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Background

Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis.

Results

To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice.

Conclusion

Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

The genome sequence framework provided by the human genome project allows us to precisely map human genetic variations in order to study their association with disease and their direct effects on gene function. Since the description of tumor suppressor genes and oncogenes several decades ago, both germ-line variations and somatic mutations have been established to be important in cancer—in terms of risk, oncogenesis, prognosis and response to therapy. The Cancer Genome Atlas initiative proposed by the NIH is poised to elucidate the contribution of somatic mutations to cancer development and progression through the re-sequencing of a substantial fraction of the total collection of human genes—in hundreds of individual tumors and spanning several tumor types. We have developed the CancerGenes resource to simplify the process of gene selection and prioritization in large collaborative projects. CancerGenes combines gene lists annotated by experts with information from key public databases. Each gene is annotated with gene name(s), functional description, organism, chromosome number, location, Entrez Gene ID, GO terms, InterPro descriptions, gene structure, protein length, transcript count, and experimentally determined transcript control regions, as well as links to Entrez Gene, COSMIC, and iHOP gene pages and the UCSC and Ensembl genome browsers. The user-friendly interface provides for searching, sorting and intersection of gene lists. Users may view tabulated results through a web browser or may dynamically download them as a spreadsheet table. CancerGenes is available at .

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein–protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

The human EB1 gene product was recently found, by a yeast two-hybrid screening, to be associated with the carboxy terminus of the APC (adenomatous polyposis coli) protein, the product of a tumour-suppressor gene thought to act as a gatekeeper in colorectal carcinogenesis. Because virtually all of the APC mutations result in the synthesis of carboxy-terminal truncated proteins, mutant APC proteins are expected to lose their ability to interact with EB1 gene product. Thus, the interaction between APC and EB1 proteins may be important for the tumour-suppressor activity of APC protein, and raises the hypothesis that EB1 is also involved in sporadic colorectal tumorigenesis. To investigate this hypothesis, somatic mutations in the entire coding sequence of EB1 cDNA were searched by reverse transcriptase single-strand conformational polymorphism (SSCP) analysis in 21 sporadic colorectal cancers and seven adenomas. None of these tumours contained somatic mutation, whereas a silent cDNA variant was identified in 14% of alleles. Furthermore, to investigate whether EB1 locus was included within a region subjected to losses of heterozygosity, four polymorphism markers surrounding EB1 locus were surveyed. Only one out of 28 colorectal tumours contained a loss of heterozygosity at the D20S107 marker. In conclusion, the present findings strongly suggest that EB1 gene is not involved in somatic colorectal carcinogenesis.

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Tumor suppressor genes and oncogenes are both commonly altered during carcinogenesis. For oncogenes and other genes that drive growth, targeting mutated or activated forms (such as the EGFR-Her2/Nneu pathway) has been shown to be an effective anti-cancer approach. Pharmacologically targeting tumor suppressor genes has not been as fruitful, as many tumor suppressor genes are irreversibly silenced through somatic mutation or entirely deleted during carcinogenesis, thereby making it difficult to restore gene function. BRM, a key SWI/SNF complex subunit and a putative tumor suppressor gene, is inactivated in 15–20% of many solid tumor types. Unlike other tumor suppressor genes, the loss of BRM has been shown to be a reversible epigenetic change, rather than an irreversible genetic alteration. Using a high throughput drug screen, we identified a number of compounds that could effectively restore BRM expression and function. Two of these compounds, RH (RH02032) and GK (GK0037), were found to be such reactivating agents. Both compounds led to robust re-expression of BRM, induced downstream expression of BRM-dependent genes and inhibited BRM-dependent growth across a wide range of BRM-deficient cancer cell lines of different origins. We therefore show, for the first time, that pharmacologic reversal of epigenetic changes of the SWI/SNF chromatic remodeling complex subunit, BRM, is a potentially viable and novel therapeutic approach.

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFκB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision.

