Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease

CELL DIVISION/DEATH: CELL CYCLE

Contents Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease Cyclins and Cyclin-Dependent Kinases The Restriction Point CDK Inhibitors in Normal and Malignant Cells The INK4a/ARF Locus S Phase Traveling through Mitosis with the Chromosomal Passenger Complex Mitosis in Animal Cells Regulating Cytokinesis Regulation of the p53 Pathway Cellular Senescence

Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease SJ Parsons, University of Virginia Health System, Charlottesville, VA, USA JO DaSilva, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA J Thomas Parsons, University of Virginia Health System, Charlottesville, VA, USA r 2016 Elsevier Inc. All rights reserved.

Glossary AKT Serine-threonine kinase activated by PI3K. AMPK Serine-threonine kinase activated by AMP. BRCA1,2 Breast cancer tumor suppressor genes 1 and 2. ERK Serine-threonine kinase activated by MEK. FYN Cytoplasmic tyrosine kinase of the SRC family. LKB Serine-threonine kinase, enhancer of AMPK activity. MEK Multi-specificity kinase activated by RAF. mTOR Serine-threonine kinase indirectly activated by AKT. MYC Transcription factor.

Introduction: Scope and Intent of Article Over the past five decades, the concept that mutation of discrete cellular genes can drive the formation and progression of human cancers has evolved. These genes, termed oncogenes and tumor suppressor genes, constitute a defining paradigm for our understanding of cancer biology. The elucidation of their function in both normal and malignant cell physiology has provided a rational platform for the development of therapeutic strategies to treat the many forms of the disease. This article provides a brief overview of (1) the role of oncogenes and tumor suppressor genes in human cancers, (2) how the functional interaction between proteins encoded by oncogenes and tumor suppressor genes alters normal cellular

Encyclopedia of Cell Biology, Volume 3

doi:10.1016/B978-0-12-394447-4.30056-6

NF1 GAP for RAS-GTP. PTEN Lipid (PI-3) phosphatase. PTP1B Cytosolic protein tyrosine phosphatase. PTPN11 RTK phosphatase. RAF Serine-threonine kinase activated by RAS-GTP. RAS Small guanine nucleotide binding protein. RB Retinoblastoma tumor suppressor – transcriptional regulator. SRC Cytoplasmic tyrosine kinase. TP53 Tumor suppressor and transcription factor.

processes such as proliferation, metabolism/energetic, and cell survival, and (3) the role of oncogenes and tumor suppressor genes in diseases other than cancer, namely human developmental syndromes.

Oncogenes and Tumor Suppressor Genes and Human Cancer During the first half of the twentieth century, two parallel tracks of research emerged to provide evidence for mutations of defined genes as major causes of cancer: one was the identification of animal viruses that induced rapid formation of cancers in chickens and rodents (retroviruses), and the other

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was the discovery of chemical compounds that caused mutations in cellular genes (carcinogens), which would in turn promote cancer formation. In the 1970s these two lines of research merged, when it was discovered that tumor-forming retroviruses carried mutated genes (oncogenes) that were derived from normal cellular genes (proto-oncogenes) (Bishop, 1983, 1985), and that human cancers contained mutated proto-oncogenes that arose in the absence of any known viral etiology (Barbacid, 1987). These findings gave rise to the concepts that cancers can derive from multiple mechanisms and by mutation of one or more cellular genes. As cancer cell biologists started to catalogue and compare the properties of normal and cancer cells, it became clear that cancer cells exhibited a set of cellular and physical properties, known as the ‘transformed’ phenotype, that were distinct from normal cells (e.g., altered morphology, unrestricted cell growth, altered metabolism, and multiple and varied chromosomal aberrations). Surprisingly, it was observed that if one ‘fused’ a normal cell with a cancer cell, the resultant hybrid cell containing a complete complement of host and donor chromosomes exhibited a ‘normal’ cellular phenotype (Harris et al., 1969; Stanbridge, 1976). Acquisition of the ‘transformed’ phenotype required the loss of certain cellular chromosomes from the normal host, leading to the idea that certain normal cellular genes could ‘suppress’ the tumorigenic properties of cancer cells. Hence the concept of tumor suppressor genes emerged in the cancer literature.

Identification of Specific Oncogenes and Tumor Suppressor Genes in Human Cancers Oncogenes ‘Genes that, when activated by mutation, increase the selective growth advantage of the cell in which they reside.’ One of the first oncogenes to be discovered is harbored in the chicken retrovirus, Rous sarcoma virus, that was first described by Peyton Rous in the early 1900s (Martin, 2004). This virus causes transformation of avian and rodent cells in cell culture and induces tumors in birds and rodents. The oncogenic powers of Rous sarcoma virus reside in the viral gene denoted SRC, which is not truly a viral gene but a mutated form of a cellular gene denoted c-SRC, present in all vertebrate species (Stehelin et al., 1976). This discovery led to the hypotheses that retroviruses can ‘capture’ and mutate cellular genes during their replication in host cells and that expression of these mutated cellular genes during subsequent rounds of host cell infection is sufficient to induce transformation of cells and tumor formation. This discovery led immediately to the realization that expression of other cellular genes altered by carcinogens or X-rays may mimic the effects of virus infection and lead to cancer formation. Evidence that human cancers contain oncogenes came from observations that introduction of DNA purified from human cancer cells induced the transformation of normal rodent cells (Krontiris and Cooper, 1981; Perucho et al., 1981; Shih and Weinberg, 1982). The molecular characterization of one of the genes responsible for this transformation unveiled a mutated version of the cellular RAS gene, which was found to encode a protein that exhibited constitutive enzymatic activity,

providing the first clue that oncogenes, of either viral or cellular origin exhibit unregulated activities (see below).

