Abstract P5-03-01: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. TNBC initially respond well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Paclitaxel causes defects in mitotic spindle formation and centrosome function, as well as improper chromosome segregation leading to cell death. While being some of the most effective drugs for this disease, taxanes are associated with high toxicity. Thus discovering new therapeutic targets that are selective for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and associated with poor survival. We further found that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across nine breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, enforced overexpression of LIN9 increases multi- and micronucleation, indicators of chromosomal instability. Conversely, LIN9 silencing also results in multi- and micronucleation, and supernumerary centrosomes. Most importantly, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and MDA-MB-468) resistance. We have previously reported that treating TNBC cells with Bromodomain and ExtraTerminal protein inhibitors (BETi) reduces LIN9 expression, thus we determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, and paclitaxel caused a greater induction of apoptosis compared to either drug alone. Dual treatment also resulted in a potentiation of abnormal centrosomes, multinucleation, and micronucleation compared to that caused by either BETi or JQ1 alone. To identify the mechanism(s) by which genetic or therapeutic suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs in MDA-MB-231 and HCC70 cell lines. We further narrowed the list of candidate LIN9 targets by identifying genes that were bound by LIN9 in a published ChIP-Seq dataset from HeLa cells whose expression is also correlated with LIN9 expression and associated with reduced breast cancer patient survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis as a potential mediator of the effects of LIN9 suppression. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor survival. In addition, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by contributing to centrosome dysfunction through upregulation of NEK2. They also indicate that targeting LIN9 expression in addition to paclitaxel treatment may be a viable therapeutic approach for TNBC patients. Citation Format: Shively MS, Gayle SS, Sahni JM, Keri RA. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-01.