A prognostic signature of G₂ checkpoint function in melanoma cell lines

Abstract

As DNA damage checkpoints are barriers to carcinogenesis, G(2) checkpoint function was quantified to test for override of this checkpoint during melanomagenesis. Primary melanocytes displayed an effective G(2) checkpoint response to ionizing radiation (IR)-induced DNA damage. Thirty-seven percent of melanoma cell lines displayed a significant defect in G(2) checkpoint function. Checkpoint function was melanoma subtype-specific with "epithelial-like" melanoma lines, with wild type NRAS and BRAF displaying an effective checkpoint, while lines with mutant NRAS and BRAF displayed defective checkpoint function. Expression of oncogenic B-Raf in a checkpoint-effective melanoma attenuated G(2) checkpoint function significantly but modestly. Other alterations must be needed to produce the severe attenuation of G(2) checkpoint function seen in some BRAF-mutant melanoma lines. Quantitative trait analysis tools identified mRNA species whose expression was correlated with G(2) checkpoint function in the melanoma lines. A 165 gene signature was identified with a high correlation with checkpoint function (p < 0.004) and low false discovery rate (≤ 0.077). The G(2) checkpoint gene signature predicted G(2) checkpoint function with 77-94% accuracy. The signature was enriched in lysosomal genes and contained numerous genes that are associated with regulation of chromatin structure and cell cycle progression. The core machinery of the cell cycle was not altered in checkpoint-defective lines but rather numerous mediators of core machinery function were. When applied to an independent series of primary melanomas, the predictive G(2) checkpoint signature was prognostic of distant metastasis-free survival. These results emphasize the value of expression profiling of primary melanomas for understanding melanoma biology and disease prognosis.

Mechanisms of chromosomal instability in melanoma

“…C . A 593 gene QTA + Bayes signature that was correlated with G2 checkpoint function [Omolo et al, ] was marginally not prognostic ( P = 0.055).…”

“…A 496 gene QTA1 Bayes signature that was correlated with G1 checkpoint function in melanoma cell lines [Carson et al, 2012] was prognostic of metastasis-free survival (P 5 0.018). C. A 593 gene QTA1 Bayes signature that was correlated with G2 checkpoint function [Omolo et al, 2013] was marginally not prognostic (P 5 0.055).…”

“…A p53‐independent G2 checkpoint was dysfunctional in one‐third of melanoma lines [Omolo et al, ]. Attenuation of signaling in this pathway of DDR was attributed in part to mutations in BRAF and NRAS , and in part to a complex signature of gene expression that repressed many regulators of mitosis such as CKS1B and WDHD1 (also known as AND1 ) [Omolo et al, ]. Melanoma cell lines grow with high levels of DNA damage and expression of DDR biomarkers because p53‐dependent and p53‐independent DNA damage checkpoint functions are attenuated.…”

“…CIN was not correlated with DDR. Melanoma cell lines were scored for expression of gH2AX and P-CHEK2 as biomarkers of DNA damage (double-strand breaks and replication stress) [Nikolaishvilli-Feinberg et al, 2014], for G1 and G2 checkpoint function [Carson et al, 2012;Omolo et al, 2013], NER [Gaddameedhi et al, 2010] and for sensitivity to inactivation of colony formation by UVC and IR [Sambade et al, 2011]. A. Maximal values for each parameter were expressed at 100% and all other values expressed as a percentage of the maximum.…”

“…The melanoma cell lines in the CIN dataset have been examined for a variety of features of DDR including IR [Sambade et al, 2011] and UV radiosensitivity, NER [Gaddameedhi et al 2010], and DNA damage G1 [Carson et al 2012], and G2 [Omolo et al, 2013] checkpoint functions. Immunofluorescence microscopy also quantified the basal levels of expression of two markers of DDR, gH2AX and P-CHEK2 [Nikolaishvilli-Feinberg et al, 2014].…”