2.4 Products of Oncogenes

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Oncogenes, whether gained through viral infection or a gain of function mutation, can be classified based on the cellular functions that they are involved in. Some of these categories include signal transduction modulators, transcription factors, apoptosis regulators and epigenetic reprogramming enzymes. Essentially, to become an oncogene, a mutation must occur in expression regulator, promoter, or within the gene itself such that cell growth does not occur at a controlled or regular rate. This section will discuss the main groups of oncogenes and their protein products exploited by cancer cells to promote uncontrolled growth.

 

 

Signal Transduction Modulators

 

Signal transduction is crucial for the progression of cancer from a single cell to a large tumor with the ability to metastasize to distant locations. Even though they do not contribute directly to the transformation of cancer cells, factors involved in signal transduction are necessary for subsequent steps in cancer progression such as clonal expansion, angiogenesis, tissue invasion and metastasis (1). Key factors in signal transduction are growth factors (GF), growth factor receptors (GFR), and signal transducers; all of which are crucial for tumor growth (2). Together they act by constitutively activating signalling pathways in the cells which will permanently activate or inhibit certain cellular processes.

There are several well-studied families of growth factors, such as epithelial growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF) (2). In normal cells, these growth factors are expressed upon receiving a signal to proliferate through ligand-receptor interactions. However, growth factor genes can acquire mutations that result in constitutive expression despite the lack of an activating receptor-substrate interaction. Alternatively, an oncogene may be created through mutation of a cell surface receptor or downstream signalling proteins involved in cell proliferation.  Many well studied signal transducers are protein kinases, which when mutated may constantly phosphorylate their targets resulting in an inappropriate signal cascade (2). In many cancer phenotypes, several growth factors are inappropriately activated, leading to dysregulated cellular processes.

 

Insulin growth factors (IGF) are fundamental in normal development, cell growth, and metabolic responses to nutrients; in an effort to promote self-renewal, pluripotency, and EMT, cancer cells often exploit IGFs and their receptors.  EMT is a process by which epithelial cells transition into much more mobile, and invasive, mesenchymal cells (15), and is a key step in the metastasis of primary tumors.  Exposure of IGF-I to cancer cells causes EMT through a number of different downstream pathways.  These include, the activation of NF-κB and subsequent upregulation of Snail.  Snail induces EMT by binding to the E-box promoter of E-cadherin (15), effectively inhibiting the transcription of this key protein required for cell-cell adhesion (14).  Furthermore, increases in IGF expression have been shown to cause a downregulation of GSK3β (14), which is a kinase response for the inhibition of EMT through targeting the Wnt/β-catenin pathway (15).  Interestingly, EMT may produce a positive feedback loop by inducing autocrine production of more IGF-I, further stimulating the EMT process (14).  Together, these pathways indicate that cancer cells can utilize IGF signaling as an oncogenic method of metastasis.

 

Transforming growth factor- β (TGF-β) is a signaling molecule that interacts with TGF- β receptors (TβR1) and plays a regulatory role in cellular proliferation, differentiation, development and angiogenesis.  In cancer it is utilized to increase canonical Smad signaling, which induces an epithelial-to-mesenchymal transition favoring metastasis and invasion (16).  Furthermore, aberrant TGF- β signaling can inhibit both CD4+ and CD8+ T cells by repressing the function of antigen presenting cells (16), this allows for a shift from differentiated anti-tumor cells into progenitor cells that release more TGF-β into the tumor environment.  Effectively, abnormal TGF- β signaling causes a positive feedback loop through immune suppression, where increased levels of TGF-β are associated with increased angiogenesis (16).  Together, these cause increase tumor growth and mobility, in combination with the ability to move away from the primary tumor and into metastasized locations around the organism.

