Pygo2 Activates MDR1 Expression and Mediates Chemoresistance in Breast Cancer Via the Wnt/Β-Catenin Pathway

Oncogene (2016) 35, 4787–4797 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Pygo2 activates MDR1 expression and mediates chemoresistance in breast cancer via the Wnt/β-catenin pathway Z-M Zhang1,2,5, J-F Wu2,3,5, Q-C Luo1,5, Q-F Liu2,4,5, Q-W Wu1, G-D Ye2,4, H-Q She1 and B-A Li2,4 The Wnt/β-catenin pathway has important roles in chemoresistance and multidrug resistance 1 (MDR1) expression in some cancers, but its involvement in breast cancer and the underlying molecular mechanism are undefined. In this study, we demonstrated that the Wnt/β-catenin pathway is activated in chemoresistant breast cancer cells. Using a Wnt pathway-specific PCR array screening assay, we detected that Pygo2, a newly identified Wnt/β-catenin pathway component, was the most upregulated gene in the resistant cells. Additional experiments indicated that Pygo2 activated MDR1 expression in the resistant cells via the Wnt/β-catenin pathway. Moreover, the inhibition of Pygo2 expression restored the chemotherapeutic drug sensitivity of the resistant cells and reduced the breast cancer stem cell population in these cells in response to chemotherapy. Importantly, these activities induced by Pygo2 were mediated by MDR1. We also determined the effect of Pygo2 on the sensitivity of breast tumors resistant to doxorubicin in a mouse model. Finally, RNA samples from 64 paired patient tumors (before and after chemotherapy) highly and significantly overexpressed Pygo2 and/or MDR1 after treatment, thus underlining a pivotal role for the Pygo2-mediated Wnt/β-catenin pathway in the clinical chemoresistance of breast cancer. Our data represent the first implication of the Wnt/β-catenin pathway in breast cancer chemoresistance and identify potential new targets to treat the recurrence of breast cancer. Oncogene (2016) 35, 4787–4797; doi:10.1038/onc.2016.10; published online 15 February 2016 INTRODUCTION ubiquitin-mediated proteasomal degradation.8 Stimulation by Wnt Breast cancer is the leading female malignancy and cause of ligands suppresses the phosphorylation and degradation of β- cancer death in developed countries. Despite recent advances in catenin, which then enters the nucleus and binds to the LEF1/TCF combined therapies, recurrent disease due to treatment failure in family of transcription factors. In turn, the β-catenin–LEF1/TCF patients remains a major clinical problem. This recurrence is complex regulates the expression of downstream target genes mainly attributed to the development of chemoresistance during involved in diverse cellular processes.9,10 Although the Wnt/β- treatment.1 Cancer cells can acquire chemoresistance via the catenin pathway has been revealed to have important roles in overexpression of drug efflux transporters, such as multidrug chemoresistance and MDR1 expression in colon cancer11 and resistance 1 (MDR1), and multidrug resistance-associated neuroblastoma,12 its involvement in breast cancer and the proteins,2–5 which facilitate the efflux of therapeutic drugs from underlying molecular mechanism are undefined. cells to prevent the accumulation of the cytotoxic agents in cells. Pygopus has been identified as a novel component of the MDR1 (P-glycoprotein, ABCB1), the best-characterized drug efflux Wingless (Wg) pathway in Drosophila.13,14 Pygo proteins (Pygo1 pump, has been found to participate in the chemoresistance of and Pygo2 in mammals) are thought to promote β-catenin/LEF/ breast cancer.5 However, the molecular mechanisms of the TCF transcriptional activation via the regulation of β-catenin regulation of MDR1 expression in breast cancer remain poorly nuclear retention and/or by facilitating β-catenin to recruit – understood. transcriptional co-activators.15 20 Moreover, multiple findings have The Wnt/β-catenin pathway has a central role in normal demonstrated that Pygo2 promotes β-catenin activity in a – development and tumorigenesis.6,7 In the absence of Wnt gene- and tissue-dependent manner in mammalian cells.21 24 In