Tumor suppressor genes ‘Genes that, when inactivated by mutation, increase the selective growth advantage of the cell in which they reside.’ In human cancers, inactivation of tumor suppressor genes occurs via two prominent mechanisms, loss of a defined region of a chromosome leading to complete loss of protein expression or expression of truncated forms of the protein, or mutations within a tumor suppressor gene altering protein function (Hinds and Weinberg, 1994; Weinberg, 1991). Direct evidence for the former in human cancer emanated from the study of retinoblastoma, a rare, inheritable, childhood tumor of the eye. Genetic studies showed that tumors arising in afflicted children either lack the RB gene or carry a mutated form of it. The RB gene encodes a transcription regulator (pRB) involved in normal growth control (see below) (Hollingsworth et al., 1993). Subsequent genetic studies have identified a number of inheritable cancers that are linked to the loss of function of defined cellular genes (e.g., Wilm’s tumor and the NF1 gene, familial adenomatous polyposis and the APC gene) (Cichowski and Jacks, 2001; Fearon and Vogelstein, 1990). The gene encoding the p53 protein, TP53, is an example of how mutations alter normal protein function, leading to tumor initiation and progression (Hinds and Weinberg, 1994; Rivlin et al., 2011; Vousden and Prives, 2009). TP53 is a transcription factor that regulates a cell’s response to stress by inducing cell cycle arrest, DNA repair, or apoptosis, or by regulating cell metabolism. The importance of p53 in human cancers is underscored by the fact that a very large number of human cancers exhibit TP53 mutations and by the finding that the Li–Fraumeni syndrome, a rare cancer predisposition syndrome, is characterized by germline mutations in TP53. Interestingly, studies of rodent and human oncogenic DNA viruses have revealed that normal p53 function is neutralized in virus-infected cells by binding to discrete virus-encoded proteins. For example, the binding of the human papilloma virus-encoded protein, E6, to p53 inhibits p53 activity and is essential for human papilloma virus transformation of human cells (Moody and Laimins, 2010).

Number of mutations in a human cancer In the past 10 years, genome-wide DNA sequencing has cataloged millions of mutations that arise in different human cancers. For example, solid tumors of the colon, breast, brain, or pancreas display somatic mutations that are predicted to alter the structure of as many as 60–70 proteins (Vogelstein et al., 2013; Wood et al., 2007). Interestingly, some tumors, such as melanomas and lung cancers, contain significantly more somatic mutations (approximately 200), consistent with the well-documented roles of the carcinogens, UV light, and cigarette smoke, in the etiology of these cancers. How do we know which mutations are important for development and progression of a given cancer? It has been suggested that mutated genes which confer selective growth to the cancer are referred to as ‘driver’ genes, whereas mutations that do not affect function are referred to as ‘passenger’ mutations. An analysis of more than 18 000 mutated genes

Cell Division/Death: Cell Cycle: Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease

containing over 400 000 subtle mutations in over 3000 tumors has defined 125 ‘driver’ genes (Vogelstein et al., 2013). Of these, 54 appear to function as oncogenes and 71 as tumor suppressor genes. Interestingly, a large number of these genes were previously identified as oncogenes or tumor suppressor genes using more classical approaches.

Functional Roles of Key Oncogene and Tumor Suppressor Gene Products Oncogenes and tumor suppressor genes regulate the balance between cell proliferation, metabolism and energetics, and cell survival. They do this by encoding proteins that transmit signals from the extracellular milieu, which in turn stimulates the transcriptional apparatus of the cells. The changes in gene expression mediated by such intracellular signals trigger the synthesis of a host of cellular proteins that drive cell cycle progression, modulate the metabolic and energetic state of the cell, and control cell death. Some of the most important pathways regulated by oncogenes and tumor suppressors are briefly described below.

Transmission of extracellular cues The ability of cells to sense and respond to their environmental milieu is dependent on a wide variety of transmembrane cell surface receptors. One class of such receptors, the receptor tyrosine kinases (RTKs), specifically engages ligands, such as polypeptide growth factors, cytokines, and hormones, with high affinity and transmits signals dependent on this binding to the inside of cells, thus initiating a cascade of biochemical reactions (pathways) that control the cell’s response to its environment (Hubbard and Miller, 2007; Figure 1(a)). In the human genome over 50 unique genes encode RTKs, including receptors for the epidermal growth factor (EGF), platelet-derived growth factor, fibroblast growth factor, vascular endothelial growth factor (VEGF), hepatocyte growth factor/scatter factor, and insulin/insulin-like growth factors (IGF). Because of their central roles in promoting cell proliferation, many RTKs have been implicated as etiological agents in various cancers, either through receptor gain-offunction mutations (EGFRs) or through receptor/ligand overexpression (EGFR, VEGFR, IGFR) (Blume-Jensen and Hunter, 2001). In cancer cells, the mutational activation of RTKs and/ or their over-expression lead to elevated and constitutive downstream signaling, resulting in uncontrolled cell proliferation (Hubbard and Miller, 2007).

