Systematic analyses of the cancer genome: lessons learned from sequencing most of the annotated human protein-coding genes

PURPOSE OF REVIEW: The availability of a reference human genome sequence has enabled unbiased mutational analyses of tumor genomes to identify the mutated genes that cause cancer. This review discusses recent insights from such analyses of protein-coding genes in breast and colorectal cancers. RECENT FINDINGS: Mutational analyses of approximately 18,000 human protein-coding genes in breast and colorectal cancers have identified 280 candidate cancer genes. These include known cancer genes, but most had not previously been linked to cancer. There are few frequently mutated cancer genes among hundreds of less frequently mutated candidate cancer genes, and the compendium of mutated genes differs among tumors of the same tissue origin. SUMMARY: Recent work has shown the feasibility of coding cancer genome sequencing, and new technologies promise to facilitate these mutational analyses. Whereas cancer genetics can identify candidate genes in a rapid and scalable fashion, careful functional studies of mutated genes are required for ultimate proof of cancer gene status and translation into clinical utility. The rapid progress of cancer genetics has yielded novel diagnostic and therapeutic modalities, and cancer genome sequencing will accelerate this development to the benefit of cancer patients.     

The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers

Genomic instability, a hallmark of cancer, is commonly caused by failures in the DNA damage response. Here we conducted a bioinformatical screen to reveal DNA damage response genes that are upregulated by estrogen and highly mutated in breast and ovarian cancers. This screen identified 53 estrogen-dependent cancer genes, some of which are novel. Notably, the screen retrieved 9 DNA helicases as well as 5 nucleases. DNA2, which functions as both a helicase and a nuclease and plays a role in DNA repair and replication, was retrieved in the screen. Mutations in DNA2, found in estrogen-dependent cancers, are clustered in the helicase and nuclease domains, suggesting activity impairment. Indeed, we show that mutations found in ovarian cancers impair DNA2 activity. Depletion of DNA2 in cells reduces their tumorogenicity in mice. In human, high expression of DNA2 correlates with poor survival of estrogen receptor-positive patients but not of estrogen receptor-negative patients. We also demonstrate that depletion of DNA2 in cells reduces proliferation, while addition of estrogen restores proliferation. These findings suggest that cells responding to estrogen will proliferate despite being impaired in DNA2 activity, potentially promoting genomic instability and triggering cancer development.     

Novel somatic and germline mutations in cancer candidate genes in glioblastoma, melanoma, and pancreatic carcinoma

A recent systematic sequence analysis of well-annotated human protein coding genes or consensus coding sequences led to the identification of 189 genes displaying somatic mutations in breast and colorectal cancers. Based on their mutation prevalence, a subset of these genes was identified as cancer candidate (CAN) genes as they could be potentially involved in cancer. We evaluated the mutational profiles of 19 CAN genes in the highly aggressive tumors: glioblastoma, melanoma, and pancreatic carcinoma. Among other changes, we found novel somatic mutations in EPHA3, MLL3, TECTA, FBXW7, and OBSCN, affecting amino acids not previously found to be mutated in human cancers. Interestingly, we also found a germline nucleotide variant of OBSCN that was previously reported as a somatic mutation. Our results identify specific genetic lesions in glioblastoma, melanoma, and pancreatic cancers and indicate that CAN genes and their mutational profiles are tumor specific. Some of the mutated genes, such as the tyrosine kinase EPHA3, are clearly amenable to pharmacologic intervention and could represent novel therapeutic targets for these incurable cancers. We also speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition.     

A new frontier in personalized cancer therapy: mapping molecular changes

Mutations in the genome of a normal cell can affect the function of its many genes and pathways. These alterations could eventually transform the cell from a normal to a malignant state by allowing an uncontrolled proliferation of the cell and formation of a cancer tumor. Each tumor in an individual patient can have hundreds of mutated genes and perturbed pathways. Cancers clinically presenting as the same type or subtype could potentially be very different at the molecular level and thus behave differently in response to therapy. The challenge is to distinguish the key mutations driving the cancer from the background of mutational noise and find ways to effectively target them. The promise is that such a molecular approach to classifying cancer will lead to better diagnostic, prognostic and personalized treatment strategies. This article provides an overview of advances in the molecular characterization of cancers and their applications in therapy.     

