The Philadelphia chromosome was initially described initially as a minute chromosome and assumed to be the product of a loss of genetic material from chromosome 22 (Nowell and Hungerford, 1960). Improvements in cytogenetics permitted more careful examination of chromosome defects, allowing Janet Rowley to determine that the deletion of genetic material in chromosome 22 was matched by a comparable insertion in chromosome 9 (Rowley, 1973). She speculated that the chromosomal defects observed in CML cases were the result of a translocation exchange between chromosomes 9 and 22. By 1985, it had been determined that cells of most patients with CML feature reciprocal translocation events between the long arms of the 2 chromosomes, resulting in the creation of a BCR-ABL hybrid gene (see Fig 7–8). The same translocation event has been found to occur in 25% to 30% of adult and 2% to 10% of pediatric ALL, and occasionally in cases of acute myelogenous leukemia (AML) (De Klein et al, 1986).
The ABL kinase is the human homolog of the transforming sequence found in the Abelson murine leukemia virus. Whereas the protein normally shuttles between the nucleus and cytoplasm, the product of the BCR-ABL fusion gene is a constitutively active tyrosine kinase permanently localized in the cytoplasm. The addition of the BCR gene product appears to promote extensive multimerization of the fusion protein, facilitating self-activation by trans-autophosphorylation. During the chronic phase of CML, the BCR-ABL kinase activates a number of downstream pathways while also increasing genomic instability. In the absence of therapeutic intervention, additional genomic aberrations ultimately lead to a transition from the chronic to the advanced CML phase known as blast crisis that is more difficult to treat. At a cellular level, the blast phase is marked by increased proliferation accompanied by impaired apoptosis and differentiation, which together increase the population of blast progenitors. One noteworthy molecular change is a moderate increase in BCR-ABL levels, enhancing throughput of the relevant signaling pathways and begetting still more genetic damage.
The correlation of a disease state with a particular genetic lesion offered the possibility for targeted therapeutic intervention. The subsequent development and successful application of imatinib, which binds in the vicinity of the adenosine triphosphate (ATP)-binding site and locks BCR-ABL in an inhibited conformation, remains one of the most celebrated achievements in targeted cancer therapy.
MYC family members are global transcription factors (see Chap. 8, Sec. 8.2.6) that activate some target genes while repressing others, thereby affecting numerous cellular processes including proliferation, growth, apoptosis, and angiogenesis (Fig. 7–10) (Meyer and Penn, 2008; Albihn et al, 2010). Roughly 15% of all human genes are thought to be regulated by MYC proteins, so it is difficult to pinpoint the specific MYC targets that are responsible for oncogenesis. For example, MYC promotes cell-cycle progression by influencing a variety of targets, including downregulation of CDK inhibitors and upregulation of CYCLIN D1, CDK4, CDC25A, and E2F transcription factors (see Chap. 9, Sec. 9.2.2). Numerous genes, associated with the production of the constituent building blocks required for cell growth, are also controlled by the MYC proteins. Importantly, MYC deregulation appears to correlate with general chromosomal instability, although there is not yet agreement about the responsible mechanisms. Paradoxically, MYC upregulation also triggers apoptosis, and additional oncogenic mutations capable of blocking cell death are required for tumor progression. Thus, MYC overexpression results in pleiotropic downstream effects that collectively promote oncogenic transformation.
MYC targets and the promotion of tumorigenesis. The MYC transcription factor is thought to regulate the expression of roughly 15% of all human genes and is frequently upregulated in a variety of cancers. MYC upregulates the expression of some genes while repressing the expression of other targets. Unsurprisingly, given the number of genes affected by MYC, its deregulation contributes to the acquisition of many of the characteristic properties of cancer cells, as defined by Hanahan and Weinberg (2011). One notable exception is the paradoxical induction of apoptosis by MYC overexpression, necessitating the disengagement of cell death pathway(s) by additional oncogenic hits so as to foster transformation. Some of the MYC-regulated genes involved in tumorigenesis are denoted, although the particular MYC targets critical to oncogenesis will undoubtedly vary in different tumors.
