Cancer of the large bowel may be looked upon as a model of stepwise tumor development, as it has morphological distinct benign and malignant stages paralleling a preferential order of genetic and epigenetic aberrations. Despite being a well-investigated disease, close to half of the patients diagnosed with colorectal cancer die within 5 years. Testicular cancer on the other hand, which is the most common cancer among young males, represents a model for successful cancer treatment, due to its effective response to chemotherapy. The malignant tumors are in the triploid range but the underlying molecular changes initiating and promoting cancer in the male germ cell lineage remain mostly unknown. Malignant peripheral nerve sheath tumors are rare malignancies, which exhibit highly complex genomes. The disease is aggressive, with surgery as the only consensus for therapy. These three cancer types develop from three distinct germ layers, and changes common among them may be important for cancer in general.
In the present thesis, we aimed to establish whether factors upstream of central signaling pathways, like the MAPK-, and PI3K-AKT-pathways , are commonly altered in the abovementioned malignancies, and possibly also mutually exclusive, hence qualifying as master keys in cancer. KRAS, BRAF, PIK3CA, and PTEN were investigated for mutations by direct sequencing, whereas methylation-specific PCR was utilized to determine the promoter methylation status of RASSF1A.In colorectal cancer, 80 % of the tumors examined were altered in one or more of the genes analyzed, and we identified a number of mutations in PTEN, previously not reported for this cancer type. Additionally, these mutations were associated with mutations in BRAF and microsatellite unstable tumors, and inversely associated with mutations in TP53.
In testicular germ cell tumors only few mutations were identified, all in seminomas, whereas several non-seminomas contained promoter methylation of RASSF1A. Thus, our findings are in line with the view that gene promoter methylation is a more common mechanism than gene point mutations are during the development of this cancer type. However, large sequence changes such as chromosomal amplifications and deletions are also commonly found.
Also among the malignant peripheral nerve sheath tumors, were few mutations found. Strikingly, however, about half of the tumors harbored promoter methylation of RASSF1A. Only few studies address promoter hypermethylation as a mechanism to inactivate genes in this malignancy, thus it remains to be seen if this is a more general mechanism in the development of the disease.
Mutually exclusive events are found in key signaling pathways, suggesting that such alterations will have the same functional effect in the pathway. Here we have shown that mutations in KRAS and BRAF are inversely associated, implying that the mutations are equivalent when it comes to the tumorigenic effect. Furthermore, mutations in PTEN are also found to be inversely associated with alterations in KRAS, as well as with mutations in TP53. Promoter methylation of RASSF1A has previously been shown to be mutually exclusive to KRAS mutations. Our findings suggest the opposite, as we observed concomitant gene promoter methylation and mutations in the respective genes.
The MAPK- and PI3K-AKT pathways can be referred to as master keys, as they are found frequently altered in cancer. Indeed, we show that colorectal carcinomas are commonly altered in approximately 80% in one or more of the five genes analyzed from these two pathways. Furthermore, we support the hypothesis that the mechanism for development in TGCTs is gene promoter methylation, and finally, we have shown that RASSF1A is a factor with master key features in-as-much as it is altered in a significant portion of all three tumor diseases. However, further investigations needs to be done before conclusions can be drawn about how commonly the MAPK and PI3K-AKT pathways are affected, through which upstream factors and by which mechanisms this occurs in TGCT and MPNST.