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.
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.
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.
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