PROGRAMMED CELL DEATH AND SENESCENCE

Apoptosis (or programmed cell death) is an important mechanism that counterbalances cell proliferation, and escape from normal apoptotic mechanisms plays a critical role in oncogenesis. Apoptosis is characterized by distinctive features that include chromatin compaction, condensation of cytoplasm, and mild convolution of the nucleus and cytoplasm. These changes are followed by nuclear fragmentation and marked convolution of the cell surface. Eventually, membrane-bound apoptotic bodies that represent the cellular residue are produced and phagocytosed. Apoptosis is distinguished biochemically by cleavage of double-stranded DNA, which results in fragmented DNA.

Studies of the roundworm Caenorhabditis elegans have led to the initial identification of the gene ced-3, a protease that is the major effector of apoptosis. Two key regulators of ced-3, designated ced-9 and ced-4, were found to prevent or induce apoptosis, respectively.⁹ The mammalian oncogene bcl-2 shares homology with ced-9 and protects lymphocytes and neurons from apoptosis¹⁰; bcl-2 complexes with bax, a protein that by itself contributes to apoptosis.¹¹ Of note, both bcl-2 and bax are part of larger gene families, and the stoichiometric relationships among different combinations of the encoded proteins can determine the balance between cell survival and cell death.¹²

Two well-defined pathways that trigger apoptosis have been described in detail. One pathway is mediated through membrane-bound death receptors, which include tumor necrosis factor (TNF) receptors, Fas, and DR5, whereas the other pathway involves activation of TP53 expression by environmental insults such as ionizing radiation, hypoxia, or growth factor withdrawal, with a subsequent increase in the bax-to-bcl-2 ratio. Both pathways converge to disrupt mitochondrial integrity and release of cytochrome c (Fig. 3-2). The so-called apoptosome complex (cytochrome c, caspase 9, and Apaf1) then activates downstream caspases, such as caspase 3, eventuating in cell death. Activation of caspases, intracellular cysteine proteases that cleave their substrates at aspartate residues, is a key step in programmed cell death in mammalian cells.

Figure 3-2. Apoptosis (programmed cell death) counterbalances cellular proliferation to regulate overall tissue growth. A complex interplay of proapoptotic and antiapoptotic molecules results in the downstream activation of caspases that mediate cell death. Some of these signals are initiated through environmental insults that activate the TP53 tumor suppressor gene, and some are initiated through death receptors, including TNF-R1, Fas, and DR5. Death receptors activate caspase 8, which in turn activates BID. In addition, there is an interplay between proapoptotic (bax, bak) and antiapoptotic (bcl-2, bcl-xl) molecules. Both pathways converge on the mitochondria, resulting in the release of cytochrome c and formation of the apoptosome (Apaf1, caspase 9, and cytochrome c). This leads to activation of multiple caspases, in particular caspase 3, and ultimately to cell death. BID, bcl-2 interacting domain; DD, death domain; FLICE, FADD-like IL-1β-converting enzyme; FLIP, FLICE (also known as caspase 8) inhibitory protein; TNF-R1, tumor necrosis factor receptor 1.

Replicative senescence also plays a role in determining overall growth in cell populations. Most primary cells when grown in vitro have a limited replicative potential and eventually undergo senescence.¹³ In contrast, malignant cells can replicate indefinitely. Up-regulation of the telomerase enzyme is essential to escape from replicative senescence. Telomeres are repetitive DNA sequences at the ends of all chromosomes that regulate chromosomal stability. Telomeres shorten with each cell division and, when they have been reduced to a certain critical length, senescence occurs. Cancer cells are able to maintain their telomere length despite multiple cell divisions through the reactivation of telomerase enzyme activity.¹⁴

SIGNALING PATHWAYS THAT REGULATE CELLULAR GROWTH

Cellular proliferation is achieved through transition of cells from G₀ arrest into the active cell cycle (see Fig. 3-1). Although progression through the cell cycle is controlled by the regulatory proteins just described, overall proliferation is modulated by external stimuli. Growth factors that bind to specific transmembrane receptors on the cell surface may be especially important. The cytoplasmic tails of these transmembrane receptor proteins produce an intracellular signal after ligand binding.