The RAS pathway The RAS pathway (Figure 1(a)) is a major intracellular signaling pathway in normal and cancerous cells of adult organisms and developing embryos alike (Bos, 1989). It is activated downstream of RTKs by guanine nucleotide exchange proteins (GEFs), which mediate the exchange of guanine nucleotide diphosphate (GDP) for guanine nucleotide triphosphate (GTP) on the small G protein, RAS (Bos et al., 2007). Once activated by GTP, RAS stimulates the catalytic activity of the serine/threonine protein kinase RAF (Dhillon et al., 2007). GAPs (GTPase-activating proteins) promote the return of RAS to the inactive GDP-bound state (McCormick, 1989).

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Activated RAF leads to the stimulation of a multi-specificty kinase, MEK, which in turn phosphorylates and activates the serine/threonine kinase, ERK. Activated ERK translocates to the nucleus, where it catalyzes the phosphorylation of a myriad of nuclear substrates, including transcription factors, transcriptional regulatory proteins, and other substrates involved in DNA replication and cell division. RAS proteins were among the first oncogenes to be identified, and mutated RAS is found in about 15% of human cancer (Davies et al., 2002). Mutation of RAF occurs in 60–70% of malignant melanomas and at a lower frequency in a wide range of other human cancers (Davies et al., 2002). Interestingly few, if any, mutations in MEK or ERK have been identified in human cancers.

The PI3K pathway A second important signaling pathway coupled to RTK activation is the phosphatidylinositol-3-kinase (PI3K) pathway (Cantley, 2002; Figure 1(b)). PI3Ks are lipid kinases that play central roles in cell cycle progression, apoptosis, DNA repair, and cellular metabolism. PI3Ks transduce signals from cell surface receptors by generating phosphorylated phosphatidylinositols, which in turn activate effector kinase pathways, principally those involving AKT and mTOR (Figure 1). These pathways play key roles in promoting cancer cell survival and proliferation (Cantley, 2002; Osaki et al., 2004). The activity of the PI3K pathway in normal cells is tightly regulated by the phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase, PTEN (phosphatase and tensin homolog deleted from chromosome 10). PTEN dephosphorylates phosphatidylinositol-3,4,5-trisphosphate, thereby negatively regulating signaling through all the downstream targets of PI3K, including AKT and mTOR (Chalhoub and Baker, 2009; Di Cristofano and Pandolfi, 2000; Osaki et al., 2004). In human cancers the deregulation of the PI3K signaling pathway is linked to the development of approximately one-third of human cancers (Samuels and Waldman, 2010). Somatic mutations in the PTEN gene are among the most prevalent of these genetic changes (Yin and Shen, 2008).

Transcriptional regulation Oncogenes encode a large repertoire of transcription factors, which can be activated by chromosomal rearrangements, gene fusions, gene amplifications, and to a lesser extent somatic mutations. A prime example is the MYC oncogene (Dang, 2012). Altered expression of the MYC gene occurs in over half of human cancers (Vita and Henriksson, 2006). In many cancers the level of MYC becomes elevated as a consequence of chromosomal rearrangements that place the MYC gene under the control of a constitutively activated promoter, such as an immuglobulin gene promoter (Burkett’s lymphoma). MYC can also be the direct target of signal transduction pathways activated by RTKs in response to nutrients, growth factors, and mitogenic stimuli. ERK, the downstream effector of the RAS/ RAF/MEK/ERK pathway, stabilizes MYC protein by phosphorylation (Gustafson and Weiss, 2010), thus preventing its turnover within the nucleus. Thus activation of this pathway either by RTK activation or mutation of RAS or RAF can lead to the stabilization of MYC and increased transcription of MYC target genes (Figure 1(a)).

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Figure 1 Key intracellular signaling pathways regulated by oncogenes and tumor suppressor genes. (a) Growth factor/mitogen binding to cell surface receptors (RTKs) leads to intracellular activation of the RAS-RAF-MEK-ERK pathway which culminates in transcriptional stimulation of genes required for cells to advance through the cell cycle. Progression through the cell cycle is dependent upon the activities of cyclin-CDKs which are needed for movement through ‘restriction’ points. Many pathways that stimulate the cell to divide are populated with oncogenes whose actions are counteracted by tumor suppressors (notably pRB and p53) (see text for details). The balance between these two classes of cell cycle regulators determines whether a cell completes division. (b) Growth factor/mitogen binding to RTKs also stimulates the PI3K pathway, which leads to the activation of AKT-mTOR and stimulation of protein synthesis. AKT and mTOR also play major roles in metabolism by regulating intracellular levels of glucose and AMP/ATP ratios. Filled ovals denote oncogenes, and filled rectangles represent tumor suppressors.

Interplay between Oncogenes and Tumor Suppressor Genes in Human Cancer In cancer, the activation of oncogenes and compromised function of tumor suppressor genes alter the finely tuned balance between positive and negative signals that regulate cell proliferation, metabolism, and cell survival. This section describes key examples of how proteins encoded by oncogenes and tumor suppressor genes functionally interact with one another, the effects these interactions have on normal

physiological processes of the cell, and how the changes in such processes provide a growth advantage to the cancer cell.

