cells to produce tumors in animals. Furthermore, individual cells of malignant tumors growing in animals or in humans exhibit marked biochemical heterogeneity, as reflected in their cell surface composition, enzyme levels, immunogenicity, response to anticancer drugs, and so on. This has made it extremely difficult to identify the essential changes that produce the malignant phenotype. Recently, however, Hahn and colleagues28 showed that ectopic expression of the human telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) in combination with the oncogenes h-ras and SV40 virus large-T antigen can induce tumorigenic conversion in normal human epithelial and fibroblast cells, suggesting that disruption of the intracellular pathways regulated by these gene products is sufficient to produce a malignant cell. Table 9-1 lists the properties of transformed malignant cells growing in cell culture or in vivo.29

Table 9-1. Properties of Transformed Malignant Cells Growing in Cell Culture and/or in Vivo.

Table 9-1

Properties of Transformed Malignant Cells Growing in Cell Culture and/or in Vivo.

Some of these characteristics may be seen both in transformed cells in culture and in tumors growing in vivo in experimental animals or patients. Some of the characteristics listed in Table 9-1 may also be observed in rapidly proliferating tissues or stem cell populations of undifferentiated phenotypes. In addition, hyperproliferative conditions in patients, such as inflammatory bowel disease or psoriasis, may have some of these characteristics. Thus, for diagnostic purposes, it is important to use a number of characteristics that define the malignant state. The evidence that these phenotypic properties found in transformed cells are related to malignant neoplasia is discussed below.

Immortality of Transformed Cells in Culture

Most normal diploid mammalian cells have a limited life expectancy in culture. For example, normal human fibroblast lines may live for 50 to 60 population doublings (the “Hayflick index”), but then viability begins to decrease rapidly unless they transform spontaneously or are transformed by oncogenic agents. However, malignant cells, once they become established in culture, will generally live for an indefinite number of population doublings, provided the right nutrients and growth factors are present. It is not clear what limits the life expectancy of normal diploid cells in culture, but it may be related to the continual shortening of chromosomal telomeres each time cells divide. Transformed cells are known to have elevated levels of telomerase that maintain telomere length. Transformed cells that become established in culture also frequently undergo karyotypic changes, usually marked by an increase in chromosomes (polyploidy), with continual passage. This suggests that cells with increased amounts of certain growth-promoting genes are generated and/or selected during continual passage in culture. The more undifferentiated cells from cancers of animals or patients also often have an atypical karyology, suggesting that the same selection process may be going on in vivo with progression over time of malignancy from a lower to a higher grade.

Decreased Requirement for Growth Factors

Other properties that distinguish transformed cells from their nontransformed counterparts are decreased density-dependent inhibition of proliferation30 and the requirement for growth factors for replication in culture. Cells transformed by oncogenic viruses have lower serum growth requirements than do normal cells.31 For example, 3T3 fibroblasts transformed by SV40,32 polyoma,33 murine sarcoma virus,33 or Rous sarcoma virus34 are all able to grow in a culture medium that lacks certain serum growth factors, whereas uninfected cells are not.

Cancer cells may also produce their own growth factors that may be secreted and activate proliferation in neighboring cells (paracrine effect), or, if the same malignant cell type has both the receptor for a growth factor and the means to produce the factor, self-stimulation of cell proliferation (autocrine effect) may occur. One example of such an autocrine loop is the production of tumor necrosis factor (TNF) and its receptor TNFR1 by diffuse large cell lymphoma.35 Coexpression of TNF-α and its receptor are negative prognostic indicators of survival, suggesting that autocrine loops can be powerful stimuli for tumor aggressiveness and thus potentially important diagnostic and therapeutic targets.

Loss of Anchorage Dependence

Most freshly isolated normal animal cells and cells from cultures of normal diploid cells do not grow well when they are suspended in fluid or a semisolid agar gel. If these cells make contact with a suitable surface, however, they attach, spread, and proliferate. This type of growth is called anchorage-dependent growth. Many cell lines derived from tumors and cells transformed by oncogenic agents are able to proliferate in suspension cultures or in a semisolid medium (methylcellulose or agarose) without attachment to a surface. This is called anchorage-independent growth. This property of transformed cells has been used to develop clones of malignant cells.36 This technique has been widely used to compare the growth properties of normal and malignant cells. Another advantage that has been derived from the ability of malignant cells to grow in soft agar (agarose) is the ability to grow cancer cells derived from human tumors to test their sensitivity to chemotherapeutic agents and to screen for potential new anticancer drugs.37

Loss of Cell-Cycle Control and Resistance to Apoptosis

Normal cells respond to a variety of suboptimal growth conditions by entering a quiescent phase in the cell division cycle, the G0 state. There appears to be a decision point in the G1 phase of the cell cycle, at which time the cell must make a commitment to continue into the S-phase, the DNA synthesis step, or to stop in G1 and wait until conditions are more optimal for cell replication to occur. If this waiting period is prolonged, the cells are said to be in a G0 phase. Once cells make a commitment to divide, they must continue through S, G2, and M to return to G1. If the cells are blocked in S, G2, or M for any length of time, they die. The events that regulate the cell cycle, called cell-cycle checkpoints, are defined in more detail in the section “Alterations of Cell-Cycle Regulation and Apoptosis”. This loss of cell-cycle checkpoint control by cancer cells may contribute to their increased susceptibility to anticancer drugs. Normal cells have mechanisms to protect themselves from exposure to growth-limiting conditions or toxic agents by calling on these checkpoint control mechanisms. Cancer cells, on the other hand, can continue through these checkpoints into cell-cycle phases that make them more susceptible to the cytotoxic effects of drugs or irradiation. For example, if normal cells accrue DNA damage caused by ultraviolet (UV) or X-irradiation, they arrest in G1 so that the damaged DNA can be repaired prior to DNA replication. Another checkpoint in the G2 phase allows repair of chromosome breaks before chromosomes are segregated at mitosis. Cancer cells, which exhibit poor or absent checkpoint controls, proceed to replicate the damaged DNA, thus accounting for persisting and accumulating mutations.

Changes in Cell Membrane Structure and Function

The cell surface membrane (plasma membrane) plays an important role in the “social” behavior of cells, that is, communication with other cells, cell movement and migration, adherence to other cells or structures, access to nutrients in the microenvironment, and recognition by the body's immune system. Alterations of the plasma membrane in malignant cells may be inferred from a variety of properties that characterize their growth and behavior, for example, the loss of density-dependent inhibition of growth, decreased adhesiveness, loss of anchorage dependence, and invasiveness through normal tissue barriers. In addition, a number of changes in the biochemical characteristics of malignant cells' surfaces have been observed. These include appearance of new surface antigens, proteoglycans, glycolipids, and mucins, and altered cell-cell and cell-extracellular matrix communication.

Alterations in Cell Surface Glycolipids, Glycoproteins, Proteoglycans, and Mucins

Aberrant glycosylation was first suggested as the basis for the tumor-associated determinants of glycolipids by the finding of a remarkable accumulation of fucose-containing glycolipids found in human adenocarcinomas, some of which were identified as lactofucopentaose-III-ceramide, lactofucopentaose-II-ceramide (Lewis A blood group glycolipid), and lactodifucohexose and lactodifucooctose ceramide (Lewis B glycolipid).38 These identifications were confirmed once monoclonal antibodies were used to identify antigens definitively. A number of monoclonal antibodies with preferential reactivity for tumor cells over normal cells react with Lewis blood group antigens, such as Lex, Lea, Leb or their analogs.38

The biochemical characterization or the aberrant glycosylation of glycoproteins was also demonstrated in earlier studies. Early studies detected the presence of high-molecular-weight glycopeptides with altered glycosylation patterns on transformed cells before they were clearly chemically identified.39,40 Later, the chemical basis for some of the changes in tumor cell glycoproteins was attributed to the fact that the N-linked oligosaccharides of tumor cells contain more multiantennary structures than the oligosaccharides derived from normal cells.41

Tumor-associated carbohydrate antigens can be classified into three groups38: (1) epitopes expressed on both glycolipids and glycoproteins; (2) epitopes expressed only on glycolipids; and (3) epitopes expressed only on glycoproteins. To the first group belongs the lacto series structure that is found in the most common human cancers, such as lung, breast, colorectal, liver, and pancreatic cancers. The common backbone structure for these epitopes is Ga1β1→3G1cNAcβ1→3Ga1 (type 1 blood group) or Ga1β1→4G1cNacβ1→3Ga1 (type 2 blood group). The second group of epitopes, expressed exclusively on glycolipids, is mostly on the ganglio or globo series structures. This series of epitopes are expressed abundantly only on certain types of human cancers, such as melanoma, neuroblastoma, small-cell lung carcinoma, and Burkitt lymphoma. The third group of epitopes, seen only on glycoproteins, is the multiantennary branches of N-linked carbohydrates and the alterations of O-linked carbohydrate chains seen in some mucins.

