Brian Gastman, MD
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Cutaneous melanocytes in people with a history of melanoma react differently to sunburn-causing levels of solar radiation than do melanocytes in healthy people, Cleveland Clinic and Case Western Reserve University researchers have found.
Intense ultraviolet radiation (UVR) alters the balance of melanocytes’ microRNA (miRNA) expression, inducing changes in cellular regulatory and immune-response pathways, the researchers discovered.
In healthy subjects, UVR exposure up-regulates miRNAs, switching melanocytes to a protective mode.
In contrast, UVR down-regulates miRNAs in the melanocytes of subjects who have had melanoma. That suppression triggers a network of genes involved in epithelial–mesenchymal transition and immune-response evasion, which the researchers speculate may represent the key first step in progression to melanoma. The UVR-mediated immune changes may help melanoma-prone melanocytes escape elimination while they accumulate the genetic and epigenetic alterations of malignancy.
The miRNA signature of this transitional phase could serve as the basis for a biomarker and help guide future treatment and prevention strategies, the investigators believe.
The research marks the first time that scientists have observed the influence of UVR on melanocytes in their natural microenvironment, using tissue samples obtained by laser capture microdissection and analyzed with advanced genetic techniques.
The results suggest that melanocytes in melanoma-prone people have a defective miRNA biogenesis program and can acquire a cancer-promoting phenotype when tipped by environmental pressures such as UVR.
“We’re finally getting to the bottom of why some people become more susceptible to melanoma while others do not,” says study co-author Brian Gastman, MD, Director of Cleveland Clinic Cancer Center’s Melanoma Program. “And we’re building the basis from which one could develop treatment plans for prevention, so that we won’t have to deal with melanoma after it becomes a deadly process.”
UVR previously had been shown to permanently alter melanocyte homeostasis and to have a direct mutagenic role in melanoma, but the molecular mechanism is unknown.
Environmental exposures such as UVR affect the level of regulatory miRNAs, which are small, non-coding RNAs that control the signal output of almost all cellular pathways. More than 1,000 miRNAs have been identified in humans, and they direct the expression of one-third of the human genome and affect a wide variety of processes.
MiRNAs dictate cellular responses by blocking translation or destabilizing messenger RNA, and are thus responsible for a type of gene silencing. Up- or down-regulation of miRNAs respectively reduces or increases protein concentration in shared pathways or signaling cascades, including those involved in cellular differentiation, proliferation, inflammation, DNA repair, apoptosis, and epithelial-to-mesenchymal transition (EMT).
In this way, miRNAs may help to restore cellular processes in response to external stresses such as UVR. Conversely, miRNAs also have been implicated in the development of many cancers, including melanoma.
The Cleveland Clinic/Case Western Reserve team wanted to better understand UVR’s impact on melanocytes and miRNAs and the oncogenic transition.
Previous studies of UVR’s effects on melanocytes mostly were conducted using in vitro cell cultures, which may not reflect the way miRNAs respond in their normal in vivo microenvironment.
To address that, Dr. Gastman and his colleagues worked with skin tissue samples obtained from study participants and flash-frozen.
The participants were 17 women between the ages of 31 and 46 with Fitzpatrick skin types of I or II. Eight subjects had no medical or dermatologic history; nine had one prior primary melanoma.
A 6 mm circular area of skin on the posterior shoulder of each volunteer was irradiated with simulated solar UVR produced by a xenon arc lamp. The UVR dose was four times the amount previously determined to produce slight skin reddening. Twenty-four hours later, the irradiated area and an adjacent non-exposed site were punch-biopsied and cryopreserved. Using laser capture microdissection, investigators extracted melanocytes from the irradiated and non-irradiated samples for miRNA expression analysis.
The analysis found a striking difference in observed UVR-induced miRNA changes in the healthy women compared with subjects with a history of melanoma. A much higher proportion of miRNAs controlling key regulatory networks were down-regulated in the melanoma patients than in the healthy subjects. In contrast, UVR miRNAs were predominantly up-regulated in the healthy subjects, suggesting that UVR exposure led to cancer-protective processes in their melanocytes.
The UVR-miRNA repression in the melanocytes of subjects with a history of melanoma released genes from inhibition, enabling probable gains of function. Further analysis of the affected gene/UVR-miRNA networks showed that they corresponded with well-known regulatory modules involved in controlling EMT and immune-evasion processes. The functional outputs of these UVR-miRNA-regulated networks include critical immune-evasive genes such as PD-L1 and PD-L2.
Although EMT and immune response-evasion are intrinsic to melanoma’s invasion-metastasis cascade, before this research they had not been connected to the effects of UVR exposure on miRNA expression. “Our results strongly indicate that some form of immunosuppression is prematurely occurring in the UV-irradiated melanocytes of patients as opposed to those of healthy persons,” Dr. Gastman says.
In essence, the findings show UVR-induced miRNA down-regulation is capable of initiating phenotypic changes in melanocytes of melanoma patients that are not apparent in healthy subjects’ melanocytes. These phenotypic changes — such as apoptosis resistance and the ability to migrate — have the potential to confer stem cell-like properties to melanoma-prone melanocytes in subjects with a history of the disease, and may allow the cells to accumulate mutations that result in malignant transformation.
While the research involved a relatively small number of volunteers, it is significant in that most studies reporting on UVR-associated molecular changes in melanoma refer to mouse or in vitro models. The analysis of human melanocytes obtained from their microenvironment after controlled UVR exposure was a complex undertaking that adds significantly to the knowledge base, Dr. Gastman says.
The decision to conduct the study in young women involved practical, enrollment-related considerations as well as issues of epidemiologic and scientific interest, according to Dr. Gastman. Epidemiologic data have suggested that gender and genetics may influence the distribution of melanoma on the skin surface, and histopathologic characteristics of the lesion and age-specific melanoma incidence rates are greater among women than men younger than 40 at diagnosis.
With the improved understanding of how melanocyte miRNAs respond to UVR, researchers are closer to developing a predictor of melanoma risk, which may ultimately lead to targeted prevention and novel early therapies for high-risk populations.
“Having an miRNA signature indicating that a genetic switch has been flipped, which is what we see here, might change the way we evaluate patients,” Dr. Gastman says.
Being able to stratify patients as high-, intermediate- or low-risk is important in order to appropriately utilize medical resources. “It’s not just about treating melanoma, it’s also about preventing melanoma, which is a huge burden in the United States,” he says.
Photo Credit: The National Cancer Institute