Epigenetic Processes in Cancer Research

Catalyst Volume 9

Abstract

Epigenetics is the study of the phenotypic variation caused by external factors (e.g. diet, nicotine use, and carcinogenic chemical exposure) that influence the mechanisms through which cells read and interpret genes. Epigenetic modifications are independent of genetic mutations. Currently, epigenetics offers significant promise in novel noninvasive cancer therapies and early diagnostic tools. Key epigenetic processes including DNA methylation and histone modification are reversible, unlike most genetic mutations. Reversing these processes in tumor suppressor genes can restore normal behavior in tumor cells. This review discusses the biological basis and treatment potential of these processes and provides a brief analysis of their potential application in cancer treatment.

Introduction

DNA is a double-helical structure packed into the nuclei of eukaryotic cells in the form of chromatin. The basic unit of DNA packing in chromatin is the nucleosome, a complex of eight histone proteins and approximately 140 DNA base pairs. Further coiling of repeating nucleosome fibers eventually yields a chromatid, a key component of a chromosome.

Gene expression is epigenetically regulated through alterations in chromatin structure and the double helix of DNA by addition of functional groups. DNA methylation and histone modification are the most understood mechanisms of such epigenetic modulation. Errant modification of functional group patterns in DNA or histone tails could result in unintended silencing of critical regulatory genes, a process which contributes to the development of several diseases, including cancer. Understanding these complex mechanisms is essential for the development of cancer therapies that reverse the inhibition of tumor-suppressor genes. This review seeks to outline the biochemical basis of the gene silencing effected by DNA methylation and histone modification, as well as the current developments in reversing these mechanisms for the prevention of cancer.

Histone Modification & Its Implications on Cancer

Histone modifications are reversible modifications at the N-terminals of histones in a nucleosome. These include, but are not limited to, acetylation, methylation, phosphorylation, and ubiquitination. Depending on the group added or removed from a given histone, local gene transcription can be upregulated or downregulated. Histone acetylation, for instance, modifies chromatin into a less-condensed conformation that is more transcriptionally active. Conversely, histone deacetylases (HDACs) remove acetyl groups, increasing the ionic attractions between positively charged histones and negatively charged DNA and tightening chromatin structure. In order to further condense chromatin, HDACs can also recruit K9 histone methyltransferases (HMTs) to methylate H3K9 histone residues, providing the condensing agent heterochromatin protein 1 (HP1) with a binding site. As a result of the condensed heterochromatin conformation, the cell has limited transcriptional access to the genome.

Recent research has established histone modifications as useful biomarkers in cancer diagnosis. HDACs, specifically HDAC1, can often be identified in elevated quantities in prostate and gastric cancer patients. Prostate cancer tumors have been characterized by general hypomethylation of histone residues H4K20me1 and H4K20me2, as well as hypermethylation of residue H3K27 and increased activity of its specific methyltransferase. These conditions are now associated with progression and metastasis of prostate cancer. Histone modifications play a multifaceted role in cancer diagnosis and treatment. In addition to serving as diagnostic biomarkers, they are also potential therapeutic agents. Drugs that specifically inhibit HDACs are currently being manufactured and assessed for clinical application after recent FDA approval.⁵

DNA Methylation & Its Implications on Cancer

In DNA methylation, a methyl group is covalently added to a cytosine ring in the DNA sequence, forming a 5-methylcytosine. Molecules of 5-methylcytosine are found primarily at cytosine-guanine dinucleotides (CpGs). Some areas of high CpG density, such as CpG islands, are characteristically unmethylated and are located in or near the promoter regions of genes, allowing them to play a role in gene expression. Enzymes termed DNA methyltransferases (DNMTs) facilitate the methylation of CpG residues. Three key members of this family include Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 is labeled as the “maintenance” DNMT due to its methylation of DNA near replication forks, which preserves the epigenetic inheritance of methylation patterns across cellular generations. The other two DNMTs serve to directly methylate other CpG residues. These enzymes can either be recruited by a transcription factor bound to the promoter region of a given gene to methylate a specific CpG island or simply methylate all CpG sites across a genome not protected by a transcription factor. Several families of proteins, such as the UHRF proteins, the zinc-finger proteins, and most notably, the MBD proteins, are responsible for the interpretation of these methylation patterns. For example, MBD proteins possess transcriptional repression domains that allow them to bind to methylated DNA and silence nearby genes.

For a malignant cancer to develop, critical tumor-suppressor genes such as p53 must be knocked out. This process can be accomplished through hypermethylation of CpG islands in certain promoter regions and genes. For example, when the DNA repair gene BRCA1 becomes hypermethylated, it is rendered inactive, which can lead to the development of breast cancer. The reversibility of methylation implies that these tumor-suppressor genes could be reactivated to effectively treat tumors. Infiltration of a cell by pharmaceutical agents that inhibit methylation-mediated suppression could restore the function of a silenced tumor-suppressor gene.⁶

One recent study demonstrated that azacitidine, an agent that can be incorporated into DNA, is capable of inducing hypomethylation. The chemical modification of this agent’s diphosphate form by a ribonucleotide reductase and subsequent phosphorylation creates a triphosphate form that displaces cytosine bases in DNA. As a result, DNMTs are limited in methylation functionality since they are isolated on a substituted DNA strand.⁷ Such manipulation of naturally occurring DNA methylation processes in cancer cells appears to be a promising method of restoring the normal function of tumor-suppressor genes.