Tumor-associated carbohydrate antigens can also be classified by the cell types expressing them as those (1) expressed on only certain types of normal cells (often only in certain developmental stages) and greatly accumulated in tumor cells; (2) expressed only on tumor cells, for example, altered blood group antigens or mucins; and (3) expressed commonly on normal cells but present in much higher concentrations on tumor cells, for example, the GM3 ganglioside in melanoma and Lex in gastrointestinal cancer.38

A variety of chemical changes in tumor cells have been identified that can explain altered glycosylation patterns. These result from three kinds of altered processes: (1) incomplete synthesis and/or processing of normally existing carbohydrate chains and accumulation of the resulting precursor form; (2) “neosynthesis” resulting from activation of glycosyltransferases that are absent or have low activity in normal cells; and (3) organizational rearrangement of tumor cell membrane glycolipids.42

Moreover, the glycosyl epitopes found in glycolipids and glycoproteins make up micro-domains that are involved in cell adhesion and signal-transduction events. They function as a “glycosynapse” (analogous to the “immunologic synapse”) in mediating these events.43 The cell motility, altered adhesive properties, and invasiveness observed in cancer cells are regulated by these glycosynapse complexes.43

Interest in the carbohydrate components of cell surface glycolipids, glycoproteins, and proteoglycans has been heightened by the fact that many of the monoclonal antibodies developed to tumor-cell-associated antigens recognize these carbohydrate moieties or peptide epitopes exposed by altered glycosylation. Moreover, many of these have turned out to be blood group-specific antigens or modifications of blood group-specific antigens, some of which are antigens that are seen at certain stages of embryonic development and thus fit the definition of oncodevelopmental antigens. Thus, the field of “chemical glycobiology” is making significant contributions to our understanding of the cell surface biochemistry of normal and malignant cells.44

Role of Glycosyl Transferases and Oligosaccharide-Processing Enzymes

The substitution of additional carbohydrate moieties on blood group-related structures is not the only aberrant modification of glycoproteins or glycolipids observed in cancer cells. Increased branching of asparagine-linked oligosaccharides and incomplete processing of these oligosaccharides have also been noted in certain cell surface as well as secretory glycoproteins.45,46 The increased activity of specific N-acetylglucosaminyl transferases in tumor cells appears to be responsible for the appearance of tri- and tetra-antennary structures, whereas the analogous glycoprotein in normal cells is often a biantennary structure. Unusually high expression of N-acetylglucosaminyltransferase IVa has been observed in human choriocarcinoma cell lines and may be the enzymatic basis for the formation of abnormal biantennary sugar chains on HCG (human chorionic gonadotropin).47 Similarly, the extra fucosylations that appear on membrane glycoproteins and glycolipids are associated with the induction of an unusual α-fucosyltransferase in chemical carcinogen-induced precancerous rat liver and in the resulting hepatomas.48 These investigations strongly suggest that the regulation of glycosyltransferase genes is important in malignant transformation. Other changes in glycosyl transferase activities include a decrease in β1,3-galactosyltransferase β3 Gal-T5 in human adenocarcinomas, as compared to normal colon.49

All these data strongly support the idea that glycosylation patterns change during transformation of normal cells into malignant ones. Because cell-cell interactions, adhesion to extracellular matrices, regulation of cell proliferation, and recognition by the host's immune system are all profoundly affected by the composition of the cell surface, the entire social behavior of a cell could be altered by such changes.

Additional evidence for the importance of glycosylation patterns of cell surface glycoproteins and glycolipids in the malignant phenotype comes from the use of glycosylation inhibitors and oligosaccharide-processing inhibitors. For example, tunicamycin, an inhibitor of addition of N-linked glycans to nascent polypeptide chains, castanospermine, an inhibitor of glucosidase, and KI-8110, an inhibitor of sialyltransferase activity, all reduce the number of lung metastases in murine experimental tumor models.50–52 In addition, swainsonine was shown to reduce the rate of growth of human melanoma xenografts in athymic nude mice,53 and castanospermine was observed to inhibit the growth of v-fms oncogene-transformed rat cells in vivo.54 These results support the hypothesis that the synthesis of highly branched complex-type oligosaccharides are associated with the malignant phenotype and may provide tumor cells with a growth advantage.


Mucins are a type of highly glycosylated glycoproteins that a variety of secretory epithelial cells produce. They are 50%; to 80%; carbohydrate by weight and function to lubricate and protect ductal epithelial cells. They contain O-linked glycans (serine- and threonine-linked) of various lengths and structures, depending on the tissue type in which they are produced. They are made in a wide variety of tissues, including the gastrointestinal tract, lung, breast, pancreas, and ovary, and tumors arising in these organs may have an altered glycosylation pattern that distinguishes them from the normal mucins and renders them immunogenic.

Total expression of the mucins is increased in many cancers and upregulated in some normal tissues under different physiologic states (eg, lactating mammary gland).55 Increased expression of muc1 has been observed in most adenocarcinomas of the breast, lung, stomach, pancreas, prostate, and ovary. Although muc1-encoded mucin has been the most extensively studied, cancer-related alterations in other mucins have been observed. Moreover, it appears that some cells, both normal and cancer, can express more than one mucin. Focal aberrant expression of muc2 and muc3 has been frequently observed in a variety of adenocarcinomas.56 However, in general, mucin genes appear to be independently regulated and their expression is organ and cell-type specific.56

There is evidence for host immune recognition of the breast cancer mucin, in that cytotoxic T lymphocytes isolated from breast cancer patients recognize a mucin epitope expressed on the breast cancer cells.57 The immune-recognized epitope involves the core protein that appears to be selectively exposed on breast, ovarian, and other carcinomas. It has also been demonstrated that patients can produce antibodies to cancer mucins,55 and this is the basis for the proposal that glycopeptides, on the basis of the aberrantly processed mucins of cancer cells, may have some utility as tumor vaccines. Clinical trials of mucin-derived vaccines are underway.58 Some mucin antigens are shed from tumor cells and can be detected in the sera of patients with pancreatic, ovarian, breast, and colon cancers. These include CA19-9, CA125, CA15-3, SPan-1, and DuPan-2 that are currently being used as tumor markers.59


The proteoglycans are high-molecular-weight glycoproteins that have a protein core to which are covalently attached large numbers of side chains of sulfated glycosaminoglycans as well as N-linked and/or O-linked oligosaccharides. They are categorized on the basis of their glycosaminoglycans into several types, including heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate.60 The glycosaminoglycans have different repetitive disaccharide units bound to the core protein through a common glycosaminoglycan linkage region: G1cNAcβ1→3Ga1β1→3Ga1β1→4Xy1β1-O-Ser. The structure of the sulfated glycopeptides from the carbohydrate-protein linkage region of some of the proteoglycans has been determined.61

Proteoglycans interact via their multiple binding domains with many other structural macromolecules, giving them the capacity “to function as a multipurpose ‘glue’ in cellular interactions.”62 They bind together extracellular matrix (ECM) components, such as hyaluronic acid, collagen, laminin, and fibronectin; mediate binding of cells to the ECM; act as a reservoir for growth factors; and “present” growth factors to growth factor receptors on cells. The proteoglycans also act as cell adhesion factors by promoting organization of actin filaments in the cell's cytoskeleton. Proteoglycans have been shown to undergo both quantitative and qualitative changes during malignant transformation, and alterations have been reported in breast, colon, and liver carcinomas, in glioma cells, and in transformed murine mammary cells and 3T3 fibroblasts.

