Abstract 1556: CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells

Abstract

Abstract Background: Paclitaxel (PTX) is one of the key drugs in clinical practice for esophageal squamous cell carcinoma (ESCC), however, therapeutic effects of PTX alone are not sufficient. PTX causes activation of spindle assembly checkpoint resulting in mitotic arrest of cancer cells. Recently, the antitumor effect of PTX has been shown to depend on aberrant chromosome segregation and abnormal spindle formation. Whereas, recent reports showed that cyclin dependent kinase inhibitor 3 (CDKN3) was often overexpressed in various human cancer tissues which was attributed to the higher mitotic activity in cancer cells, and loss of CDKN3 induced abnormal mitosis and centrosome overduplication. Therefore, we hypothesized that the combination of PTX and CDKN3 inhibition may promote mitotic errors in cancer cells. The aim of this study is to examine the combined effect of PTX and CDKN3 inhibition on ESCC. Methods: The expression levels of CDKN3 were compared between ESCC cell lines (TE1, TE5, TE6, TE8, TE10, and TE11) and normal esophageal epithelial cell line (EPC2-hTERT). CDKN3 knockdown ESCC cells using shRNA were created. Cell viability assay (WST-1 and clonogenic assay), immunofluorescence microscopy for measuring the number of centrosomes or spindle microtubules, and time-lapse images for assessing mitosis and cell fate were performed. Results: The expression levels of CDKN3 in ESCC cells were high compared to normal esophageal epithelial cells. Here, TE6 and TE10 ESCC cells were selected for further experiments based on higher CDKN3 expression. WST-1 assays showed an increased sensitivity to PTX after CDKN3 knockdown. Clonogenic assay showed reduced clonogenicity with CDKN3 knockdown, which was further reduced when exposed to 1 nM PTX (no interaction by two-way ANOVA analysis). Quantification of centrosomes revealed an increased frequency of centrosome overduplication in cells with CDKN3 knockdown, and it was further increased when treated with 1 nM PTX. Quantification of microtubules revealed CDKN3 knockdown cells with 1nM PTX had irregular multipolar spindle formation in almost half of the cells in mitosis. In time-lapse images, mitotic errors were more frequent ( > 80% in combination vs 54% in PTX vs 32% in CDKN3 knockdown) and most of the daughter cells after abnormal mitoses resulted in cell death in interphase or did not undergo another cell division. The cumulative cell death was significantly increased in combination treatment with PTX and CDKN3 inhibition compared to PTX treatment alone (Hazard ratio 2.22; 95% CI 1.43 - 3.45, p = 0.0004 ) and CDKN3 inhibition alone (Hazard ratio 3.09; 95% CI 1.92 - 4.98, p < 0.0001). Conclusions: CDKN3 knockdown increased the frequencies of centrosome overduplication and multipolar spindle formation when combined with PTX exposure. These mitotic errors caused multipolar divisions and subsequent cell death. Targeting CDKN3 could be a novel anti-cancer strategy. Citation Format: Shigeki Kataoka, Junichi Matsubara, Trang H. Vu, Tomohiro Kondo, Tomoki Saito, Yosuke Mitani, Osamu Kikuchi, Atsushi Yamada, Shinya Ohashi, Manabu Muto. CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1556.