The transformation of a normal cell into a cancer cell takes place through a sequence of a small number of discrete genetic events, somatic mutations: thus, cancer can be regarded properly as a genetic disease of somatic cells. The analogy between evolution of organisms and evolution of cell populations is compelling: in both cases what drives change is mutation, but it is Darwinian selection that enables clones that have a growth advantage to expand, thus providing a larger target size for the next mutation to hit. The search for molecular lesions in tumors has taken on a new dimension thanks to two powerful technologies: the micro-arrays for quantitative analysis of global gene expresssion (the transcriptome); and ‘deep’ sequencing for the global analysis of the entire genome (or at least the exome). The former offers the most complete phenotypic characterization of a tumor we could ever hope for – we could call this the ultimate phenotype; the latter can identify all the somatic mutations in an individual tumor – we could call this the somatic genotype. However, there is definitely the risk that while we are ‘drowned by data, we remain thirsty for knowledge’. If we want to heed the teachings of Lorenzo Tomatis, I think the message is clear: we ought to take advantage of the new powerful technologies – not by becoming their slaves, but remaining their masters. Identifying somatic mutations in a tumor is important not because it qualifies for ‘oncogenomics’, but because through a deeper understanding of the nature of that particular tumor it can help us to optimize therapy or to design new therapeutic approaches.

Summary

Most clinically distinguishable malignant tumors are characterized by specific mutations, specific patterns of chromosomal rearrangements and a predominant mechanism of genetic instability but it remains unsolved whether modifications of cancer genomes can be explained solely by mutations and selection through the cancer microenvironment.

It has been suggested that internal dynamics of genomic modifications as opposed to the external evolutionary forces have a significant and complex impact on Darwinian species evolution. A similar situation can be expected for somatic cancer evolution as molecular key mechanisms encountered in species evolution also constitute prevalent mutation mechanisms in human cancers. This assumption is developed into a systems approach of carcinogenesis which focuses on possible inner constraints of the genome architecture on lineage selection during somatic cancer evolution. The proposed systems approach can be considered an analogy to the concept of evolvability in species evolution.

The principal hypothesis is that permissive or restrictive effects of the genome architecture on lineage selection during somatic cancer evolution exist and have a measurable impact. The systems approach postulates three classes of lineage selection effects of the genome architecture on somatic cancer evolution: i) effects mediated by changes of fitness of cells of cancer lineage, ii) effects mediated by changes of mutation probabilities and iii) effects mediated by changes of gene designation and physical and functional genome redundancy. Physical genome redundancy is the copy number of identical genetic sequences. Functional genome redundancy of a gene or a regulatory element is defined as the number of different genetic elements, regardless of copy number, coding for the same specific biological function within a cancer cell. Complex interactions of the genome architecture on lineage selection may be expected when modifications of the genome architecture have multiple and possibly opposed effects which manifest themselves at disparate times and progression stages.

Dissection of putative mechanisms mediating constraints exerted by the genome architecture on somatic cancer evolution may provide an algorithm for understanding and predicting as well as modifying somatic cancer evolution in individual patients.

Summary

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.

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Graphical Abstract

Highlights

► Whole-genome sequences of the Tasmanian devil and two distant cancer subclones ► The Tasmanian devil cancer lineage originated recently in a female devil ► The devil cancer genome is relatively stable despite ongoing evolution ► Clonal divergence and geographic spread elucidated through patterns of mutation

Whole-genome sequences of the Tasmanian devil and two devil cancer subclones suggest that the cancer first arose from a female devil and that the clone has subsequently genetically diverged during its spread across Tasmania.

The origin of genetic instability in tumors is a matter of debate: while the prevailing model postulates a mutator phenotype resulting from an alteration in a caretaker gene as a prerequisite for genetic alterations leading to tumor formation, there is evidence against this model in the majority of cancers. A model for chromosomal instability should take into account the role of oncogenes in directly stimulating DNA and cellular component replication, creating aberrant structures when overexpressed. I will distinguish here two distinct mechanisms for the genetic instability of tumors: primary and secondary. Primary genetic instability is dependent on the inactivation of genes involved in maintaining genetic stability (caretaker genes), whereas secondary genetic instability is dependent on genes involved in tumor progression, i.e. oncogenes and tumor suppressor genes of the gatekeeper type. Secondary genetic instability, the most frequent condition, can be explained by the fact that some of the genes involved in tumor progression control replication of cell structures from within, leading to replication unbalance.

Background

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.

Methodology/Principal Findings

Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.

