Amber S. Zhou, John B. Tucker, Christina M. Scribano, Andrew R. Lynch, Caleb L. Carlsen, Sophia T. Pop-Vicas, Srishrika M. Pattaswamy, Mark E. Burkard, Beth A. Weaver
Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents.
Microtubule poisons are one of the most commonly used therapies for numerous malignancies, including primary and metastatic breast cancers of all subtypes [1–3]. Improved understanding of mechanisms that dictate response to microtubule poisons is urgently needed, as a substantial proportion of patients derive no benefit from this cornerstone of treatment and suffer needless toxicity and delays in effective treatment. Although the best-selling chemotherapy drug paclitaxel (Taxol) is considered highly effective, only 41% to 58% of patients respond . Similar fractions of metastatic breast cancer patients respond to other microtubule poisons, including docetaxel (30% to 63%), eribulin (12% to 29%), and vinorelbine (15% to 50%) [5–12]. Combination therapies that could sensitize the large number of tumors that are resistant to microtubule poisons would have a profound clinical impact.
Materials and methods
Microtubule poison study design
Patients who volunteered to participate in this study were enrolled in a prospective trial at the UW Carbone Cancer Center specifying the treatment, biopsy, and analysis plan. The protocol was approved by the UW Health Sciences Institutional Review Board, assigned UWCCC protocol number UW16151, conducted in accordance with the ethical standards established in the 1964 Declaration of Helsinki and registered on clinicaltrials.gov (NCT03393741). Patients were enrolled if they had metastatic or incurable breast cancer, for which antimicrotubule chemotherapy was indicated. Enrolled patients provided written, informed consent. Patients received standard-of-care microtubule poison treatment (either taxane, eribulin, or vinorelbine). There were no major complications from protocol-mandated research biopsy. Response was assessed based on RECIST 1.1 criteria .
Clinically useful microtubule poisons induce multipolar spindles
To determine whether the clinically relevant mechanism of paclitaxel is conserved among microtubule poisons, we tested whether clinically useful antimicrotubule agents are capable of inducing multipolar spindles without mitotic arrest. Because low nM concentrations of paclitaxel generate clinically relevant intracellular concentrations, we tested whether comparable concentrations of other microtubule poisons exerted similar effects in cell culture. Triple negative (negative for expression of estrogen receptor, progesterone receptor, and overexpression of HER2) breast cancer cell lines dervied from metastatic cancer (Cal51 and MDA-MB-231) were treated with increasing concentrations of microtubule poisons classically considered to be stabilizers (docetaxel, ixabepilone, epothilone B) or destabilizers (vinblastine, vinorelbine, eribulin). All were capable of inducing multipolar spindles very similar to those caused by paclitaxel at low (0.1 to 10) nM doses (Fig 1A–1D and Fig A in S1 File). The lowest concentrations that induced multipolar spindles did so without causing the large increase in mitotic index produced by high doses of drug, which cause mitotic arrest (Fig B in S1 File).
Microtubule-targeting drugs are a mainstay of treatment for various cancers. For decades, these agents were expected to exert their anticancer activity by inducing mitotic arrest in patient tumors. Because of this, substantial drug discovery efforts were directed at developing agents that could arrest cells in mitosis without affecting microtubules. The failure of numerous such agents to exert efficacy in clinical trials was disappointing and caused some to speculate that the efficacy of microtubule poisons is due to effects on interphase microtubules rather than mitosis [45,46]. Others have continued to focus on mitotic arrest as the clinically relevant mechanism of these drugs . It is now clear that the concentration of microtubule poisons achieved in breast cancers has a considerable effect on mitosis, without causing mitotic arrest. Clinically relevant concentrations of diverse microtubule poisons cause an at least transient increase in multipolar spindle poles. Maintenance of multipolar spindles throughout mitosis causes high rates of CIN and sensitivity to the antimicrotubule agent, but focusing of multipolar spindles can occur at any point during mitosis. Focusing reduces chromosome missegregation and causes resistance. The timing of focusing dictates the magnitude of these effects, with the earliest focusing resulting in the most profound resistance.
We thank S. Godinho and D. Pellman for the inducible Plk4 MCF10A cell line, D. Compton for NuMA antibody, T. Kinoshita in the Translational Research Initiatives in Pathology (TRIP) lab for histology services, our patients for their participation in this research, and members of the Weaver, Burkard, and Suzuki laboratories for insightful discussions.
Citation: Zhou AS, Tucker JB, Scribano CM, Lynch AR, Carlsen CL, Pop-Vicas ST, et al. (2023) Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles. PLoS Biol 21(10): e3002339. https://doi.org/10.1371/journal.pbio.3002339
Academic Editor: Jonathon Pines, The Institute of Cancer Research, UNITED KINGDOM
Received: April 4, 2023; Accepted: September 18, 2023; Published: October 26, 2023
Copyright: © 2023 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported, in part, by National Institutes of Health grants P30 CA014520 (UW Carbone Cancer Center); R01CA234904 (to B.A.W. and M.E.B.), T32 GM008688 (to A.S.Z.), T32 CA009135 (J.B.T, C.M.S), F31CA254247 (to A.R.L), and T32 GM141013 (to C.L.C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: M.E.B. declares the following: Medical advisory board of Strata Oncology; Research funding from Abbvie, Arcus, Apollomics, Elevation Oncology, Endeavor, Genetech, Puma, and Loxo Oncology, Seagen. All other authors declare that they have no conflict of interest.
Abbreviations: CENP-Ei, CENP-E inhibition; CIN, chromosomal instability; dox, doxycycline; NEBD, nuclear envelope breakdown; Plk4, Polo-like kinase 4; shRNA, short hairpin RNA.