In addition to peptide growth factors, extracellular matrix and cell-cell adhesion molecules have a significant impact on cell proliferation. Although the full spectrum of molecules that play a role in cell-matrix and cell-cell adhesion is still not defined, it is known to include integrins, cadherins, selectins, and proteoglycans. Interactions with these adhesion molecules lead to changes in the cell cytoskeleton, indirectly modulating external growth stimuli. Alterations in cell-matrix or cell-cell interactions are particularly important in contributing to the invasive phenotype characteristic of malignant cells.

Interaction of ligands with their receptors at the cell surface induces intracellular signals that ultimately result in alterations in gene transcription. Three important receptor subtypes appear to initiate cellular signaling through ligand-receptor interaction at the cell surface: (1) tyrosine kinases; (2) serine and threonine kinases; and (3) G protein–coupled receptors.

The receptors for many peptide growth factors contain intrinsic tyrosine kinase activity within their intracellular tail. After ligand binding, tyrosine kinase activity is stimulated, leading to phosphorylation of tyrosine residues in target proteins within the cell. The full spectrum of proteins phosphorylated by each tyrosine kinase remains to be determined. Most receptors also autophosphorylate tyrosine residues present in the receptors themselves to initiate signaling and, in some cases, this also causes attenuation of their own activity to effect an intramolecular feedback regulatory mechanism. The receptors for many peptide growth factors, including epidermal growth factor (EGF), belong to this receptor class.

Other receptors on the cell surface possess kinase activity directed toward serine or threonine residues rather than tyrosine. These receptors also phosphorylate a variety of cellular proteins, leading to a cascade of biological responses. Multiple sites of serine and threonine phosphorylation are present on many growth factor receptors, including the tyrosine kinase receptors, suggesting the existence of significant interactions among various receptors present on a single cell.¹⁵ The transforming growth factor-β (TGF-β) receptor complex is one important example of a serine-threonine kinase–containing transmembrane receptor.

Many receptors are members of the so-called seven-membrane–spanning receptor family. These receptors are coupled to guanine nucleotide binding proteins, and are designated G proteins. G proteins undergo a conformational change that is dependent on the presence of guanosine phosphates.¹⁶ Activation of G proteins can trigger a variety of intracellular signals, including stimulation of phospholipase C and the generation of phosphoinositides (most importantly, inositol 1,4,5-triphosphate) and diacylglycerol through hydrolysis of membrane phospholipids, as well as modulation of the second messengers cyclic AMP and GMP.¹⁷ Somatostatin receptors exemplify a G protein–coupled receptor prevalent in the GI tract.

Binding of growth factors and cytokines to cell surface receptors typically produces alterations in a variety of cellular functions that influence growth. These functions include ion transport, nutrient uptake, and protein synthesis. However, the ligand-receptor interaction must ultimately modify gene expression within the nucleus to affect cell proliferation. The regulation of the content and activity of transcriptional factors within the nucleus is the final step in pathways that translate an external stimulus to a change in cell proliferation. These transcriptional factors modulate the expression of genes that control cell proliferation and phenotype.

The Wnt pathway is one important example of a signaling pathway that regulates the cell cycle machinery to control the proliferation of intestinal epithelial cells (Fig. 3-3). Although the details of the specific interactions between the Wnt ligand and its receptor Frz, a member of the seven-membrane receptor family, in the GI tract are not fully clarified, an active Wnt signal ultimately results in the accumulation of β-catenin in the nucleus, where it binds with the transcription factor TCF-4 to activate a set of target genes.¹⁸ Inhibition of the Wnt signal in mice can be achieved by deletion of TCF-4 or overexpression of a Wnt inhibitor designated Dickkopf1, which results in dramatic hypoproliferation of the intestinal epithelium.^19,²⁰ This hypoproliferation appears to be mediated by decreased expression of the TCF-4 target gene c-MYC, which directly represses p21^CIP1/WAF1.²¹ Thus, a Wnt signal stimulates proliferation of intestinal epithelial cells by repressing the cell cycle inhibitor p21^CIP1/WAF1.