The suppression of colon cancer cell growth in nude mice by targeting beta-catenin/TCF pathway

The adenomatous polyposis coli (APC) or beta-catenin genes are frequently mutated in colorectal cancers, leading to activation of downstream genes with beta-catenin/T-cell factor (Tcf)-responsive promoters. We have developed a gene therapy approach selectively targeting colorectal cancer cells in which beta-catenin/Tcf4 pathway is activated by using a recombinant adenovirus AdTOP-CMV-TK, which carries a herpes simplex virus thymidine kinase gene (HSV TK) under the control of a beta-catenin/Tcf-response promoter linking to a minimum CMV promoter. AdTOP-CMV-TK and ganciclovir (GCV) treatment significantly suppressed the growth of human DLD-1 colon cancer cells in nude mice. Furthermore, no significant tumor suppression effect was observed in human hepatoma cell line SK-HEP-1, in which the beta-catenin/Tcf pathway is not activated, as a control experiment. In summary, we demonstrated the selective targeting of colorectal cancers with activated beta-catenin by AdTOP-CMV-TK and GCV treatment in animal models, as well as its therapeutic potential for colon cancer metastasized to liver.     

Cancer as an evolutionary process at the cell level: an epidemiological perspective

Germ-line mutations (present in all cells) in genes that are crucial for the cell cycle cause cancer only in specific cell lines (e.g. mismatch repair genes in the colon; BRCA1-2 in breast and ovary; other cancers in Bloom syndrome, neurofibromatosis and xeroderma pigmentosum). The mutation rate of genes other than mismatch repair or p53 is the same in colon cancer and in normal cells, indicating that a 'mutator phenotype', increasing the rate of mutations in many genes, is not an essential feature of sporadic cancers; conversely, fusion genes, TEL-AML1/AML1-ETO, typical of leukemia, are 100 times more frequent at birth than in overt leukemia in children, indicating that further selective events are needed to cause malignancy. The devastating impairment of immunity, as in AIDS patients, does not cause cancer other than Kaposi's sarcoma and non-Hodgkin's lymphoma, although immunological control is considered to be an essential mechanism in preventing the spread of cancer cells. These observations suggest that cell-specific additional events are needed to explain carcinogenesis. Carcinogenesis has been traditionally interpreted as the sequence of initiation (mutation) and promotion (clone expansion), with an interesting similarity with the neo-Darwinian theory of evolution, based on a first stage of genetic change (including recombination) and a second stage of selection. I propose that carcinogenesis consists in two general phases (not necessarily stages), i.e. genetic change followed by clone expansion (selective advantage). As in neo-Darwinian theory selection is chiefly represented by the elimination of the less fit, the selection of mutated cells would mainly consist in resistance to apoptosis or other types of 'bottlenecks' that hamper a cell's survival; an example of such a bottleneck is the autoimmunity that induces paroxysmal nocturnal hemoglobinuria in individuals with PIG-A mutations. Cancer rates show great variation in different countries around the world, a variation only marginally explained by genetic differences. More interestingly, migrants change their risk of cancer by adapting to that of the population into which they move: as these changes are not likely to be entirely due to mutagens in the environment, we have to invoke selective pressure over mutated cells to explain them. My theory is that mutated cells adapt to environmental 'niches' better than normal cells, in a 'gene-environment interaction' that involves the history of the genetic changes the cell has undergone and the kind of environment in which it happens to live.     

Kinase mutations in cancer: chinks in the enemy's armour?

