Question

Using one example of each class of gene, explain how mutations in tumour suppressor genes and oncogenes can lead to alterations in cell signalling pathways, and contribute to cancer development.

Answer 1

The mutations incurred in the normal-functioning genes can be classified as

• Gain of function mutation: mutation leading to the ability of new functions suppressing the normal functioning of the gene.
• Loss of function mutation: mutation leading to losing the ability of the gene to perform the normal functioning.

Mutations in TP53 - Tumor suppressor gene

TP53 encodes the p53 protein, an important tumor suppressor protein. Many different types of cancer show a high incidence of TP53 mutations, leading to the expression of mutant p53 proteins. TP53 is the most commonly mutated gene in human cancer. Alterations have been found in virtually every region of the protein, but only a handful of the most frequently occurring mutations have been studied in depth for their contribution to cancer progression. In some cases, frameshift or nonsense mutations result in the loss of p53 protein expression, as seen with other tumor suppressors. However, more frequently, the tumor-associated alterations in p53 result in missense mutations, leading to the substitution of a single amino acid in the p53 protein that can be stably expressed in the tumor cell. These substitutions occur throughout the p53 protein, but most commonly cluster within the DNA binding region of p53, with six “hotspot” amino acids that are most frequently substituted. These mutations generally lead to a loss or diminution of the wild-type activity of p53, and because p53 normally acts as a tetramer, these mutant proteins may also function as dominant negative inhibitors over any remaining wild-type p53.

p53 is a transcription factor able to regulate several intracellular pathways involved in cell survival, DNA-repair, apoptosis, and senescence. p53 is also known as “the guardian of the genome”, being able to preserve DNA integrity in response to a number of stimuli, such as ionizing radiations, genotoxic insults and oxidative stress. Several studies have demonstrated that many mutant p53 proteins, not only lose their tumor suppression functions but also gain new oncogenic functions. This phenomenon is termed “the gain of function of mutant p53”. More specifically, mutant p53 interacts with proteins that normally partner with wild type p53. This new association deprives them of their anticancer activities and in place, they are corrupted to act as cancerogenesis promoters.

PML stabilizes and activates wild type p53 only in presence of stressed conditions, while, when p53 is mutated its association with PML is constitutive. Mutant p53-PML stabilized interaction leads p53 to aberrantly transcribe its targets. The major consequences of the gain of functions mutant p53 affect the metabolism and the response to oxidative stress of cancer cells. Rapid cell proliferation strongly needs a ready supply of energy and basic macromolecules, so mutant p53 proteins have been reported to facilitate the supply of cancer cells.

Mutant p53 is able to modify lipid metabolism in a way that encourages rapid cell proliferation and intracellular signaling. In fact, mutant p53 engages the SREBPs (sterol regulatory element-binding protein) and leads to FAs synthesis, β-oxidation arrest, and cholesterol synthesis, favoring so, the generation of a robust supply of lipids for cell membranes formation.

The strongest effect of mutant p53 on the metabolism regards the glucose. Mutant p53 constitutively stimulates glycolysis acting in various ways. First of all, it up-regulates GLUT1 and GLUT 4 favoring the rapid uptake of glucose in tumor cells, moreover, mutant p53 stimulated genes involved in glycolysis activation such as TIGAR, and finally, it stimulates the PPP acting on G6PD. The final result is a wide supply of glucose which can be consumed in order to produce energy also in aerobic conditions.

Rapid cell proliferation produces high levels of ROS, which may dampen cell membranes, protein structures and DNA inducing apoptosis. Mutant p53, as previously seen, can constitutively stimulate genes involved in antioxidant cell response. The greater response to the activation of these genes is the synthesis of molecules able to neutralize ROS, such as glutathione.

Mutations in Ras - proto-oncogene

Proto-oncogenes are a group of genes that cause normal cells to become cancerous when they are mutated. Mutations in proto-oncogenes are typically dominant in nature, and the mutated version of a proto-oncogene is called an oncogene. Often, proto-oncogenes encode proteins that function to stimulate cell division, inhibit cell differentiation, and halt cell death. All of these processes are important for normal human development and for the maintenance of tissues and organs. Oncogenes, however, typically exhibit increased production of these proteins, thus leading to increased cell division, decreased cell differentiation, and inhibition of cell death; taken together, these phenotypes define cancer cells.

All mammalian cells express three closely related Ras proteins: H-Ras, K-Ras, and N-Ras that promote oncogenesis when mutationally activated at codons 12, 13 or 61. Despite a high degree of similarity between the isoforms, K-Ras mutations are far more frequently observed in cancer and each isoform displays preferential coupling to particular cancer types. Ras proteins are proto-oncogenes that are frequently mutated in human cancers. They are encoded by three ubiquitously expressed genes: HRAS, KRAS, and NRAS. These proteins are GTPases that function as molecular switches regulating pathways responsible for proliferation and cell survival. Ras proteins are normally tightly regulated by guanine nucleotide exchange factors (GEFs) promoting GDP dissociation and GTP binding and GTPase-activating proteins (GAPs) that stimulate the intrinsic GTPase activity of Ras to switch off the signaling. Aberrant Ras function is associated with hyper-proliferative developmental disorders and cancer and in tumors is associated with a single mutation typically at codons 12, 13 or 61.

One of the most frequent ways in which the MAPK is set to overdrive is by a mutation in the RAS protein; mutations in the KRAS protein are found in about 40% of all colorectal cancer (CRC) cases. These mutations are sometimes present in early adenomas and in cells with the minimal potential to develop a malignancy. In molecular terms, mutations in these three KRAS/NRAS codons may lead to conformational changes so that the RAS-GAP protein cannot activate the inherent GTPase enzyme anymore. As a result, the GTP molecules are not hydrolyzed and instead they maintain RAS continuously in its active state, thus causing protumorigenic effects by amplifying signaling in the MAPK pathway.