Figure 3-3. The Wnt signaling pathway is an important regulator of intestinal epithelial cell proliferation and tumorigenesis. In the absence of a Wnt signal (left panel), β-catenin forms a cytoplasmic complex with APC, Axin, and glycogen synthase kinase-3β (GSK-3β). This β-catenin destruction complex phosphorylates (-P) β-catenin and targets it for degradation via the ubiquitin-mediated proteasomal pathway. In the presence of an active Wnt signal (right panel), β-catenin is stabilized, and excess cytoplasmic β-catenin is translocated to the nucleus, where it interacts with the TCF-4 transcription factor to regulate the expression of many key target genes. VEGF, vascular endothelial growth factor.

Cyclin D1 has an extremely short half-life (<20 minutes) and is a rate-limiting factor for progression through the G₁ phase of the cell cycle (see Fig. 3-1). Consequently, it is one of the most tightly regulated of all cell cycle proteins. Extracellular signals from growth factors, including EGF, colony-stimulating factor 1 (CSF-1), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF), can regulate cellular proliferation by rapidly inducing the expression of the cyclin D1 gene.²²

Tissue homeostasis is also maintained by growth-inhibiting signals that counterbalance proliferative signals. TGF-β is a potent growth-inhibiting factor that mediates arrest of the cell cycle at the G₁ phase. TGF-β not only induces the transcription of the cell cycle inhibitors p15^INK4B and p21^CIP1/WAF1, but also enhances the inhibitory activity of p27KIP1 on the cyclin E/cdk2 complex (see Fig. 3-1).²³ These effects are mediated intracellularly through the Smad family of proteins.

INTESTINAL TUMOR DEVELOPMENT: MULTISTEP FORMATION AND CLONAL EXPANSION

Multiple sequential genetic alterations are required for the transformation of normal intestinal epithelium to frank malignancy. This multistep nature of tumorigenesis is most directly illustrated by the changes that accrue in the development of colonic neoplasia (see Chapters 122 and 123). The accumulation of genetic alterations roughly parallels the progression from normal epithelium through adenomatous polyps (or, in the case of ulcerative colitis, flat dysplastic mucosa) to malignant neoplasia. Studies on the molecular pathogenesis of colon cancer have served as a paradigm for the elucidation of genetic alterations in other GI cancers. For example, a similar progression is also seen in the transition from normal squamous epithelium to metaplastic mucosa (Barrett’s esophagus) through dysplasia to adenocarcinoma of the esophagus. Gastric and pancreatic oncogenesis are each thought to proceed through similar multistep pathways.

Models of the multistep or multiple hit process of tumor formation have largely superseded earlier concepts of oncogenesis that discriminated between tumor initiation and subsequent promotion. Initiation was attributed to a single change in a cell that converted it from a normal to a malignant cell. Promotion reflected all the factors that acted after the initiating event to enhance tumor growth. However, oncogenesis occurs through a series of events that result in incremental changes in cell behavior until the cell eventually passes some threshold associated with the malignant phenotype. Nevertheless, there is still some merit in a more limited concept of tumor promotion. A number of factors promote the likelihood of malignant transformation through the stimulation of increased cellular turnover, which increases opportunities for somatic mutations to occur.²⁴ In the GI tract, these promoting factors include dietary constituents (see later) as well as chronic inflammation, which are associated with increased cell proliferation. Thus, a number of chronic inflammatory conditions increase the site-specific risk of cancer, such as ulcerative colitis (see Chapter 112), chronic gastritis (see Chapters 51 and 54), chronic pancreatitis (see Chapters 59 and 60), Barrett’s esophagus (see Chapters 44 and 46), and chronic hepatitis (see Chapter 94). Although the mechanisms whereby inflammatory processes elicit eventual tumor development are incompletely understood, cytokines produced by inflammatory cells can stimulate tumor cells, leading to activation of nuclear factor-κB (NF-κB) in the tumor cells that can serve to inhibit apoptosis and stimulate proliferation.²⁵

Clonal expansion is also essential to tumor development.²⁶ Whereas germline mutations may lead to altered expression of a gene in all cells in a tissue, subsequent additional somatic mutations generally occur only in a small, largely random subpopulation of cells. Clonal expansion occurs if a specific gene mutation results in a survival advantage for the cell. A second round of clonal expansion occurs when a cell within this population sustains still another genetic alteration, which further enhances its growth properties. After several iterations, a final genetic alteration eventually confers a property that, together with the preceding genetic alterations, makes a cell malignant.