Co-Application of DNA Methylation & Histone Modification

The discovery of the protein MeCP2 has validated the hypothesis that DNA methylation regulates histone acetylation patterns. MeCP2 recruits histone HDACs to the promoter regions of methylated CpG islands. The resulting hypoacetylated histones yield a highly condensed heterochromatin structure that represses transcription of nearby genes. Clinical studies have thus emphasized the utilization of both demethylating agents and histone deacetylase inhibitors for transcriptional activation of tumor-suppressor genes.⁸

Conclusion

The field of epigenetics has opened a door to novel, promising cancer therapies unimaginable just a decade ago. Manipulating DNA methylation and histone modification can reverse tumor-suppressor gene silencing. Reactivating tumor-suppressor genes through epigenetic modifications can halt and reverse cancer proliferation.

References

1. Kornberg, R.D. Science. 1974, 184, 868-871.
2. Fahrner, J.A. et al. J Cancer Res. 2002, 62, 7213-7218.
3. Alelú-Paz, R. et al. J Sign Transduc. 2012, 8 pg.
4. Bártová, E. et al. J Histochem Cytochem. 2008, 56, 711-721.
5. Chervona, Y. et al. Am J Cancer Res. 2012, 5, 589-597.
6. Baylin, B.S. Nature Clin Prac Onco. 2005, 2, S4-S11.
7. Jütterman, R. et al. Proc Natl Acad Sci USA. 1994, 91, 11797-11801.
8. Herranz, M. et al. In Target Discovery and Validation Reviews and Protocols, 1; Sioud, M., Eds.; Humana Press: New York, NY, 2007, 2, pp 25-62.

Dangers of DNA Profiling

Catalyst Volume 9

DNA profiling has radically changed forensics by providing an objectively verifiable method for linking suspects to crimes. Currently, many states collect the DNA of felons in order to ensure that repeat offenders are caught and convicted efficiently.¹ Over the past few years, situations in which law enforcement officials can collect DNA from suspects have increased drastically. In 2013, President Obama strongly supported the creation of a national DNA database that included samples from not only people who are convicted, but also those arrested.¹ In Maryland v. King (2013), the Supreme Court declared that law enforcement officials are justified in collecting DNA prior to conviction if it aids in solving a criminal case.² In the years since this decision, the creation of a national DNA database has become a particularly polarizing and contentious issue. Proponents argue that a database would dramatically improve the ability of law enforcement to solve crimes. However, detractors argue that the potential for misuse of genetic information is too great to warrant the creation of such a system.

DNA is popularly referred to as the “blueprint of life” and contains extremely sensitive information such as an individual’s susceptibility to genetic disorders. One of the major arguments against the creation of a national DNA database is that such information could be hacked. Yaniv Erlich, a geneticist at MIT, illustrated this when he used “genome mining” to find the true identities of individuals in a national genome registry. In his study, Erlich obtained genomes from the 1000 Genomes Project, a large database used for scientific research. He then used a computer algorithm to search for specific DNA sequences known as short tandem repeats (STRs) on the Y chromosome of males. These STRs are remarkably invariable from generation to generation. Erlich was able to use the Y chromosome’s STR marker to identify the last names of the individuals to whom the DNA belonged by using easily accessible genealogy sites.³ With just a computer and access to genome data, Erlich could identify personal information in DNA registries. Clearly, the creation of a national DNA database could give rise to widespread privacy concerns. Though there are large fines associated with unauthorized disclosure or acquisition of DNA data, current federal regulations do not technically limit health insurance companies from using genome mining in order to determine life insurance or disability care.⁴

Hacking of federal databases is not an unreasonable scenario—just this past July, sensitive information including the addresses, health history, and financial history of over 20 million individuals was stolen in a massive cyber-attack.⁴ That attack uncovered information about every single individual who has attempted to work or has worked in the United States government. A similar abuse of genetic information by third parties is undoubtedly a danger associated with a national DNA database. Despite advances in federal protections such as the Genetic Information Nondiscrimination Act, there are still numerous instances where genetic information regarding disease is used in employment decisions.⁵

Another potential issue associated with the creation of a DNA database is the notion of genetic essentialism. Genetic essentialism argues that the genes of an individual can predict behavioral outcomes.⁶ Critics of a national DNA database argue that certain factors—such as the extra Y chromosome—may lead law enforcement officials to suspect certain individuals more than others, which sets up a dangerous precedent.

