Role of Oncogenes and Tumor-suppressor Genes in Carcinogenesis: A Review

Oncogenes and Cancer

In the normal cell, there are proto-oncogenes which are key regulatory factors of biological processes. Proto-oncogenes may function as growth factors, transducers of cellular signals and nuclear transcription factors (Table I). Mammalian and avian genomes contain a range of proto-oncogenes which control normal cell differentiation and proliferation (3). Changes to these genes that influence either the control of their behavior or the way that their encoded proteins are structured can show up in cancer cells as enacted oncogenes. When such oncogenes are formed, they go on to drive cell multiplication and assume a pivotal role in the pathogenesis of cancer. The physical mutations that lead to the activation of proto-oncogenes can be distinguished into two types: those that lead to differences in the structure of the encoded protein and those that cause deregulation of protein expression (4, 5). Mutations influencing structure include point mutations of RAS proto-oncogenes and chromosomal translocations that produce hybrid genes, such as the Philadelphia translocation (BRC-ABL). Increased expression occurs in human tumors via the amplification of genes or translocation of chromosomes, such as those that put the MYC gene under the control of the immunoglobulin enhancer sequences (5-8).

Proto-oncogene activation brings about their change into oncogenes; to date 50 to 60 oncogenes have been recognized (9). Although various proto-oncogenes have been identified in actuated, oncogenic form in human tumor genomes, the exact hereditary modifications that caused any of these actuations remain unclear. In a typical cell, proto-oncogene expression is controlled by its own transcriptional promoter – the sequence of DNA that controls the degree of transcription. Each proto-oncogene promoter enables the gene to react to an assortment of physiological signals. A proto-oncogene can be expressed at quite low levels depending on the cell's metabolic needs; nevertheless, on specific events, when necessary, the expression of the gene may be strongly induced (9).

The activation mechanisms of proto-oncogenes are as follows (5, 9):

Chromosomal translocation of a proto-oncogene from a location that cannot be transcribed to an adjacent location where it can be transcribed (e.g. the chromosomal translocation of the MYC oncogene in human Burkitt's lymphoma).

Point mutation of a proto-oncogene, where substitution of a single base by another base is translated by substituting an amino acid in the oncoprotein (e.g. a point mutation at codon 12 of the RAS oncogene).

Gene amplification, by incorporating multiple copies of an oncogene, resulting in increased production of oncoprotein (e.g. c-MYC in neuroblastoma).

Incorporation of a gene that promotes the transcription (promoter gene) near the proto-oncogene resulting in overexpression of the gene (e.g. the retrovirus carcinogenicity mechanism).

Example of the chromosomal translocation of the MYC oncogene and its signaling pathway. The three nuclear phosphoproteins N-MYC, C-MYC and L-MYC are highly related and constitute the so-called MYC family encoded by cellular proto-oncogenes. These proteins originate from genes located on chromosomes 2, 8 and 1, respectively. Constituent parts of MYC are the first exon non-encoding for protein and the protein-encoding third exon. The cell cycle is regulated through a nuclear DNA-binding protein that originates from the MYC gene. Transformation, dedifferentiation, immortalization and cell proliferation are promoted by the MYC gene family (10).

C-MYC transcription factor can be dimerized with MAX dimerization protein 1 (MAX), which is involved in the transcriptional regulation of target genes associated with apoptosis and cell growth (11). The transactivation resulting from C-MYC deletions during nuclear translocation are involved in the negative regulation of adenomatous polyposis coli (APC) gene. This is regulated with the WNT pathway through β-catenin. Knockdown of APC can lead to carcinogenic expression of C-MYC. The activation of P53 or alternative reading frame (ARF) checkpoints are regulated by acute sustained oncogenic expression of C-MYC when C-MYC is deregulated (12).

MYC suppression is mediated by MYC–MAX with a regulation of the transcription of target genes using the ZBTB17 gene (MIZ1) line with the promoter Initiator element (Inr). Inr may be a MIZ1 cofactor or a silent C-MYC replacement of nuclear phosphoprotein (NPM) (13, 14).

