Question

The p53 protein can activate genes involved in apoptosis, known as programmed cell death. Discuss how mutations in genes coding for proteins that function in apoptosis could contribute to cancer.

Answer 1

Inactivation of the p53 tumor suppressor is a frequent event in tumorigenesis. In most cases, the p53 gene is mutated, giving rise to a stable mutant protein whose accumulation is regarded as a hallmark of cancer cells. Mutant p53 proteins not only lose their tumor suppressive activities but often gain additional oncogenic functions that endow cells with growth and survival advantages. Interestingly, mutations in the p53 gene were shown to occur at different phases of the multistep process of malignant transformation, thus contributing differentially to tumor initiation, promotion, aggressiveness, and metastasis.

Mutations in p53 Are a Frequent Event in Cancer:

The evolution of a normal cell toward a cancerous one is a complex process, accompanied by multiple steps of genetic and epigenetic alterations that confer selective advantages upon the altered cells. The alterations underlying tumorigenesis are considered to endow the evolving tumor with self-sufficiency of growth signals, insensitivity to antigrowth signals, evasion from programmed cell death, unlimited replicative potential, sustained angiogenesis, and finally, the ability to invade and metastasize.

Despite massive research efforts and the very impressive progress made over the past several decades, full molecular understanding of cancer still remains a major challenge to the biomedical community. Back in 1947, Isaac Berenblum and Philippe Shubik discovered that chemical carcinogenesis consists of two stages: initiation and promotion. More than 2 decades later, Knudson proposed a theory for tumor development known as the “Knudson two hit hypothesis.” This theory suggested a genetic model for retinoblastoma development, according to which the inherited RBgene mutation is described as the first hit and the tumor-restricted mutation as the second hit. This model was later expanded to include additional genetic aberrations, such as inactivation of a tumor suppressor and activation of an oncogene, as hits.

Despite the huge diversity in the genes implicated in tumorigenesis, the p53 transcription factor (encoded by the human gene TP53) stands out as a key tumor suppressor and a master regulator of various signaling pathways involved in this process. The many roles of p53 as a tumor suppressor include the ability to induce cell cycle arrest, DNA repair, senescence, and apoptosis, to name only a few. Indeed, TP53 mutations were reported to occur in almost every type of cancer at rates varying between 10% (e.g., in hematopoietic malignancies) and close to 100% (e.g., in high-grade serous carcinoma of the ovary). For further information, see the IARC TP53 mutation database version R15, November 2010. The importance of p53 as a cardinal player in protecting against cancer development is further emphasized by Li-Fraumeni syndrome (LFS), a rare type of cancer predisposition syndrome associated with germline TP53 mutations. Unlike the majority of tumor suppressor genes, such as RB, APC, or BRCA1, which are usually inactivated during cancer progression by deletions or truncating mutations, the TP53 gene in human tumors is often found to undergo missense mutations, in which a single nucleotide is substituted by another. Consequently, a full-length protein containing only a single amino acid substitution is produced. The cancer-associated TP53 mutations are very diverse in their locations within the p53 coding sequence and their effects on the thermodynamic stability of the p53 protein. However, the vast majority of the mutations result in loss of p53’s ability to bind DNA in a sequence-specific manner and activate transcription of canonical p53 target genes.

TP53 mutations are distributed in all coding exons of the TP53 gene, with a strong predominance in exons 4-9, which encode the DNA-binding domain of the protein. Of the mutations in this domain, about 30% fall within 6 “hotspot” residues (residues R175, G245, R248, R249, R273, and R282) and are frequent in almost all types of cancer. The existence of these hotspot residues could be explained both by the susceptibility of particular codons to carcinogen-induced alterations and by positive selection of mutations that render the cell with growth and survival advantages.

In addition to the loss of function that a mutation in TP53 may cause, many p53 mutants are able to actively promote tumor development by several other means. In a heterozygous situation, where both wild-type (WT) and mutant alleles exist, mutant p53 can antagonize WT p53 tumor suppressor functions in a dominant negative (DN) manner. The inactivation of the WT p53 by the mutant p53 in a DN mechanism stems from the fact that the transcriptional activity of WT p53 relies on the formation of tetramers, whose DNA binding function may be interfered by mutant p53. However, such a heterozygous state is often transient, as TP53 mutations are frequently followed by loss of heterozygosity (LOH) during cancer progression. LOH is often seen in the case of tumor suppressors where, at a particular locus heterozygous for a mutant and WT allele, the WT allele is either deleted or mutated. The LOH of the short arm of chromosome 17, where TP53 is located, implies a selective force driving the inactivation of the remaining WT allele, suggesting that the DN activity of mutant p53 is not sufficient to completely inactivate WT p53.

Furthermore, accumulating evidence supports the concept that many mutant p53 isoforms can exert additional oncogenic activity by a gain-of-function (GOF) mechanism. This term refers to the acquisition of oncogenic properties by the mutant protein, compared with the mere inactivation of the protein. Both the DN and GOF effects may play a significant role in the positive selection of missense mutations in TP53 during tumorigenesis.

When Is p53 Inactivated in Malignant Transformation?

The notion that mutations in TP53may occur at different stages along the process of malignant transformation raises the possibility that mutated p53 may contribute differently to various steps of this process. It is still an open question whether TP53 mutations are involved in the initiation of malignant transformation or perhaps only at more advanced stages of cancer, leading to additional growth and aggressiveness advantages. It appears, however, that the timing of the mutation during tumorigenesis is extremely variable from one cancer to another.