Oncogenes- lecture-3

Oncogenes in Human Cancer

• Oncogenes are cancer susceptibility genes.
• Proto-oncogenes are normal genes that are present in normal cells and are involved in normal growth and development.
• Under certain circumstances due to the effect of certain processes protooncogenes are converted to Oncogenes.

Significance of Proto-oncogenes

In the normal cellular environment, proto-oncogenes have crucial roles in cell proliferation and differentiation (Figure 1). The normal growth and differentiation of cells are controlled by growth factors that bind to receptors on the surface of the cell. The signals generated by the membrane receptors are transmitted inside the cells through signaling cascades involving kinases, G proteins, and other regulatory proteins (See figure 2). Ultimately, these signals affect the activity of transcription factors in the nucleus, which regulate the expression of genes crucial in cell proliferation, cell differentiation, and cell death.  Proto-oncogene products have been found to function at critical steps in these pathways and inappropriate activation of these pathways can lead to tumorigenesis (Figure-1).

Figure -1– Under the effect of Chemical carcinogens, radiations or viruses or in persons with a genetic predisposition, the proto-oncogenes are transformed into oncogenes. The oncogenes have either abnormal or more gene products resulting in altered cellular functions and malignant transformation of a normal cell to cancer cells.

Mechanisms of Oncogene Activation

Mechanisms that up-regulate (or activate) cellular oncogenes (Proto-oncogenes) fall into four broad categories: point mutation, gene amplification, chromosomal rearrangement, and insertional mutagenesis.

1) Point Mutation

Point mutation is a common mechanism of oncogene activation. For example, mutations in one of the RAS genes (HRAS, KRAS, or NRAS) are present in up to 85% of pancreatic cancers and 50% of colon cancers but are relatively uncommon in other cancer types. Most of the activated RAS genes contain point mutations in codons 12, 13, or 61.(figure 2)

Mechanism ofRasactivation- The gene product (p21) is related to a G protein that modulates the activity of Adenylate cyclase and thus plays a key role in cellular responses to many hormones and drugs. p21 has GTPase activity also to terminate the hormonal action. The mutations in p21 appear to affect its conformation and to diminish its activity as a GTPase. The lowered activity of GTPase results in chronic stimulation of the activity of Adenylate cyclase, which normally is diminished when GDP is formed from GTP. The continuous stimulation of the activity of Adenylate cyclase can result in a number of effects on cellular metabolism exerted by the increased amount of c AMP affecting the activities of various cAMP-dependent protein kinases. These events shift the balance of cellular metabolism towards a state favoring malignant transformation.

Figure 2- showing the effects of activates RAS protein. Growth factor- Receptor binding stimulates tyrosine kinase activity that stimulates, Ras protein, a G protein that is active when bound to GTP. Active Ras protein activates adenylate cyclase and a phosphorylation cascade is triggered. Mutated Ras protein remains active due to the inability to replace GTP by GDP resulting in phosphorylation of cellular proteins and more synthesis of cell cycle regulatory proteins and thereby malignant transformation of the cell.

2) Gene Amplification

The second mechanism for activation of oncogenes is DNA sequence amplification (Figure-3), leading to overexpression of the gene product.(Figure 3) Gene amplification is observed in tumors in patients on Methotrexate, an anticancer drug, an inhibitor of an enzyme dihydrofolate reductase. Tumor cells can become resistant to the action of this drug. The basis of this is that the gene for dihydrofolate reductase becomes amplified, resulting in an increase in activity of the enzyme up to 400 folds.

Certain cellular oncogenes can also be amplified in this manner and can get activated. An increased amount of the products of certain oncogenes, produced by gene amplification may play a role in the progression of tumor cells to a more malignant state.

Figure-3–showing gene amplification

3) Chromosomal Rearrangement

• Chromosomal alterations provide important clues to the genetic changes in cancer.
• The chromosomal alterations in human solid tumors such as carcinomas are heterogeneous and complex and likely reflect selection for the loss of tumor-suppressor genes on the involved chromosome.
•  In contrast, the chromosome alterations in myeloid and lymphoid tumors are often simple translocations.
• The basis of translocation is that a piece of one chromosome is split off and joined to another chromosome.
• If the second chromosome donates material to the second, the translocation is said to be ‘Reciprocal’.
• The breakpoints of recurring chromosome abnormalities usually occur at the site of cellular oncogenes.

Examples

a) Burkitt’s lymphoma, a B cell tumor characterized by a reciprocal translocation between chromosomes 8 and 14. The segment of chromosome 8 that breaks off and moves to chromosome 14 contains C-MYC. As shown in the figure-4, the transposition places the previously inactive C-MYC under the influence of the enhancer sequences the genes coding for the heavy chains of immunoglobulins. This juxtaposition results in the activation of transcription of C-MYC. There is a greatly increased synthesis of C-MYC coded DNA binding protein that acts to drive or force the cell towards becoming malignant, perhaps by an effect on the regulation of mitosis.

