Abstract
BackgroundDespite continuous efforts to identify genes that are pivotal regulators of advanced melanoma and closely related to it, to determine which of these genes have to be blocked in their function to keep this highly aggressive disease in check, it is far from clear which molecular pathway(s) and specific genes therein, is the Achilles’ heel of primary and metastatic melanoma. In this report, we present data, which document that the DEAD-box helicase DDX11, which is required for sister chromatid cohesion, is a crucial gatekeeper for melanoma cell survival.MethodsPerforming immunohistochemistry and immunoblot analysis, we determined expression of DDX11 in melanoma tissues and cell lines. Following transfection of melanoma cells with a DDX11-specific siRNA, we conducted a qPCR analysis to determine downregulation of DDX11 in the transfected melanoma cells. In subsequent studies, which focused upon an analysis of fluorescently labeled as well as Giesma-stained chromosome spreads, a proliferation analysis and apoptosis assays, we determined the impact of suppressing DDX11 expression on melanoma cells representing advanced melanoma.ResultThe findings of the study presented herein document that DDX11 is upregulated with progression from noninvasive to invasive melanoma, and that it is expressed at high levels in advanced melanoma. Furthermore, and equally important, we demonstrate that blocking the expression of DDX11 leads not only to inhibition of melanoma cell proliferation and severe defects in chromosome segregation, but also drives melanoma cells rapidly into massive apoptosis.ConclusionTo date, little is known as to whether helicases play a role in melanoma development and specifically, in the progression from early to advanced melanoma. In this report, we show that the helicase DDX11 is expressed at high levels in primary and metastatic melanoma, and that interfering with its expression leads to severe chromosome segregation defects, telomere shortening, and massive melanoma cell apoptosis. These findings suggest that DDX11 could be an important candidate for molecular targeted therapy for advanced melanoma.
Highlights
Despite continuous efforts to identify genes that are pivotal regulators of advanced melanoma and closely related to it, to determine which of these genes have to be blocked in their function to keep this highly aggressive disease in check, it is far from clear which molecular pathway(s) and specific genes therein, is the Achilles’ heel of primary and metastatic melanoma
Status and pattern of DDX11 expression in normal skin, nevus and melanoma tissues, and melanoma cell lines To identify genes that are upregulated with progression from noninvasive melanoma in situ (MIS) to radial growth phase (RGP) melanoma, which is the first stage of invasive melanoma, we recently subjected RNAs isolated from archived, formalin-fixed paraffin-embedded (FFPE) tissue samples representing these two stages of early melanoma development to whole-genome cDNA-mediated Annealing (DASL) HT BeadChip arrays
DDX11 expression was not detected in epidermal melanocytes of normal skin (NS) (Figure 1A) - a finding that is in agreement with the data of a previous study, which showed that DDX11 is expressed at extremely low levels in normal human skin [4]
Summary
Despite continuous efforts to identify genes that are pivotal regulators of advanced melanoma and closely related to it, to determine which of these genes have to be blocked in their function to keep this highly aggressive disease in check, it is far from clear which molecular pathway(s) and specific genes therein, is the Achilles’ heel of primary and metastatic melanoma. In line with our long-term effort to identify genes that are upregulated with progression from early to advanced melanoma, we recently profiled archived, formalin-fixed paraffin-embedded tissue samples representing early as well as advanced melanoma on DASL BeadChip arrays. The data from this whole-genome expression analysis revealed that one of the identified genes, upregulated to substantial levels with progression from noninvasive melanoma in situ > to invasive radial growth phase melanoma > primary melanoma > metastatic melanoma is the gene DDX11, which has never before been associated with melanoma. Biallelic mutations in DDX11 have been identified as the cause of the Warsaw breakage syndrome cohesinopathy, which among other clinical manifestations is associated with abnormal skin pigmentation [8]
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