PURPOSE OF REVIEW: Over the past few years, a revolution has transformed the oncology field. This revolution is characterized by two main features. The first is the introduction of the concept of individualized cancer therapy. The second is the development of drugs targeting molecules selectively altered in tumours. This review analyses these aspects by looking at the role that altered kinases and their inhibitors have played in this historical process. RECENT FINDINGS: Tumour progression is the result of the sequential accumulation of mutations in genes monitoring the rates of cell birth and cell death. The molecular profiling of cancers has shown that protein and lipid kinases are frequently altered in tumour cells. In most cases, these alterations translate in constitutively active proteins, which are amenable of therapeutic targeting. Intriguingly, even 'established' cancer cells remain somewhat 'addicted' to the deregulated activity of mutated kinases. This feature appears to be the basis for the ability of kinase inhibitors in controlling the development of a number of cancers. The therapeutic efficacy of kinase inhibitors is impaired by the emergence of tumour cells carrying 'resistance' mutations. SUMMARY: Many oncogenes are mutated kinase genes. In most cases, the mutations result in the constitutive activation of the affected kinase that can be pharmacologically inhibited. Unfortunately, upon treatment with kinase inhibitors, resistant clones develop rapidly, impairing their therapeutic effect. Strategies to overcome resistance are discussed as well as the possibility to target kinases regulating cancer stem cells.     

Aneuploidy and cancer

Numeric aberrations in chromosomes, referred to as aneuploidy, is commonly observed in human cancer. Whether aneuploidy is a cause or consequence of cancer has long been debated. Three lines of evidence now make a compelling case for aneuploidy being a discrete chromosome mutation event that contributes to malignant transformation and progression process. First, precise assay of chromosome aneuploidy in several primary tumors with in situ hybridization and comparative genomic hybridization techniques have revealed that specific chromosome aneusomies correlate with distinct tumor phenotypes. Second, aneuploid tumor cell lines and in vitro transformed rodent cells have been reported to display an elevated rate of chromosome instability, thereby indicating that aneuploidy is a dynamic chromosome mutation event associated with transformation of cells. Third, and most important, a number of mitotic genes regulating chromosome segregation have been found mutated in human cancer cells, implicating such mutations in induction of aneuploidy in tumors. Some of these gene mutations, possibly allowing unequal segregations of chromosomes, also cause tumorigenic transformation of cells in vitro. In this review, the recent publications investigating aneuploidy in human cancers, rate of chromosome instability in aneuploidy tumor cells, and genes implicated in regulating chromosome segregation found mutated in cancer cells are discussed.     

Role of miRNAs in breast cancer

miRNAs belong to an important class of endogenous molecules which are present in a wide range of organisms including animals, plants and viruses. They are involved in regulating expression of several genes inside a cell due to presence of complementary region against specific mRNA molecules. Altered expression patterns cause progression of multiple diseases inside an organism. They have also been confirmed to be involved in different cancers including breast cancer. In this review, we discuss role of miRNAs with respect to uncontrolled division of cells, promotion, progression and metastasis in breast cancer.     

Multiple primary cancers as a guide to heritability

There are approximately 100 genes which when mutated are known to predispose to one or more forms of cancer. Currently, genetic testing is offered for many of these, either as single genes or as multi‐gene panels. Features of hereditary cancer include a positive family history of cancer, early age of onset and the appearance of multiple primary cancers in one individual. In some cases multiple cancers may be of the same site (e.g., bilateral breast cancer) and in other cases they may be at different sites. Various combinations of cancer sites may be indicative of specific cancer syndromes such as the breast ovarian cancer syndrome. Genetic testing should be offered to individuals who have experienced multiple primary cancers in some circumstances, the genetic counselor should review the ages of sites of cancer, their pathologic features and the family history of cancer as part of the pre‐test evaluation.     

Pancreatic cancer: promise for personalised medicine?

Sleeping Beauty (SB) is a genetically engineered insertional mutagenesis system. Its ability to rapidly induce cancer in SB-transgenic mice as well as the ease of identification of the mutated genes suggest important roles for SB in the discovery of novel cancer genes as well as the generation of models of human cancers where none currently exist. The range of SB-related tumors extends from haematopoietic to solid cancers such as hepatocellular carcinoma. This review follows the refinement of SB for different cancers and assesses its potential as a model for all cancers and a tool for cancer gene discovery.     

