Two papers authored by researchers at the University of California, San Francisco described the genetic changes that turn harmless moles into malignant melanomas and the experiment they devised to recreate the step-by-step evolution of normal skin cells into cancer cells [1], [2].

Summary ([1]) We elucidated genomic and transcriptomic changes that accompany the evolution of melanoma from pre-malignant lesions by sequencing DNA and RNA from primary melanomas and their adjacent precursors, as well as matched primary tumors and regional metastases. In total, we analyzed 230 histopathologically distinct areas of melanocytic neoplasia from 82 patients. Somatic alterations sequentially induced mitogen-activated protein kinase (MAPK) pathway activation, upregulation of telomerase, modulation of the chromatin landscape, G1/S checkpoint override, ramp-up of MAPK signaling, disruption of the p53 pathway, and activation of the PI3K pathway; no mutations were specifically associated with metastatic progression, as these pathways were perturbed during the evolution of primary melanomas. UV radiation-induced point mutations steadily increased until melanoma invasion, at which point copy-number alterations also became prevalent. Summary ([2]) Loss of the CDKN2A tumor suppressor is associated with melanoma metastasis, but the mechanisms connecting the phenomena are unknown. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we discovered that a lineage-restricted transcription factor, BRN2, is downstream of CDKN2A and directly regulated by E2F1. In a cohort of melanocytic tumors that capture distinct progression stages, we observed that CDKN2A loss coincides with both the onset of invasive behavior and increased BRN2 expression. Loss of the CDKN2A protein product p16INK4A permitted metastatic dissemination of human melanoma lines in mice, a phenotype rescued by inhibition of BRN2. These results demonstrate a mechanism by which CDKN2A suppresses the initiation of melanoma invasion through inhibition of BRN2.

Melanomas

Melanomas are a type of skin cancer that develops from melanocytes, pigment cells contained in the skin. Melanomas develop primarily as a consequence of ultraviolet light exposure, which damages cellular DNA, inducing abnormal skin growth. Melanomas can develop from existing moles, although most moles remain benign; those with irregular borders, or that exhibit color changes or increase in size, are more at risk of malignancy, i.e. spreading beyond their initial site.

Thicker moles are more likely than thin ones to have already metastasized, and their surgical removal might not be enough to fully eliminate the cancer. Measuring the thickness of a melanoma is a a way to assess its stage of progression, but it’s not a completely accurate method.

Even though melanoma is generally treated successfully if caught early, it remains the most dangerous kind of skin cancer, resulting in nearly 60,000 deaths in 2015 alone.







The first paper

The first study looked at samples from malignant melanoma patients; these samples included tissues from malignancies and tissues from the benign moles that became those malignancies. Samples of primary and metastatic tumors—that is, of both original tumors and their spawned tumors—were also available and examined. This allowed the scientists to investigate the differences between different states of melanoma progression within the same patients.

For the first time ever, researchers were thus able to identify the mutations that occur in the genome of cells as they change into malignant melanoma cells. Their findings suggest that the mechanisms controlling tumor suppression, cell growth, and DNA regulation are slowly changed by several independent mutations occurring throughout the progression of the disease; eventually, the cancer defense mechanisms of the cells become sufficiently impaired to allow malignancies to emerge. This contradicts the previous belief that molecular pathway are turned off somewhat abruptly, like the flip of a switch.

The second paper

Using the CRISPR-Cas9 gene editing technique, the researchers further proceeded with an experiment to reproduce the same mutations observed in the previous study in healthy human skin cells in the laboratory. This step-by-step execution allowed them to more accurately study the specific effects of each individual mutation involved—which is much more difficult to do in a sample of metastatic melanoma tissue, as many mutations have already occurred, making for a rather fuzzy picture.







As described in the abstract of the paper, this method has allowed the scientists to discover that, as mutations disrupt the activity of the CDKN2A tumor suppressor gene, both the onset of invasive behavior and an increase in the expression of the BRN2 transcription factor are observed; the latter is due to the loss of the p16INK4A protein encoded by CDKN2A. As the researchers discovered, the increased expression of BRN2 drives melanoma invasion.

Conclusion

Knowing which mutations contribute to inducing malignancy may provide us with useful biomarkers for more precise diagnosis as well as new therapeutic avenues. Furthermore, this study reminds us that CRISPR has great potential as a research tool besides its clinical applications.

Literature

[1] Shain, A. H., Joseph, N. M., Yu, R., Benhamida J., Liu, S., Prow, T., Ruben, B., North, J., Pincus, L., Yeh, I., Judson, R., Bastian, B. C. (2018). Genomic and Transcriptomic Analysis Reveals Incremental Disruption of Key Signaling Pathways during Melanoma Evolution. Cancer Cell, 34, p45–55.e4.







[2] Zeng, H., Jorapur, A., Shain, A. H., Lang, U. E., Torres, R., Zhang, Y., … & Bastian, I. N. (2018). Bi-allelic loss of CDKN2A initiates melanoma invasion and metastasis via E2F1-BRN2 axis. Cancer Cell, 34, p56-68.e9.