Minimal blood flow to the basement membrane and a lack of epidermal oxygen diffusion results in a hypoxic melanocyte microenvironment. This setting causes unique gene regulation patterns, including Hypoxia-Inducible Factor 1α over-expression. Based on this knowledge, we assessed hypoxia-induced transcriptional changes specific to melanocytes and melanoma. We determined that several members of DNA repair pathways were down-regulated by hypoxia in melanoma, including MutS Homologue 6 (MSH6).
We examined MSH6 mRNA and protein expression across a melanoma progression panel. While hypoxia caused strong MSH6 up-regulation in melanocytes, it also caused a significant transcriptional down-regulation in early melanomas; late vertical and metastatic phases were unaffected. There was no evidence of hypoxia-induced genomic instability in either cell subset. These results suggest alternative mechanisms of hypoxic MSH6 regulation in melanocytes, and compensation by other genes for MSH6 down-regulation.
We also observed inconsistent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels under varying oxygen conditions—hypoxic mRNA up-regulation was seen in all melanoma cell lines tested. While these alterations were uncorrelated with changes in translation or proliferation, this was the first known identification that hypoxia affects GAPDH mRNA levels in melanoma.
Secondly, we evaluated the role of microenvironments on cellular response to irradiation and inhibition of melanoma-related pathways. Clonigenicity was assessed in melanoma cells cultured alone or with endothelial cells to determine the effects of hypoxia, irradiation, and drug treatments. While rapamycin decreased clonigenicity of melanoma and endothelial cells equally, sorafenib increased clonigenicity of melanoma cells independently of their BRAF status despite decreasing cellular proliferation by 50-60%. This may help explain sorafenib's failure in melanoma treatment. Additionally, tumor microenvironments had significant effects on clonigenic responses of melanoma cells in various settings.
We conclude that hypoxic microenvironments cause global gene expression changes in melanoma, including genes responsible for DNA damage repair. Despite this fact, it appears that other mechanisms are responsible for maintaining DNA fidelity. We also conclude that proliferation and clonigenicity are independent events, and that tumor microenvironments, including hypoxia and cellular cross-talk, dictate chemotherapy and radiotherapy responses. We expect these results will lead to greater understanding of factors that result in clinical failures of melanoma treatments.
|School||THE JOHNS HOPKINS UNIVERSITY|
|Subjects||Molecular biology; Pharmacology; Oncology|
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