Two putative tumor-suppressor genes are glycosyl transferases required for the biosynthesis of the proteoglycan heparan sulfate.63 Mutations of these genes, called ext1 and ext2, have been associated with the development of skeletal dysplasias, and these findings suggest that alterations in the synthesis of heparan sulfate precursor polysaccharide are involved in dysregulation of heparan sulfate production and function in tumor formation.

Modification of Extracellular Matrix Components

The ECM plays a key role in regulating cellular proliferation and differentiation. In the case of tumors, it is now clear that development of a blood supply and interaction with the mesenchymal stroma on which tumor cells grow are involved in their growth, invasive properties, and metastatic potential. This supporting stromal structure is continuously remodeled by the interaction between the growing tumor and host mesenchymal cells and vasculature.

The ECM components include collagen, proteoglycans, and glycoproteins, such as fibronectin, laminin, and entactin. The ECM forms the milieu in which tumor cells proliferate and provides a partial barrier to their growth. Basement membranes are a specialized type of ECM. These membranes serve as a support structure for cells, act as a “sieving” mechanism for transport of nutrients, cellular metabolic products, and migratory cells (eg, lymphocytes), and play a regulatory role in cell proliferation and differentiation.64 Basement membranes also prevent the free passage of cells across them, but there are mechanisms that permit the passage of inflammatory cells. It is also clear that basement membranes act as regulators of cell attachment, through cellular receptors called integrins (see below). There is also “cross-talk” between epithelial cells and their ECM to create a microenvironment for accurate signal transduction for growth factors and other regulatory molecules. It has been shown, for example, that exogenous reconstituted basement membranes stimulate specific differentiation of a variety of cell types, including mammary cells, hepatocytes, endothelial cells, lung alveolar cells, uterine epithelial cells, Sertoli cells, and Schwann cells.65

The basement membrane barrier can be breached by tumor cells that release a variety of proteases, glycosidases, and collagenases that have the ability to degrade various components of the matrix and thus allow tumor cells to invade through tissue barriers and blood vessel and lymph channel walls. In addition, malignant cells themselves have receptors for and/or can produce certain components of the matrix; this capability enables them to bind to the vascular endothelium and may be involved in their ability to metastasize. Tumor cells may also release polypeptide factors that can modulate the type of proteoglycans produced by host mesenchymal cells. For example, normal fibroblasts produce proteoglycans containing an unusual amount of chondroitin sulfate when they are exposed to conditioned growth medium from cultured human colon cancer cells.66

Cell-ECM and Cell-Cell Adhesion

Cells in tissues are attached to one another and to the ECM. Disruption of these adhesion events leads to increased cell motility and potential invasiveness of cells through the ECM. In addition, most cell types require attachment to the ECM for normal growth, differentiation, and function. This attachment is responsible for what was termed “anchorage dependence.” Normal cells that are detached from their binding to the ECM undergo apoptosis, whereas tumor cells that are less dependent on this attachment are free to proliferate, wander, and invade tissues.

Cell adhesion to the ECM is mediated by cell surface receptors called integrins. Integrins are a family of proteins consisting of αβ heterodimers that are integral membrane proteins with a specific arginine, glycine, aspartic acid (RGD) amino acid sequence involved in binding to the ECM.67 Integrins also link the external ECM cytoskeleton to the intracellular actin cytoskeleton, and via this connection a linkage to control of gene expression in the cell nucleus is established. In this way, cell-ECM interactions can control gene read-out involved in cell differentiation and function. Cell-ECM interactions occur via focal adhesions that consist of clusters of ECM-bound integrins, and these, in turn, connect to actin fibrils and the signal-transduction machinery inside the cell. These signaling pathways include the focal adhesion kinase (FAK) pathway that participates in the control of anchorage dependence, and growth factor signaling pathways, such as the ras-raf-mitogen-activated kinase, protein kinase C, and phosphatidylinositol 3-kinase pathways.68 Thus, integrins cooperate with growth factors to enhance mitogenic signaling. Alterations in integrin receptor expression have been observed in chemically transformed human cells and in human colon and breast cancer tissue.69

There are in vivo data in human cancers that indicate the importance of the local environment in tumor invasion. For example, lymph nodes in patients with head and neck squamous cell carcinomas had identical detectable genetic defects as “normal” mucosa in the areas where head and neck cancers would be expected to occur based on the lymphatic drainage.70 Cancers did eventually develop in some of the histologically normal-looking epithelium, suggesting that the local epithelial environment moderated expression of the malignant phenotype and that this moderating influence was lost once the tumor cells escaped and spread to local lymph nodes.

Cell-cell interactions are also important for the normal regulation of cell proliferation and differentiation. These interactions are mediated by a family of molecules called cell adhesion molecules (CAMs), which act as both receptors (on one cell) and ligands (for another cell). The expression of CAMs is programmed during development to provide positional and migratory information for cells. A large family of CAMs has been identified. One group of these, called cadherins, comprise a superfamily of Ca2+-dependent transmembrane glycoproteins that play an essential role in the initiation and stabilization of cell-cell contacts. Regulation of cadherin-mediated cell-cell adhesion is important in embryonic development and maintenance of normal tissue differentiation.71,72

The extracellular domain of various cadherins is responsible for cell-cell homotypic binding (a given cadherin domain for a given cell type), and the conserved cytoplasmic domains interact with cytoplasmic proteins called catenins. Each cadherin molecule can bind to either β-catenin or γ-catenin, which, in turn, bind α-catenin. α-Catenin links the cadherin complex to the actin cytoskeleton. Cell lines that lack α-catenin lose normal cell-cell adhesiveness, and tumor cells with mutated or downregulated α-catenin have increased invasiveness.73

E-cadherin is the predominant type of cadherin expressed in epithelial tissue. Alterations of E-cadherin expression and function have been observed in human cancers.74 In addition, downregulation of E-cadherin correlates with increased invasiveness, metastasis, and poor prognosis in cancer patients. Suppression of this invasive phenotype can be achieved by transfection of E-cadherin cDNA (complementary deoxyribonucleic acid) into carcinoma cells, and contrarily, invasiveness of E-cadherin gene-transfected cells can be restored by exposure of the cells to E-cadherin antibodies or an E-cadherin antisense ribonucleic acid (RNA).74 Germ line mutations of the E-cadherin gene (cdh1) have been found in New Zealand Maori families with a dominantly inherited susceptibility to gastric cancer.75

The cellular binding protein for E-cadherin is β-catenin, the intracellular location of which can be cell membrane associated, cytoplasmic, or nuclear. Early mutations in the human colon cancer progression pathway affect the cellular distribution of β-catenin. In patients with colon cancer, the normal colonic epithelial cells adjacent to neoplastic lesions had mostly cell surface membrane-associated expression of β-catenin, whereas cytoplasmic expression of β-catenin was observed in aberrant crypt foci.76 Nuclear expression was observed in more advanced dysplasias and increased as adenomas progressed to carcinomas. These latter changes are also observed in less-well-differentiated areas of tumors and are accompanied by loss of E-cadherin expression at the invasive front of breast carcinomas, possibly as a consequence of hypermethylation of the E-cadherin promoter.77

Production of Lytic Enzymes

Transformed malignant cells in culture and human cancer cells in vivo produce a variety of lytic enzymes that degrade the ECM and allow cancer cells to invade tissues, lymphatic channels, and the vasculature. These proteases include plasminogen activator, cathepsins, adamalysin-related membrane proteases, and a number of matrix metalloproteases (MMPs).78 The MMPs are a large family of proteases that includes collagenases (MMPs 1, 2, and 9) and stromelysins (MMPs 3 and 11). Collagenases have been found at elevated levels in melanoma and in cancers of the colon, breast, lung, prostate, and bladder. Usually, these elevated levels correlate with higher tumor grade and invasiveness. MMP-2 levels are significantly elevated in the serum of patients with metastatic lung cancer, and in those patients with high levels, response to chemotherapy is diminished.79

Genetic Alterations in Cancer Cells

Suffice it to say here that cancer at the cellular level is essentially a genetic disease, in that all cancer cells have some alteration of gene expression. These genetic alterations include chromosomal translocations and inversions, gene deletions, gene amplifications, point mutations, and duplications or losses of whole chromosomes. Much of the information about genetic alterations in cancer has been gleaned from studies of leukemias and lymphomas because it is easier to obtain relatively pure populations of cells. Nevertheless, a significant amount of information has been obtained about genetic changes in solid tumors, in which gene deletions (eg, loss of tumor-suppressor gene function) and oncogene activation (eg, K-ras mutations) are a common phenomenon.