Enhancer activation by translocation, although not universal, appears to play an important role in malignant progression. In addition to transcription factors and signal transduction molecules, translocation may result in the overexpression of cell cycle regulatory proteins such as cyclins and of proteins that regulate cell death such as bcl-2.

Figure-4- showing chromosomal translocation in Burkitt’s lymphoma

b) Philadelphia chromosome

The first reproducible chromosome abnormality detected in human malignancy was the Philadelphia chromosome detected in CML (Chronic myelogenous leukemia). This cytogenetic abnormality is generated by a reciprocal translocation involving the ABL oncogene, a tyrosine kinase on chromosome 9, being placed in proximity to the BCR (breakpoint cluster region) on chromosome 22. (Figure -5). The consequence of expression of the fused BCR–ABL gene product is the activation of signal transduction pathways leading to cell growth independent of normal external signals.

Normally C-ABL encodes a protein kinase. The juxtaposition results in chimeric BCL-ABR m RNA, which encodes a fusion protein displaying the increases tyrosine kinase activity. The increased activity transforms the normal cell to a leukemic cell.

Imatinib, a drug that specifically blocks the activity of BCR–ABL has shown remarkable efficacy with little toxicity in patients with CML.

 

Figure-5- showing Philadelphia chromosome in Chronic Myelogenous Leukemia, Chromosome 9 and 22 are involved in this translocation forming the BCR-ABL fusion gene.

4) Insertional mutagenesis- This process occurs in viral Oncogenesis. Certain viruses lack oncogenes but may cause cancer over a longer period of time. When these viruses infect cells, a DNA copy (c DNA ) of their genome is synthesized by the activity of reverse transcriptase enzyme and the cDNA is integrated into the host genome. The integrated double-stranded c DNA is called “Provirus”. Based on the site of their insertion, two mechanisms are involved –

a) Promoter Insertion– The cDNA copies of retroviruses are flanked at both ends by sequences named as long terminal repeats. These sequences are important in proviral integration and they can act as promoters of transcription (Figure-6).

For example-following infection of chicken B lymphocytes by certain avian leukemia viruses, the provirus becomes integrated near the myc gene. The myc gene is activated by an upstream, adjacent long terminal repeat acting as a promoter, resulting in transcription of its product in such cells. A B cell tumor is formed. By a similar mechanism, the human C-MYC gene is activated causing colorectal carcinoma.

Figure-6- Proto-oncogene activation by insertional mutagenesis of MMTV-(a) Insertion of viral genomic DNA into somatic cellular DNA in close proximity of a silent oncogene. (b) Inserted proviral DNA induces the transcription of the oncogene.

b) Enhancer Insertion- In some cases, the provirus is inserted downstream from the myc gene, or upstream from it but oriented in the reverse direction, nevertheless the myc gene becomes activated. Such activation can not be due to promoter insertion since a promoter sequence must be upstream of the gene whose transcription it increases and the and the sequence must be in the correct 5’-3’ direction. Instead, the enhancer sequences present in the long terminal repeat sequences of the retroviruses are involved.

Of the mechanisms described above, the mechanisms like-promoter insertion, enhancer insertion, chromosome translocation, and gene amplification, cause an increase in the amount of the gene product due to increased transcription of the oncogene. Thus, an increased amount of the product of an oncogene may be sufficient enough to push a cell towards becoming malignant.

Figure-7- Summary of the mechanisms involved in the conversion of Proto-oncogenes to Oncogenes. If the point mutation takes place in the regulatory genes, the excess gene product is formed but if the point mutation takes place in the structural genes, the abnormal gene product is formed.

Point mutation, on the other hand, involves a change in the structure of the gene product, but the amount mostly remains the same unless it is in the regulatory genes (Figure-7). Thus it implies that the presence of a structurally abnormal key regulatory protein in a cell may also shift the equilibrium of a cell towards malignant transformation.

Activation of oncogenes alone is not the sole pathway of malignancy. A combination of activation of oncogenes and inactivation of tumor suppressor genes is involved in the malignant transformation of cells in certain types of cancers(Figure-8).

Figure-8- Highlighting the role of oncogenes and tumor suppressor genes in carcinogenesis

Product of oncogenes

1) They act on the key intracellular pathways involved in growth control.

2) They act as DNA binding proteins to affect the control of the cell cycle

3) The product of certain oncogenes act as growth factors or imitate the action of an occupied growth factor receptors.