Oncogenes, Tumor Suppressor Genes, and Cell Cycle Regulation Cell proliferation is a highly controlled process that involves passage through the four main phases of the cell cycle (Ho, 2009): (1) G1, or interphase, during which cellular constituents (other than DNA) are increased in preparation for the

Cell Division/Death: Cell Cycle: Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease

generation of two new daughter cells, (2) S phase, DNA synthesis, (3) G2, the second interphase stage in which the integrity of the newly synthesized DNA is ascertained, among other things, and (4) M phase (mitosis) in which equal distribution of cellular components into two daughter cells and cell division takes place. To pass from one phase to another, the cell must traverse one or more checkpoints (Weinberg, 2007). Not unexpectedly, these checkpoints are frequent sites of regulation by proteins encoded by oncogenes and tumor suppressor genes. If cells exit the cell cycle, they assume a resting or quiescent state, termed G0. The majority of cells in adult organisms, cells that form the fully differentiated organs of the human body, reside in the G0 state. When injury occurs and/or cell replacement is needed, growth factors (such as those that activate RTKs) initiate the exit of cells from G0 into G1 and their continued presence is required for passage through the first checkpoint within G1 (termed the restriction point). Ligand activation of growth factor receptors stimulates intracellular signaling cascades (Figure 1(a)), which culminate in new gene transcription. Among the genes activated by this process is the cyclin D gene family, which is expressed in early G1. Cyclin D binds and activates the cyclin-dependent protein kinases (cdks), 4 and 6, which phosphorylate pRB, leading to the release of the transcription factor, E2F, and progression through the cell cycle. pRB is a transcriptional repressor that blocks progression through early to mid-G1 and must be inactivated by phosphorylation for passage through the mid-G1 restriction point. Specifically, in early G1, unphosphorylated pRB binds to and inhibits the transcription factor, E2F. Phosphorylation of pRB by cyclinD/cdk4/cdk6 leads to the release of E2F and renders E2F active transcriptionally. E2F, in turn, stimulates production of cyclin E, which binds and activates cdk2. The cyclin E/cdk2 complex completes inactivation of Rb by fully phosphorylating it, thereby allowing the cell to complete G1 and enter S phase. In cancer, pRB is inactivated by deletion/mutation or by DNA tumor viruses, such as papillomaviruses, which produce viral proteins that bind and sequester pRB (Moody and Laimins, 2010), leading to the release of E2F and activation of the transcriptional machinery. Without pRB functioning as the gate-keeper of the early checkpoint, cancer cells are free to divide without submitting themselves to quality controls throughout G1. Such a situation fosters accumulated mistakes in DNA replication (S phase), uncontrolled growth, and tumor development. Although pRB itself is rarely mutated in human cancers, its pathway is disregulated in a large majority of patients, stressing the importance of the pathway to oncogenesis. One of the most common alterations is overexpression of Cyclin D, cdk 4 and cdk 6. Furthermore, growth factor pathways are frequently hyperactivated in the same cells that harbor pRB pathway disruptions, resulting in amplified dysregulation of G1 transit. The transcription factor p53 monitors the strength and/or fidelity of events related to cell cycle progression, including strength of oncogenic signaling, hypoxia, DNA damage, and transcription (Weinberg, 2007). When abnormalities are sensed, a variety of pathways can trigger signals that increase levels of p53 (Harris, 1996). In turn, p53 activates pathways that regulate whether the cell should arrest progression while

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repairing cellular damage (DNA) or undergo programmed cell death or apoptosis. If the cell is able to repair the abnormality sensed by p53, it can reenter and complete the cell cycle or enter a state of dormancy/senescence from which it does not return. When damage is too great for the cell to survive, p53 initiates apoptosis. The TP53 gene is the most commonly mutated tumor suppressor in human cancers, a testament to its importance. Loss of p53 function results in continual cycling of cells even in the presence of metabolic imbalances, DNA damage, and transcriptional irregularities, giving rise in many instances to cells that accumulate mutations in key cell signaling pathways, that harbor chromosomal abnormalities and that have altered their metabolic equilibrium – all hallmarks of malignancy.

Oncogenes, Tumor Supressor Genes and Cell Metabolism Fully differentiated, nondividing cells have low nutrient requirements and derive the majority of their ATP from catabolism of glucose to pyruvate, which is further processed through the TCA cycle and the electron transport chain (oxidative phosphorylation) (Vander Heiden et al., 2009). In contrast, rapidly dividing cells (normal or cancerous) have high nutrient requirements and an increased need for synthesis of macromolecules (proteins, RNA and DNA, carbohydrates, and complex lipids) to support cell division (Figure 2(a)). To satisfy these needs, rapidly dividing cells take up more glucose and glutamine than nondividing cells, and process them through glycolysis and glutamine catabolism, thereby generating both ATP and macromolecules, and reducing their dependence on oxidative phosphorylation. Cancer cells accomplish this conversion from oxidative phosphorylation to glycolysis even in the presence of high oxygen tension, termed aerobic glycolysis, a hallmark of the Warburg effect. Oncogenes and tumor suppressor genes antagonistically regulate this conversion through altered production of catabolic and anabolic enzymes, posttranslational modifications, and protein–protein interactions (Dang and Semenza, 1999; Iurlaro et al., 2014). We will briefly discuss three examples of such genes, namely, PI3K, MYC, and p53.