 

The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase, carries mutations in many different types of cancers (3). Normally, EGFR is involved in cellular proliferation and migration and has important roles during development (3, 4). This transmembrane receptor is activated by binding of a ligand, which induces dimerization (4). Dimerization results in activation of the kinase domain and stimulates a signalling cascade (4). There are multiple ways through which constitutive activation of EGFR may be achieved including overexpression, truncation, and mutations in the kinase domain, all of which can lead to activation independent of ligand binding (3). The most common EGFR variant in glioblastoma, EGFRvIII, lacks a large portion of its extracellular domain, so is unable to bind ligands and remains constitutively active (3, 4). 

                                                                              Figure 2.4.1. How a signal can alter cellular processes. Released under the Creative Commons Attribution-ShareAlike 4.0 International license (CC BY-SA 4.0).

                                                                 

Transcription Factors

 

Transcription factors (TF) are a large family of proteins downstream of the signal transduction pathway that bind to regulatory regions of genes and control their expression. Depending on the region and properties of the TF, this association can either enhance or suppress gene expression. Tight regulation of gene expression by transcription factors is essential for controlled cell growth and cellular functionality. Targets of TFs are extremely diverse; they can be found in almost every cellular process from apoptosis to DNA replication to cytoskeleton remodelling (5). Alterations of transcription factors can lead to many problems, such as underexpression or overexpression of gene products, which may in turn lead to cancer (5). 

                                         
 

One example of an oncogenic TF is the Y-Box Binding Protein (YB-1). Its wildtype function is to activate genes during embryonic development at the correct time in order for cells to differentiate. In adult cells, YB-1 is usually silent (6). When inappropriately expressed in adult cells, like in many breast and prostate cancers, YB-1 induces the expression of oncogenes by transporting mRNA between the nucleus and the cytoplasm. This results in activation of genes such as MHCII, EGFR, Her2, and c-Myc. These help neoplastic cells to evade immunosurveillance and also promote growth and proliferation (6).

Another example of an oncogenic TF is the c-Myc protein. c-Myc forms a heterodimer with another protein called Max, which can then bind to the E-boxes of DNA (12). Full length c-Myc on its own is unable to bind to DNA. Myc activates gene expression by creating a heterodimer with the Max protein and various other co-factors, including transcription factor 2H, and the transformation/transcription domain-associated protein (TRRAP) protein (13). The E-boxes to which c-Myc binds can be bound by other transcription factors, so competitive binding can affect the level of gene regulation (13). 

 

Apoptosis Regulators

 

Apoptosis is programmed cell death; an important step in maintaining normal cell functions and deterring cancer formation. Apoptosis regulators may function either as oncogenes or tumor suppressor genes. If a cell loses the capacity to undergo apoptosis, it will have increased potential to become immortalized, and therefore be at a higher risk of becoming cancerous. In a normal cell, apoptotic signals can come from two sources: stress from the environment and death signals from other cells (7). For a tumor to survive, it must be able to ignore signals coming from both of these pathways - even uncontrolled cellular proliferation alone creates a stressful and toxic milieu, and the host immune system attempts to prevent further proliferation via death signals (7).

Two well known genes that help cancer cells evade apoptosis are the genes encoding Protein Kinase B (AKT) and the B-Cell Lymphoma 2 (Bcl-2) family of proteins. Both of these proteins have been found to be upregulated in many different cancers. AKT inactivates the FOXO transcription factor which normally transcribes pro-apoptotic genes, while at the same time activates NFkB, a TF which transcribes anti-apoptotic genes (8). Bcl-2 inhibits apoptosis by maintaining the mitochondrial membrane potential as well as inhibiting the release of cytochrome C from the mitochondria (7).

 

 

Epigenetic Reprogramming Enzymes

 

Epigenetics is the study of heritable genetic processes (i.e chromatin modifications / chromatin state) that alter temporal and spatial gene expression without changing the genetic sequences. The structure of chromatin is modified to expose the DNA strand such that proteins such as RNA polymerase and transcription factors can bind to and activate or inhibit the genes, in order to guide downstream protein expression (10). Different modification mechanisms include direct modification of the DNA strand such as DNA nucleotide methylation, and DNA binding protein modifications such as histone acetylation and deacetylation, as well as phosphorylation of other proteins associated with the DNA strand (5). During these processes, various enzymes add or remove these markers on to the chromatin at certain locations. If the epigenetic code is dysregulated or modified incorrectly due to misfolded, malfunctioning, or absent enzymes, it can lead to cancer.

