Genetics and Epigenetics of Childhood Cancers

Functional Genetics of Childhood Cancer Predisposition Syndromes

* At the time of this writing, the following publicly accessible databases of cancer-associated mutations and structural aberrations are available: http://target.cancer.gov ; http://pediatriccancergenomeproject.org ; http://www.sanger.ac.uk/genetics/CGP ; and http://cansar.icr.ac.uk .

The study of childhood cancer predisposition syndromes is an important source of information about the genetic basis of human cancers. Indeed, the retinoblastoma 1 (RB1) gene, which is mutated in familial retinoblastomas, was the first human tumor suppressor gene identified. What follows is an overview of known genes mutated in childhood cancer predisposition syndromes based on the major cell biologic pathways thought to be perturbed by the inherited gene mutations.

Dysregulation of Cell Division and Growth

A large number of childhood cancer predisposition syndromes involve mutations of genes that regulate cell growth and division. One of the first to be described, the RB1 gene, is mutated in children who are predisposed to the development of retinoblastoma, pinealoblastoma, and osteosarcoma. The RB1 gene is part of the G1 cell cycle checkpoint, where its product directly binds and regulates the activity of the E2F family of transcription factors and the DREAM chromatin remodeling complex in response to stress. Cancer-causing deletions and nonsense mutations of RB1 can cause entry into the cell cycle by otherwise quiescent cells and mitigation of senescence that limits cellular life span. Whereas loss of RB1 in cellular and mouse models causes defects in mitotic chromosome segregation that lead to aneuploidy, most human retinoblastomas are largely diploid, suggesting that the canonic chromosome segregation effects of RB1 inactivation in human retinoblasts are functionally compensated. Human retinoblastomas invariably exhibit biallelic inactivation of RB1 , but otherwise often have a comparatively low mutation rate. One study found that RB1 was the only known cancer gene mutated in these tumors. However, additional subclonal DNA mutations were observed, with some mutations located outside of coding regions, which may cooperate with RB1 loss to drive retinoblastoma formation. In addition, epigenetic alterations have also been implicated in cooperating with RB1 mutation.

The adenomatous polyposis coli (APC) gene, which is mutated in children with familial adenomatous polyposis who are predisposed to hepatoblastoma and colon carcinoma, also controls the G1 cell cycle checkpoint. APC serves as a ubiquitin ligase that regulates the protein stability of β-catenin, which controls transcription of cyclins, including cyclin D, which in turn associates with cyclin-dependent kinase (CDK)4/6 to negatively regulate RB1 activity. Disease-associated dominant mutations of APC tend to occur in distinct regions of the gene, leading to the generation of truncated protein products, consistent with the requirement of residual or neomorphic activity of the mutant proteins. Because APC regulates the stability of many proteins—and because, in addition to the cell cycle progression, β-catenin also regulates cell adhesion and differentiation—the mechanism of tumorigenesis induced by APC mutations is thought to be pleiotropic.

In addition to mutations of genes whose products regulate cell division, cancer-causing mutations also directly dysregulate mitogenic signaling and cell growth. The most common cancer predisposition syndrome, neurofibromatosis, is predominantly caused by autosomal-dominant inheritance of inactivating mutations of the neurofibromin 1 (NF1) gene, with the less frequent form caused by mutations of neurofibromin 2 (NF2) or merlin . Children with mutations of NF1 are predisposed to the development of benign and malignant tumors, including gliomas, peripheral nerve sheath tumors, pheochromocytomas, and leukemias. NF1 is a large protein that is not completely understood but is known to regulate the activity of rat sarcoma (Ras). In turn, NF1 mutant tumors exhibit increased Ras guanosine triphosphate hydrolase activity, which stimulates the mitogen-activated protein and AKT kinase cascades that, among other effects, result in the transcription of genes involved in cell growth and stimulation of protein synthesis. Although most patients with germline NF1 mutations experience the development of neurofibromas, which harbor biallelic inactivation of the gene, only a minority of these tumors evolve to become malignant. This finding indicates that disease-associated NF1 mutations have limited transforming ability, and that additional lesions are required for carcinogenesis.

The complex relationship between cancer-causing gene mutations and their effects on tumorigenesis is also evident from mutations of ras genes themselves. Genes related to ras , including Kirsten rat sarcoma (KRAS) , neuroblastoma rat sarcoma ( NRAS) , and Harvey rat sarcoma (HRAS) , are some of the first human protooncogenes identified, based on the involvement of the corresponding viral oncogenes in the pathogenesis of rat sarcomas. However, whereas NF1 mutations stimulate Ras activity and cause neurofibromatosis, patients with inherited mutations of the ras genes instead experience the development of cardiofaciocutaneous, Noonan, LEOPARD, and Costello syndromes. Inherited mutations of the ras genes are missense activating mutations, analogous to the oncogenic viral ras mutations, yet the spectrum of tumor phenotypes in these patients is distinct. In addition to gliomas these phenotypes also include leukemias, neuroblastomas, and rhabdomyosarcomas. These differences between NF1- and ras -mutant cancer predisposition syndromes are not simply due to differences in tissue expression of the two genes, because both appear to be expressed in neuronal, mesenchymal, and hematopoietic tissues. Rather, they may reflect the developmental consequences of Ras activation as a result of the inactivation of an endogenous negative regulator as opposed to direct mutational activation of an oncogenic signaling pathway, a property that may inform the development of strategies to target these lesions therapeutically.

