Published OnlineFirst September 25, 2012; DOI: 10.1158/1535-7163.MCT-12-0441-T Molecular Cancer Preclinical Development Therapeutics Inhibiting Aurora Kinases Reduces Tumor Growth and Suppresses Tumor Recurrence after Chemotherapy in Patient-Derived Triple-Negative Breast Cancer Xenografts Angela Romanelli1, Anderson Clark1, Franck Assayag2, Sophie Chateau-Joubert5, Marie-France Poupon2, Jean-Luc Servely5,6, Jean-Jacques Fontaine5, Xiaohong Liu1, Edward Spooner1, Samantha Goodstal1, Patricia de Cremoux3, Ivan Bieche 7, Didier Decaudin2,4, and Elisabetta Marangoni2 Abstract Triple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan- inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin–cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment. Mol Cancer Ther; 11(12); 2693–703. Ó2012 AACR. Introduction responsible for a large proportion of breast cancer deaths, Triple-negative breast cancers (TNBC), which lack despite its relatively small proportion among all breast expression of estrogen receptors (ER), progesterone cancers, due to its generally aggressive clinical course. The receptors (PR), and EGF receptor 2 (HER2), account for standard-of-care is chemotherapy, although recent 15% of breast tumors in Europe and an even higher research suggests a sound rationale for the use of targeted percentage of breast cancer in women of African descent. agents with antitumor and/or antiangiogenic activity TNBC tumors have a relatively high rate of recurrence, such as receptor tyrosine kinase inhibitors. The absence distant metastases, and poor overall survival (1). TNBC is of tumor-specific treatment options in this cancer subset underscores the critical need to develop a better under- standing of the biology of this disease, as well as to Authors' Affiliations: 1EMD Serono Research Institute, Billerica, Massa- chusetts; 2Preclinical Investigation Unit, Institut Curie - Translational advance treatment strategies for these patients (1). Research Department, Hopital;^ 3APHP Hopital^ Saint-Louis, Unite d'Onco- Signaling pathway abnormalities commonly reported logie Moleculaire - Universite Paris-Diderot, Sorbonne Paris Cite, CNRS in TNBCs involve the regulatory mechanisms of cellular UMR7212/INSERMU944; 4Medical Oncology Department, Institut Curie, Paris; 5National Veterinary School of Maisons Alfort, Maisons-Alfort; 6INRA, proliferation, differentiation, p21-mediated cell signaling, Phase Department, Nouzilly; and 7Oncogenetics Department, Institut and G –S checkpoint controls (2, 3). ^ 1 Curie, Hopital Rene Huguenin, Saint-Cloud, France The Aurora kinase proteins are serine/threonine Note: Supplementary data for this article are available at Molecular Cancer kinases that act as regulators of mammalian cell division. Therapeutics Online (http://mct.aacrjournals.org/). Aurora A localizes to centrosomes/spindle poles and is Current address for A. Romanelli: Translational and Experimental Medicine, required for spindle assembly, whereas Aurora B is a Sanofi Oncology, Cambridge, MA; and current address for E. Spooner: chromosome passenger protein required for phosphory- Merck Research Labs, Oncology, Boston, MA. lation of histone H3, chromosome segregation, and cyto- Corresponding Author: Elisabetta Marangoni, Preclinical Investigation kinesis (4–7). Unit, Translational Research Department, Institut Curie, Hopital^ St Louis, Quadrilatere historique, Porte 13, 1, Ave Claude Vellefaux, Par- Aurora A and B have been implicated in tumor forma- is 75010, France. Phone: 331-5319-7422; Fax: 331-5319-7418; E-mail: tion and progression (6, 8) and are overexpressed in a [email protected] variety of cell lines (4, 9). Relatively high expression of doi: 10.1158/1535-7163.MCT-12-0441-T Aurora A and B has been shown in small patient cohorts Ó2012 American Association for Cancer Research. in several tumor types, including breast, lung, colon, www.aacrjournals.org 2693 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst September 25, 2012; DOI: 10.1158/1535-7163.MCT-12-0441-T Romanelli et al. prostate, pancreas, liver, skin, stomach, rectum, esopha- grown in Dulbecco’s Modified Eagle’s Medium F12 with