Recent evidence has led to the suggestion that cancer stem cells in tumors may play a central role in tumorigenesis. These cells are defined by the capacity for self-renewal and the ability to generate progeny that lack this capacity but manifest the characteristics of the heterogeneous lineages that comprise the tumor.²⁷ Failure to eradicate the cancer stem cell population is posited to underlie tumor recurrences after chemotherapy. Precise identification of the cancer stem cell compartment has been possible for hematologic malignancies. However, comparable definitive proof of their presence in solid tumors, such as intestinal cancers, remains a challenge.²⁸

A genetically unstable environment was thought to be necessary for the development of the multiple alterations that ultimately result in cancer. Genomic instability is observed in almost all cancers, regardless of organ site. Instability of the genome may result from several mechanisms. In colon cancer, there are at least two well-recognized forms of genetic instability, and they have been termed chromosomal instability and microsatellite instability.²⁹ Chromosomal instability results in tumor cells that display frequent aneuploidy, large chromosomal deletions, and chromosomal duplications. In contrast, tumors that display microsatellite instability are often diploid or near-diploid on a chromosomal level but harbor frequent alterations in smaller tracts of microsatellite DNA (see later discussion on DNA repair). Thus, there are at least two distinct routes to the formation of a colorectal cancer, depending on the nature of the underlying genetic instability (Fig. 3-4).

Figure 3-4. Multistep model of colorectal cancer based on underlying genetic instability. There are two major pathways, chromosomal instability (top half of figure) and microsatellite instability (bottom half). The progression from normal colonic epithelium to carcinoma is associated with the acquisition of several genetic alterations. In the chromosomal instability pathway, these alterations include the concomitant activation of oncogenes (e.g., K-ras) through a point mutation and inactivation of tumor suppressor genes (e.g., APC, TP53) through a point mutation or deletion. In addition, DNA hypomethylation may be important in this process. An increasing aggregate number of mutations can be correlated with progression from early benign polyp to cancer, as reflected by analysis of polyps by size. In the microsatellite instability model, mutations in DNA mismatch repair genes such as MSH2 and MLH1 create a mutator phenotype in which mutations accumulate in specific target genes (see section on DNA mismatch repair). Tumors develop much more rapidly through this pathway than through the chromosomal instability pathway (horizontal arrows).

NEOPLASIA-ASSOCIATED GENES

The genes that collectively play an important role in oncogenesis generally lead to disruption of the orderly mechanisms of normal cell proliferation. Insofar as normal cell proliferation appears to depend on a wide variety of genes, it is not surprising that alterations in diverse genes confer part or all the phenotypic features of transformation. Despite this diversity, all these genes that become altered appear to belong to one of three distinct groups: (1) oncogenes, which actively confer a growth-promoting property; (2) tumor suppressor genes, the products of which normally restrain growth or proliferation; and (3) DNA repair genes, which contribute to transformation by fostering genomic instabilbity and facilitating mutations in other genes. Activation of oncogenes or inactivation of tumor suppressor genes and DNA repair genes contributes to malignant transformation (Table 3-1).

Table 3-1 Oncogenes, Tumor Suppressor Genes, and DNA Repair Genes Altered in Gastrointestinal Tumors*

ONCOGENES

Typically, oncogenes are genes that encode a normal cellular protein expressed at inappropriately high levels or mutated genes that produce a structurally altered protein that exhibits inappropriately high activity. For example, several genes that encode tyrosine kinase–containing growth factor receptors become oncogenes after a mutation results in unregulated tyrosine kinase activity that is no longer dependent on the presence of the appropriate ligand. The normal cellular genes from which the oncogenes derive are designated proto-oncogenes or cellular oncogenes.