Alterations in Chromatin Structure and Function

Chromatin in higher organisms is organized into nucleosomes that are tuna-fish-can-shaped structures made up of two molecules each of the core histones H2A, H2B, H3, and H4, forming an octamer core around which approximately two turns of DNA are wrapped. In a tightly wrapped conformation, DNA transcription into messenger ribonucleic acid (mRNA) is inhibited. The initiation of gene transcription requires a partial unwrapping of this octamer core, which is regulated by biochemical alteration of the core histones. The mechanism involved in this is still only partially understood, but it involves chemical modifications that regulate the acetylation, methylation, phosphorylation, and ubiquitination states of histones. Combinations of these covalent histone modifications have been called the “histone code,” by which is meant that specific alterations of histones at specific times in the cell cycle “mark” histone tails by these chemical modifications in a way that enables them to recruit or “de-recruit” other chromatin modifying proteins such as transcriptional coactivators or repressors.80 Some of the histone modifications appear to involve reciprocal alterations that affect DNA transcription. For example, Nakayama and colleagues81 have shown in fission yeast that histone H3 methylation on lysine-9 is linked to H3 deacetylation on lysine-14, both of which events are necessary for formation of heterochromatin, the form that is inactive in transcription. In contrast, the “on” state of chromatin active in gene transcription is related to acetylation of lysine-14 and phosphorylation of serine-10.80

Other similar reciprocal chromatin activating and deactivating events have been observed in mammalian (HeLa) cells, in which methylation of arginine-3 of histone H4 facilitates subsequent acetylation of H4 amino acid “tails,” leading to transcriptional activation of nuclear hormone receptor.82

Some gene-silencing events require both histone deacetylation and DNA methylation. Methylation of DNA by DNA methyltransferase recruits methyl-binding proteins and histone deacetylases. This coupling of DNA methylation and histone deacetylation correlates with silent transcriptional regions in chromatin.83 These processes of controlling chromatin structure and function are key to understanding cell differentiation and the altered gene expression that occurs in malignant transformation.

Some of the genes involved in the acetylation and deacetylation of histones have been identified.84 There are two categories of acetylation genes: hat1 and hat2. Acetylation of histone H4, for example, reduces the affinity of the histone aminoterminal tail for DNA and allows a reduction of DNA wrapping around the histone octamer and a subsequent decrease in the tightness of nucleosome packaging. This makes more DNA sequences available for transcription. Deacetylation of histone H4 by deacetylases (HDAC1 and HDAC2), on the other hand, increases affinity of H4 for DNA and results in tighter packing of nucleosomes and less transcription. Mutations in yeast deacetylases have been identified that allow H3 and H4 acetylation to be maintained. This would be expected to result in constitutively unfolded regions of chromatin and increased gene transcription. Disruption of deacetylase activity that alters expression of many genes in yeast, as well as in mammalian cells, has been observed.84 Mutations in histone acetylases, deacetylases, and components of these complexes have significant effects in yeast cells, and similar mutations may have implications for human disease, including cancer. Recent data have shown that members of the HDAC1 and HDAC2 family of genes belong to a network of genes coordinately regulated and involved in chromatin remodeling during cell differentiation.85

In addition to acetylation, phosphorylation of histones is also important for chromatin structure and function.86 A fifth histone, H1, interacts with DNA, links adjacent nucleosome cores, and further condenses chromatin structure. Phosphorylation of H1 is thought to play a role in increased gene transcription. Phosphorylation of histone H3, on the other hand, is required for proper chromosome condensation and segregation during mitosis.86 In addition, during the immediate-early response of mammalian cells to mitogens, histone H3 is rapidly and transiently phosphorylated by a kinase called Rsk-2.87 This suggests that chromatin remodeling is part of the cascade involved in mitogen-activated protein kinase-regulated gene expression.

A “cancer-chromatin connection” is implicated by the observations relating to the role of the tumor-suppressor gene rb in the regulation of the histone deacetylase HDAC1.88 RB acts as a strong transcriptional repressor by forming a complex with the transcriptional activating factor E2F and HDAC1, tethering these activities to E2F-responsive promoters, including the cyclin E promoter region. Repression of E2F-bound promoters by RB is released by mitogenic signals that activate cyclin-dependent kinase phosphorylation of RB, thereby releasing RB from the complex and allowing histone acetylation to occur. This increases accessibility of gene promoter sequences to transcriptional activators. Point mutations of the rb gene observed in some tumors abolish RB-induced repression and RB-associated deacetylase activity, allowing increased E2F-mediated gene expression. Viral oncoproteins can disrupt the interaction between RB and HDAC1. In addition, nonliganded retinoic acid receptors (RARs) have been shown to repress transcription of target genes by recruiting the histone deacetylase complex to these genes.89 Mutant forms of RAR-α result from chromosomal translocations seen in human acute promyelocytic leukemia (APL). These mutant forms prevent appropriate deacetylase activity and result in dysregulated gene activation. This dysregulation can be diminished by all-trans-retinoic acid, at doses that induce APL cell differentiation. These findings suggest that oncogenic alterations in RARs mediate leukemogenesis via aberrant regulation of the histone acetylation state.

DNA Methylation

Methylation of DNA on cytosine in CpG islands is another mechanism for regulating gene expression. In general, though not always, hypermethylated DNA sequences are less expressed, and hypomethylated sequences are more expressed. CpG islands are short sequences rich in CpG dinucleotides found in the 5'-regulatory regions of about half of all human genes. Alterations in DNA methylation patterns have been observed in tumor cell lines, animal tumor models, and primary human cancers. Feinberg and colleagues90 observed an average of 8%; to 10%; reduction in genomic 5-methylcytosine content in colon adenomas and adenocarcinomas. Interestingly, three patients with the highest 5-methylcytosine content in their normal colon appeared to have a germ line predisposition to cancer (Lynch syndrome). Hypermethylation of DNA is postulated to be involved in the loss of tumor-suppressor gene function. Hypermethylation of the regulatory sequences of some of those genes, including p53, p15, p16, E-cadherin, vhl, and hmlh1, has been observed, but whether this is a cause of tumor-suppressor gene silencing is still unclear. Aberrantly methylated CpG sequences have been detected in serum and tissue of patients with colorectal,91 non-small-cell lung,92 liver,93 and prostate94 cancers.

DNA methyltransferase activity has been reported to be overexpressed in a number of human cancer cell lines and tissues, although the incidence and extent of this is still being debated.95 So far, three DNA methyltransferases have been detected in mammalian cells,95 and the activity of one of these, DNMT1, is threefold higher in fos oncogene-transformed fibroblasts than in normal fibroblasts, and the transformed cells contain more 5-methylcytosine than normal fibroblasts.96 Transfection of the dnmt1 gene into fibroblasts induces transformation, whereas inhibition of dnmt1 expression by an antisense oligonucleotide reverses fos-induced transformation. These results suggest that oncogene-induced malignant transformation is mediated through alterations in DNA methylation.