gus, endometrium, cervix, bladder, ovary, and thyroid 12 mg/mL sodium bicarbonate, 10% FBS, 10 mmol/L cancers (4–6, 10). In breast cancer, the expression of HEPES, 10 mg/mL insulin (all from Life Technologies), À Aurora kinase A has been found to be predictive of and 10 12 mol/L estradiol (Sigma-Aldrich). The HBCx-17, survival in a cohort of more than 600 primary tumors (7). HBCx-15, and HBCx-8 TNBC cell lines were obtained Given their pivotal role in mitosis and overexpression from 3 patient-derived breast cancer xenografts (15, 16). in cancers, Aurora kinases have become attractive targets. A number of inhibitors displaying differential inhibitory Cell proliferation assay activities toward the 3 family members have since then The ATPlite assay was used as a surrogate for detection been developed and used to understand the functional of cell count/proliferation. Intracellular ATP concentra- role of Aurora kinases in mitotic progression. Aurora tions were determined using the ATPlite Luminescence kinase inhibitors induce tretrapoloidy and polyploidy as ATP Detection Assay System (Perkin Elmer), following a result of aberrant mitosis. The effect of the Aurora manufacturer’s instructions. Briefly, cells were incubated kinases inhibitors is therefore unique in that tumor cells in the presence of serial dilutions of AS703569 (range from do not undergo cell-cycle arrest after drug treatment. 0.001 to 100 mmol/L). After 96 hours in culture, the growth Rather, they are catastrophically driven forward and from medium was replaced with 100 mL PBS. Cell lysis buffer aberrant mitosis, which leads to cell death. (50 mL) was added and mixed for 5 minutes, 50 mLof In vivo studies with several agents targeting Aurora ATPlite substrate reagent was then added and mixed for 5 kinases have shown promising results, with tumor growth minutes. After incubating for 10 minutes in the dark, being inhibited in a number of models (8, 11, 12). The plates were measured for luminescence on a Victor-5 therapeutic potential of Aurora-based targeted therapy is 1428 Multilabel HTS counter (Perkin Elmer). Results were also being assessed in clinical trials (13), although none of displayed as relative light units. them have specifically addressed TNBCs. In the current studies, we investigated the efficacy of a Cell-cycle and apoptosis analysis pan-Aurora kinase inhibitor, AS703569 (14), in preclinical Cells were plated on 6-well tissue culture dishes and models of human breast cancers. We report that AS703569 allowed to grow overnight to a density of 40% to 50% has potent antitumor activity in TNBC cell lines, associ- confluency. They were then treated with various concen- ated with endoreduplication and apoptosis. This antitu- trations of inhibitor and harvested after 48, 72, and 96 mor activity was confirmed in vivo using patient-derived hours. At the time of harvest, cells were washed once with breast cancer xenografts. PBS and trypsinized. Cells were harvested in 1 mL of complete medium and pelleted for 5 minutes at 600 Â g. Materials and Methods Cells were fixed and permeabilized with the addition of 100% ice-cold methanol and incubated on ice for 30 Compound minutes. Cells were pelleted and washed twice in 1% Inhibition of Aurora kinases was achieved using the bovine serum albumin (BSA; Fisher Scientific)/PBS and small-molecule pan-Aurora kinase inhibitor, AS703569 split into 2 tubes, one to measure cell cycle by propidium (previously named R763), an orally potent ATP compet- iodide (PI) staining and the other to measure apoptosis by itive inhibitor. In vitro biochemical assays showed that staining of cleaved caspase-3. Cells for cell-cycle analyses AS703569 inhibited Aurora kinases A, B, and C with IC50 were resuspended in PI/RNase (Becton Dickenson) and values of 4.0, 4.8, and 6.8 nmol/L, respectively (Fig. 1A; incubated for 15 minutes at room temperature and then ref. 14). The selectivity of AS703569, assessed in a panel of analyzed by flow cytometry using a Guava EasyCyte cell-based kinase assays, was previously published (14). instrument (Guava). Cells to be analyzed for the presence In vitro experiments were