Role of the CDKN2A and related cell cycle regulatory genes in melanoma and other human cancers

Abstract: The main objective of this thesis has been to investigate the involvement of the CDKN2A (p16INK4a and p14ARF) and related cell cycle regulatory genes in melanoma and other types of human cancer. The CDKN2A gene represents a unique locus in the entire human genome. It encodes two separate cell cycle regulators from distinctive exon 1 alpha and exon 1 beta, that splice with common exons 2 and 3 and are translated in different reading frames. By inhibition of the cyclin D-CDK4/6 complex, p16 INK4a prevents phosphorylation of the RB protein, in turn leading to block of cell cycle progression. The p14 A F protein participates in the p53 pathway and restrains cell growth by p53 stabilization by sequestering HDM2. The locus carrying the CDKN2A gene on chromosome 9p21 is commonly altered in a number of human cancer types. This study has assessed the status of the CDKN2A (p16INK4a and p14ARF) gene in malignant melanoma and squamous cell carcinoma of esophagus (ESCC). In addition, mutations were determined in the CDKN2B, CDKN2C, CDK4, p53 and p53R2 genes. The results showed alterations of the CDKN2A gene in both primary and metastatic melanomas. In the primary melanomas a high frequency of loss of heterozygosity was detected at chromosome 9p21, while the rates of mutations and homozygous deletions in the CDKN2A gene were relatively low. A significantly increased frequency of loss of heterozygosity was found at the marker D9S736, which is located telomeric to the CDKN2A gene, suggesting the presence of an additional tumour suppressor gene. An interesting correlation was found between the frequency of loss of heterozygosity on chromosome 9 and increased patient age at diagnosis. Analysis of metastatic melanoma likewise showed low frequencies of mutations and homozygous deletions of the CDKN2A gene. The results showed an association between polymorphisms in the 3´untranslated region of the CDKN2A gene and progression of disease. One of those polymorphisms was also significantly overrepresented in sporadic primary melanomas compared to healthy controls. No mutations were found in the CDKN2B, CDKN2C, CDK4 or p53R2 genes, suggesting a limited role in melanoma. Similarly no evidence was found for the involvement of the p53 gene in malignant melanoma. The study of esophageal squamous cell carcinomas showed a very high proportion of genetic and epigenetic alterations in the CDKN2A (p16INK4a and p14ARF) and p53 genes. Of 21 cases analysed, 86% were found to have at least one alteration in the CDKN2A gene. No major alterations were detected in the CDKN2B, CDKN2C, CDK4 or p53R2 genes. However, in the 5´untranslated region of the p53R2 gene a novel polymorphism was detected. Additionally another novel polymorphism, an 8 base pair insertion in the same region was detected in control population, which was subsequently genotyped for association with different cancers. Altogether, the cancer types studied showed a complex pattern of genetic alterations, indicating the contribution of various disrupted cell cycle regulatory mechanisms. Involvement of the CDKN2A gene was found in a subset of sporadic melanoma cases. The mutation rate of the CDKN2A gene was low, albeit allelic loss at 9p21 was frequent. A prevailing theory is the existence of other tumour suppressor genes within the locus. However, candidate genes remain to be identified. Alterations of the CDKN2A (p16INK4a and p14ARF) and p53 genes were found to be major events in esophageal squamous cell carcinomas.