Loss of Heterozygosity

Deletion of genetic material is a very common event in human cancer. Indeed, it is the most frequently observed genetic abnormality in solid tumors. These deletion events often involve loss of heterozygosity (LOH) of the expression of either the maternal or paternal alleles of a gene. If this is accompanied by mutation of the remaining allele, as is sometimes the case for a tumor-suppressor gene such as p53, an important mechanism to regulate cell proliferation and differentiation is lost. An early observation of LOH in human cancer was by Solomon and colleagues,97 who showed that about 20%; of human colorectal cancers had undergone allelic loss on chromosome 5q. Fearon and his colleagues subsequently reported how a series of genetic alterations, including LOH of alleles at chromosomal regions 5q (apc gene), 17p (p53 gene), and 18q, are involved in progression of colorectal cancer.98

It is now recognized that LOH occurs in most, if not all, human solid tumors and may involve up to 20%; of the genome. In some cancers, including lung, ovarian, and colorectal cancers, LOH is an early event and may occur at the stage of dysplasia or carcinoma in situ. The prevalence of LOH differs at different positions within the genome and is more prevalent at certain “hot spots.” Frequently involved allelic loss occurs in cancer cells on chromosomes 3p, 5q, 7q, 8q, 9p, 13q, 17p, and 18q. These losses often involve regions containing tumor-suppressor genes. The tumor-suppressor gene functions contained in these regions include p53, brca1, rb, brca2, apc, vhl, and p16. LOH is detected using molecular genetic techniques such as restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR). It is of interest that the same genes that have undergone LOH in hereditary cancers frequently also undergo LOH in “spontaneous” cancers.

Loss of Genomic Imprinting

Genomic imprinting is an epigenetic modification of the genome that allows only the maternal or paternal allele of a gene to be expressed. So far, approximately 30 mammalian genes are known to be imprinted.99 In a number of human cancers, loss of imprinting (LOI) occurs, allowing both the maternal and paternal alleles to be expressed. If this occurs for a growth factor, such as insulin-like growth factor-2 (IGF-2), cells get a double dose of a growth stimulatory signal. LOI of IGF-2 has been observed in approximately 45%; of a series of patients with colorectal cancer.100 Interestingly, this LOI could also be detected in patients' circulating leukocytes, suggesting that this is an alteration that precedes the onset of neoplasia and could be used as a screening test for cancer susceptibility. Somewhat paradoxically, LOI can be reversed by drugs that are DNA methyltransferase inhibitors, such as 5-aza-2-deoxycytidine, suggesting that an aberrant DNA methylation event induces LOI.101 LOI of the igf2 gene appears to be involved in tumor progression, leading to a more invasive phenotype.102

Telomeres and Telomerase

Normal human cells undergo a finite number of cell divisions when grown in culture and ultimately stop dividing and undergo what is called “replicative senescence.” For human cells, the number of cell divisions attained before senescence ensues is approximately 50.103 One difference between young, replicating cells and their senescent counterparts is the length of specialized “tails” at the end of chromosomes, called telomeres. In human cells, telomeres are made up of an average of 5,000 to 15,000 base-pair repeats containing the sequence (TTAGGG)n together with telomere-binding proteins.104 Younger cells have the longer telomeres. Every time a cell divides, 50 to 100 base pairs are lost, and a cellular signal is eventually triggered to stop cell division.

Cells of higher eukaryotic organisms maintain telomere length by the activity of an enzyme complex called telomerase. This is a ribonucleoprotein complex that contains several proteins and RNA. The catalytic component of this complex is a reverse transcriptase, hTERT, that uses the RNA contained in the complex as a template for reverse transcription to replicate the DNA sequences in the telomere. Germ cells and pluripotent tissue stem cells have telomerase activity; however, telomerase is turned off in cells from most tissues as they differentiate. Most human cancers appear to be able to reactivate telomerase activity, thus rejuvenating their proliferative capacity105; however, 10%; to 15%; of human cancers do not express telomerase and apparently maintain telomere length by a different mechanism.106 Telomerase has been a hot target for both diagnostic and therapeutic approaches to cancer. A problem with the use of telomerase inhibitors for cancer therapy is the slow onset of action of such agents because tumor cells can continue to proliferate until telomere length reaches a critical length. Moreover, normal stem cells, such as those involved in hematopoiesis and wound healing, are negatively affected by telomerase inhibition.106 There are also data indicating that restoring telomerase in human cells extends their lifespan,107 suggesting that senescence can be overcome and perhaps providing a way to maintain human stem cells for replacement of aging or damaged tissues.

DNA Repair

DNA repair mechanisms are covered extensively in Chapters 2 and 5. It is sufficient to note here that a number of biochemical mechanisms are invoked by human cells when their DNA is damaged by internal metabolic events (eg, oxidative stress, cytosine deamination) or exogenous factors (eg, chemical carcinogens, irradiation). These repair mechanisms include (1) photoactivation repair for removal of UV-induced pyrimidine dimers; (2) strand-break repair for excision and repair of a length of DNA sequence; (3) base-excision repair producing apurinic or apyrimidinic sites in DNA; (4) nucleotide excision repair; and (5) O6-alkylguanine-DNA alkyltransferase that recognizes and removes small alkyl adducts from DNA.

DNA repair is usually very accurate, but if repair cannot occur prior to or during DNA replication, it may be error prone, potentially leading to a mutagenic and carcinogenic event. A number of inherited defects in DNA repair systems predispose individuals to getting cancer. These syndromes include xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, Bloom syndrome, and Cockayne syndrome.

Alterations of Cellular Differentiation in Cancer

A cancer develops from cells that are capable of dividing. All tissues in the body contain some cells that can divide and renew themselves. A subset of the cell population in any tissue can differentiate into the functional cells of that tissue. The normal process of cellular differentiation ultimately leads to an adult, fully differentiated, “dead-end” cell that cannot, under ordinary circumstances, divide again. These fully differentiated cells are the workhorse cells in most tissues in the body. Under circumstances that are not clearly understood, cells that have the potential to divide can be changed by interaction with carcinogenic agents into a cell type that is capable of continued proliferation and thereby is prevented from achieving the normal state of complete differentiation. The carcinogen-altered cell is said to have undergone malignant transformation. Somehow, the genes controlling cell proliferation are locked in the “on” position when they should be in the “off” position, and the genes controlling differentiation are either not expressed or are expressed only imperfectly. What we need to know to understand carcinogenesis and to develop ways of preventing or curing cancer, then, is contained in the mechanisms of normal cellular differentiation. Only by understanding these mechanisms can the manner in which cells are altered during malignant transformation be ascertained.

Differentiation is the sum of all the processes by which cells in a developing organism achieve their specific traits. By acquisition of these special traits, progeny cells are distinguishable from their parent cells and from each other. Somatic cells that share a set or a subset of structural and functional characteristics become organized into tissues in higher organisms. Indeed, cellular differentiation is the sine qua non of multicellular life.

The process of differentiation appears to be fairly permanent, in that as tissues develop, some cells retain the capacity to divide, whereas others divide and then differentiate into cells with a more restricted phenotype.108 These latter cells are then said to be pluripotent rather than totipotent, that is, they are now committed to develop into one of the cell types peculiar to their tissue of origin. Embryologists have traditionally defined the commitment of a cell to one general pathway of differentiation rather than another as determination. They reserve the term differentiation for the final events in which a terminally differentiated cell arises from a pluripotent one. However, biochemically, this is probably an artificial distinction because the total process most likely represents a continuum of biochemical and molecular events leading from a totipotential cell to a terminally differentiated one. The final characterization of differentiation requires the identification of the particular biochemical events that lead to the uniquely specialized adult cell. By definition, the process of differentiation requires a heritable alteration in the pattern of gene readout in one of the two progeny cells arising from the same parent cell. Because all the cells in the body are derived from a single cell, the fertilized ovum, this process must entail the expression of characteristics in one progeny cell that are not expressed in the other progeny cell from the same parent cell, and this process must continue to occur throughout embryonic development to generate the wonderful diversity of cell types present in the adult organism.

The discovery of human embryonic stem cells27 and embryonic germ cells,109 and recent data showing that even adult tissues such as brain, liver, and muscle contain stem cells that can be induced to differentiate into a variety of cell types,110 has drastically altered the whole question: What is a stem cell? These findings indicate that the plasticity of certain cells in adult tissues is far beyond what was imagined a few years ago. It also raises more cogently the question of what is the target cell for the malignant transformation process. If these pluripotent stem cells are lurking in all tissues in the body, why aren't all tissues subject to the same propensity to undergo carcinogenesis? Yet we know that brain tumors and sarcomas, although not rare, occur much less often than epithelial cell tumors (carcinomas) and that cancers of heart muscle are very rare. One could, of course, argue that muscle cells and brain cells are less exposed to environmental agents and hormonal influences that can activate the carcinogenic process. There are data, however, that damage to these tissues can activate endogenous stem cells to proliferate and move to areas of the lesion.111 These data imply that these same cells would also be susceptible to carcinogenic damage because they are the cells that have the proliferative capacity to undergo mutagenic damage as they replicate their DNA. This also raises the question of the ability of cells in adult tissues to dedifferentiate in response to tissue damage and the question of whether stem cells in adult tissues are really dedifferentiated adult cells. This isn't just an academic question because how one approaches the field of cancer prevention, initiation, and progression depends on the cell type being targeted for prevention or antiprogression strategies. The answer to this question also determines the ability to identify early, initiated cells, or dormant vs aggressive tumor cells. For example, the genes that are up- or downregulated and the timing of altered gene expression would likely be different in initiated, undifferentiated stem cells, as opposed to dedifferentiated adult cells.