The PI3K pathway AKT, a major effector of PI3K (Figure 1(b)), is an important stimulator of the tumor glycolytic phenotype. Activation of AKT increases the expression and translocation of glucose transporters, phosphorylation of key glycolytic enzymes, and activation of mTOR through phosphorylation of negative inhibitors of mTOR (Cairns et al., 2011). The serine/threonine kinase mTOR, in turn directly stimulates mRNA translation and new protein synthesis by phosphorylating/activating S6 kinase and phosphorylating/inactivating 4EBP1. Through an indirect transcriptional mechanism, mTOR also increases levels of HIF1 (Denko, 2008), a key sensor of oxygen levels and a transcription factor whose levels are augmented under hypoxic conditions, conditions frequently observed in centers of human tumors. HIF1 increases synthesis of multiple glycolytic enzymes, thereby significantly contributing to the conversion from oxidative phosphorylation to glycolysis.

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Figure 2 Oncogenes and tumor suppressor genes in metabolism and cell survival/apoptosis. (a) Glycolysis and oxidative phosphorylation (OXPHOS) are major pathways for energy generation, principally via the utilization of glucose and glutamine. The major points of regulation by oncogenes (ovals) and tumor suppressors (rectangles) are indicated (see text for details). (b) Cell survival is mediated by two major pathways, the extrinsic pathway, which is stimulated by extracellular death ligands, and the intrinsic pathway, which is activated by intracellular cues that regulate the balance between pro- and anti-apoptotic Bcl-2 family proteins. In normal cells, RTK signaling upregulates anti-apototic Bcl-2 family proteins, whereas the tumor suppressors p53 and pTEN upregulate the activity of pro-apoptotic Bcl-2 family members. In cancer cells, the normal control of these pathways is perturbed (see text for details). Filled ovals denote oncogenes, and filled rectangles represent tumor suppressors.

mTOR also plays a central role in the integration of growth factor signals and nutrient availability through regulation by adenosine monophosphate activated kinase (AMPK), a sensor of adenosine triphosphate (ATP) concentrations available to the cell (Figure 1(b)). A high AMP:ATP (Hardie, 2007) ratio activates AMPK, which then inactivates mTOR, thereby shifting

from glycolysis to ATP production through the oxidative phosphorylation pathway. AMPK activity itself is positively regulated by LKB1, a tumor suppressor mutated in liver and lung cancers. LKB1 is a serine-threonine kinase that phosphorylates AMPK to enhance its negative effect on mTOR (Shaw et al., 2004). Thus the LKB1/AMPK axis functions

Cell Division/Death: Cell Cycle: Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease

in opposition to PI-3 kinase to regulate cellular energetics. Mutation of LKB1 and high levels of oncogenic signaling can override the negative control of LKB1/AMPK through hyperactivation of PI3K, permitting cells to divide under unfavorable energetic conditions.

MYC The MYC oncogene regulates cell metabolism in multiple ways, including increased expression of enzymes involved in glutamine metabolism (Dang and Semenza, 1999; Figure 2 (a)). MYC also collaborates transcriptionally with HIF1 to elevate levels of glucose transporters and glycolytic enzymes, thereby, reinforcing glycolysis, particularly under hypoxic conditions. Under normoxic conditions, HIF1 protein levels are reduced by oxygen-dependent hydroxylation and ubiquitination, mediated in part by the tumor suppressor, von Hippel–Lindau (VHL) protein, a ubiquitin ligase. Thus, HIF1 is regulated positively by oncogenes such as tyrosine kinases and MYC and negatively by VHL (Kim and Dang, 2006).

TP53 The tumor suppressor p53 plays a major role in regulating metabolism largely through its transcriptional activity (Levine and Puzio-Kuter, 2010). In normal cells, p53 helps to maintain appropriate levels of signaling proteins and metabolic enzymes that favor oxidative phosphorylation and limit glycolysis (Figure 2(a)). In cancer cells that harbor p53 mutations, conversion from oxidative phosphorylation to the glycolytic phenotype is favored. Specifically, mutated p53 results in increased expression of glucose transporters, enhanced nucleotide biosynthesis, and reduced synthesis of cytochrome c oxidase and negative regulators of PI3K. Thus, the rigorous control p53 exercises over metabolism in normal cells (opposing growth factor action) is diminished in cancer cells by mutational inactivation, which permits conversion of the cell to a metabolic phenotype that favors rapid growth.

Oncogenes, Tumor Suppressor Genes, and Apoptosis Oncogenes and tumor suppressor genes play major roles in determining whether cells survive or die by regulating the balance between pro-survival and apoptosis-inducing pathways, regulated by the Bcl-2 family (Adams and Cory, 2007). This large family of genes (total of 25) can be either proapoptotic (e.g., Bax, Bad, Bak, and Bok) or anti-apoptotic (e.g., Bcl-2, Bcl-xL, and Bcl-w) and together regulate the major pathways (extrinsic and intrinsic) that activate apoptosis (Figure 2(b)). Members of this family of proteins associate with one another at the mitochondrial membrane and regulate the organelle’s redox potential and membrane permeability. An overabundance of pro-apoptotic proteins at the membrane results in pore formation, release of cytochrome c into the cytoplasm, formation of the apoptosome, and activation of the apoptotic process. Pro-survival oncogenes, such as growth factors, their receptors, and components of their signaling pathways, increase levels of anti-apoptotic Bcl-2 family members, whereas tumor suppressors, such as p53, PTEN, and IGF1 binding proteins, increase levels of pro-apoptotic family members.

