Downregulation of BRCA1 Protein in BCR-ABL1–positive Cells Depends on Tiar-Mediated Repression of BRCA1 mRNA Translation
Authors: Podszywalow-Bartnicka Paulina , Wolczyk Magdalena , Kusio-Kobialka Monika , Wolanin Kamila , Skowronek Krzysztof , Nieborowska-Skorska Margaret , Dasgupta Yashodhara , Skorski Tomasz , Piwocka Katarzyna 
Published: October 2019

Abstract
Expression of the BCR-ABL1 kinase causes chronic myeloid leukemia in chronic phase (CML). BCR-ABL1 modulates DNA repair mechanisms leading to genomic instability causing resistance to the tyrosine kinase inhibitors and/or disease transition from chronic phase to more malignant stages. BCR-ABL1 –mediated downregulation of breast cancer type 1 susceptibility (BRCA1) protein, a key element in homologous recombination repair (HRR) of DNA double-strand breaks (DSB) was first observed by Deutsch et al, Blood 2003 and later confirmed in our laboratories (Wolanin et al, Mol Cancer Ther 2010; Cramer-Morales et al, Blood 2013). In addition to HRR, BRCA1 protein has been implicated in a broad range of cellular processes, including cell cycle control, cell division and gene transcription (Wu et al, Protein Cell 2010; Moiola et al, Cell Physiol Biochem 2012). We previously reported that downregulation of BRCA1 protein affected the function of spindle assembly checkpoint and postmitotic checkpoint leading to aneuploidy and resistance to spindle poisons in CML cells (Wolanin et al). Recently we also showed that BRCA1-dependent defects in DSB repair in CML cells can sensitize them to synthetic lethality induced by RAD52 inhibitor (Cramer-Morales et al).
The mechanism of BRCA1 downregulation in CML cells remained unknown. We observed that it was not due to decreased BRCA1 mRNA expression or shortened protein half-life (Piwocka et al, InTech Press 2011), and it was partially reversible by inhibition of BCR-ABL1 kinase by imatinib (Deutsch et al; Wolanin et al). In addition, mutations abrogating the expression of BRCA1 protein have not been detected in CML (Friedenson, BMC Cancer 2007).
Here we report that downregulation of BRCA1 protein is paradoxically associated with enhanced half-life and increased levels of BRCA1 mRNA in a BCR-ABL1 transformed cell line and in CML primary cells. Using polysome profiling and luciferase-BRCA1 3’UTR reporter system we demonstrated that downregulation of BRCA1 protein in BCR-ABL1 expressing cells is caused by inhibition of BRCA1 mRNA translation. We show that this is not accompanied by increased protein degradation. Altogether, these results implicated the involvement of mRNA binding proteins such as Hu protein R (HuR) and TIA1 cytotoxic granule-associated RNA-binding protein-like 1 (TIAR) proteins because of their capability to bind the AU-Rich Element (ARE) sites in 3’UTR of human BRCA1 mRNA. It has been reported that in breast cancer HuR can regulate BRCA1 mRNA stability by binding to its 3’UTR (Saunus et al, FEBS Lett 2007; Cancer Res 2008). The other ARE-site binding protein – TIAR – is a well characterized translation repressor protein activated by stress which is known to inhibit protein synthesis transiently by formation of translational silent stress granules (Anderson & Kedersha, J Cell Sci 2002). We have described that BCR-ABL1 expression causes ER stress and thus activation of Unfolded Protein Response (UPR) stress-induced pathway (Kusio-Kobialka et al, Cell Cycle 2012) which stimulates TIAR (Kedersha & Anderson, Biochem Soc Trans 2002).
We observed that BCR-ABL1 kinase promoted cytosolic localization and formation of the TIAR-HuR complex, which facilitated the association with BRCA1 mRNA. We found that HuR protein positively regulated BRCA1 mRNA stability and translation. Conversely TIAR, which was localized predominantly in the cytosolic stress granules in CML cells, abrogated BRCA1 mRNA translation and downregulated BRCA1 protein level. Altogether, we postulate that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein levels in BCR-ABL1 –positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in “BRCAness” CML and BCR-ABL1 –positive ALL cells.
Acknowledgments: We acknowledge Prof. Anne Willis and Dr. Lindsay Wilson for technical help and advice. Prof. Myriam Gorospe for providing plasmid with HuR cDNA. This work was supported by the National Science Centre research grant 2011/01/B/NZ3/02145 to K.P. and the Polish Ministry of Science and Higher Education grants IP2011 043071 and IP2010 032870 to P.P-B. Skorski’s lab was supported by NIH/NCI R01 CA123014.

Disclosures
No relevant conflicts of interest to declare.