Revisiting Non-BRCA1/2 Familial Whole Exome Sequencing Datasets Implicates NCK1 as a Cancer Gene

Abstract

Through linkage and candidate gene screening, many breast cancer (BC) predisposition genes have been identified in the past 20 years. However, the majority of genetic risks that contribute to familial BC remains undetermined. In this study, we revisited whole exome sequencing datasets from non-BRCA1/2 familial BC patients, to search for novel BC predisposition genes. Based on the infinite mutation model, we supposed that rare non-silent variants that cooccurred between familial and TCGA-germline datasets, might play a predisposition contributing role. In our analysis, we not only identified novel potential pathogenic variants from known cancer predisposition genes, such as MRE11, CTR9 but also identified novel candidate predisposition genes, such as NCK1. According to the TCGA mRNA expression dataset of BC, NCK1 was significantly upregulated in basal-like subtypes and downregulated in luminal subtypes. In vitro, NCK1 mutants (D73H and R42Q) transfected MCF7 cell lines, which attributed to the luminal subtype, were much more viable and invasive than the wild type. On the other side, our results also showed that overall survival and disease-free survival of patients with NCK1 variations might be dependent on the genomic context. In conclusion, genetic heterogeneity exists among non-BRCA1/2 BC pedigrees and NCK1 could be a novel BC predisposition gene.

Keywords: NCK1; breast cancer; invasion; non-BRCA1/2; predisposition gene.

FIGURE 1

NCK1 mutation diagram and potential functional effect. (A) Mutation diagram of NCK1 collected in cBioportal (Pan-Cancer). (B) Mutation diagram of germline mutations in NCK1, including all TCGA-germline variants and NCK1 D73H, identified in familial breast cancer pedigree (F2887). (C) Multiple sequence alignment of sequence flanking NCK1 D73 residue. (D) Distribution of substitution score (ΔS) of NCK1 based on Position Specific Score Matrix. (E and F) The cell viabilities in all groups of mutant over-expression assay about R42Q (E) and D73H (F) at different time points (0, 1, 2, 3, 4, 5, and 6 days). Data were expressed as mean ± standard deviation (SD) of experiments with triplicates. Asterisks indicate significant increasing of cell viability in mutant (R42Q and D73H) transfected MCF7 cells compared with wild type transfected MCF7 cells (Student’s t-test; P < 0.01). Model: random mutations generated by in silico, non-TCGA germline: variants collected in ExAC non-TCGA dataset; TCGA-germline: variants collected in ExAC, but not in the ExAC non-TCGA dataset; Somatic: somatic variants collected in cBioportal.

FIGURE 2

Expression spectrum of NCK1 in 99 tumor-normal paired samples across different stage and subtypes. (A) All; (B) Stage I; (C) Stage II; (D) Stage III and IV; (E) basal-like; (F) Her2; (G) LumA (LuminalA); and (H) LumB (LuminalB).

FIGURE 3

Roles of NCK1 in tumor progression might be context dependent. (A) Images of MCF7 cells migrated from transwell membrane (B) Cell count and quantitative analysis of the migrated MCF7 cells. Patients with both NCK1 aberrations and TP53 mutations showed a much poorer overall survival (C) and disease-free survival (D). Selected patients: patients with both NCK1 aberrations and TP53 mutation. Unselected patients: patients with only NCK1 aberrations. Scale bar: 200 μm. Data are depicted as mean ± standard deviation (SD).