Conclusions/Significance

This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

Neurospora comprises a primary model system for the study of fungal genetics and biology. In spite of this, little is known about genome evolution in Neurospora. For example, the evolution of synonymous codon usage is largely unknown in this genus. In the present investigation, we conducted a comprehensive analysis of synonymous codon usage and its relationship to gene expression and gene length (GL) in Neurospora tetrasperma and Neurospora discreta. For our analysis, we examined codon usage among 2,079 genes per organism and assessed gene expression using large-scale expressed sequenced tag (EST) data sets (279,323 and 453,559 ESTs for N. tetrasperma and N. discreta, respectively). Data on relative synonymous codon usage revealed 24 codons (and two putative codons) that are more frequently used in genes with high than with low expression and thus were defined as optimal codons. Although codon-usage bias was highly correlated with gene expression, it was independent of selectively neutral base composition (introns); thus demonstrating that translational selection drives synonymous codon usage in these genomes. We also report that GL (coding sequences [CDS]) was inversely associated with optimal codon usage at each gene expression level, with highly expressed short genes having the greatest frequency of optimal codons. Optimal codon frequency was moderately higher in N. tetrasperma than in N. discreta, which might be due to variation in selective pressures and/or mating systems.

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Background

The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. We hypothesize that key tumor suppressor genes in cancer may be subject to mutation or hypermethylation.

Methods and Findings

Here, we show that combined genetic and epigenetic analysis of these genes reveals many with a higher putative tumor suppressor status than would otherwise be appreciated. At least 36 of the 189 genes newly recognized to be mutated are targets of promoter CpG island hypermethylation, often in both colon and breast cancer cell lines. Analyses of primary tumors show that 18 of these genes are hypermethylated strictly in primary cancers and often with an incidence that is much higher than for the mutations and which is not restricted to a single tumor-type. In the identical breast cancer cell lines in which the mutations were identified, hypermethylation is usually, but not always, mutually exclusive from genetic changes for a given tumor, and there is a high incidence of concomitant loss of expression. Sixteen out of 18 (89%) of these genes map to loci deleted in human cancers. Lastly, and most importantly, the reduced expression of a subset of these genes strongly correlates with poor clinical outcome.

Conclusions

Using an unbiased genome-wide approach, our analysis has enabled the discovery of a number of clinically significant genes targeted by multiple modes of inactivation in breast and colon cancer. Importantly, we demonstrate that a subset of these genes predict strongly for poor clinical outcome. Our data define a set of genes that are targeted by both genetic and epigenetic events, predict for clinical prognosis, and are likely fundamentally important for cancer initiation or progression.

Stephen Baylin and colleagues show that a combined genetic and epigenetic analysis of breast and colon cancers identifies a number of clinically significant genes targeted by multiple modes of inactivation.

Editors' Summary

Background.

Cancer is one of the developed world's biggest killers—over half a million Americans die of cancer each year, for instance. As a result, there is great interest in understanding the genetic and environmental causes of cancer in order to improve cancer prevention, diagnosis, and treatment.

Cancer begins when cells begin to multiply out of control. DNA is the sequence of coded instructions—genes—for how to build and maintain the body. Certain “tumor suppressor” genes, for instance, help to prevent cancer by preventing tumors from developing, but changes that alter the DNA code sequence—mutations—can profoundly affect how a gene works. Modern techniques of genetic analysis have identified genes such as tumor suppressors that, when mutated, are linked to the development of certain cancers.

Why Was This Study Done?

However, in recent years, it has become increasingly apparent that mutations are neither necessary nor sufficient to explain every case of cancer. This has led researchers to look at so-called epigenetic factors, which also alter how a gene works without altering its DNA sequence. An example of this is “methylation,” which prevents a gene from being expressed—deactivates it—by a chemical tag. Methylation of genes is part of the normal functioning of DNA, but abnormal methylation has been linked with cancer, aging, and some rare birth abnormalities.

Previous analysis of DNA from breast and colon cancer cells had revealed 189 “candidate cancer genes”—mutated genes that were linked to the development of breast and colon cancer. However, it was not clear how those mutations gave rise to cancer, and individual mutations were present in only 5% to 15% of specific tumors. The authors of this study wanted to know whether epigenetic factors such as methylation contributed to causing the cancers.

What Did the Researchers Do and Find?

The researchers first identified 56 of the 189 candidate cancer genes as likely tumor suppressors and then determined that 36 of these genes were methylated and deactivated, often in both breast and colon (laboratory-grown) cancer cells. In nearly all cases, the methylated genes were not active but could be reactivated by being demethylated. They further showed that, in normal colon and breast tissue samples, 18 of the 36 genes were unmethylated and functioned normally, but in cells taken from breast and colon cancer tumors they were methylated.