Transforming viruses can direct oncogenesis by proviral DNA integration in the proximity of protooncogenes or tumor-suppressor genes, leading to altered expression or splicing (see Chap. 6, Sec. 6.3). An interesting example is the mechanism of chicken B-cell lymphogenesis by the slowly transforming avian leukosis virus (ALV). The ability of ALV to induce lymphomas was initially puzzling because of the apparent absence of any transforming sequence in the viral genome. Nevertheless, a careful analysis of lymphomas from independently infected birds revealed proviral integration at highly specific sites, giving rise to similar viral–host hybrid RNAs (Hayward et al, 1981). The viral coding segments were often altered in ways that precluded their transcription, indicating that viral protein expression was not required for transformation. Remarkably, the hybrid RNAs were found to include Myc coding sequences leading to increased expression of myc protein, suggesting that proviral integration promoted oncogenesis through upregulation of myc levels. Lymphomas in birds caused by ALV infection are slow growing as the oncogenic mechanism involves clonal selection of the rare proviral integration events in the vicinity of the myc protooncogene. This constituted the first observation of neoplastic transformation caused by the upregulation of a nonmutated cellular gene. Interestingly, the acutely transforming avian myelocytomatosis retrovirus (MC29), from which MYC derives its name, induces leukemia in birds through expression of v-Gag-Myc already encoded in the viral genome (Reddy et al, 1983).
Upregulation of Myc expression is observed in some mouse plasmacytomas as a result of a recombination between the Myc and immunoglobin (Ig) heavy-chain genes. Burkitt lymphomas, as well as other human B-cell lymphomas, frequently feature chromosome translocation events between chromosome 8, where the MYC gene is located, and chromosomes 14, 2, or 22, where the Ig heavy- and light-chain genes reside. These rearrangements have the effect of bringing the MYC gene under the transcriptional control of Ig gene enhancer elements, leading to its overexpression. Whereas genomic translocations involving MYC genes are common in hematopoietic cancers, heightened MYC levels are often attained in solid tumors through gene amplification. The MYCN gene, which is normally expressed during development, has been found amplified in neuroblastomas, and increased copy numbers of the related MYCL1 gene have been observed in several cancers, including ovarian tumors.
The involvement of epidermal growth factor (EGF) signaling in tumorigenesis has been recognized for 3 decades. The EGF family is comprised of 11 related ligands that stimulate intracellular signaling by binding to the EGF receptors, ERBB1 (EGFR), ERBB3, and ERBB4 (Baselga and Swain, 2009). A fourth member of the EGF receptor family, ERBB2 (HER2), cannot bind ligands itself, but is activated by dimerization with other family members (see also Chap. 8, Sec. 8.2.1). The EGF receptors are transmembrane proteins consisting of an N-terminal ligand-binding ectodomain, an intracellular kinase domain, and a C-terminal regulatory tail that contains docking sites for a number of downstream effectors. Ligand binding to the ectodomain induces receptor dimerization, transmitting conformational changes that promote kinase activation. Active receptor signaling complexes can be formed by both homodimerization and certain heterodimer combinations. The ERBB3 receptor binds ligands but has an inactive kinase domain and therefore can only transmit the EGF signal as part of a heterodimer with an active family member (Fig. 7–11).
EGF receptor family. The EGF receptor family consists of 4 transmembrane proteins that couple a ligand-binding domain that responds to external stimuli with intracellular tyrosine kinase domains responsible for signal transduction. ERBB1 (EGFR), ERBB3, and ERBB4 each respond to a particular set of EGF family ligands, with some overlap, whereas ERBB2 appears to be an orphan receptor that must dimerize with ERBB1 or ERBB3 in order to engage downstream pathways. In addition, ERBB3 has an inactive kinase domain and transmits signals by dimerizing with ERBB2 or ERBB4. Collectively, these receptors regulate pathways that impact a number of cell processes, and are deregulated in a number of cancers.