Getting to Know All the Players

The process of early development is a complicated one, and there are some similarities and some differences among various multicellular organisms. The biochemical signals and genes involved, however, show a lot of evolutionary conservation. Various polypeptide growth factors have been shown to play a role in early morphogenesis.112 For example, in early Xenopus development, there are a series of inductive events that involve growth factors, whose actions lead to differentiation of mesoderm at the interface between the animal and vegetal poles of the embryo. This induction is most efficiently achieved by a combination of members of the fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β) families of growth factors. In Xenopus, Drosophila, and developing chick limb buds, members of the FGF and TGF-β families of polypeptide growth factors appear to act in early development by regulating expression of hox genes.112–115 For example, growth factors regulate expression of a hox gene called xhox3 in Xenopus that is required for anterior-posterior patterning. Similar observations have been made in Drosophila. Because hox genes themselves code for transcriptional regulators that can turn genes on or off, some of which may code for growth factor-like substances, one can visualize a cascade of events in which a local concentration of growth factor turns on a hox gene, which, in turn, activates another growth factor that turns on another hox gene in a responding cell, suggesting a way that pattern formation could be transmitted from one cell region to another.

The activation of hox genes, however, does not clearly explain how, for example, within a given mesodermal area, different mesodermal cell types arise because hox genes are expressed, albeit perhaps at different times and levels, throughout the mesodermal layer. Thus, additional genes must be expressed in a carefully regulated way to lead to further “subspecialization” or differentiation events. One well-studied example of this is the expression of genes involved in the muscle differentiation pathway, for example, the myogenic genes myo D and myogenin.

Another example is limb bud formation, which has been studied in vertebrates.116 This occurs in several stages. The first phase involves the establishment of signaling centers within the bud primordium. These signaling centers have positional determinants in the embryo: anterior-posterior, dorsal-ventral, and medial-lateral. The second phase is usually associated with increased cell proliferation that is mediated by various mitogens such as members of the FGF and Sonic hedgehog family of gene products. Ultimately, limb bud outgrowth ceases as a consequence of decreased release of mitogens, and a balance between cell proliferation and programmed cell death is achieved. How the genes involved in this late phase are regulated is not totally clear, but it determines what regulates final organ size and the relationship of organ size to the overall size of the developing embryo.

The relationship of these processes to cancer is intriguing. Alterations of these events occur in malignancy: a turn-on of genes leading to cell proliferation, an alteration in the balance of cell proliferation and apoptosis, and a lack of feedback controls to limit organ size. Thus, understanding the regulation of these developmental events should go a long way toward understanding what goes wrong in the biochemistry of the cancer cell.

Other important parameters of morphogenesis include the ability of like cells to cluster together and “talk to each other” and the ability of cells to produce and interact with a specific tissue-type ECM. Thus, the ability to regulate cell-cell and cell-ECM (cell-substratum) interactions is also key to normal development and cellular differentiation. Two families of adhesion molecules are involved: cell-CAMs and cell-substratum adhesion molecules (SAMs).117,118 CAMs produce cell-cell contact between like-minded cells that foster their interactions and cell sorting into homogeneous populations. As noted above in the section entitled “Cell-ECM and Cell-Cell Adhesion”, CAMs, or cadherins as they are also called, are large transmembrane proteins that interact through cytoplasmic connections called catenins that link cadherins to the cell cytoskeleton, thus providing an internal signaling process for CAMs that are in contact with the extracellular environment. These interactions are capable of modulating formation of actin cables in the cytoplasm and, thus, of affecting cell migration and cell surface polarity.

Thus, a number of key interactions among growth factors, hox genes, CAMs, SAMs, the ECM, and specific genes involved in cell-lineage-specific pathways occur during early development and early differentiation. Although mostly studied in lower organisms, all these genes have homologous counterparts in mammalian, including human, cells.

Induction of Differentiation in Cancer Cells

There are a number of examples of animal malignant tumors or human cancer cells in culture that can be induced to lose their malignant phenotype by treatment with certain differentiation-inducing agents. These include induction of differentiation of the Friend virus-induced murine erythroleukemia by dimethyl sulfoxide (DMSO); differentiation of murine embryonal carcinoma cells by exposure to retinoic acid, cAMP analogs, hexamethylene bisacetamide, or sodium butyrate; and differentiation of human acute promyelocytic (HL-60) cells in culture by a number of anticancer drugs, sodium butyrate, DMSO, vitamin D3, phorbol esters, or retinoic acid analogs.118

Being able to treat cancer through induction of cellular differentiation is an attractive idea because the therapy could be target-cell specific and most likely be much less toxic than standard chemotherapeutic agents. The best example of this is the treatment of acute promyelocytic leukemia in patients with all-trans-retinoic acid. A more recent example is induction of solid tumor differentiation by the peroxisome proliferator-activated receptor γ (PPARγ) ligand troglitazone in patients with liposarcoma.119 PPARγ is a nuclear receptor that forms a heterodimeric complex with the retinoid X receptor (RXR). This complex binds to specific recognition sequences on DNA and, after binding ligands for either receptor, enhances transcription differentiation-inducing genes, including those for the adipocyte-specific pathway. PPARγ appears to act as a tumor suppressor in the prostate and thyroid gland, but not in the colon, where its actions are more complex.120 Nevertheless, agents that can exploit the proliferation-inhibiting effects of PPARγ in cancer tissue and have minimal metabolic side effects may be good targets for drug discovery.

Alterations in Signal-Transduction Mechanisms

This is covered in detail in Chapter 5. The only point to be made here is that a large number of growth factors, cytokines, hormones, and exogenous chemicals can trigger cellular responses via receptor-mediated events that foster cellular proliferation and/or differentiation. Sometimes these factors do both. The intracellular signaling pathways that accomplish this are varied and complex. Frequently, these pathways are inappropriately activated in cancer cells by either inappropriate expression of an oncogene coding for a growth factor, a growth factor receptor, or components of intracellular signaling pathways. A key point to keep in mind is that there is significant cross-talk between these signaling pathways such that up- or downregulation of one of them may trigger coordinate responses in another one. Thus, inhibition of one component of a signal-transduction pathway may be compensated for in the cell by upregulation of another pathway. This has important therapeutic implications because a drug that blocks an early or upstream component of a given pathway may be circumvented by activation of another parallel pathway. This is seen, for example, in the development of resistance to some chemotherapeutic agents. A goal, then, is to try to target the downstream events where transduction pathways converge in their ability to stimulate gene activation events.

An example of the cross-talk among ligand-receptor triggered events is the binding of the growth factor β platelet-derived growth factor (βPDGF) to its receptor βPDGFR.121 This induces dimerization of the receptor, which, in turn, triggers signal-transduction pathways. The βPDGF receptor becomes autophosphorylated on multiple tyrosines by activation of its receptor tyrosine kinase. This fosters binding to specific Src homology 2 domain (SH2)-containing proteins that are part of the Grb2-Sos-Ras-Raf-Mek-Erk pathway. In addition, there is cross-talk with the phosphatidyl inositol kinase (PI3K) pathway. PI3K can also stimulate Rac GTPase, which can activate JAK/STAT signaling events. Activation of the SH2 domain protein PLC-γ1 can also potentially stimulate protein kinase C (PKC) signaling pathways. Thus, cytoplasmic signaling proteins form networks of interactions rather than simple linear pathways.121 These diverse signaling pathways, in turn, induce broadly overlapping sets of genes.122

Guanosine triphosphate (GTP)-binding protein (G-protein) signaling events are another ubiquitous pathway for gene activation, some of which are mediated by cAMP that has protean effects on cellular processes.123 Mutations in components of G-protein coupled pathways have been observed, some of which appear to be involved in a number of human diseases, including tumor formation.123,124

Alterations of other signal-transduction pathways also correlate with malignant transformation. For example, cellular transforming events induced by the viral oncogene ν-fps correlate with activation of the endogenous STAT3 signal-transduction pathway.125 TGF-β signaling is mediated via the SMAD family of transducer proteins, and somatic mutations of one of these, SMAD4, are frequently observed in pancreatic cancers and, less frequently, in colon, breast, and lung cancers.126 Functionally disruptive mutations of SMAD2 have been observed in colorectal and lung cancers.