Analysis of the candidate target genes for mutation in microsatellite instability-positive cancers of the colorectum, stomach, and endometrium

Microsatellite instability (MSI) in human carcinoma DNA is a characteristic phenotype observed in hereditary non-polyposis colorectal cancer and also in some human sporadic cancers including multiple primary carcinomas. In this study, we analyzed mutations in the hCHK1, E2F4, hMSH3, and hMSH6 genes in MSI+ human cancers arising in colorectum, stomach and endometrium. The E2F4 and hMSH3 genes were mutated in all tumor types. Interestingly, the hMSH6 gene was mutated in colorectal and gastric cancers but not in endometrial cancer; this is similar to the TGFbetaRII gene. It is notable that the mutation status of the secondary mutators, hMSH3 and hMSH6, did not influence slippage-related frameshift mutations in genes harboring simple tandem-repeats, which suggests that the MSI phenotype may be affected mainly by abnormalities in the primary mutator genes, not by the secondary mutator genes. No mutations were observed in the cell cycle checkpoint gene hCHK1; mutations of this gene are thought to have a limited role, if any, in at least the tumor types analyzed in this study.     

Glycodelin reduces breast cancer xenograft growth in vivo

Malignant growth is characterized by loss of cell differentiation, uncontrolled proliferation and resistance to apoptosis. Many tumor suppressor genes that protect cells against malignant transformation regulate cell differentiation. Here, we show for the first time that glycodelin, a differentiation-related protein, reduces breast cancer tumor growth in vivo. We found that glycodelin cDNA-transfected MCF-7 breast cancer cells showed a differentiated phenotype and produced smaller tumors in mouse mammary fat pads compared with control-transfected cells. Glycodelin-induced differentiation was associated with reduced expression of oncogenes and increased expression of tumor suppressor genes. Our results suggest that glycodelin acts as a tumor suppressor in breast cancer. This may explain its reported association with a more favorable prognosis in some cancers.     

hBUB1 defects in leukemia and lymphoma cells

Tumorigenesis is a multi-step process involving a series of changes of cellular genes. Most solid tumors and hematopoietic malignancies often show abnormal chromosome numbers, the aneuploidy. The chromosomal aneuploidy keeps cells in the state of chromosomal instability (CIN) that will increase the mutation rate of cells affected and thus push them deeper into the process of tumorigenesis. The yeast genetic studies showed that normal distribution of chromosome during mitosis is under the surveillance of a set of genes, the spindle assembly checkpoint genes, that include the BUB and MAD gene groups and MPS. In some colorectal cancers with CIN it was found to have hBUB1 gene mutated and the mutated gene functions dominantly. We have examined a series of breast cancer cell lines with or without CIN for the hBUB1 gene mutation and found none. However, we detected various degrees of deletion in the coding sequences of the hBUB1 gene in cells from T lymphoblastic leukemia cell lines, Molt3 and Molt4, and cells from some acute lymphoblastic leukemia and Hodgkin's lymphoma patients. So far the lesions of deletion are in the kinetochore localization domain of the hBUB1 gene that may explain why the deletion lesions in the BUB1 gene cause aneuploidy in lymphoma and lymphoma cells. The deletions are heterozygous in nature. Like the mutated hBUB1 gene in colorectal cancer, the mutant hBUB1 cDNA from lymphoblastic leukemia cells behaves dominantly.     

Mutated Fanconi anemia pathway in non-Fanconi anemia cancers

An extremely high cancer incidence and the hypersensitivity to DNA crosslinking agents associated with Fanconi Anemia (FA) have marked it to be a unique genetic model system to study human cancer etiology and treatment, which has emerged an intense area of investigation in cancer research. However, there is limited information about the relationship between the mutated FA pathway and the cancer development or/and treatment in patients without FA. Here we analyzed the mutation rates of the seventeen FA genes in 68 DNA sequence datasets. We found that the FA pathway is frequently mutated across a variety of human cancers, with a rate mostly in the range of 15 to 35 % in human lung, brain, bladder, ovarian, breast cancers, or others. Furthermore, we found a statistically significant correlation (p < 0.05) between the mutated FA pathway and the development of human bladder cancer that we only further analyzed. Together, our study demonstrates a previously unknown fact that the mutated FA pathway frequently occurs during the development of non-FA human cancers, holding profound implications directly in advancing our understanding of human tumorigenesis as well as tumor sensitivity/resistance to crosslinking drug-relevant chemotherapy.     

p53: Balancing tumour suppression and implications for the clinic

It is well accepted today that cancer develops through a multi-step process that involves normal cells being led by well-defined phases into cancer cells. Along this process cells lose their natural cancer defence system that is mediated by tumour suppressor genes and accumulate genetic instabilities that permit the expression of the specific oncogenic networks. Remarkable is the p53 tumour suppressor which is mutated in more than 50% of human cancers. In turn, various mutant p53 proteins with an oncogenic activity are accumulated in the cells and contribute to malignancy. This chapter overviews the p53 field with respect to the history behind the discovery of the p53 tumour suppressor, the structure and function of p53, the oncogenic activities of the various p53 mutants and the clinical significance of a tailor-made p53-based gene therapy.     