The microRNA network may be also be regulated by C-MYC. This happens by suppressing dozens of miRs, such as miR-17-92. For example, lactate dehydrogenase expression is shown to be affected by miR-34a glutaminase expression, miR-23a/b and insulin signaling by LET-7. MicroRNAs include miR-17 that targets phosphatase and tensin homolog gene (PTEN) and continuously induces protein kinase B (AKT). Additionally, it activates transcription factor E2F1 and pro-apoptotic recombinant human BCL2 apoptosis regulator (BCL2)-like protein 11 (BIML). Epithelial–mesenchymal transition and angiogenesis are associated with miRNAs downstream of C-MYC (10).

Relationship with tumor. Deregulation of C-MYC has been found in the majority of human tumors. Augmented C-MYC expression is the consequence of either dysregulation of the signal transduction pathway for its expression or by mutations in the C-MYC locus and C-MYC is the most commonly mutated gene in tumors. New therapeutic approaches to anticancer treatment target C-MYC since in many cases the cause of tumorigenesis is its overexpression. A second mutation that deactivates the apoptotic pathway (e.g. p53) is regulated by C-MYC oncogene when normal cell apoptotic pathways lack adequate survival factors. Apoptotic inhibition and cell survival are stimulated by two synergistic oncogenes, such as BCL2, BCL-xL, and RAS (10, 11).

Example of oncogene to proto-oncogene conversion after a point mutation. For human pancreatic cancer, the most common causative event is a single missense mutation that is acquired in transforming protein 21 gene (KRAS) that modifies the 12th codon leading ultimately to either valine or aspartate substitution for glycine, finally causing protein activation.

Pancreatic tumor progression and initiation involves activation of oncogenic KRAS by means of metastasis, metabolism alteration, resistance to drugs and alterations in signal transduction pathways (1). Astonishingly, in distal metastasis, advanced or precursor lesions of KRAS disappearance can lead to rapid regression of the tumor (15-17). This suggests that tumor initiation and maintenance, as well as progression are all affected by the presence of oncogenic KRAS (1).

There is a close correlation between NRAS, HRAS and KRAS A small membrane GTPase is encoded by KRAS in KRAS signaling. RAS is converted to an active form by the binding of GTP under the regulatory influence of Guanine exchange factors (GEFs). Conversely RAS is converted to its inactive form after hydrolysis of GTP to GDP, mediated by GTPase-activating proteins. Finally, a number of cellular processes, including cell proliferation are triggered by RAS proteins, through a number of different downstream signaling pathways. There is specific activity on different cell types for each member of the RAS family via gene regulation. KRAS is necessary for the development in addition to its donative effects on pancreatic colon and lung cancer, by causing the GTP to GDP hydrolysis after oncogenic mutations induce RAS activation. Different tumor types are associated with different regulation of KRAS (18).

The activated small membrane GTPase RAS is the cornerstone of the KRAS pancreatic signaling cascade, which in turn leads to the activation of the phosphoinositide 3-kinase (PI3K), rapidly accelerated fibrosarcoma (RAF)/methyl-ethyl-ketone (MEK)/extracellular signal-regulated kinases (ERK) and RAS-related protein B (RALA/B) signaling branches. There are many direct and indirect interactions among these branches, as well as several inhibitory and regulatory feedback loops.