These observations increase the long list of signal-transduction components that are known to be altered in cancer, such as Ras, Myc, Src, and Erb B.123 Thus, it is clear that disruption of signal-transduction pathways is a commonly observed event in human cancer and provides a target for therapeutic intervention.

It should also be noted that the use of DNA microarray technology is providing a way to trace what happens to multiple pathways when cells are altered by external stimuli or malignant transformational events. This has fostered the new field of “pathway biology” in which we are learning that stimulation of a cell (eg, by a growth factor) or damage to a cell (eg, by oxidative stress) up- or downregulates the expression of a wide variety of genes that code for proteins in multiple pathways that, heretofore, we had no idea were linked.

Phosphorylation/Dephosphorylation Events

As noted in the section just above, many signal transduction events involve phosphorylation steps. These steps include (1) receptors coupled to tyrosine kinase activity; (2) receptors coupled to guanine nucleotide-binding proteins, which, in turn, may activate or inhibit adenylate cyclase, activate phosphoinositide hydrolysis leading to protein kinase C activation and intracellular Ca2+ release, or modulate cell membrane ion channels; and (3) intracellular receptors, such as those for steroid hormones, thyroid hormone, and retinoic acid, all of which have DNA-binding domains, as well as ligand-binding domains, and can interact directly with DNA to modulate gene transcription. All these receptor-mediated signal-transduction mechanisms are potential sites for upregulation or deregulation in cancer cells, for example, by oncogene activation or overexpression or by tumor-suppressor gene inactivation.

Tyrosine Kinases

The tyrosine kinasecoupled receptors mentioned in the section entitled “Alterations in Signal Transduction Mechanismsæ are one potential target for carcinogenic alteration. Activation of these receptors can lead to phosphorylation of a number of key substrates. Many growth factor receptors mediate their cellular effects by intrinsic tyrosine kinase activity, which, in turn, may phosphorylate other substrates involved in mitogenesis. A number of transforming oncogene products have growth factor or growth factor receptor-like activities that work via a tyrosine kinase-activating mechanism. For example, the v-src gene product is itself a cell membrane associated tyrosine kinase. The v-sis oncogene product is virtually homologous to the B chain of PDGF. The v-erb product is a truncated form of the epidermal growth factor (EGF) receptor. The fms gene product is analogous to the receptor for CSF-1. The met and trk protooncogene products are receptors for hepatocyte growth factor (HGF) and nerve growth factor (NGF), respectively.

Some of the key substrates for receptor- tyrosine kinase-coupled activity are (1) phospholipase Cγ (PLCγ), which, in turn, activates phosphatidyl inositol hydrolysis, releasing the second messengers diacylglycerol (DAG) and inositol triphosphate (INSP3) that activate PKC and mobilize intracellular calcium release (a number of tumor promoters also activate PKC); (2) the GTPase (guanosine triphosphatase)-activating protein (GAP) that modulates ras protooncogene protein function; (3) src-like tyrosine kinases; (4) PI3K that associates with and may modulate the transforming activity of polyoma middle T antigen and the v-src and v-abl gene products; and, (5) the raf protooncogene product that is itself a serine/threonine protein kinase.

Thus, activation of protein kinases is a key mechanism in regulating signals for cell proliferation. The substrates of these kinases include transcription regulatory factors, such as those linked to mitogenic signaling pathways, for example, proteins encoded by the jun, fos, myc, myb, rel, and ets protooncogenes.

There has been considerable interest in the ErbB (HER) family of receptor tyrosine kinases (RTKs) as therapeutic targets.127–129 Four members of this family of RTKs have been identified: epidermal growth factor receptor (EGFR, or ErbB1, HER1), ErbB2 (HER2, Neu), ErbB3 (HER3), and ErbB4 (HER4). These receptors share an amino acid homology of 40%; to 50%; and have common functional characteristics, yet differences in expression in various tissues and different phenotypes observed in knockout mice indicate that they have nonredundant functions. At least 25 ligands, with different binding affinities, are known for various members of this family. However, no high-affinity ligand specific for ErbB2 is known. When these RTKs are activated, they, in turn, activate a number of downstream signaling partners that induce a variety of cellular events that regulate cell proliferation and differentiation.

Dysregulated function and/or overexpression of the ErbB family are observed in a wide variety of human cancers, including breast, prostate, lung, ovarian, and renal cancers, as well as glioblastomas. Therapeutic modalities aimed at blocking the dysregulated activities of these receptors include small molecules such as the anilinoquinazoline analog ZD1839 (“Iressa”)127,128 and antibodies such as Herceptin, targeted at ErbB2 in breast cancer.130

A recent observation links various levels of activation of EGFR to different cellular functions. It was observed that the EGFR-mediated effects on DNA synthesis, cell adhesion, and cell motility differ depending on levels of saturation of the receptor.129 For example, even though exogenously added EGF has no additional effects on DNA synthesis that is already maximally stimulated by subsaturating levels of EGFR occupation by endogenous ligand, added EGF can still induce integrin α2 expression, integrin-mediated adhesion, and motility in cultured human colon cancer cells. These data suggest that these activities may be additional targets for EGFR-modulating agents.

A lot of excitement has been generated by an inhibitor of another constitutively activated RTK in chronic myelogenous leukemia (CML). In this case, the BCR-ABL translocation that causes activation of this RTK in CML can be inhibited by a specific inhibitor known as ST1571 (Gleevec).131 Because this agent has quite specific activity vs BCR-ABL (and vs the oncogene c-kit), the hope has been raised that other specific, molecularly targeted agents could be found for other tumors. This could dramatically improve the therapeutic index of anticancer chemotherapy.

Protein Phosphatases

Although it has been known for a long time that protein phosphatases play a regulatory role in certain cellular metabolic functions, for example, in the activation-inactivation steps for glycogen synthase and phosphorylase, it has only recently been demonstrated that phosphatases play a role in the activity of various receptors and in the function of certain cell-cycle regulating genes. For example, expression of a truncated, abnormal protein tyrosine phosphatase in BHK cells produces multinucleated cells, possibly by dephosphorylating the cyclin-dependent kinase p34cdc2. Activation of p34cdc2 requires dephosphorylation of a tyrosine residue, and this activation drives the cell from the G2 into the M phase. The truncated phosphatase apparently interferes with the normal synchrony between nuclear formation and cell division.

Protein tyrosine phosphatases (PTPases), it is now known, are a diverse family of enzymes that exist in cell membranes. Some of them are associated with receptors that have tyrosine kinase activity. Phosphatases are also in other intracellular locations. The aberrant phosphorylation state of tyrosine in certain key proteins, such as c-Src or c-Raf, that can lead to cellular transformation could theoretically come about as a result of deregulation of a protein kinase or underexpression of a protein phosphatase. For example, cells treated with vanadate, a PTPase inhibitor, have increased protein phosphotyrosine levels and a transformed phenotype.132 Further evidence that PTPases are involved in cancer is the observation that receptor-linked PTPase γ (one of the PTPase isozymes) is located on chromosome 3, which has a deletion in renal cell and lung carcinomas, suggesting that the PTPase γ gene may act as a tumor-suppressor gene. Thus, one could predict that a high level of expression of specific PTPases may be able to reverse the malignant phenotype, and one can think of strategies, then, to transfect these genes into tumor cells or deliver inducers of the enzymes to tumor cells.

