CELLULAR PROLIFERATION

Neoplasia is the ultimate result of the disruption of exquisite mechanisms regulating normal cell growth. Growth is determined by the balance of cellular proliferation, differentiation, senescence, and programmed cell death. Proliferation occurs as cells traverse the cell cycle (Fig. 3-1). In preparation for cell division, there is a period of deoxyribonucleic acid (DNA) synthesis, designated the S phase. After an intervening gap period, designated the G₂ phase, actual mitosis occurs in the M phase. After another intervening gap period, the G₁ phase, DNA replication can begin again.

Figure 3-1. Regulation of the cell cycle by cyclins, cdks, and cdk inhibitors. In the normal cell cycle, DNA synthesis (in which chromosomal DNA is duplicated) occurs in the S phase, whereas mitosis (in which nuclei first divide to form a pair of new nuclei, followed by actual cellular division to form a pair of daughter cells) takes place in the M phase. The S and M phases are separated by two gap phases, the G1 phase after mitosis and before DNA synthesis, and the G2 phase following the S phase. During these gap phases, the cell is synthesizing proteins and metabolites, increasing its mass, and preparing for the S phase and M phase. Cell cycle progression is regulated primarily at two points, the G2/M and G1/S boundaries, through the coordinated activities of cyclins and cyclin-dependent kinases (cdks), which in turn are negatively regulated by cdk inhibitors (Ink4 and Cip/kip families). The mid-G1 phase is characterized by the interaction between cyclin D1 and cdk4/6. This complex hyperphosphorylates the retinoblastoma protein (pRb) and its family members (e.g., p130). Another important complex at the G1/S boundary is that of cdk2 and cyclin E (cyc E). The result is to release transcription factors such as E₂F that are complexed with pRb. In turn, E₂F binds to and activates the promoters of genes important in DNA synthesis.

The commitment to proceed through DNA replication and cell division occurs during the G₁ phase at the so-called start or restriction (R) point. Cells may exit this cycle of active proliferation before reaching the R point and enter a quiescent phase, G₀. Cells can subsequently re-enter the cell cycle from the G₀ state (see Fig. 3-1). The duration of each cell cycle phase as well as the overall length of the cycle vary among cell types.

Regulation of cell cycle progression appears to be achieved principally by cyclins and cyclin-dependent kinase activity at the G₁/S and G₂/M phase transitions. Cyclin proteins are classified on the basis of their structural features and temporal expression patterns during the cell cycle (see Fig. 3-1). Cyclins A and B are expressed predominantly during the S and G₂ phases. In contrast, cyclins D and E proteins are most active during the G₁ phase.¹ Overexpression of cyclin D1 in fibroblasts results in more rapid entry of cells into the S phase. Cyclin D1 is frequently overexpressed in a number of GI and non-GI malignancies, including those originating from the oral cavity, esophagus, breast, and bladder.²

Each cyclin forms a complex with a cyclin-dependent kinase (cdk) in a cell cycle–dependent fashion. Cyclins function as catalysts for cdk activity (see Fig. 3-1). Cdks physically associate with cyclins through their catalytic domains. The cyclin-cdk complexes regulate cell cycle progression through phosphorylation of key target proteins, including the retinoblastoma gene product (pRb) as well as the Rb family members p130 and p107.³ The final result is progression out of G₁ into the S phase of the cell cycle.

The cell cycle is also regulated by multiple cdk inhibitors; p21^CIP1/WAF1 and p27^KIP1 are inhibitors of cyclin E/cdk2. Originally discovered to be part of the complex containing cyclin D1 and cdk4/6, p21^CIP1/WAF1 is transcriptionally activated by the TP53 tumor suppressor gene product (see Fig. 3-1).⁴ p16^INK4A is another cdk inhibitor that specifically inhibits cdk4 and cdk6⁵ and is part of a larger family of related inhibitors that includes p14, p15, and p18. p16^INK4A is frequently inactivated in esophageal squamous cell cancers and pancreatic ductal adenocarcinomas, a finding that is consistent with its function as a tumor suppressor gene.^6,7 p16^INK4A disrupts the complex of cyclin D1 and cdk 4/6, thereby freeing p21^CIP1/WAF1 and p27^KIP1 to inhibit the activity of cyclin E/cdk2.⁸

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.

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.