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Another function of pro-survival oncogenes is to activate the PI3K and NFκB pro-survival pathways. PI3K activation leads to AKT phosphorylation and inactivation of the Bad, caspase 9, and IκB pro-apoptotic proteins (Manning and Cantley, 2007), whereas NFκB activation leads to enhanced expression of many pro-survival genes, most notably Bcl-2 (Cogswell et al., 2000). PI3K and NFκB effects are opposed by PTEN and IκB, respectively. The tumor suppressor p53 can directly induce apoptosis if the cell has incurred too much damage to divide (Fridman and Lowe, 2003). This is accomplished by transcriptionally regulating many pro-apoptotic genes, such as increasing expression of the Fas death-inducing receptor, IGFBP3 (which sequesters IGF-1 and prevents it from binding its receptor), Bax (which oligomerizes and induces cytochrome c release from the mitochondria), and NOXA (which binds and inhibits the anti-apoptotic protein Bcl-2). P53 also inhibits the expression of pro-survival genes, such as Smac/DIABLO, which bind and inhibit inhibitors of apoptosis (IAPs). Finally, p53 can act by directly binding members of the Bcl-2 family to promote apoptosis (Haupt et al., 2003). Loss of p53 function in cancer can lead to survival of cells that have sustained multiple mutations and damage, thereby permitting unregulated growth.

Role of Oncogenes and Tumor Suppressor Genes in Human Developmental Diseases Sustained proliferative signaling resulting from activated oncogenes and inactivated tumor suppressor genes are fundamental traits of cancer. However, oncogenes and tumor suppressors also play important roles during embryonic development, as highlighted by the diverse set of human syndromes resulting from congenital mutations affecting not only the predisposition to neoplasms but also bone growth, neurodegeneration, and glucose homeostasis.

Developmental Syndromes of RAS-ERK and PI3K-AKT Dysregulation Noonan/LEOPARD, neurofibromatosis type1, PTEN hamartomatous, and Proteus/Cowden syndromes are autosomally inherited familial syndromes that involve members of the RAS-ERK or PI3K-AKT signaling pathways (Figure 3(a)). Dysregulation of these pathways during embryonic development and neural crest differentiation results in common features, including craniofacial dysmorphism, cardiac malformations and cutaneous, musculoskeletal and ocular abnormalities, neurocognitive delay, and in some syndromes a predisposition to the development of cancer.

Noonan syndrome Noonan syndrome (NS) is an autosomal dominant disorder that is characterized by postnatally reduced growth, distinctive craniofacial features, congenital cardiac defects, bleeding disorders, and variable cognitive deficits (Tartaglia et al., 2011). Mutation of PTPN11, which encodes the non-receptor protein tyrosine phosphatase SHP2, accounts for the majority of cases. SHP2 is a negative regulator of RTKs and the RAS-ERK pathways, which when mutated and inactivated in NS patients

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Figure 3 Oncogenes and tumor suppressor genes in developmental and metabolic disorders. (a) Developmental abnormalities associated with the RAS-ERK and PI3K-AKT pathways. Components of the RAS-ERK and PI3K-AKT pathways activated downstream of RTK are dysregulated in various human developmental syndromes. (b) Factors that affect the FYN-LKB1-AMPK pathway regulation of cellular metabolism and diabetes. The FYNLKB1-AMPK pathway activity is increased by diverse factors, such as low circulating glucose, sedentary lifestyle, and AICAR (an analog of AMP and activator of AMPK) to regulate multiple metabolic processes. See text for details.

leads to increased signaling through the RAS-ERK pathway (Keilhack et al., 2005). SOS1 missense mutations are the second most common cause of NS occurring in approximately 13% of cases. SOS1 is a RASGEF that becomes mutationally activated in NS, resulting in increased GTP-bound Ras and enhanced downstream signaling (Roberts et al., 2007).

Neurofibromatosis The main characteristics of Neurofibromatosis type1 (NF1) include cutaneous, subcutaneous and plexiform neurofibromas,

café-au-lait spots, and Lisch nodules in the iris. Changes of the skeletal system, vascular defects, learning disabilities, poor social skill, and predisposition for the development of malignant neoplasia are also observed (Friedman and Birch, 1997). NF1 is caused by heterozygous loss-of-function mutations or deletions of the NF1 gene, encoding a GTPase-activating protein that functions as a negative regulator of RAS GTPase. Loss of NF1 results in hyperactivation of Ras signaling pathways, which contributes to the development of the defects observed in neurofibromatosis.

Cell Division/Death: Cell Cycle: Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease PTEN-opathies Germline mutations of the tumor suppressor, PTEN, have been identified in patients with diverse clinical features ranging from overgrowth to malignancy that are collectively classified as PTEN hamartomatous syndromes (PHS). These include Cowden syndrome (CS) and Bannayan–Riley–Ruvalcaba syndrome (BRRS).

