Mitotic catastrophe is a tumor suppressive mechanism that arrests cells experiencing a defective M phase, ultimately leading to apoptosis or other forms of cell death. DNA damage and aberrant chromosome segregation are two of the major triggers of mitotic catastrophe, and this latter event is frequently caused by an improperly formed mitotic spindle. Microtubule targeting agents (vinca alkaloids and taxanes), inhibitors of mitotic checkpoint kinases, and disruption of the multi-protein anaphase promoting complex/cyclosome (APC/C) or the spindle assembly checkpoint (SAC) cause mitotic catastrophe. However, there are significant liabilities from each of these approaches that have prevented clinical success. In contrast, targeting nuclear receptor proteins has been a very successful therapeutic strategy. Estrogen-related receptor beta (ERRbeta) is an orphan member of the nuclear receptor superfamily. Though it lacks a natural ligand, synthetic ligands or endogenous partner proteins can modulate endogenous ERRbeta activity. ERRbeta function may also depend upon which of the three alternatively spliced forms (beta2, delta10, or the short form) is expressed. The short form of ERRbeta has well-established tumor suppressor activities in prostate cancer, but the function of the other ERRbeta splice variants in this and other cancers is unknown. We evaluated a synthetic small molecule activator of ERRbeta (DY131) and identified several cancer cell lines that undergo a robust G2/M arrest, a previously unreported finding. These G2/M arresting cancer cells display classic markers of apoptotic cell death as well as perturbed nuclear/chromatin structure, which are highly suggestive of mitotic catastrophe. Employing splice variant-selective shRNAs in the T98G glioblastoma cell line, we discovered that silencing of ERRbeta2, but not the short form, completely abrogates DY131-induced G2/M arrest and cell death. ERRbeta2, which has an extended C-terminus with 68 additional amino acids, essentially lacks transcriptional activity when compared to the short form. We considered that transcription-independent functions of ERRbeta2, perhaps driven by protein-protein interactions at its unique C-terminus, might be responsible for the observed G2/M arrest and mitotic catastrophe phenotype. Sequence analysis identified a proline-rich region that closely matches the consensus binding site(s) for the SH3 domains of the non-receptor tyrosine kinase c-Src and the actin binding protein cortactin, and preliminary co-immunoprecipitation experiments demonstrate an ERRbeta2/cortactin interaction in unstimulated and DY131-treated cells. Importantly, tyrosine phosphorylated cortactin localizes to the spindle poles during mitosis, and its presence there is required for proper centrosome separation. We therefore hypothesize that activation of the ERRbeta2 splice variant specifically induces G2/M cell cycle arrest and mitotic catastrophe by disrupting normal chromosome segregation, and that it does so at least in part through its interactions with centrosome-associated cortactin. We will test this hypothesis in two Specific Aims.
Specific Aim 1 : To determine whether ERRbeta2 localizes to centrosomes during mitosis under unstimulated and DY131-treated conditions.
Specific Aim 2 : To test whether ERRbeta2/cortactin interactions are required for DY131-induced G2/M arrest leading to mitotic catastrophe. Successful completion of these studies will illuminate the novel cellular and molecular functions of the ERRbeta2 splice variant. In the long term, selective activation of ERRbeta2 may ultimately prove to be a rational augmentation to enhance anti-mitotic therapy in multiple malignancies.
The studies proposed in this application are highly relevant to public health. Mitotic catastrophe is a unique tumor suppressive mechanism that arrests cancer cells experiencing a defective M phase, ultimately leading to apoptosis or other forms of cell death. However, therapeutic approaches designed or discovered to enhance this process have significant liabilities that have prevented widespread clinical success. Our work will provide proof-of-concept for a new strategy - selective activation of the orphan nuclear receptor ERRbeta2 splice variant - to enhance anti-mitotic therapy in multiple malignancies.
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|Heckler, Mary M; Zeleke, Tizita Zewde; Divekar, Shailaja D et al. (2016) Antimitotic activity of DY131 and the estrogen-related receptor beta 2 (ERRÎ²2) splice variant in breast cancer. Oncotarget :|
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