As discussed throughout this chapter, oncogenes arise from normal genes within the genome (proto-oncogenes) that have been altered in a way that leads to transformation of the cell. This change from a normal proto-oncogene to a cancer-causing oncogene is called oncogene activation. Previously we discussed how viruses could activate oncogenes by insertional mutagenesis, or by incorporating active versions of oncogenes into the genome. In the absence of viral influences, oncogenes are primarily activated in one of three ways: chromosomal rearrangements, gene mutations, and gene amplification (1) (Fig. 2.5.1).
Chromosomal rearrangements (including chromosome inversion and translocation) occur when segments of a chromosome are moved and recombine in a novel location, either within the same chromosome or to a non-homologous one (2). These rearrangments can result in either the fusion of a proto-oncogene to regulatory regions that increase its expression, or the fusion of two genes that now code a novel protein with oncogenic function (2).
One example of a chromosome translocation event occurs in Chronic Myelogenous Leukemia (CML) and results in the production of a fusion protein known as the Philadelphia chromosome. This rearranged chromosome is found in over 90% of CML cases (3). It is created by a reciprocal translocation between a segment from chromosome 9, which contains the ABL gene and a segment on chromosome 22 containing the BCR gene (4). The outcome is a shortened chromosome 22, also known as the Philadelphia chromosome, that contains a BCR-ABL fusion gene encoding the fusion protein. Abl is a member of the Src family of kinases that normally function in signal transduction pathways which are under tight regulatory control (3). Bcr-Abl performs similar functions to the wildtype Abl protein but is constitutively active, and thus drives cell growth and transformation (3).
Oncogenic fusion genes produced by chromosomal rearrangements are common in many cancers, especially haematological cancers, sarcomas and prostate cancer (5). While some chromosomal rearrangements are caused by translocations (like the Philadelphia chromosome), others are caused by inversion events. One such example is the tropomyosin receptor kinases (trk) oncoprotein. The neurotrophic tyrosine receptor kinase (NTRK) family are receptors that regulate cytoskeleton assembly, axonal and dendritic growth, synaptic and protein channel functions, cell survival and proliferation, retrograde signaling and receptor communication (6). The second component of this fusion gene, Tropomyosin 3 (TPM3), is a non-muscle, actin binding protein with a coiled-coil structure (7). A chromosomal inversion in chromsome 1 between the NTRK gene and the TPM3 gene results in a chimeric protein, trk, in which 7 out of 8 exons from TPM3 have replaced the transmembrane and cytoplasmic domains of the NTRK protein (6). Though not well understood, this chimeric protein is predicted to be constitutively expressed by the transphosphroylation of tropomyosin kinase domains that are exposed cytoplasmically, or dimerization caused by the added tropomyosin coiled-coil structure (7). Figure 2.5.2 diagrams the inversion between NTRK and TPM3 which causes formation of the trk oncogene.
Aside from the creation of fusion proteins, chromosomal rearrangments can also activate oncogenes by driving over-expression of a normal protein. An example of this can be found in Burkitt’s lymphoma, which was previously discussed in this chapter. A segment from chromosome 8 carrying the c-MYC gene is translocated to a region on chromosome 14 encoding immunoglobulin lymphocyte-specific genes and is positioned near an Ig regulating enhancer which drives the expression of c-MYC (8, 9). This results in increased expression of c-MYC, a transcription factor that drives cell proliferation pathways, thus causing excessive growth of B lymphocytes (8).
Point mutations (the alteration of a single base pair) can be caused by environmental carcinogens or occur spontaneously, and these small genetic changes can alter the conformation of encoded proteins enough to confer oncogenic activity (10). One example of this is seen in the RAS family of oncogenes. Ras proteins have two states: the inactive form which is bound to the guanine nucleotide GDP, and the active form which is bound to GTP (11). The active GTP-bound form is normally inactivated relatively quickly by GTPase-activating proteins (GAPs) which cause the hydrolysis of GTP to GDP (11). Many Ras mutations interfere with this process, producing a constitutively active oncoprotein that triggers signaling pathways for continuous cell growth (1). Point mutations at codons 12, 13 or 61 within the RAS gene are found in many cancers, the highest incidence being 90% in pancreatic cancer cases (12). Another example of an oncogene which can be activated by point mutations is the epidermal growth factor (EGF) receptor, which is commonly mutated in lung cancers. When this cell surface receptor binds its ligand, it homodimerizes and triggers downstream signaling. Multiple point mutations can produce a constitutively active version of this protein, often by deleting the extracellular domain (13). Other types of local DNA rearrangements, such as insertions, deletions, and transpositions may also activate oncogenes. However, these types of mutations usually disable a gene and thus are found more frequently in the activation of tumor suppressor genes.