Activation of a receptor-linked tyrosine kinase such as epidermal growth factor receptor may activate RAS in an upstream signaling cascade. In such cases, a phosphorylation cascade can be activated after homodimerization of epidermal growth factor receptors upon their binding to extracellular growth factors. This in turn leads to the activation of GEFs and growth factor receptor-bound 2 adaptor protein acting on RAS and allowing the substitution of GDP to GTP, ultimately leading to the formation of KRAS-GTP. RAF serine/threonine kinases are attracted toward the cellular membrane and activated by KRAS-GTP. RAS homolog enriched in brain (RHEB) is negatively regulated by a complex of proteins encoded by several genes, such as TSC complex subunit genes TSC1 and TSC2, after transcription stimulation by ERK. PI3K containing p110 subunit (consisting of p110 and p85) is activated by KRAS-GTP, stimulating the PI3K signaling pathway which leads to the formation of phosphatidylinositol-4,5-tri-phosphate (PIP3) from phosphatidylinositol-4,5-bi-phosphate. Either mechanistic target of rapamycin kinase complex 2 (mTORC2) or phosphoinositide-dependent kinase-1 (PDK1) found on the plasma membrane can be activated by AKT after its stimulation by PIP3. PTEN, which acts as negative regulator of adenylate kinase 3 (AK3) is inhibited by the stimulation of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) under the influence of AKT. Additionally, mTORC1 bearsthe inhibitory TSC1/TSC2 complex that is blocked by AKT. Finally, the binding of GTP to the small RAL A/B GTPases cause its activation driven by the KRAS-GTP activation of RAL-GEF. TRAF family member-associated NFKB activator (TANK) inhibits the release of NF-kB when RALA/B binds TANK-binding kinase 1. In summary, protein translocation, proliferation and differentiation of cells are affected by the three signaling cascade branches (1).

It is now known that oncogenes can be formed or transferred to cells by means of viruses. Both DNA viruses and RNA retroviruses can transform the cells they infect (19, 20). DNA oncogenic viruses have as a common feature the interaction of their viral oncoproteins with specific regulatory proteins of the host cell, resulting in the onset of carcinogenicity. E7 protein encoded by human papilloma virus oncogenic types, adenovirus E1A protein, and SV40 virus large T-antigen all have the ability to bind to the peroxidase (pRB5) protein (19, 20). Retroviruses are oncogenic RNA viruses which can convert RNA into DNA by means of reverse transcriptase and then integrate into the DNA of the cell. Some retroviruses upon incorporation into the DNA of the cell ‘capture’ a proto-oncogene from the host and convert it to an oncogene, resulting in oncogenic transformation of the cell. Other retroviruses do not carry an oncogene but create tumors by their incorporation into the DNA at a location near a proto-oncogene, resulting in the activation of the proto-oncogene and its conversion to an oncogene (21). Several different oncogenes, derived from cell proto-oncogenes, have been identified by their homology with various transformed retroviral genes. Listed below are some examples of cellular oncogenes recognized by retroviruses: Simian sarcoma (SIS), McDonough feline sarcoma (FMS), erythroblastosis (ERBB), ABL, Harvey murine sarcoma (Ha-RAS), Kirsten murine sarcoma (K-RAS), avian sarcoma virus 17 (JUN), FBJ osteosarcoma (FOS), avian MC29 myelocytomatosis (MYC). Chemical carcinogens can also activate proto-oncogenes and are recognized as carcinogens with direct or indirect action. All chemical carcinogens have a common feature: the creation of electrophilic centers (22-24).

Tumor-suppressive Genes and Blockage of Carcinogenesis

In the normal cell, in addition to oncogenes, there are tumor-suppressive genes, which play a major role in the normal growth and differentiation of the cell and block the development of cancer. Tumor-suppressor genes form a huge group that exhibit one common characteristic: somehow, every single one of these genes protects the organism from neoplasia. Both copies of a tumor-suppressor gene must be in an inactive state before a cancer cell can proliferate or survive further. Lack or inactivation due to mutations of tumor-suppressor genes leads to cancer (1). Various tumor-suppressor genes are expected to lie spread around the genome of the humans and play a role in the advancement of various types of human neoplasia (25).

Examples of tumor-suppressor genes are retinoblastoma 1 protein (RB1) and P53. P53 protein, which is the product of the tumor-suppressor P53 gene, acts as a tetramer. Mutations of one allele in a cell can suppress the action of the P53 protein because all the tetramers will contain at least one mutant P53 protein. Mutations of the P53 tumor-suppressor gene are therefore prevalent. Retinoblastoma protein, on the other hand, acts as a monomer and hence its mutations are residual (4, 26).

Loss of heterozygosity may be caused by intermediate deficiencies, deletions of chromosomes, or abnormal mitotic divisions and is thought to reflect the loss of one or more tumor-suppressor genes (27).