AKT1 and PIK3CA mutations in Proteus and Cowden syndrome Approximately 11% of CS patients carry germline AKT1 or PI3K mutations. Cell lines generated from patients that have these mutations display increased phosphorylation of AKT1 and PIP3 levels, providing evidence that mutation of AKT1 or PI3K is a predisposition for CS (Orloff et al., 2013). An activating AKT1 mutation was also found to cause Proteus syndrome (Lindhurst et al., 2011), characterized by segmental overgrowth of the brain, bone, skin, and other tissues. Overall, the relatively high prevalence of some of these disorders and the frequency of defects in RAS-ERK and PI3KAKT signaling associated with them suggest that these pathways represent one of the most common events affecting the developmental process.

Oncogenes and Tumor Suppressor Genes in Neurodegenerative Diseases In mammals, most neurons are generated before birth, but it is now well accepted that neurogenesis continues into adulthood. Accumulating evidence indicates that impaired neurogenesis is associated with dysregulation of oncogenic signaling pathways and tumor suppressor function leading to development of diseases, such as Alzheimer’s and Parkinson’s diseases. In these neurodegenerative disorders, one of the main etiological features is enhanced cell death, accompanied by dysregulation of the tumor suppressor, p53.

Parkinson’s disease In Parkinson’s disease (PD) the main locus of degeneration is a small area in the ventral midbrain, the pars compacta of the substantia nigra that is mostly composed of dopaminergic neurons (Barzilai and Melamed, 2003). PD-affected substantia nigra harbors intracellular inclusions named Lewy bodies containing α-synuclein. Autosomal dominant inherited cases of PD are linked with mutations in α-synuclein that abrogate its ability to protect neuronal cells challenged with pro-apoptotic effectors by lowering p53 expression and transcriptional activity (da Costa et al., 2000). Transgenic mice expressing α-synuclein with the pathogenic A53T mutation exhibit accumulation of p53 in the mitochondria and induction of cell death, suggesting a critical role of p53 in the development of the disease (Giaime et al., 2006). Several lines of evidence indicate that proteins associated with autosomal recessive cases of PD also functionally interact with p53. PD cases of recessive inheritance result from mutation in three genes encoding parkin, PINK-1, or DJ-1. Parkin physically interacts with the p53 promoter and represses p53 transcription (da Costa and Checler, 2010). Mutation of Parkin prevents repression, allowing elevated transcription of p53 and induction of apoptosis. Mutations in DJ-1 also result

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in accumulation of p53, as well as p53’s transcriptional target Bax (Fan et al., 2008).

Alzheimer’s disease The pathology of Alzheimer’s disease (AD) is characterized by two histological lesions, the senile plaques and the neurofibrillary tangles. The severity of these lesions correlates with the level of cognitive decline. Tangles result from the intracellular accumulation of hyperphosphorylated tau protein, while senile plaques are extracellular deposits mainly due to the aggregation of various hydrophobic amyloid-β peptides (Mandelkow, 1999; Masters et al., 1985). That cell death in AD is p53-dependent is supported by several observations. APPderived fragments can modulate p53 by directly transactivating its promoter. Additionally, oxidative DNA damage induces the extracellular translocation of APP, concomitantly with increased p53 mRNA levels. Finally, in both mouse models of AD and in AD brains, neurons with altered morphology display both APP and p53 accumulation (Ohyagi et al., 2005). Comparison of p53 immunoreactivity in postmortem ADaffected brains revealed a consistent increase in p53 expression in the temporal cortex areas as compared to normal controls (Kitamura et al., 1997). The increased expression of p53 coincides with increased DNA fragmentation and Fas expression, indicating that p53 can contribute to cell death in AD brains (de la Monte et al., 1997). The tumor suppressors p21, p27, PTEN, and BRCA1 have also been implicated in the pathology of neurodegenerative disease. BRCA1, for example, co-localizes with neurofibrillary tangles. Analysis of clinically normal, aged brain tissue also revealed less BRCA1 than in cases of AD. This finding suggests that neurofibrillary tangles of normal aging differ in origin from those present in neurodegenerative disease. In addition to the suppression of cell cycle, BRCA1 is implicated in the maintenance of genomic integrity. In cells with BRCA1 deletion, a loss of telomere repeats was observed, a feature present in degenerating neurons of AD (McPherson et al., 2006). Studies of patients and animal models of PD have also demonstrated a direct role for PTEN in neurodegeneration associated with oxidative stress. Oxidative stress in rat hippocampal cells leads to mitochondrial accumulation of PTEN resulting in the induction of apoptosis. Conversely, knockdown of PTEN prevents neuronal apoptosis (Morris et al., 2010). Oncogenic signaling pathway activation leading to aberrant cell cycle entry of terminally differentiated neuronal cells has been proposed as a model of pathogenesis in neurodegenerative disorders. Cell cycle reentry driven by adenoviralexpression of c-MYC and RAS oncogenes in post-mitotic primary cortical neurons leads to tau protein phosphorylation and conformational changes similar to those observed in AD (McShea et al., 2007). Evidence of cell cycle reentry is also present in mutant APP and tau transgenic mice, occurring prior to the development of pathological features such as neurofibrillary tangles and senile plaques (Lee et al., 2009).