All 3 portions of the ERBB1 receptor are subject to oncogenic mutations (Pines et al, 2010). Ligand-independent dimerization occurs as a result of point mutations or, more commonly, deletions within the ectodomain that leave the receptors in a constitutively activated conformation. Many such deletion mutants have been characterized, with one notable example being the variant III mutant, lacking the exons 2 to 7 coding sequences, which is particularly common in gliomas, but also found in lung, breast, ovarian, and other cancers. Interestingly, these deletion mutants mimic the avian erythroblastosis virus v-ErbB oncoprotein, from which the EGFR family members took their names. Numerous point mutations are also found in the kinase domain, especially in non–small cell lung cancers, which promote constitutive activation, even in the absence of ligand-induced dimerization. Again, the effects of the point mutations are also closely replicated by small insertions and deletions within the kinase domain.
Activation of the EGFR receptor results in phosphorylation of tyrosine residues in the C-terminal tail, which serve as docking sites for the recruitment of downstream effectors (see Chap. 8, Sec. 8.2). Generally, activating point mutations result in the receptor tail attaining a phosphorylation status intermediate between that of unstimulated and growth factor-stimulated wild-type receptors. An important consequence of this "partial" activation is a resulting shift in the downstream signaling output from the mutant receptors. Whereas stimulated wild-type epidermal growth factor receptors (EGFRs) activate a number of downstream pathways, the mutant receptors tend to prominently activate mitogen-activated protein (MAP) kinase and phophatidylinositol-3 (PI3) kinase signaling at the expense of the other pathways. Normally, EGF signaling is attenuated by a feedback mechanism that involves the removal of the receptors from the cell membrane by endocytosis, followed by lysosomal degradation or recycling. Some point mutations have been found to disrupt this regulatory device, resulting in hyperactivation of the EGF pathway.
EGF signaling has an important physiological role in mammary development, and deregulation of downstream pathways feature prominently in many breast tumors (Schmitt, 2009). The ERBB2/ERBB3 heterodimeric receptor complex is a potent signal transducer, particularly of the PI3 kinase pathway, because the ERBB3 receptor recruits the PI3 kinase directly rather than through intermediary adaptor proteins. The ERBB2(HER-2) gene is amplified or overexpressed in roughly one-quarter of all human breast cancers, as well as a smaller percentage of ovarian and gastric cancers. Overexpression of the ERBB2 receptor has also been correlated with elevated ERBB3 levels. Although ERBB2 somatic mutations have now been identified in some lung tumors, gene amplification is the most common mechanism for oncogenic upregulation of the receptor. Amplification of the ERBB2 gene is generally an early event in breast cancer progression and is associated with poor prognosis in breast and gastric cancers.
In response to specific upstream signals, the PI3 kinases phosphorylate the 3′ hydroxyl group of phosphatidylinositols, generating membrane-embedded lipid secondary messengers that activate downstream pathways (Fig. 7–12; see also Chap. 8, Sec. 8.2.5) (Engelman et al, 2006). Although the superfamily includes a number of isoforms, the preponderance of research has focused on the Class IA PI3 kinases, which consist of p110 catalytic and p85 regulatory subunits. These preferentially convert phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), which recruits Pleckstrin homology (PH) domain-containing proteins, such as the PKB/AKT serine/threonine kinase, to the plasma membrane for activation. The involvement of PI3 kinase signaling in oncogenesis was evident in the 1980s from the discovery of an interaction between the transforming polyoma viral middle T antigen and the p85 subunit of the kinase (Whitman et al, 1985; Otsu et al, 1991). In parallel, a constitutively active version of the p110 subunit was isolated from the genome of the ASV16 avian retrovirus, which was known to transform chicken fibroblasts (Chang et al, 1997).
