Identification of a Locus Near ULK1 Associated With Progression-Free Survival in Ovarian Cancer

Abstract

Background: Many loci have been found to be associated with risk of epithelial ovarian cancer (EOC). However, although there is considerable variation in progression-free survival (PFS), no loci have been found to be associated with outcome at genome-wide levels of significance.

Methods: We carried out a genome-wide association study (GWAS) of PFS in 2,352 women with EOC who had undergone cytoreductive surgery and standard carboplatin/paclitaxel chemotherapy.

Results: We found seven SNPs at 12q24.33 associated with PFS (P < 5 × 10^-8), the top SNP being rs10794418 (HR = 1.24; 95% CI, 1.15-1.34; P = 1.47 × 10^-8). High expression of a nearby gene, ULK1, is associated with shorter PFS in EOC, and with poor prognosis in other cancers. SNP rs10794418 is also associated with expression of ULK1 in ovarian tumors, with the allele associated with shorter PFS being associated with higher expression, and chromatin interactions were detected between the ULK1 promoter and associated SNPs in serous and endometrioid EOC cell lines. ULK1 knockout ovarian cancer cell lines showed significantly increased sensitivity to carboplatin in vitro.

Conclusions: The locus at 12q24.33 represents one of the first genome-wide significant loci for survival for any cancer. ULK1 is a plausible candidate for the target of this association.

Impact: This finding provides insight into genetic markers associated with EOC outcome and potential treatment options.See related commentary by Peres and Monteiro, p. 1604.

Figure 1.

Manhattan Plot of results of the meta-analysis of association of PFS with patients (all histologies) who had standard chemotherapy. The top red line indicates the genome-wide significance threshold; the lower line indicates a suggestive significance level.

Figure 2.

Association of rs10794418 with PFS in patients who had standard chemotherapy, stratified by histotypes in the OncoArray results. Hazard Ratios, along with upper and lower 95% confidence intervals are plotted. Sample sizes are indicated in brackets.

Figure 3.

Association of expression of ULK1 and PFS using Kaplan-Meier plotter (http://kmplot.com/), an online tool to assess the expression levels of microarray-quantified genes using data from publically available datasets²². (A) Women diagnosed with ovarian cancer of any histotype (n = 1,435) treated with any chemotherapy. (B) Women diagnosed with serous ovarian cancer (n = 1,104) treated with any chemotherapy. (C) Women diagnosed with serous ovarian cancer (n = 616) treated with taxol and platin. Cases were divided into high and low expression using auto best cut-off and censored at 10 years.

Figure 4.

PFS-associated variants lie in an enhancer of the ULK1 gene (A) Chromosome conformation capture (3C) interactions between the ULK1 promoter and regions containing PFS-associated variants in endometrioid (A2780, IGROV1 and TOV112Dluc) and serous (OVCAR8 and CaOV3) cells. 3C interactions were quantified by ddPCR on three independent 3C libraries using primers designed against the HindIII restriction fragments across the 12q24.33 locus. (B) Luciferase reporter assay showing the protective and risk associated haplotypes containing rs12301971 (T allele associated with risk), rs112786120 (A allele associated with risk) and rs11246872 (A allele associated with risk), cloned upstream of the ULK1 promoter-driven luciferase reporter constructs. Cells were transiently transfected and assayed for luciferase activity 24 hours later. Error bars denote 95% confidence intervals from three independent experiments performed in triplicate. P-values were determined by two-way ANOVA followed by Dunnett’s multiple comparisons test (** p < 0.01, *** p < 0.001) on log transformed data; for ease of interpretation back transformed data have been graphed.

Figure 5.

Knockdown of ULK1 showing increased sensitivity to carboplatin (A) Relative ULK1 mRNA level in TOV112D (left panel) and OVCAR (right panel) NTC and ULK1 knockout lines. (B) ULK1 protein level in TOV112D (left panel) and OVCAR (right panel) NTC and ULK1 knockout lines. (C) Relative IC₅₀ of Carboplatin (top panel) or Paclitaxel (bottom panel) in TOV112D (left panel) or OVCAR (right panel) NTC and ULK1 knockout lines.