A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer

Abstract

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or 'BRCAness', is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies.We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

Keywords: BRCA1; NFkB; microenvironment; predictive biomarker; triple negative breast cancer.

Figure 1

A. NFκB Luciferase Activity Assay of (i) HCC1937 (BRCA1 mutant) cells stably transfected with either empty vector (HCC EV) or full length BRCA1 (HCC BR), (ii) MDA468 (BRCA1 low) cells stably transfected with either empty vector (MDA EV) or full length BRCA1 (MDA BR), or (iii) 184A1 (normal breast) cells stably transfected with empty vector (EV) or BRCA1 shRNA (BRsh2). Cells were transfected with either NFkB reporter construct (NFκB) or the empty vector control (pGL3). Renilla was used to normalise for transfection efficiency. Values are expressed as relative luciferase units (RLU) normalised to pGL3 and Renilla. B. Real time PCR of NFκB target genes in (i) HCC EV and BR, (ii) MDA EV and BR and (iii) 184A1 EV and SH2 cells. β-tubulin was used as a housekeeper. Expression was then normalised to HCC EV, MDA EV and 184A1 SH2 respecively (iv) Real time PCR analysis of CXCL1 mRNA in HCC EV and BR, MDA EV and BR and 184A1 EV and SH2 cells transiently transfected with either scrambled control (scr) or p65 specific (p65si) siRNA for 72hrs. β-tubulin was used as a housekeeper.

Figure 2

A. NFκB Luciferase Activity Assay of i) HCC EV and BR, (ii) MDA EV and BR and (iii) 184A1 EV and SH2 cells pre-treated for one hour with vehicle control (Ctrl), 250nM Gamma Secretase Inhibitor (GSI), 3.3μM ATM inhibitor (ATMi), 10 μM NAC or 10μM Parp inhibitor (Pi). Cells were then transfected with either NFkB reporter construct (NFκB) or the empty vector control (pGL3) with the relevant treatment. Renilla was used to normalise for transfection efficiency. Values are expressed as relative luciferase units (RLU) normalised to pGL3 and Renilla. B. Flow cytometry based analysis of Reactive Oxygen Species (ROS) using Carboxy-H2DCFDA in (i) HCC EV and BR cells treated with or without 10μM N-acetyl-L-cysteine (NAC), (ii) MDA EV and BR and (iii) 184A1 EV and SH2 cells.

Figure 3. Kaplan Meier Curves of (i) the in- house Triple negative dataset and publically available (ii) GSE58812, (iii) GSE21653 and (iii) GSE2034 datasets stratified using the identified BRCA1-/NFκB+ (NFkB on) and non-BRCA1-/NFκB+ (NFkB off) groups

Log-rank p-values are shown.

Figure 4

A. Box and Whisker plots of microarray derived M2/M1 Gene Expression Signature scores in (i) in house BRCA1 mutant dataset, (ii) in house TNBC dataset and publically available (iii) GSE58812, (iv) GSE21653 and (v) GSE2034 datasets. B. Box and Whisker plots of microarray derived CD68/CD8 expression ratios in (i) in house BRCA1 mutant dataset, (ii) in house TNBC dataset and publically available (iii) GSE58812, (iv) GSE21653 and (v) GSE2034 datasets.

Figure 5

A. (i) NFκB Luciferase Activity Assay of 2 “NFκB-on” cell lines (HCC1937, HCC1395) and 2 “NFκB-off” cell lines (MDA231, MDA453). Cells were transfected with either NFkB reporter construct (NFκB) or the empty vector control (pGL3). Renilla was used to normalise for transfection efficiency. Values are expressed as relative luciferase units (RLU) normalised to pGL3 and Renilla. (ii) Flow cytometry based analysis of Reactive Oxygen Species (ROS) using Carboxy-H2DCFDA in the same cell lines as (i). B. Real time PCR analysis of M1 and M2 macrophage markers in THP-1 cells co-cultured with media from “NFkB on” (HCC1937, HCC1395) and “NFkB off” (MDA231, MDA453) cells for 24 hours. β-tubulin was used as a housekeeper. C. Dose response curve of “NFkB on” (HCC1937, HCC1395) and “NFkB off“(MDA231, MDA453) treated with (i) IGF2 inhibitor or (ii) Bicalutamide. Cells were treated with the indicated range of concentration of drug for 72hrs before cell viability was assessed by MTT. Cell survival was normalised to vehicle control (100%).