Abstract 3364: RAI2 maintains genome stability of breast cancer cells

Abstract

Abstract Genome instability is considered as an enabling hallmark of cancer formation and progression as well as a source of intratumoral heterogeneity. Retinoid acid-induced protein 2 (RAI2) was initially identified as a new metastasis-associated protein especially related with the presence of disseminated tumor cells in the bone marrow of breast cancer patients. In primary breast tumors low RAI2 gene expression correlates with mutant TP53 status and moreover, RAI2 depleted luminal breast cancer cell lines show decreased expression of key regulators of G2/M transition. We hypothesize that RAI2 has an influence on mitotic fidelity and is involved in maintaining genomic stability in breast cancer cell lines. To analyze consequences of RAI2 depletion on mitotic fidelity in luminal breast cancer cells, we followed mitotic cells by live cell imaging and analyzed chromosomal segregation by immune fluorescence staining and karyotyped RAI2 depleted cells. For analysis of cell cycle dependent RAI2 protein expression we executed Western blot analysis after cell synchronization and treatment with genotoxic agents. Localization of ectopically expressed HA-tagged RAI2 protein was analyzed in breast cancer cells treated with genotoxic agents using immune fluorescence staining. Finally, we performed in silico validation in different breast cancer data sets. Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability, associated with loss of acentric chromosomal fragments during ana- and metaphase as well as a prolonged mitosis. Furthermore, karyotyping revealed a significant reduction of chromosome number from 78 to 73 in RAI2 depleted cells. In synchronized parental breast cancer cells we could show an increased RAI2 expression during S/G2 phase compared to unsynchronized cells. Interestingly, genotoxic treatment resulted in a significant upregulation of RAI2 protein, which can also be seen as an increased formation of nuclear speckles (p<0.05). Finally, in silico analysis of breast cancer data sets revealed a significant correlation between low RAI2 expression and an expression signature of chromosomal instability (p>0,001). Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability. Our results show that RAI2 depletion causes pre-mitotic DNA lesions. Moreover, in breast cancer patients a low RAI2 expression was significantly associated with chromosomal instability. We conclude that the RAI2 protein preserves mitotic fidelity and chromosomal integrity in luminal breast cancer cell lines by affecting either the DNA damage response or DNA replication during S- and/or G2-phase. Citation Format: Lena Boettcher, Bernd Zobiak, Kerstin Borgmann, Alexandra Zielinski, Antonio Virgilio Failla, Klaus Pantel, Harriet Wikman, Stefan Werner. RAI2 maintains genome stability of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3364.