Mechanism Based Precision Diagnostics for BRCA1-Associated TNBC

BRCA1 gene mutations result in an increased risk for TNBC, which has a high mortality rate, and currently, there are no targeted therapies nor biomarkers for early detection. Various BRCA1 mutations have been found throughout the BRCA1 coding region; some are germline pathogenic variants, and others are known as variants of uncertain significance (VUS), whose cancer risk is unknown. Our previous work suggested that BRCA1/1a proteins interact with a nuclear chaperone Ubc9 and function as a growth/tumor suppressor in TNBC cells, unlike the pathogenic variants. This work is based on the hypothesis that BRCA1 may contain driver mutations that may cause loss of function and TNBC development or passenger mutations that maintain WT BRCA1 function. We tested this hypothesis by transfecting BRCA1/1a and its various mutant (K109R, C61G, I26A) plasmids into patient derived BRCA1 mutant TNBC cells. We studied the various functions such as in vivo association of BRCA1 with Ubc9 by immunofluorescence analysis, induction of apoptosis using chromatin condensation, growth/tumor suppression activity using colony suppression and scratch migration assay. Our results demonstrate for the first time BRCA1/1a I26A variant to function like WT BRCA1/1a, unlike K109R and C61G mutants. The I26A mutant (which lacks E3 Ubiquitin ligase activity possesses homologous recombination (HR) activity and induces SIRT1 expression) was found to bind Ubc9, inhibit growth/migration of TNBC cells and trigger apoptosis. This is the first study demonstrating the physiological link between Ubc9 binding, HR activity, loss of BARD1- dependent E3 Ubiquitin ligase activity, growth/ tumor suppression, apoptosis, and SIRT1 induction by I26A mutant BRCA1/1a protein in TNBC cells. Clinically, the ability to predict which of these mutations can result in TNBC offers unprecedented prospects for early detection and cancer prevention. This study in the future will accelerate precision oncology, provide guidelines for early detection, prevention, and reduction in cancer health disparities.