DNA Repair and Genome Instability

The prevention of de novo cancer-causing mutations ultimately depends on efficient DNA repair and maintenance of genome stability. Indeed, several cancer predisposition syndromes involve defective DNA repair. For example, inherited mutations of the tumor protein 53 ( TP53) tumor suppressor gene cause most cases of Li-Fraumeni syndrome, which is associated with the predisposition to sarcomas, leukemias, and breast carcinomas. The genotype-phenotype relationship of TP53 cancer-causing mutations is complex, with some mutations having apparent loss-of-function, dominant-negative, or gain-of-function properties in experimental models. p53 regulates the expression of genes involved in cell division, apoptosis, and DNA repair, and its mutation in cells leads to chromosomal instability. This process is mediated partly by direct effects of p53 on the transcription and translation of target genes, as well as via feedback interaction with its ubiquitin ligase murine double minute 2 (MDM2), which also has oncogenic effects independent of its negative regulation of p53. As a result, the dysregulation of cellular DNA damage and stress response by TP53 mutations is profoundly tumorigenic, contributing to the somatic pathogenesis of most human cancers. The p53-MDM2 feedback regulation also complicates therapeutic targeting, because inhibition of MDM2 aimed at restoring p53 tumor suppression may in effect enhance the deleterious activity of gain-of-function p53 mutant proteins.

Defective DNA damage response is also a hallmark of Fanconi anemia (FA), a cancer predisposition syndrome characterized by bone marrow failure and development of myelodysplasia, leukemia, and carcinomas. Although mutations of more than 15 different genes can cause FA, most of the known FA-mutant gene products physically interact with the components of a conserved DNA damage response mechanism, breast cancer 1 (BRCA1), ataxia telangiectasia mutated (ATM), Nijmegen breakage syndrome 1 (NBS1), and radiation-sensitive 51 (RAD51), and are required for the repair of chromosome defects that occur during homologous recombination. FA pathway–deficient cells are hypersensitive to DNA-damaging agents, and over time they accumulate deleterious gene mutations (usually segmental deletions).

Several other chromosome-breakage syndromes with cancer predisposition involve defects in closely related biochemical pathways of DNA repair of homologous recombination errors, such as ataxia telangiectasia (due to mutations of ATM ), Nijmegen breakage syndrome (due to mutations of NBS1 ), and Bloom syndrome (due to mutations of BLM ). Genes involved in nucleotide excision or transcription-coupled DNA repair can also be mutated and cause the cancer predisposition syndromes xeroderma pigmentosum and Cockayne syndrome, usually due to accumulation of ultraviolet radiation–induced mutations (typically nucleotide substitutions).

Aberrant Gene Expression

Another hallmark of cancer cells is expression of genes in inappropriate developmental states, often driven by mutations of gene products that regulate gene expression. For example, patients with inherited mutations of the Wilms tumor 1 (WT1) gene experience development of the Denys-Drash syndrome (due to loss-of-function missense mutations) or the Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome (due to gene deletions), both with predisposition to the development of bilateral Wilms tumors. In spite of years of study, the function of the WT1 protein is incompletely understood. WT1 is a key regulator of mesenchymal cell development, including kidney mesenchyme, in part through its functions as a transcriptional repressor. However, WT1 also interacts with messenger RNA (mRNA) and splicing factors, consistent with possible posttranscriptional functions in regulation of gene expression. In addition, although recessive mutations of WT1 appear consistent with its tumor suppressive functions in Wilms tumorigenesis, in other developmental and tissue contexts, particularly myeloid leukemias, WT1 can enhance cell survival and proliferation and as a result can act as an oncogene or haploinsufficient tumor suppressor gene. Such tissue-dependent phenotypes are characteristic of cancer-associated genes that affect gene expression and cell fate determination, insofar as the functional consequences of mutations depend on the spectrum of expressed genes themselves and differentiation state in general.

Tumorigenesis due to aberrant regulation of gene expression can also occur posttranscriptionally, as in the case of inherited mutations of the DICER1 gene, which predispose to the development of pleuropulmonary blastoma, cystic nephroma, and rhabdomyosarcoma. DICER1 is a ribonuclease that is required for the generation of endogenous microRNAs (miRNAs), which negatively regulate gene expression by inducing degradation and translational blockade of specific mRNAs. Cancer-associated mutations affect conserved, enzymatically required residues in DICER1 and cause defective miRNA processing, leading to increased expression of target genes. The reason inherited DICER1 mutations predispose specifically to the development of pleuropulmonary blastoma, cystic nephroma, and rhabdomyosarcoma is not understood, but it is reasonable to hypothesize that this predisposition is due to the specific requirements for miRNA-mediated gene expression in these tissues or expression of specific target protooncogenes.