A protein tyrosine phosphatase called PTEN is mutated in human brain, breast, and prostate cancers.133 This was discovered by mapping homozygous deletions on human chromosome 10q23 that occur at high frequency in human cancers. Mutations of the pten gene were detected in 17%; of primary glioblastomas, as well as in human-derived cancer cell lines and xenografts of glioblastoma (31%;), prostate cancer (100%;), and breast cancer (6%;). PTEN is a protein tyrosine phosphatase that dephosphorylates PIP3 in the phosphatidyl inositol pathway. Loss of PTEN activity increases PIP3 phosphorylation and leads to cellular transformation. Thus, PTEN is considered to have tumor suppressor function, and this protein and its substrates are potential targets for new therapeutic agents.

An effect of phosphatases opposite to that of PTEN has been observed in metastatic human colon cancer. Saha and colleagues134 have observed that the PRL-3 protein tyrosine phosphatase gene was overexpressed in each of 18 colon cancer metastases, as compared to nonmetastatic tumors and normal colorectal epithelium. This somewhat counterintuitive observation reminds us that the dysregulated state of phosphorylation events can have inhibitory or stimulatory effects on the cancer process depending on the cell type and the microenvironment. Nevertheless, it does suggest that enzymes such as that encoded by PRL-3 can be targets for yet another approach to anticancer drug discovery.

Alterations of Cell-Cycle Regulation and Apoptosis

Cell-Cycle Regulation

Cell-cycle checkpoints occur at key transitions in the cell cycle and provide go/no go decision points that determine whether or not a cell progresses to the next cell-cycle phase. The biochemical mechanisms involved in these checkpoint controls have been identified. Most of what we know about cell-cycle regulation originally came from lower organisms, including yeast.135 One of the first genes to be identified as an important cell-cycle regulator in yeast is cdc2/cdc28. Activation of this gene requires association with a regulatory subunit called cyclin A. It is now known that sequential activation and inactivation of cyclin-dependent kinases (cdks) is the primary means of cell-cycle regulation. Thus, this is another example of the importance of phosphorylation/dephosphorylation in the biochemistry of cell regulation.

The role of various cdks, cyclins, and other gene products in regulating checkpoints at G1 to S, G2 to M, and mitotic spindle segregation have been described in detail elsewhere135–137 and in Chapter 3. Alterations of one or more of these checkpoint controls occur in most, if not all, human cancers at some stage in their progression to invasive cancer. Examples of some of these alterations are given in the paragraph just below.

Alteration of the G1/S checkpoint occurs in many human cancers. Cyclin D1 gene amplification occurs in a subset of breast, esophageal, bladder, lung, and squamous cell carcinomas. Cyclins D2 and D3 are overexpressed in some colorectal carcinomas. In addition, the cyclin D-associated kinases cdk4 and cdk6 are overexpressed or mutated in some cancers. Mutations or deletions in the cdk4 and cdk6 inhibitor INK4 have been observed in familial melanomas, and in biliary tract, esophageal, pancreatic, head and neck, non-small-cell lung, and ovarian carcinomas. Inactivating mutations of cdk4 inhibitory modulators p15, p16, and p18 have been observed in a wide variety of human cancers. Cyclin E is also amplified and overexpressed in some breast and colon carcinomas and leukemias.

A key player in the G1/S checkpoint system is the retinoblastoma gene rb. Phosphorylation of RB by cyclin D-dependent kinase releases RB from the transcriptional regulator E2F and activates E2F function. Inactivation of rb by genetic alterations occurs in retinoblastoma and is also observed in other human cancers, for example, small-cell lung carcinomas and osteogenic sarcomas.

The p53 gene product is an important cell-cycle checkpoint regulator at both the G1/S and G2/M checkpoints but does not appear to be important at the mitotic spindle checkpoint because gene knockout of p53 does not alter mitosis. The p53 tumor-suppressor gene is the most frequently mutated gene in human cancer, indicating its important role in conservation of normal cell-cycle progression. One of p53's essential roles is to arrest cells in G1 after genotoxic damage, to allow for DNA repair prior to DNA replication and cell division. In response to massive DNA damage, p53 triggers the apoptotic cell death pathway. Data from short-term cell killing assays, using normal and minimally transformed cells, have led to the conclusion that mutated p53 confers resistance to genotoxic agents; however, data from clonogenic assays suggest that p53 status plays little or no role in sensitivity of cells to the killing effects of anticancer drugs or radiation.138

The spindle assembly checkpoint machinery involves genes called bub (budding uninhibited by benomyl) and mad (mitotic arrest-deficient).137 There are three bub genes and three mad genes involved in the formation of this checkpoint complex. A protein kinase called Mps1 also functions in this checkpoint function. The chromosomal instability, leading to aneuploidy in many human cancers, appears to be caused by defective control of the spindle assembly checkpoint. Mutant alleles of the human bub1 gene have been observed in colorectal tumors displaying aneuploidy. Mutations in these spindle checkpoint genes may also result in increased sensitivity to drugs that affect microtubule function because drug-treated cancer cells do not undergo mitotic arrest and go on to die.


Apoptosis (sometimes called programmed cell death) is a cell suicide mechanism that enables multicellular organisms to regulate cell number in tissues and to eliminate unneeded or aging cells as an organism develops. The biochemistry of apoptosis has been well studied in recent years, and the mechanisms are now reasonably well understood.139–141 The enzymatic machinery for this was first discovered in the nematode C. elegans, and later the homologs of these genes and their products were identified in mammalian cells, including human cells. The apoptosis pathway involves a series of positive and negative regulators of proteases called caspases, which cleave substrates, such as poly-ADP-ribose-polymerase (PARP), actin, fodrin, and lamin. In addition, apoptosis is accompanied by the intranucleosomal degradation of chromosomal DNA, producing the typical DNA ladder seen for chromatin isolated from cells undergoing apoptosis. The endonuclease responsible for this effect has now been identified.142,143

A number of “death receptors” have also been identified.139 Death receptors are cell surface receptors that transmit apoptotic signals initiated by death ligands. The death receptors sense signals that tell the cell that it is in an uncompromising environment and needs to die. These receptors can activate the death caspases within seconds of ligand binding and induce apoptosis within hours. Death receptors belong to the tumor necrosis factor receptor gene superfamily and have the typical cystine-rich extracellular domains and an additional cytoplasmic sequence termed the death domain. The best characterized death receptors are CD95 (also called Fas or Apo1) and TNF receptor TNFR1 (also called p55 or CD120a).

The importance of the apoptotic pathway in cancer progression is seen when there are mutations that alter the ability of the cell to undergo apoptosis and allow transformed cells to keep proliferating rather than dying. Such genetic alterations include the translocation of the bcl-2 gene in lymphomas that prevents apoptosis and promotes resistance to cytotoxic drugs. Other genes involved as players on the apoptosis stage include c-myc, p53, c-fos, and the gene for interleukin-1β-converting enzyme (ICE). Various oncogene products can suppress apoptosis. These include adenovirus protein E1b, Ras, and ν-Abl.

Mitochondria play a pivotal role in the events of apoptosis by at least three mechanisms: (1) release of proteins, for example, cytochrome c, that triggers activation of caspases; (2) alteration of cellular redox potential; and (3) production and release of reactive oxygen species after mitochondrial membrane damage.144 Another mitochondrial link to apoptosis is implied by the fact that Bcl-2, the antiapoptotic factor, is a mitochondrial membrane protein that appears to regulate mitochondrial ion channels and proton pumps. The apoptotic protein Bax can interact with Bcl-2 in its mitochondrial conformation to alter the protective function of Bcl-2.

Apoptosis occurs in most, if not all, solid cancers. Ischemia, infiltration of cytotoxic lymphocytes, and release of TNF may all play a role in this. It would be therapeutically advantageous to tip the balance in favor of apoptosis over mitosis in tumors, if that could be done. It is clear that a number of anticancer drugs induce apoptosis in cancer cells. The problem is that they usually do this in normal proliferating cells as well. Therefore, the goal should be to manipulate selectively the genes involved in inducing apoptosis in tumor cells. Understanding how those genes work may go a long way to achieving this goal.

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Copyright © 2003, BC Decker Inc.
Bookshelf ID: NBK12516


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