Introduction

Breast cancer 2, early onset (BRCA2) is involved in DNA-damage repair via homologous recombination (HR). Defective HR leads to severe genomic instability and homozygous deletion of BRCA2 is therefore deleterious for survival of normal cells. In apparent contrast, inherited heterozygous mutations in BRCA2 increase the risk to develop cancer, which goes along with somatic loss of the wild type allele. So paradoxically, BRCA2 loss is lethal in normal cells, but can be tolerated in cancer cells. It is currently unclear how tumor cells can survive without BRCA2. To address this question, we performed a genome-wide functional genetic screen and identify Tumor Necrosis Factor-alpha (TNFα) signaling, via TNF receptor 1 (TNF-R1) and Src-associated-substrate-during-mitosis-of-68kDa (Sam68), as an important determinant of cell death induction upon BRCA2 inactivation.

Material & Methods

A genetic screen was performed in human near-haploid KMB-7 cells. Two monoclonal KMB-7 lines were engineered to express independent doxycycline-inducible BRCA2 short hairpin RNAs (shRNAs). Cells were randomly mutagenized using retrovirus carrying a strong splice-acceptor, resulting in gene knock-outs. Deep sequencing of surviving colonies revealed gene mutations that prevented shBRCA2-mediated cell death. Validation of TNF-R1 and Sam68 was done by shRNAs in multiple breast- and leukemic cell lines. Cell viability was determined by MTT and clonogenic assays. Competition assays with vectors expressing shRNA including IRES-driven mCherry were analyzed with flow cytometry. Biochemical analyses were assessed by western blotting and ELISA. Immunoprecipitation with FLAG-tagged TNFα was performed to study TNF-R1 complex formation.

Results

From our genetic screens, loss-of-function mutations involved in TNFα signaling were found to significantly rescue cell death in BRCA2-depleted cells. To validate these findings, BRCA2 was stably depleted in a panel of cancer cell lines, which decreased viability. Subsequent co-depletion of TNF-R1 or Sam68 rescued survival. Notably, BRCA2 depletion resulted in the production of TNFα. In line with this finding, the majority of tested cancer cell lines were found to be sensitive to recombinant TNFα upon BRCA2 depletion. This phenomenon was not restricted to BRCA2 inactivation, since depletion of related DNA repair genes (BRCA1, FANCD2) or hydroxyurea-induced replication stress also sensitized cells to recombinant TNFα. Mechanistically, BRCA2 depletion was induced to observe differential complex formation of TNF-R1 with downstream effector proteins upon TNFα activation.

Conclusion

Our data reveal that down-regulation of TNF-R1 or its downstream signaling component Sam68 rescued cytotoxicity upon BRCA2 inactivation. These findings and mechanistic follow-up experiments describe a novel mechanism by which autocrine TNFα signaling, induced by loss of BRCA2, limits tumor cell viability.

Citation Format: Anne M. Heijink, Francien G. Talens, Anouk Baars, Marcel A. van Vugt. Tumor necrosis factor-alpha signaling determines cytotoxicity induced by BRCA2 deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2017-2488