Gene amplification is the third main way that oncogenes can be activated (Fig. 2.5.1). Gene amplification occurs when a portion of the genome is duplicated, resulting in a higher than normal copy number of a certain gene (1). This often occurs during tumor progression and can involve hundreds of kilobases of DNA containing many genes (1). This leads to increased expression of the amplified genes and, if they are involved in cell proliferation and survival, can drive transformation of the cell. Commonly amplified oncogenes include some that we have already discussed such as RAS proteins and EGF receptors (1). Rather than finding mutated versions of these proteins in the cell, the normal protein can be expressed at high levels and acheive similar oncogenicity. The MYC gene family is also often amplified in many cancers (1). Earlier we discussed c-MYC in the context of Burkitt's lymphoma, where chromosomal translocation causes increased expression of this gene. Gene amplification of c-MYC is common in other cancers including lung, breast, cervical, and ovarian cancer, and has similar oncogenic effects, though the protein is upregulated by duplication of the gene rather than a translocation event (14).
Another type of commonly amplified proto-oncogene is ERBB2, which is implicated in breast and ovarian cancers (15). This gene encodes a tyrosine kinase, the ERBB2 protein which is a transmembrane receptor, and is similar to EGF receptors (15). There has been debate as to whether gene amplification is a spontaneous or induced process (15). However, experimental evidence indicates that gene amplification is not a direct result of exposure to cytotoxic or exogenous agents. Rather, amplification occurs in the replication and repair stage, after the cell’s DNA has been damaged by internal or external agents (15). Gene amplifications give an impression of how genetically unstable tumor cells can be due to genome alteration (15).
Local DNA Rearrangement
There are also other mechanisms by which oncogenes arise. Similar to the mechanism of chromosomal rearrangements, local DNA rearrangements can also induce activation of oncogenes. However, rearrangements of local DNA involve deletion, insertion, transposition and inversion of smaller pieces of DNA on a local scale. For example, the tal-1 gene undergoes a chromosomal translocation to cause T cell acute lymphoblastic leukemia (T-ALL) in 3% of patient (16). However, there are 25% of T-ALL patients that have gene rearrangement but cannot be detected by karyotype analysis, which measure chromosomal appearance to detect translocations. It was found that these patients have a small deletion (90kb). These deletion events arise independently in patients through site-specific DNA rearrangements (16).
Insertional mutagenesis is another mechanism that can activate oncogenes. This type of activation involves the infection of host cell with viruses or transposons. The viral or transposable element must be inserted into the proper location to trigger activation of an oncogene. For example, a proto-oncogene involved in cell proliferation can acquire increased expression due to an insertion of an enhancer. This gain-of-function mutation can occur through recombination, where an enhancer is inserted either upstream or downstream of the gene and can cause the gene to become an oncogene, promoting tumor formation as cell proliferation occurs at higher than normal levels.
As discussed previously, the parasitic nature of viruses requires the need for cellular machinery for its own replication. Increasing the host cell proliferation would also be beneficial for the virus to increase its own survival. Therefore, viruses have acquired the ability to overexpress genes to drive host cell proliferation.
Activation By Epigenetic Derepression
Unlike the cases of epigenetic silencing of tumor suppressor genes in cancer cells, the epigenetic events that lead to oncogene overexpression are far less well characterized. Much like promoter DNA hypermethylation functions in the silencing of tumor suppressor genes, promoter hypomethylation is thought to contribute to the activation of genes that are normally repressed or at low transcription levels in normal cell types. The correlation between upregulation and promoter hypomethylation has been reported within several oncogenes, including the the signalling cascade activator SNGG (17), MCJ, MAL, HOXA10, and TUBB3 (18).
Evidence linking the epigenetic modification of chromatin to oncogene activation has also been reported, but remains under established as well. The upregulation of claudin-3 and claudin-4, proteins which aid in ovarian cell invasion, was shown to be associated with a loss of the repressive histone modification H3K27me3 along the gene body in ovarian cancer cells (19). Such findings open the field to the suggestion that further chromatin modifacation could influence the activation of cancer-promoting genes, independent of DNA methylation.
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