PI3 kinase pathway. The PI3 kinase pathway regulates a variety of cell functions, including migration, metabolism, survival, proliferation, and growth. It is frequently deregulated in human cancers at various points. In response to external stimuli, activated receptor tyrosine kinases recruit PI3 kinase via its p85 regulatory subunit, either directly or mediated by an adaptor protein such as IRS-1. Upon recruitment, activated PI3 kinase (PI3K) converts PIP2 (phosphatidylinositol 4,5 bisphosphate) to PIP3 (phosphatidylinositol 3,4,5 triphosphate). This reaction is antagonized by the lipid phosphatase, PTEN, which acts as a brake on the pathway. PH-domain proteins, such as the PDK1 and AKT kinases, are recruited to the plasma membrane by PIP3, whereupon PDK1 contributes to the activation of AKT, which then transmits the signal downstream by phosphorylating a range of targets. AKT also modulates the RHEB guanosine triphosphatase (GTPase)-TORC1 signaling axis by downregulating the TSC1–TSC2 complex, which is modulated by a number of upstream signals including activation by LKB1-AMPK in response to energy deprivation. The PI3 kinase pathway is generally overstimulated in cancers through aberrant activation of the receptors, activating mutations in the PI3 kinase catalytic subunit (p110), and through the loss of PTEN.
More recently, activating mutations have been identified in the PIK3CA gene, encoding the p110 subunit, in a number of tumors, including breast, prostate, endometrial, and colon cancers (Chalhoub and Baker, 2009). These mutations result in constitutive activation of the kinase in the absence of upstream signaling, a buildup of PIP3, and increased mobilization of downstream pathways that regulate cell processes such as growth, proliferation, survival, migration, and glucose metabolism. Remarkably, approximately 80% of all somatic PI3 kinase mutations occur at 3 hotspot residues: E542K and E545K within the helical domain, and H1047R in the kinase domain. All 3 mutations have been found to increase the in vitro activity of the kinase as well as the PI3 kinase signaling throughput in cells and tissues bearing the mutations. Amplifications of the PIK3CA gene are sometimes observed in cervical, head and neck, gastric, as well as lung cancers. Less frequent cancer-associated activating mutations have also been identified in the PIK3R gene, encoding the p85 subunit, as well as the AKT1 gene, encoding 1 of the more prominent downstream effectors of PI3 kinase signaling.
The importance of MAP kinase signaling in neoplastic transformation is evident from the frequency of oncogenic mutations affecting multiple proteins in the pathway, including growth factor receptors, RAS guanosine triphosphatase (GTPase), and its immediate target, RAF (Fig. 7–13) (Karreth and Tuveson, 2009). The pathway modulates a number of cellular processes, including proliferation, growth, survival, differentiation, and migration, albeit in a tissue-dependent manner (see also Chap. 8, Sec. 8.2.3). RAS genes are among the most commonly mutated genes in human cancer, with a variety of aberrations detected in at least 30% of all tumors. Among the 3 RAS genes, the preponderance of mutations is observed in the K-RAS gene, especially in pancreatic, lung, colon, endometrial, and ovarian tumors. Mutations in the H-RAS gene are more commonly observed in bladder cancers with N-RAS mutations found in neuronal, melanoma, and myeloid malignancies.
MAP kinase pathway. The mitogen-activated kinase (MAPK)/ERK pathway is frequently deregulated during oncogenesis. The pathway is stimulated by binding of ligands to their cognate receptors, ultimately resulting in activation of the ERK1/2 kinase. Activated receptors recruit adaptors, such as GRB2, and guanine nucleotide exchange factors (GEFs), such as SOS, which promotes the guanosine triphosphate (GTP) loading of RAS. RAF is then activated by RAS-GTP, triggering a kinase cascade resulting in the sequential activation of MEK1/2 and ERK1/2, whose downstream targets regulate a number of cell functions including migration, survival, proliferation, and growth. Overactivation of the pathway through activating mutations or amplification of receptors, as well as activating mutations in various RAS isoforms or B-RAF, is commonly observed in human cancers.
