Synthetic lethality between CCNE1 amplification and loss of BRCA1

Abstract

High-grade serous ovarian cancers (HGSCs) are characterized by a high frequency of TP53 mutations, BRCA1/2 inactivation, homologous recombination dysfunction, and widespread copy number changes. Cyclin E1 (CCNE1) gene amplification has been reported to occur independently of BRCA1/2 mutation, and it is associated with primary treatment failure and reduced patient survival. Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway. Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression. These events may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations. Using data from a genome-wide shRNA synthetic lethal screen, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplification. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.

Keywords: CDK2; DNA repair; RNAi; cell cycle; pan-cancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

(A) Pan-cancer copy number analysis of 6,547 tumor samples comprising 22 cancer types from TCGA. Frequency of high-level amplification of peak regions of copy number change incorporating CCNE1 is shown for cancer types with amplification. Data were obtained from the TCGA Copy Number Portal using all available data as of February of 2013. Name or total number of genes including CCNE1 within significant peak regions of amplification is indicated. (B) CCNE1 copy number assessed by qPCR in primary tumor samples from the Australian Ovarian Cancer Study (n = 193) stratified by WT, germ-line (GL), or somatic (SOM) BRCA1/2 mutation or methylation (METH) status. Bars indicate mean and SD. t test. *P value < 0.05; **P value < 0.01.

Fig. 2.

(A) Venn diagram of candidate genes essential for the survival of CCNE1-amplified (n = 474) and overexpressing (n = 486) cell lines identified in the shRNA synthetic lethal screen. Candidates were further filtered to include genes that were present in commonly amplified regions in ovarian tumors, coexpressed with CCNE1, present in significantly enriched gene pathways, or among top-ranking shRNA hits. (B) Top 25 ranking genes annotated by inclusion criteria. Statistical significance of ranking by second-best scoring shRNA or a composite score of all shRNAs (KS statistic) given (Materials and Methods). *Go term processes: cell cycle, DNA repair, or response to DNA damage.

Fig. 3.

(A) Cells were transfected with a boutique siRNA library against 142 candidate genes, and the effect on cell viability was measured 5 d after transfection. Significance (t test P value) of hits in the OVCAR-3 (CCNE1-amplified) cell line plotted against the viability ratio of OVCAR-3 to SK-OV-3 (unamplified) highlights significant hits specific to OVCAR-3. Average data from duplicate wells across three independent experiments are shown (n = 3). The vertical dotted line is at P value = 0.05. (B) Clonogenic survival after siRNA transfection in SK-OV-3 (unamplified) and OVCAR-3 (CCNE1-amplified) ovarian cell lines. Average percentage of discrete colonies formed after 7 d relative to no siRNA controls is shown (n = 3 independent experiments performed in triplicate). Statistical significance (t test) was calculated by comparison with nonsilencing (NS) siRNA in the same cell line. *P value < 0.05; ***P value < 0.001. Error bars indicate SEM.

Fig. 4.

Ovarian tumor cell line sensitivity to bortezomib ranked by average 72-h cytotoxicity assay IC50 value (n = 3 independent experiments performed in triplicate). Error bars indicate SEM. CCNE1 copy number status was determined by qPCR, where copy number gain and amplification are defined as a log2 ratio to normal > 0.5 and > 2.0, respectively. CCNE1 gene expression of each cell line above (high) or below (low) the median value of 10 parental lines is indicated. The OVCAR-3-R1 and -RD1 sublines were derived from OVCAR-3 and are resistant to CDK2 inhibitors PHA-533533 and dinaciclib, respectively (7).