Accumulation of genetic alterations in oncogenes and tumor suppressor genes

The tumor suppressor gene Adenomatous polyposis coli (APC) is mutated in more than 70% of sporadic cancers. Germline mutations in APC result in familial adenomatous polyposis (FAP) or in one of its variants (Kheirelseid et al. 2013). FAP is an autosomal dominant syndrome and accounts for approx. 0.5% of all CRCs (Haggar and Boushey 2009; Fearon 2011). The protein encoded by the APC gene is a member of the Wnt signaling pathway (Takayama et al. 2006) and has multiple functional domains that mediate oligomerization and binding to a variety of intracellular proteins, such as β-catenin, the glycogen synthase kinase (GSK)-3β and axin. One main tumor-promoting effect of the mutation in APC results in overactivation of the Wnt signaling pathway with subsequent expression of genes that promote cell growth (Kheirelseid et al. 2013). The majority of the mutations lead to a premature stop codon and thus to a truncated protein (Chung 2000; Takayama et al. 2006; Fearon 2011; Human Gene Mutation Database (HGMD® Professional 2016.1)). Powell et al. (1992) were able to

prove that mutations in APC occur as a very early event during colorectal tumorigenesis, and showed that the frequency of these mutations does not change between adenomas and carcinomas (Powell et al. 1992).

The Kirstein rat sarcoma (K-RAS) is among H-RAS (Harvey rat sarcoma viral oncogene homolog) and N-RAS (neuroblastoma RAS viral oncogene homolog) a member of the RAS family of genes (Forrester et al. 1987; Fearon 2011). RAS proteins function downstream of the receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) (Fearon 2011). K-RAS is mutated in approx. 50% of CRCs (Forrester et al. 1987; Fearon 2011). Alterations in this gene occur as activating point mutations mostly in codon 12 and 13 (Bos 1989; Fearon 2011; HGMD® Professional 2016.1), leading to continuous activation of downstream signaling pathways (Malumbres and Barbacid 2003). The percentage of mutations of intermediate-stage and late-stage adenomas is similar (Vogelstein et al. 1988), but in only 20% of small adenomas with APC mutations (Powell et al. 1992; Tsao and Shibata 1994). This leads to the suggestion that alterations of K-RAS follow APC mutations. Interestingly, mutations of K-RAS seem not to be necessary for the conversion of adenomas to malignant adenocarcinomas (Kheirelseid et al. 2013). Activated RAS regulates multiple cellular functions, such as cell proliferation, differentiation and survival (Shaukat et al. 2012).

The loss of chromosome 18q occurs in almost 50% of late adenomas and in more than 70% of CRCs (Vogelstein et al. 1988). The gene Deleted in colorectal cancer (DCC) is localized in the region 18q21. DCC is expressed in normal colonic mucosa, but its expression is reduced or even absent in the majority of CRCs (Fearon and Vogelstein 1990), leading to the idea of DCC being a tumor suppressor gene (Kheirelseid et al.

2013). Elimination of DCC is not believed to be a key genetic alteration in tumor formation, but one of a variety of changes that can promote growth of existing tumors (Kheirelseid et al. 2013). This hypothesis is supported by the observation of Krimpenfort et al. (2012). They showed, by using a mouse model of mammary carcinoma based on somatic inactivation of the Tumor-protein-53 (P53), that the loss of DCC is irrelevant for primary tumor development, but in addition to inactivation of P53 it promotes the formation of metastases (Krimpenfort et al. 2012). Importantly, this finding is contradictory to the model of Fearon and Vogelstein (1990), in which the DCC loss occurs before P53 inactivation. Interestingly, other researchers even scrutinize the role of DCC during CRC development e.g. because of the absence of a cancer phenotype of DCC-deficient mice. They rather point to the tumor suppressor genes

SMAD4/2 as the candidate genes on 18q, because inactivation of SMAD4 has been shown to associate with the progression of cancer (Takayama et al. 2006). SMAD4/2 encode proteins that are members of the TGF-β signaling pathway (Fearon 2011).

Inactivating mutations in SMAD4 were found in approx. 10 to 15% of CRCs, whereas mutations that inactivate SMAD2 occur in approx. 5% of CRCs (Fearon 2011).

Furthermore, SMAD4 was shown to be one of the eight most frequent mutated genes in CRC among e.g. APC, P53 and K-RAS (Muzny et al. 2012). These controversial data indicate that further research is needed.

The Tumor-protein-53 (P53) is a transcription factor with tumor suppressor activity.

P53 recognizes DNA damage and controls cell cycle progression and cell survival and thus is called “guardian of the genome” (Kheirelseid et al. 2013). It is known to be mutated in 50% of primary human tumors (Somasundaram 2000; Kheirelseid et al.

2013). The vast majority of mutations in P53 are missense mutations (Fearon 2011;

HGMD® Professional 2016.1). Mutations and the loss of heterozygosity in the P53 gene were detected at a low frequency in adenomas and at a high frequency in carcinomas, suggesting that alterations in P53 mediate the conversion from adenoma to carcinoma (Vogelstein et al. 1988; Ohue et al. 1994; Fearon 2011). Furthermore, two or more of the described alterations (mutations in K-RAS, APC, DCC and P53) were found in more than 90% of CRCs, while only 7% of early adenomas had more than one of the four genetic alterations. The percentage of mutations gradually increases as the adenomas progress to intermediate and late stages. In addition, all late-stage adenomas contained all four genetic alterations. These facts support the idea that the alterations of the four genes were not sufficient for the progression to malignancy (Fearon and Vogelstein 1990).

Fig. 1: Model for the multistep development of colorectal cancer.

Colorectal tumorigenesis proceeds through a series of genetic alterations involving oncogenes (K-RAS) and tumor suppressor genes (APC, DCC / SMAD4/2, P53). The loss or mutation of APC occurs

at a relatively early stage of tumorigenesis and may be responsible for the hyperproliferative epithelium.

Mutation of the K-RAS gene occurs in one cell of a pre-existing small adenoma and produces a larger and more dysplastic tumor through clonal expansion. Deletion of the APC gene occurs at a very early stage in tumorigenesis and deletions of DCC / SMAD4/2 and P53 usually appear at a later stage. Recent studies scrutinize the role of DCC during CRC development and rather SMAD4/2 is currently a candidate gene. However, the order of these alterations differs, and accumulation of these alterations, rather than their order with respect to each other, is important. The signaling pathways, in which the proteins encoded by the mutated genes are involved in, are indicated in red (modified after Fearon and Vogelstein 1990; Fearon 2011).