Lynda Chin, MD, and her colleagues found that they could increase both the number of tumors and the speed with which they formed by exposing newborn mice with an intact Rb pathway to UV radiation. (A pathway is a chain of biochemical signals that regulates cellular activity.) Those mice in which the Rb pathway was already essentially knocked out were unaffected by the dose of UV radiation. "It looks like the Rb pathway is specifically targeted by ultraviolet radiation," said Chin, the study's senior author. Karupiah Kannan, PhD, and Norman Sharpless, MD, formerly at the DFCI, are first authors on the study.
Melanoma is the deadliest form of skin cancer and, left unchecked, can spread aggressively to other parts of the body. However, if caught early, many melanoma lesions can be effectively treated surgically. The discovery that UV radiation triggers melanoma formation by dismantling a specific pathway inside the skin's pigment-producing cells, or melanocytes, offers the possibility of an efficient means of distinguishing, at an early stage, cancerous moles from non-cancerous ones.
"If you see in a sun-induced lesion that its Rb pathway has been inactivated, then the risk of it becoming a melanoma is much greater than one without such a lesion," said Chin, who is also an assistant professor of dermatology at Harvard Medical School. "You could then use that as a prognostic factor to determine which of these funny-looking moles needs to be cut out with a wide margin around it,"
Though there were hints that it might be particularly susceptible to environmental attack, the discovery that the Rb pathway is specifically targeted by UV radiation is surprising for several reasons. To begin, UV radiation was thought to be a less picky carcinogenic agent, targeting multiple pathways in the cell. When it hit, it was thought to do so in the manner of a surgical strike, causing a point mutation. Yet when Chin and her colleagues explored the DNA in the tumors of the UV-affected mice they found a more massive disruption. A whole region of chromosome 5 appeared to be expanded, or amplified, suggesting that the UV radiation had actually caused the double stranded DNA molecule to break.
"Given the data we see, people need to think of amplification, and also deletion, presumably, as a mechanism by which UV exerts its mutagenic effects," Chin said. "So far people haven't really looked at these as UV-induced changes."
Perhaps most significant, the findings suggest that Rb may be a kind of molecular dark horse in the human melanoma story. Human melanomas are known to exhibit defects in their Rb pathway, but they also exhibit mutations in other pathways such as p53 pathways. Mutation of both Rb and p53 pathways are needed for a tumor to emerge. Based on genetic studies in mice, many thought the p53 mutations were more important than the Rb mutations. Opinion began to shift a little over a year ago, when Sharpless and a team of Dana-Farber researchers reported that a mice engineered to lack only a gene in the Rb pathway, p16, developed tumors at a faster rate when exposed to carcinogenic chemicals. Prompted by the findings, the researchers suggested that the normal function of the Rb pathway might be to tell the cell to stop dividing in the presence of environmentally induced damage.
To explore this hypothesis, and to compare more generally the role of the Rb and p53 pathways in melanoma, Chin and her colleagues exposed newborns of two strains of genetically-engineered mice--one lacking a critical component of the p53 pathway, p19, and one lacking the Rb pathway protein p16--to UV radiation. (Both strains carried the Ras mutation, making them especially cancer-prone.) The p19 mutants, whose Rb pathway was intact and therefore vulnerable to attack, had a much higher chance of developing melanomas when exposed to UV radiation--85 percent compared to 50 percent in unexposed litters. They also developed more tumors, two to three compared to one in the non-exposed group. And the lesions appeared, on average, six weeks sooner.
The p16 mutants, whose Rb pathway was already defective and therefore impervious to additional UV insult, exhibited no such difference. Their chance of developing tumors was the same whether or not they were exposed to UV radiation even though their p53 pathway was theoretically susceptible to attack by UV radiation.
Puzzled as to where in the Rb pathway the UV agent was aiming its blows, the researchers compared both the DNA and the gene expression patterns of melanomas from the UV radiation-exposed p19 knockouts to the unexposed mice, which presumably developed their tumors spontaneously. While loss of p16 function showed up in the expression patterns of 50 percent of the unexposed mice tumors, it appeared in only 20 percent of exposed mutants. Upon further examination, they found another gene, cdk6, was overexpressed and amplified in the vast majority of the UV-induced tumors. In a remarkable convergence, the investigators realized that cdk6 resides on chromosome 5 within the precise region that the investigators also noted to be amplified when the tumors were examined in a genome-wide manner. These functional and genetic data suggest that cdk6, and thereby the Rb pathway, is the UV target in this model.
Why this gene, which was not turned on in any of the spontaneously developing mouse tumors, should be especially vulnerable to UV perturbation is not clear. Nor is the cdk6 gene UV radiation's only likely target. Though it was overexpressed in the majority of the UV exposed tumors, many lesions exhibited as yet unknown defects. "The data suggest that UV is really targeting the Rb pathway rather than specific components," said Chin. Such a single-minded strategy would make sense if the Rb pathway were the cell's main environmental carcinogen sensor, she added.
Turning the findings into a tool for diagnosing human melanomas will require more basic research. "People have not thought to correlate Rb status with sun exposure history and progression of pigmented lesions," Chin said. "It is not an easy study to do but at least now someone can formulate a hypothesis and design a study to answer that question."
The study was funded in part by the National Institutes of Health, the Claudia Adams Barr Program in Cancer Research and the Rockefeller Brothers Fund.
Dana-Farber Cancer Institute is a principal teaching affiliate of the Harvard Medical School and is among the leading cancer research and care centers in the United States. It is a founding member of the Dana-Farber/Harvard Cancer Center (DF/HCC), designated a comprehensive cancer center by the National Cancer Institute.