In contrast to the genetic mutations, the 18 genes were frequently methylated across a range of tumor types, and eight genes were methylated in both the breast and colon cancers. The authors found by reviewing the genetics and epigenetics of those 18 genes in breast and colon cancer that they were either mutated, methylated, or both. A literature review showed that at least six of the 18 genes were known to have tumor suppressor properties, and the authors determined that 16 were located in parts of DNA known to be missing from cells taken from a range of cancer tumors.

Finally, the researchers analyzed data on cancer cases to show that methylation of these 18 genes was correlated with reduced function of these genes in tumors and with a greater likelihood that a cancer will be terminal or spread to other parts of the body.

What Do These Findings Mean?

The researchers considered only the 189 candidate cancer genes found in one previous study and not other genes identified elsewhere. They also did not consider the biological effects of the individual mutations found in those genes. Despite this, they have demonstrated that methylation of specific genes is likely to play a role in the development of breast and/or colon cancer cells either together with mutations or independently, most likely by turning off their tumor suppression function.

More broadly, however, the study adds to the evidence that future analysis of the role of genes in cancer should include epigenetic as well as genetic factors. In addition, the authors have also shown that a number of these genes may be useful for predicting clinical outcomes for a range of tumor types.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050114.

A December 2006 PLoS Medicine Perspective article reviews the value of examining methylation as a factor in common cancers and its use for early detection

The Web site of the American Cancer Society has a wealth of information and resources on a variety of cancers, including breast and colon cancer

Breastcancer.org is a nonprofit organization providing information about breast cancer on the Web, including research news

Cancer Research UK provides information on cancer research

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins publishes background information on the authors' research on methylation, setting out its potential for earlier diagnosis and better treatment of cancer

The purpose of this review is to summarize the evidence that can be used to reconstruct the etiology of human cancers from mutations found in tumors. Mutational spectra of the tumor suppressor gene p53 (TP53) are tumor-specific. In several cases, these mutational spectra can be linked to exogenous carcinogens, most notably for sunlight-associated skin cancers, tobacco-associated lung cancers, and aristolochic acid-related urothelial tumors. In the TP53 gene, methylated CpG dinucleotides are sequences selectively targeted by endogenous and exogenous mutagenic processes. Recent high-throughput sequencing efforts analyzing a large number of genes in cancer genomes have so far, for the most part, produced mutational spectra similar to those in TP53 but have unveiled a previously unrecognized common G to C transversion mutation signature at GpA dinucleotides in breast cancers and several other cancers. Unraveling the origin of these G to C mutations will be of importance for understanding cancer etiology.

Background

Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas.

Methodology/Principal Findings

We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1–2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3–5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines.

Conclusions/Significance

This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.

Background

Current large-scale cancer sequencing projects have identified large numbers of somatic mutations covering an increasing number of different cancer tissues and patients. However, the characterization of these mutations at the structural and functional level remains a challenge.

Results

We present results from an analysis of the structural impact of frequent missense cancer mutations using an automated method. We find that inactivation of tumor suppressors in cancer correlates frequently with destabilizing mutations preferably in the core of the protein, while enhanced activity of oncogenes is often linked to specific mutations at functional sites. Furthermore, our results show that this alteration of oncogenic activity is often associated with mutations at ATP or GTP binding sites.

Conclusions

With our findings we can confirm and statistically validate the hypotheses for the gain-of-function and loss-of-function mechanisms of oncogenes and tumor suppressors, respectively. We show that the distinct mutational patterns can potentially be used to pre-classify newly identified cancer-associated genes with yet unknown function.

The evidence that links classical protein-coding proto-oncogenes and tumor suppressors, such as MYC, RAS, P53, and RB, to carcinogenesis is indisputable. Multiple lines of proof show how random somatic genomic alteration of such genes (e.g., mutation, deletion, or amplification), followed by selection and clonal expansion, forms the main molecular basis of tumor development. Many important cancer genes were discovered using low-throughput approaches in the pre-genomic era, and this knowledge is today solidified and expanded upon by modern genome-scale methodologies. In several recent studies, non-coding RNAs (ncRNAs), such as microRNAs and long ncRNAs (lncRNAs), have been shown to contribute to tumor development. However, in comparison with coding cancer genes, the genomic (DNA-level) evidence is sparse for ncRNAs. The coding proto-oncogenes and tumor suppressors that we know of today are major molecular hubs in both normal and malignant cells. The search for ncRNAs with tumor driver or suppressor roles therefore holds the additional promise of pinpointing important, biologically active, ncRNAs in a vast and largely uncharacterized non-coding transcriptome. Here, we assess the available DNA-level data that links non-coding genes to tumor development. We further consider historical, methodological, and biological aspects, and discuss future prospects of ncRNAs in cancer.