One well studied chromatin modifying enzyme that is potentially oncogenic is the Mixed-Lineage Leukimia (MLL) Histone Methyltransferase. MLL methyltransferase is found to be involved in Acute Lymphocytic Leukimia (ALL) and Acute Myelocytic Leukimia (AML) (10). The wildtype function of MLL methyltransferase is to modify chromatin around HOX gene loci - genes encoding a transcription factor that controls embryonic development along the anterior-posterior axis and determines segment structures. However, MLL can potentially translocate to more than 50 target genomic sites that are correlated with leukimia occurrence, leading to altered function of the enzyme and altered expression of HOX genes that results in leukemia phenotype (10).

An enzyme that induces epigenetic change by direct DNA alteration is the tet methylcytosine diozygenase 2 (TET2) enzyme. This enzyme converts methylated DNA, namely methylcytosines into 5-hydroxy methyl cytosines which is the rate limiting step in the cascade leading to eventual demethylation of the nucleotide (11). Demethylation caused by abberant TET2 activity in neoplastic growths causes genes previously epigenetically inactivated to become expressed, including oncogenes. Wherease reduced TET2 actvity causes overmethylation, causing some tumor supressor genes to be turned off. Furthermore, it has been hypothesized that hyper demethylation observed in gain of function TET2 mutant tumor cells is causing reactivation of dormant retroviruses. These now active retro viruses are excising and reinserting in different parts of the tumor cells genome causing even more mutations and genome instability. 

 

References

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  4. Normanno, N. et al. (2006)."Epidermal growth factor receptor (EGFR) signaling in cancer." Gene 366(1):2-16. 
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  6. Wu J., Stratford A.L., Astanehe A., and Dunn S.E. (2007). “YB-1 is a Trascriptional/Translational Factor that Orchestrates the Oncogenome by Hardwiring Signal Transduction to Gene Expression.” Transl. Onco. 2:p49-65.
  7. Lowe S.W. and Lin A.W. (2000). “Apoptosis in cancer.” Carcinogenesis. 21(3):p485-495.
  8. Altomare D.A. and Testa J.R. (2005). “Perturbations of the AKT signaling pathway in human cancer.” Oncogene. 24:p7455-7464.
  9. Critical Reviews in Oncology/Hematology 2007; 61:52
  10. Guenther M.G. et al. (2005). “Global and Hox-specific roles for the MLL1 methyltransferase.” PNAS 102(24):p8603-8608.
  11. Yang H. et. al. (2013), "Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation” Oncogene 32, 663-669.
  12. Dang C.V. et al. (1999), "Function of the c-Myc Oncogenic Transcription Factor" Experimental Cell Research 253, 63-77.
  13. Dang C.V. (2012), "MYC on the Path to Cancer" Cell 149, 22-35
  14. Malaguarnera, R and Belfiore, A (2014).  The emerging role of insulin and insulin-like growth factor signaling in cancer stem cells.  Frontiers in Endocrinology 5(10):1-16.
  15. Tsai, HA et al. (2013). 3, 5, 4' - trimethoxystilbene, a natural methoxylated analog of resveratrol, inhibits breast cancer cell invasiveness by downregulation of PI3K/Akt and Wnt/(beta)-catenin signaling cascades and reversal of epithelial-mesenchymal transition.  Toxicology Applied Pharmacology, 272(3): 746-756
  16. Sheen, YY et al. (2013). Targeting and Transforming Growth Factor-Beta signaling in Cancer Therapy. Biomolecules and Therapeutics, 21(5): 323-331.