Microsatellite DNA differs from the rest of DNA because its structure is based on the linear repetition of smaller subunits (up to six nucleotides) and does not code for proteins. Microsatellite DNA is used in genetic studies. The instability of microsatellite DNA reflects an increase in the rate of mutagenesis in microsatellite DNA sequences and thus in the entire genome and is associated with damage to the DNA-repair system (27).

The Clonal Theory of Oncogenesis

According to the clonal theory of oncogenesis, tumors start from a single cell. But the clonal origin of the tumors does not mean that a malignant tumor develops because of a single mutation. Oncogenes often act synergistically to cause malignant transformation. For example, we mention the synergistic effect of two oncogenes in transgenic mice, in which RAS and MYC oncogenes were transfected. MYC oncogene caused tumorigenesis only after 100 days and only in a small percentage of the transgenic mice. The MYC oncogene was initially discovered in the genome of an avian retrovirus and found to be activated through several alternative mechanisms in cancer cells: Proviral integration, gene amplification and chromosomal translocation. The RAS oncogene caused tumorigenesis earlier than the MYC oncogene, and after 150 days, 50% of the transgenic mice developed a tumor. In transgenic mice with both RAS and MYC oncogenes, cancer developed in 100% of the transgenic mice and in a much shorter period (28).

Programmed Cell Death (Apoptosis)

There are two forms of death of eukaryotic cells: Necrosis and apoptosis or programmed cell death. Necrosis is caused by abrupt cellular damage (e.g. ischemia) or after physical or chemical injury. In necrosis, changes occur in the protoplasm resulting in increased cell volume, cell death and finally DNA decomposition by the release of lysosomal enzymes (4). However, apoptosis is a normal occurrence of normal tissue growth and changes occur in the nucleus (nuclear breakage). In apoptosis, DNA fragmentation due to an endogenous endonuclease precedes cell death and it appears that cell death in apoptosis is not a direct result of DNA fragmentation but of the cell's inability to repair extensive DNA damage. Since the most common form of cell death is apoptosis, it seems that inhibition of apoptosis is responsible for the development of cancer (29).

Several genes have been associated with apoptosis and either stimulate or inhibit it. For example, the proteins of the MYC and BAX proto-oncogenes, the P53 tumor-suppressor gene and the E2F transcription factor activate apoptosis. On the contrary, the BCL2, ABL, and RAS oncoproteins inhibit apoptosis. Finally, RB may cause inhibition of the growth of human cancer cells (23, 29, 30).

Molecular Mechanisms of Angiogenesis in Solid Tumors

Angiogenesis and angiogenic factors. Angiogenesis is the development of new blood vessels (31). Angiogenesis naturally occurs during embryogenesis and healing of wounds, while occurring pathologically in rheumatoid arthritis, diabetic retinopathy, psoriasis, atherosclerosis and the development of solid tumors. The mechanism of angiogenesis as described in the literature involves the breakdown of the basement membrane, cell division and the migration of endothelial cells, and finally the anastomosis of the new vessel with a pre-existing one (32, 33).

Several angiogenic factors have been found that are expressed in solid tumors, such as platelet-derived endothelial cell growth factor, vascular endothelial growth factor, basic fibroblast growth factor and interleukin-8 (IL8). The activation mechanisms of angiogenic factors are under investigation, while hypoxia, and mutations in the P53 and IL8 have been found to be involved (34-37).

Natural inhibitors of angiogenesis. A large number of angiogenesis inhibitors are known and, in many cases, their genes have been isolated. The mechanisms of action of the natural inhibitors of angiogenesis to date are as follows (38, 39):

Inhibition of proteinases necessary for the basement membrane degradation of the blood vessels (e.g. plasminogen activator inhibitor and tissue inhibitor of metalloproteinase).

Inhibition of the action of growth factors on tumor vascular endothelial cells (e.g. α2 macroglobulin).

Induction of apoptosis of endothelial cells of tumors and metastases (e.g. tumor necrosis factor α, which is a cytokine, and angiostatin, which is a plasminogen fragment).