Cervical cancer (CC) is one of the most malignant tumors and the second or third most common type of cancer in women worldwide. The association between human papillomavirus (HPV) and CC is widely known and accepted (99.7% of cases). At present, the pathogenesis mechanisms of CC are not entirely clear. It has been shown that inactivation of tumor suppressor genes and activation of oncogenes play a significant role in carcinogenesis, caused by the genetic and epigenetic alterations. In the past, it was generally thought that genetic mutation was a key event of tumor pathogenesis, especially somatic mutation of tumor suppressor genes. With deeper understanding of tumors in recent years, increasing evidence has shown that epigenetic silencing of those genes, as a result of aberrant hypermethylation of CpG islands in promoters and histone modification, is essential to carcinogenesis and metastasis. The term epigenetics refers to heritable changes in gene expression caused by regulation mechanisms, other than changes in DNA sequence. Specific epigenetic processes include DNA methylation, chromotin remodeling, histone modification, and microRNA regulations. These alterations, in combination or individually, make it possible to establish the methylation profiles, histone modification maps, and expression profiles characteristic of this pathology, which become useful tools for screening, early detection, or prognostic markers in cervical cancer. This paper reviews recent epigenetics research progress in the CC study, and tries to depict the relationships between CC and DNA methylation, histone modification, as well as microRNA regulations.

Background

Reports on common mutations in neuroendocrine tumors (NET) are rare and clonality of NET metastases has not been investigated in this tumor entity yet. We selected one NET and the corresponding lymph node and liver metastases as well as the derivative cell lines to screen for somatic mutations in the primary NET and to track the fate of genetic changes during metastasis and in vitro progression.

Results

Applying microarray based sequence capture resequencing including 4,935 Exons from of 203 cancer-associated genes and high-resolution copy number and genotype analysis identified multiple somatic mutations in the primary NET, affecting BRCA2, CTNNB1, ERCC5, HNF1A, KIT, MLL, RB1, ROS1, SMAD4, and TP53. All mutations were confirmed in the patients’ lymph node and liver metastasis tissue as well as early cell line passages. In contrast to the tumor derived cell line, higher passages of the metastases derived cell lines lacked somatic mutations and chromosomal alterations, while expression of the classical NET marker serotonin was maintained.

Conclusion

Our study reveals that both metastases have evolved from the same pair of genetically differing NET cell clones. In both metastases, the in vivo dominating “mutant” tumor cell clone has undergone negative selection in vitro being replaced by the “non-mutant” tumor cell population. This is the first report of a bi-clonal origin of NET derived metastases, indicating selective advantage of interclonal cooperation during metastasis. In addition, this study underscores the importance to monitor cell line integrity using high-resolution genome analysis tools.

The classical somatic mutation theory (SMT) of carcinogenesis and metastasis postulates that malignant transformation occurs in cells that accumulate a sufficient amount of mutations in the appropriate oncogenes and/or tumor suppressor genes. These mutations result in cell-autonomous activation of the mutated cell and a growth advantage relative to neighboring cells. However, the SMT cannot completely explain many characteristics of carcinomas. Contrary to the cell-centered view of the SMT with respect to carcinogenesis, recent research has revealed evidence that the tumor microenvironment plays a role in carcinogenesis as well. In this review, we present a new model that accommodates the role of the tumor microenvironment in carcinogenesis and complements the classical SMT. Our “feedback” model emphasizes the role of an altered spatiotemporal communication between epithelial and stromal cells during carcinogenesis: a dysfunctional intracellular signaling in tumorigenic epithelial cells leads to inappropriate cellular responses to stimuli from associated stromal or inflammatory cells. Thus, a positive feedback loop of the information flow between parenchymal and stromal cells results. This constant communication between the stromal cells and the tumor cells causes a perpetually activated state of tumor cells analogous to resonance disaster.