The notion that chromosomal abnormalities can alter behavioral outcomes has generated numerous studies examining the link between criminality and changes in sex chromosomes—the genes that determine whether an individual is male or female. Normally, females will have two X chromosomes, whereas males have one X chromosome and one Y chromosome. However, in rare cases, males can either have an extra X chromosome (XXY) or an extra Y chromosome (XYY). General literature review suggests that XXY men have feminine characteristics and are substantially less aggressive than XYY or XY men.⁷ Conversely, studies like Jacobs et al. have suggested that the XYY condition can lead to increased aggression in individuals.⁸ However, Alice Theilgaard, one of the most prominent researchers on this topic, found that most behavioral characteristics associated with the XYY chromosomal abnormality are controversial.⁷ Even tests based on objective measures, like testosterone levels, have been inconclusive. Theilgaard argues that the XYY chromosomal abnormality does not cause increased aggression or propensity to commit crimes. Rather, she states that the criminality of XYY individuals might be a socially constructed phenomenon. XYY individuals often have severe acne, lowered intellect, and unusual height. This makes it difficult for people with this condition to “fit in.” As a result of their physical characteristics, XYY individuals might feel ostracized and become antisocial.⁸ Thus, it is reasonable to conclude that merely having an extra Y chromosome does not predispose someone to be violent; rather a wide variety of social factors play a role.

It is entirely plausible that law enforcement individuals could misinterpret genetic information. For example, they could mistakenly believe that an individual with the XYY condition is more likely to be a suspect for a violent crime. Such an assumption would hinder law enforcement officials from objectively evaluating the evidence involved in a crime and shift the focus to individual characteristics of particular suspects. People in favor of a national DNA database often argue that it would be a great method of solving crimes. Specifically, some officials argue that a database would prevent recidivism (a relapse in criminal behavior) and deter people from committing crimes. However, research done by Dr. Avinash Bhati suggests that the inclusion of DNA in a national registry only seems to reduce recidivism for burglaries and robberies; in other crime categories, recidivism is generally unaffected.⁹ This suggests that a convict’s knowledge that he/she is in a DNA database is not a true deterrent. The concerns raised by this study should show that databases might not be as effective a crime-fighting tools as proponents suggest.

Both genome mining and genetic essentialism present very real harms associated with the creation of a national DNA database. Having sensitive genetic information in one centralized registry could potentially lead to abuse and discriminatory behaviors by parties that have access to that information. Even if genome databases are strictly regulated, the possibility of that information being hacked still exists. Furthermore, assuming that genetics are the only determinants of behavior could lead to people with genetic abnormalities being suspected of crimes at a higher rate than “normal” individuals. Social factors often shape the way an individual acts; the possibility of law enforcement officials embracing the genetic essentialism approach is another associated harm. In the end, it seems that the negative consequences associated with the creation of a national DNA database outweigh the benefits.

References

1. Barnes, R. Supreme Court upholds Maryland law, says police may take DNA samples from arrestees. Washington Post, https://www.washingtonpost.com/politics/supreme-court-upholds-maryland-law-says-police-may-take-dna-samples-from-arrestees/2013/06/03/0b619ade-cc5a-11e2-8845-d970ccb04497_story.html (accessed 2015).  
2. Wolf, R. Supreme Court OKs DNA swab of people under arrest. USA Today, http://www.usatoday.com/story/news/politics/2013/06/03/supreme-court-dna-cheek-swab-rape-unsolved-crimes/2116453/ (accessed 2015).
3. Ferguson, W. A Hacked Database Prompts Debate about Genetic Privacy. Scientific American, http://www.scientificamerican.com/article/a-hacked-database-prompts/ (accessed 2015).
4. Davis, J. Hacking of Government Computers Exposed 21.5 Million People. The New York Times, http://www.nytimes.com/2015/07/10/us/office-of-personnel-management-hackers-got-data-of-millions.html?_r=0 (accessed 2015).
5. Berson, S. Debating DNA Collection. National Institute of Justice, http://www.nij.gov/journals/264/pages/debating-dna.aspx (accessed 2015).
6. Coming to Terms with Genetic Information. Australian Law Reform Commission , http://www.alrc.gov.au/publications/3-coming-terms-genetic-information/dangers-‘genetic-essentialism’ (accessed 2015).
7. Are XYY males more prone to aggressive behavior than XY males? Science Clarified, http://www.scienceclarified.com/dispute/vol-1/are-xyy-males-more-prone-to-aggressive-behavior-than-xy-males.html (accessed 2015).
8. Dar-Nimrod, I.; Heine, S. Genetic Essentialism: On the Deceptive Determinism of DNA. Psychological Bulletin, http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3394457/ (accessed 2015).Are XYY males more prone to aggressive behavior than XY males? Science Clarified.
9. Bhati, A. Quantifying The Specific Deterrent Effects of DNA Databases. PsycEXTRADataset. 2011. https://www.ncjrs.gov/app/publications/abstract.aspx?id=258313 (accessed May 2015).