The most common mutations are gain-of-function substitutions in codons 12, 13, and 61, with many lower-frequency mutations identified throughout the protein sequence. The RAS gain-of-function mechanism is somewhat distinct in that it is the RAS GTPase enzymatic turnover that is blocked by the oncogenic mutations, effectively locking the protein into the activated guanosine triphosphate (GTP)-bound signaling state, and resulting in the upregulation of its downstream effectors.
The primary downstream effectors of RAS are the 3 RAF family serine/threonine kinases (see also Chap. 8, Sec. 8.2.3). Although a virally transduced version of C-RAF exhibits oncogenic properties, missense mutations are infrequently observed in the C-RAF gene, and these rare substitutions do not generally appear to stimulate kinase activity. Similarly, mutations in the A-RAF gene have been detected very rarely. B-RAF is the most frequently mutated RAF family member with approximately 40% of malignant melanomas and 2% of all human cancers, including thyroid, colon, and ovarian cancers, exhibiting B-RAF mutations (Beeram et al, 2005; Maurer et al, 2011). The V600E substitution in the kinase activation loop accounts for roughly 90% of all B-RAF mutations. Development of vemurafenib, an agent that specifically targets this mutation, has led to marked improvement in outcome for patients with malignant melanoma (Chapman et al, 2011). Interestingly, mutations in the RAS and B-RAF genes are almost entirely mutually exclusive, indicative of the potency of a single oncogenic hit at either gene that evidently relieves virtually all the selective pressure for additional mutations in the pathway. Indeed, the B-RAFV600E mutant protein is constitutively activated and essentially independent of upstream signaling from growth factor receptors or RAS. An analysis of the effects of the V600E substitution on the B-RAF structure suggested that the mutation results in the activation loop adopting an open conformation than is more accessible to substrates. Typically, this conformation change is accomplished by specific regulatory phosphorylation events particularly within the kinase activation loop.
Despite extensive genome-wide sequencing efforts to produce a detailed genetic map of the cancer landscape, surprisingly few novel oncogenes and tumor-suppressor genes have been identified. The IDH1 and IDH2 genes, encoding 2 previously unglamorous isocitrate deydrogenase metabolic enzymes, are the most prominent examples of the worthiness of these large-scale projects. The mechanism by which these proteins contribute to tumorigenesis, though far from clear, appears to define a novel oncogenic paradigm. Now recognized as oncogenes, they were initially mistaken for tumor suppressors because the observed cancer mutations destroy their normal cellular activities.
Exon sequencing carried out on human glioblastoma multiforme, as well as other cancers, revealed somatic mutations in IDH1 and IDH2 at homologous arginine positions in some of the sampled tumors (Parsons et al, 2008; Yan et al, 2009). Functional characterization of the mutant proteins indicated that the amino acid substitutions led to a loss of function, destroying their ability to convert isocitrate into α-ketoglutarate, and suggesting that these were novel tumor suppressors. However, there was no evidence of a LOH at the IDH loci in human tumors, indicating that they did not conform to Knudson's 2-hit tumor-suppressor model. An alternative explanation for these findings arose from the observation that cells harboring the tumor-associated IDH1 mutation exhibit an accumulation of a particular metabolite, 2-hydroxyglutarate (2-HG) (Dang et al, 2009). An analysis of the effects of the mutation on the IDH1 structure suggested that although the active site of the mutant enzyme failed to interact with isocitrate, its normal substrate, it was capable of interacting with α-ketoglutarate and converting it to 2-HG. Thus, the arginine substitution effectively represents both a loss-of-function and gain-of-function mutation that destroys the normal enzymatic activity while creating a novel one.
On the basis of these results, it has been proposed that 2-HG represents an oncometabolite whose ability to promote oncogenesis needs to be fully elucidated. In addition to gliomas, IDH1 and IDH2 mutations have also been detected in AML genomes, including a novel mutation in the IDH2 sequence which also leads to 2-HG build up (Balss et al, 2008; Ward et al, 2010). Importantly, knockdown of IDH1 and IDH2 reduced the growth of cultured AML cells, consistent with their roles as oncogenes rather than tumor-suppressor genes.