Background

The mutation spectra of the TP53 gene and other tumor suppressors contain multiple hotspots, i.e., sites of non-random, frequent mutation in tumors and/or the germline. The origin of the hotspots remains unclear, the general view being that they represent highly mutable nucleotide contexts which likely reflect effects of different endogenous and exogenous factors shaping the mutation process in specific tissues. The origin of hotspots is of major importance because it has been suggested that mutable contexts could be used to infer mechanisms of mutagenesis contributing to tumorigenesis.

Results

Here we apply three independent tests, accounting for non-uniform base compositions in synonymous and non-synonymous sites, to test whether the hotspots emerge via selection or due to mutational bias. All three tests consistently indicate that the hotspots in the TP53 gene evolve, primarily, via positive selection. The results were robust to the elimination of the highly mutable CpG dinucleotides. By contrast, only one, the least conservative test reveals the signature of positive selection in BRCA1, BRCA2, and p16. Elucidation of the origin of the hotspots in these genes requires more data on somatic mutations in tumors.

Conclusion

The results of this analysis seem to indicate that positive selection for gain-of-function in tumor suppressor genes is an important aspect of tumorigenesis, blurring the distinction between tumor suppressors and oncogenes.

Reviewers

This article was reviewed by Sandor Pongor, Christopher Lee and Mikhail Blagosklonny.

We have analyzed synonymous codon usage in the genome of A. tamarense CCMP 1598 for protein-coding sequences from 10865 expressed sequence tags (ESTs). We reconstructed a total of 4284 unigenes, including 74 ribosomal protein and 40 plastid-related genes, from ESTs using FrameDP, an open reading frame (ORF) prediction program. Correspondence analysis of A. tamarense genes based on codon usage showed that the GC content at the third base of synonymous codons (GC3s) was strongly correlated with the first axis (r = 0.93 with P < .001). On the other hand, the second axis discriminated between presumed highly and low expressed genes, with expression levels being confirmed by the analysis of EST frequencies (r = −0.89 with P < .001). Our results suggest that mutational bias is the major factor in shaping codon usage in A. tamarense genome, but other factors, namely, translational selection, hydropathy, and aromaticity, also appear to influence the selection of codon usage in this species.

Genetic recombination is the creation of new gene combinations in a cell or gamete, which differ from those of progenitor cells or parental gametes. In eukaryotes, recombination may occur at mitosis or meiosis. Mitotic recombination plays an indispensable role in DNA repair, which presumably directed its early evolution; the multiplicity of recombination genes and pathways may be best understood in this context, although they have acquired important additional functions in generating diversity, both somatically (increasing the immune repertoire) and in germ line (facilitating evolution). Chromosomal homologous recombination and HsRad51 recombinase expression are increased in both immortal and preimmortal transformed cells, and may favor the occurrence of multiple oncogenic mutations. Tumorigenesis in vivo is frequently associated with karyotypic instability, locus-specific gene rearrangements, and loss of heterozygosity at tumor suppressor loci — all of which can be recombinationally mediated. Genetic defects which increase the rate of somatic mutation (several of which feature elevated recombination) are associated with early incidence and high risk for a variety of cancers. Moreover, carcinogenic agents appear to quite consistently stimulate homologous recombination. If cells with high recombination arise, either spontaneously or in response to “recombinogens,” and predispose to the development of cancer, what selective advantage could favor these cells prior to the occurrence of growth-promoting mutations? We propose that the augmentation of telomere-telomere recombination may provide just such an advantage, to hyper-recombinant cells within a population of telomerase-negative cells nearing their replicative (Hayflick) limit, by extending telomeres in some progeny cells and thus allowing their continued proliferation.

Elite controllers or suppressors (ES) are HIV-1 infected patients who maintain viral loads of < 50 copies/ml without antiretroviral therapy. CD8+ T cells are thought to play a key role in the control of viral replication and exert selective pressure on gag and nef in HLA-B*57 positive ES. We previously showed evolution in the gag gene of ES which surprisingly was mostly due to synonymous mutations rather than non-synonymous mutation in targeted CTL epitopes. This finding could be the result of structural constraints on Gag, and we therefore examined the less conserved nef gene. We found slow evolution of nef in plasma virus in some ES. This evolution is mostly due to synonymous mutations and occurs at a rate similar to that seen in the gag gene in the same patients. The results provide further evidence of ongoing viral replication in ES and suggest that the nef and gag genes in these patients respond similarly to selective pressure from the host.