Oncogenes and Tumor Suppressor Genes in Diabetes Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia and altered lipid metabolism

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Cell Division/Death: Cell Cycle: Interplay between Oncogenes and Tumor Suppressor Genes in Human Disease

due to peripheral insulin resistance and defects of insulin secretion by pancreatic islet β-cells. The insulin receptor activates multiple downstream pathways to control energy homeostasis; however, the PI3K-AKT pathway is considered its major effector. The insulin-independent LKB1-AMPK pathway also contributes to metabolic control. Dysfunction of these pathways can lead to impaired glucose homeostasis and diabetes.

PI3K-AKT pathway Genetic defects in loci encoding insulin pathway components, transcription factors or rate-limiting enzymes of glucose metabolism account for only 1–5% of diabetes cases (Permutt et al., 2005). Nonetheless, the effects of the genetic alterations in insulin signaling downstream of PI3K-AKT has been extensively studied. For example, the amino acid substitution R409Q in the catalytic p85a subunit of PI3K has been shown to compromise insulin-stimulated PI3K activity, while the mutation R274H within the AKT2 kinase domain results in severe autosomal dominant insulin resistance. This substitution greatly impairs AKT kinase activity and functions in a dominant-negative fashion to inhibit adipocyte differentiation in vitro. As is the case in humans, mice lacking AKT2 are insulin resistant, hyperglycemic, and hyperinsulineamic (George et al., 2004). Conversely, mice lacking negative regulators of PI3KAKT signaling exhibit improved glucose homeostasis. Both PTP1B (a phosphoserine-phosphothreonine phosphatase) deficiency and PTEN hemizygosity lead to improved glucose tolerance and insulin sensitivity in mice (Elchebly et al., 1999; Wong et al., 2007).

LKB1-AMPK pathway Although some cases of obesity and T2DM result from genetic dysfunction, the increased incidence of these disorders is strongly correlated with sedentary lifestyle and over-nutrition. Dietary changes and regular exercise are the first therapies for the management of T2DM. However, when lifestyle modifications are insufficient to control serum glucose and lipid levels, drugs that increase insulin action and/or induce body energy consumption are used for treatment. Two commonly used drugs that potentiate insulin action, metformin and thiazolidinediones, have been shown to activate the LKB1AMPK pathway (described above; Figure 3(b)). AMPK activity is increased during exercise and could, at least in part, mediate the favorable effects of physical activity on insulin sensitivity, lipid and glucose utilization in skeletal muscle, adipose tissue, and liver (Ruderman et al., 2003). Studies with pharmacological inhibitors of AMPK and in animal models harboring AMPK deletions indicate that AMPK activation positively regulates glucose uptake and metabolism. The serum glucose lowering effects of LKB-AMPK pathway activation occurs through CREB-dependent transcription of PGC-α and subsequent inhibition of the glucogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (Koo et al., 2005). In addition to its role in glucose metabolism, the LKB1AMPK pathway plays a critical role in the regulation of fatty acid oxidation in skeletal muscle and liver. AMPK increases fatty acid oxidation by phosphorylating the inhibitory site of acetyl-CoA-carboxylase, one of the main rate-controlling enzymes for the synthesis of malony-CoA, a potent inhibitor

of mitochondrial fatty acid oxidation. AMPK also represses pyruvate kinase and fatty acid synthase expression leading to decreased lipogenesis (Cook and Gamble, 1987; Leclerc et al., 1998). These actions can contribute to the prevention of obesity and insulin resistance. Although studies of LKB1-AMPK function in animal models of T2DM indicate that pathway activation may prevent disease onset, data collected to date from individuals with obesity and insulin resistance, with or without diabetes, have not detected aberrant AMPK activity in skeletal muscle of these patients (Hojlund et al., 2004). These studies suggest that impaired insulin action on glycogen synthesis and lipid oxidation in skeletal muscle of these patients is not associated with altered AMPK expression or activity. However, it has also been reported that exercise-induced activation of AMPK is impaired in muscle of obese individuals with or without T2DM suggesting that activation above basal level could be effective in the treatment of diabetes and metabolic syndromes (Steinberg et al., 2004).

FYN tyrosine kinase as a novel regulator of LKB1/AMPK Recently, FYN, a non-RTK of the SRC family, was shown to play a role in insulin sensitivity and lipid utilization by regulating LKB1 function (Figure 3(b)). FYN knockout mice exhibit marked reductions in adiposity, fasting glucose and insulin levels, as well as improved insulin sensitivity (Bastie et al., 2007). FYN deletion also improved plasma and tissue triglycerides, increased energy expenditure and enhanced fatty acid oxidation. The increased catabolism coincided with increased mitochondrial markers and AMPK phosphorylation in adipose tissue and skeletal muscle. Pharmacological inhibition of FYN in wild type mice induces weight loss, due to increased fatty acid oxidation associated with whole-body energy expenditure. FYN was also shown to modulate LKB1 subcellular localization through direct phosphorylation of LKB1 tyrosines 261 and 365. Mutation of these residues within LKB1 results in cytoplasmic localization and subsequent activation of AMPK1 in muscle cells (Yamada et al., 2010). These findings establish the proto-oncogene FYN as a novel nutrient sensor and regulator of adipose tissue mass and insulin sensitivity. Taken together these observations indicate that activation of oncogenic signaling pathways through mutations in oncogenes or loss of tumor suppressors can impair or improve metabolic control.

See also: Cell Division/Death: Cell Cycle: Regulation of the p53 Pathway

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