Identification of frequently mutated genes with relevance to nonsense mediated mRNA decay in the high microsatellite instability cancers

Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in high‐microsatellite instability (MSI‐H) cancers. Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). We have identified novel genes that are frequently mutated at their cMNR by blocking NMD in two MSI‐H cancer cell lines. After blocking NMD, we screened for differentially expressed genes using DNA microarrays, and then used database analysis to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. cMNR mutations have not been previously reported in MSI‐H cancers for 15 of the 28 genes. We analyzed the cMNR mutation of each of the 15 genes in 10 MSI‐H cell lines and 21 MSI‐H cancers, and found frequent mutations of 12 genes in MSI‐H cell lines and cancers, but not in microsatellite stable (MSS) cancers. Among these genes, the most frequently mutated in MSI‐H cell lines were MLL3 (70%), PHACTR4 (70%), RUFY2 (50%) and TBC1D23 (50%). MLL3, which has already been implicated in cancer, had the highest mutation frequency in MSI‐H cancers (48%). Our combined approach of NMD block, database search, and mutation analysis has identified a large number of genes mutated in their cMNR in MSI‐H cancers. The identified mutations are expected to contribute to MSI‐H tumorigenesis by causing an absence of gene expression or low gene dosage effects.     

Adenovirus expressing RIZ1 in tumor suppressor gene therapy of microsatellite-unstable colorectal cancers

Viral vector-mediated delivery of tumor suppressor genes represents a promising strategy of cancer therapy. Several best-studied tumor suppressor genes, such as p53 and retinoblastoma (Rb), have been evaluated for gene therapy of tumors that carry mutations in these genes. However, these genes may not be applicable to microsatellite instability positive [MSI(+)] tumors because they are rarely mutated in these tumors. The Rb-interacting zinc finger gene RIZ1 is commonly mutated in MSI(+) colorectal, gastric, and endometrial cancers and has demonstrated a capacity to induce cell cycle arrest and apoptosis. Here, we found that RIZ1 expression through adenovirus vectors suppressed growth of MSI(+) HCT116 colorectal xenograft tumors that carry RIZ1 mutations. Malignant cells in the established tumors were efficiently transduced by RIZ1 adenoviruses and underwent apoptosis in response to RIZ1 expression. In comparison, a recombinant p53 adenovirus did not induce apoptosis and tumor suppression. These results suggest that RIZ1 may be useful in gene therapy of MSI(+) colorectal cancers.     

Homologous recombination deficiency and ovarian cancer

The discovery that PARP inhibitors block an essential pathway of DNA repair in cells harbouring a BRCA mutation has opened up a new therapeutic avenue for high-grade ovarian cancers. BRCA1 and BRCA2 proteins are essential for high-fidelity repair of double-strand breaks of DNA through the homologous recombination repair (HRR) pathway. Deficiency in HRR (HRD) is a target for PARP inhibitors. The first PARP inhibitor, olaparib, has now been licensed for BRCA-mutated ovarian cancers. While mutated BRCA genes are individually most commonly associated with HRD other essential HRR proteins may be mutated or functionally deficient potentially widening the therapeutic opportunities for PARP inhibitors. HRD is the first phenotypically defined predictive marker for therapy with PARP inhibitors in ovarian cancer. Several different PARP inhibitors are being trialled in ovarian cancer and this class of drugs has been shown to be a new selective therapy for high-grade ovarian cancer. Around 20% of high-grade serous ovarian cancers harbour germline or somatic BRCA mutations and testing for BRCA mutations should be incorporated into routine clinical practice. The expanded use of PARP inhibitors in HRD deficient (non-BRCA mutant) tumours using a signature of HRD in clinical practice requires validation.