Illustration by Anasthasia Shilov.
Cancer has been one of the hottest topics in biomedical research in the past decades. In lieu of a cure, researchers have been looking for ways to treat or prevent it. Whilst cancers can occur due to genetic factors, many of are in fact caused by lifestyle-related factors. For example, you’ve probably heard that things like smoking and alcohol consumption are common causes of cancer, and that UV radiation from the sun can cause skin cancer. This is because these environmental factors either cause mutations or increase the proliferation of cells, which increases the probability of mutation.
UV Radiation and DNA Mutation
A group of researchers at the Yale School of Medicine have been looking into the mechanism underlying how UV radiation causes cancer. The effect of UV radiation starts with a photon, a particle of energy, striking the DNA. Of the four nucleotides that comprise DNA, two of them, cytosine and thymine, are known as pyrimidines. When the photon hits two adjacent pyrimidines, also known as a dipyrimidine or a PyPy site, the pyrimidines join together, creating a cyclobutane pyrimidine dimer (CPD).
Although UV radiation results in other DNA damage as well, CPDs are the main UV photoproducts and play the biggest role in cancer. Some CPDs are cancer-causing mutations in skin cells that activate or inactivate signaling pathways. Starting from one damaged cell, the process of DNA and cell replication lead to the copying of DNA containing CPDs, proliferating the mutant cell to a group of abnormal clones. Unregulated dividing cells a hallmark of cancer.
Mapping UV Hot Spots
Douglas Brash, one of the researchers working on this project, was initially interested in how DNA damage leads to cancer. After looking into UV radiation, he decided to explore whether there are specific parts of the genome that are more correlated to disease due to locally greater damage induction or slower repair. Specifically, the group decided to delve into mapping the genome for CPDs and search for specific places most associated with cancer as caused by UV radiation.
CPDs due to sun exposure usually occur about once every ten thousand bases of DNA, and occur in different places in each cell. In mapping the genome, the group found that there were certain spots, called CPD hyperhotspots, that were approximately a hundred times more likely than other spots to be affected by UV radiation. These hyperhotspots differed between cell types. In addition, a lot of these hotspots were found in promoters of genes as well, rather than just the structural genes.
Six Years, Two Methods
In total, the group’s development of methods and research to make this discovery took a lengthy six years. Eventually, they came up with two methods that could be used to understand the genome and analyze the consequences in the overall scheme of UV radiation and cancer. The first step to their analysis involves a method known as adductSeq, which involves converting the DNA damage caused by UV radiation into something that can be understood by a high-throughput DNA sequencing machine.
In adductSeq, a nick is first made in the DNA right next to where the DNA damage occurs (usually a CPD), using an enzyme that’s normally involved in repairing the damage. Then, a short segment of DNA, called a linker, is ligated to the nick to tag the CPD site to mark where the damage occurred. Thus, the DNA can be sequenced and analyzed for CPDs and other DNA damage. The amount of damage is quantified by counting the number of times the same site appears in the experiment.
The next step to understanding the outcome of DNA damage is the actual analysis of the genome that has been sequenced. This is done using a method called freqSeq, which analyzes the data from the high-throughput DNA sequencing machine and assesses its validity. This is important because, when a whole genome is being read and mapped, there are often stochastic variations in genome coverage and differences in DNA loading and sequencing quality between experiments. In addition, the reference genome used for mapping the experiment’s DNA fragments contains errors due to several genome locations having been assigned the same location. Since the experiment is looking for CPDs (which are rare outliers rather than consensus sequences), analyzing the statistics behind the experiments is incredibly important to quantifying the CPDs in the genome and therefore to successfully understanding the effects of UV radiation and locating CPD hotspots.
Predicting Skin Cancer Risk
With these techniques, the researchers were able to study the genome that resulted from UV radiation and locate hyperhotspots. One might expect that mutations are likely to occur at these sites, and indeed mutations occur there in melanoma skin cancers. In addition, they found that, even in the absence of UV, melanocytes also presented a certain degree of lesions or DNA damage. This is because, when melanin is synthesized, highly reactive unpaired electrons known as radicals are produced. These radicals often create apurinic sites, sites at which a purine (adenine or guanine) has been cleaved off the DNA, leaving no base at all. The CPD hotspots were distributed across genes and gene-rich chromosomes. In addition, CPDs were most commonly found at places where certain transcription factors bind, which corresponds to the effects CPDs have on signaling pathways that make cells clone more rapidly.
The ideal end goal of identifying cancer risk
factors and their effects on DNA is to develop methods to predict cancer risk
and prevent cancer development. With skin cancer, for example, Brash believes
that, with the methods outlined in their paper, the researchers could determine
someone’s risk of getting skin cancer based on their previous sun exposure. Ideally,
this technology would ideally allow a family physician to tell a patient that
they ought to be monitored by a dermatologist. This would allow for better
prevention methods, ranging from simple things like applying sunscreen to
curative surgery. Skin cancers start with early lesions which can be cured,
even for melanoma, so catching them early would allow doctors to remove them,
preventing the cancer from spreading in the first place.
More Mutations Moving Forward
While this research was primarily done on fibroblasts and melanocytes, the researchers who worked on this paper are looking to apply the same concepts and methods to study different types of cells. Currently, research is being done on keratinocytes and whether the same patterns exist in those cells. Being able to look at all the different types of cells and map out the different spots for DNA damage will further allow scientists to understand the effect of UV radiation and other factors on cells and cancer development.
Further research is also being done on rare mutations, which are the end result of DNA damage. Combined with the knowledge we now have on the effect of UV radiation on DNA, additional research on mutations could lead to a conclusion about major risk factors of cancer. This would further aid in the search for more knowledge about the disease and ways to prevent and treat it.
About the Author:
Catherine Zheng is a first-year interested in studying either chemical or biomedical engineering.
I’d like to thank Dr. Douglas Brash for discussing his research with me.