More than 80 oncogenes have been isolated, and additional oncogenes continue to be identified. Most of these genes are widely expressed in many different types of tumor cells. Multiple oncogenes (usually in combination with altered tumor suppressor genes) are commonly found within a single tumor.

Several mechanisms can lead to oncogene activation. These include gene transduction or insertion, point mutation, gene rearrangement, and gene amplification. Gene transduction and insertion generally result from retroviral infection. Point mutations result in constitutively active oncogene products. Gene rearrangements can result in oncogenic fusion proteins, and gene amplifications lead to uncontrolled overexpression of a normal gene product.

The proteins encoded by oncogenes comprise at least four distinct groups—peptide growth factors that may be secreted into the extracellular milieu, protein kinases, signal-transducing proteins associated with the inner cell membrane surface (membrane-associated G proteins), and transcriptional regulatory proteins located in the nucleus.

Oncogenes and Peptide Growth Factors

The transforming effects of enhanced expression of a variety of growth factors have been demonstrated both in vitro and in vivo. Several growth factor–related proteins encoded by oncogenes have now been recognized, including the family of Wnt proteins and Sis, which encodes the β chain of platelet-derived growth factor. It is axiomatic that cells that produce high levels of a growth factor must also express specific receptors activated by that growth factor to yield an autocrine growth-stimulating loop that is often present in neoplasms. For example, several colon-derived cancer cell lines express TGF-β and IGF I and II, as well as their receptors. This autocrine mechanism may be contrasted with overproduction of a growth factor that exerts its influence at a remote cellular target rather than within the tumor itself, exemplified by gastrin produced by gastrinoma cells, which exerts trophic effects on the gastric mucosa but not on the tumor itself (see Chapter 32).

Protein Kinase–Related Oncogenes

The largest family of oncogenes encodes proteins with kinase activity. These oncogenes encompass the full variety of protein kinases, including receptor and nonreceptor tyrosine kinases and cytoplasmic serine and threonine kinases. Many members of this large oncogene group are expressed by neoplasms of the GI tract.

A brief consideration of receptor protein tyrosine kinase HER2/Neu/ERBB2, which is related to the EGF receptor, is particularly illustrative. There are four EGF receptor family members (ERBB1-4). The viral v-erb-b2 encodes a truncated form of the EGF receptor that lacks most of the external EGF-binding domain.³⁰ As a result, the receptor no longer requires the presence of the ligand for activation and remains continuously activated, stimulating proliferation. The neu oncogene is derived from a rat cellular proto-oncogene closely related to the EGF receptor. The oncogene differs from its normal counterpart by a point mutation that changes a single residue (valine to glutamic acid) within the transmembrane domain, thereby causing activation of the 185-kd tyrosine kinase protein (p185neu).³¹ The human counterpart (ERBB2) of the neu oncogene is not mutated but is overexpressed or amplified in a variety of adenocarcinomas, including those arising in the stomach, breast, and prostate.³² In addition, ERBB2 expression increases progressively in the transition from normal esophageal mucosa through the dysplastic state characteristic of Barrett’s esophagus to esophageal adenocarcinoma.³³

In contrast with the receptor type of tyrosine kinase that possesses intrinsic catalytic activity, many other receptors and membrane proteins lack self-contained signaling activity. Instead, they are coupled to nonreceptor tyrosine kinases on the cytoplasmic side of the plasma membrane that act as signal transducers. A number of oncogenes associated with neoplasms of the GI tract, most notably the colon, are members of the src family of nonreceptor tyrosine kinases. Members of the src family are approximately 60-kd phosphoproteins (v-src) that possess inherent tyrosine kinase activity and associate with the inner surface of the plasma membrane. Autophosphorylation of the normal c-src leads to attenuation of its kinase activity, thereby providing inherent regulation to limit unrestrained activity.³⁴ Increased levels of c-src activity have been found in colonic cancer tissue and colon cancer–derived cell lines.³⁵ Activating mutations of c-src have been identified in a subset of advanced, metastatic colon cancers.³⁶