stimulation, cytoplasmic β-catenin is assembled into the destruc- this study, we demonstrate that the Wnt/β-catenin pathway and tion complex composed of axin, glycogen synthase kinase 3β Pygo2 are activated in chemoresistant breast cancer and that (GSK3β), adenomatous polyposis coli (APC) and CK1-α. β-catenin is Pygo2 activates MDR1 expression in chemoresistant breast cancer phosphorylated in this complex and subsequently targeted for cells via the Wnt/β-catenin pathway. Our study reveals an 1Department of Breast Surgery, The First Affiliated Hospital, Xiamen University, Xiamen, Fujian, China; 2State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China; 3Department of Bioengineering, College of Food and Bioengineering, Henan University of Science and Technology, Luoyang, China and 4Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China. Correspondence: Dr H-Q She, Department of Breast Surgery, The First Affiliated Hospital, Xiamen University, 55 Zhenhai Rd, Xiamen, 361003 Fujian, China or Dr B-A Li, Department of Biomedical Sciences, Xiamen University School of Life Sciences, D212 Huang Chaoyang Hall, Xiang’an Campus of Xiamen University, Xiamen, 361100 Fujian, China. E-mail: [email protected] or [email protected] 5These authors contributed equally to this work. Received 20 June 2015; revised 24 December 2015; accepted 27 December 2015; published online 15 February 2016 Pygo2 and MDR1 in chemoresistance of breast cancer Z-M Zhang et al 4788 association between MDR1 expression and Pygo2, which high- immunoprecipitation assay, both β-catenin and Pygo2 were found to lights the role of the Wnt/β-catenin pathway in the regulation of occupy the endogenous MDR1 promoter at the LEF1/TCF-binding chemoresistance of breast cancer. site in MCF-7/ADR cells (Figure 2e). However, the knockdown of β-catenin led to a dramatically reduced occupancy by Pygo2 (Figure 2f). Together, the above results suggest that Pygo2 activates RESULTS MDR1 expression in MCF-7/ADR cells via the Wnt/β-catenin pathway. The Wnt/β-catenin pathway and Pygo2 are activated in drug- resistant cell lines Inhibition of Pygo2 expression restores chemotherapeutic drug To determine whether the Wnt/β-catenin pathway is involved in sensitivity of the resistant cells β the chemoresistance of breast cancer, we performed Wnt/ - We hypothesized that Pygo2 activation is required for drug catenin-dependent luciferase assays in three doxorubicin-resistant resistance of the resistant cells. Therefore, we evaluated the effect cell lines (MCF7/ADR, MDA-MB-231/ADR and T-47D/ADR) com- of Pygo2 knockdown on the cell sensitivity to doxorubicin and pared with their respective counterparts (MCF7, MDA-MB-231 and paclitaxel. After Pygo2 was knocked down in MCF-7/ADR cells, the T-47D). The TopFlash assays revealed a much higher TCF cells were treated with the aforementioned drugs, and the cell transcriptional activities in the resistant cell lines than in the viability was measured using the MTS/PMS assay. As shown in non-resistant cell lines (Figure 1a). Consistent with this result, a Figures 3a and b, Pygo2 inhibition significantly restored the western blot analysis revealed that the nuclear expression of sensitivity of MCF-7/ADR cells to doxorubicin and paclitaxel in a β-catenin was increased in the resistant cells compared with the dose-dependent manner. Similar results were achieved when we non-resistant cells (Figure 1b). To define the underlying molecular performed the above experiments in MDA-MB-231/ADR cells mechanism by which β-catenin is activated, we screened the gene (Supplementary Figures S2A and B) and T-47D/ADR cells expression profiles of MCF7 versus MCF7/ADR using a Wnt (Supplementary Figures S2C and D). We next examined the effect pathway-specific PCR array. Notably, the upregulation of Pygo2, of Pygo2 on the drug efflux driven by MDR1. The rhodamine-123 a newly identified Wnt/β-catenin pathway component, was retention assay demonstrated that Pygo2 knockdown significantly highest in MCF7/ADR cells (Supplementary Table S1). Quantitative increased the intracellular accumulation of rhodamine-123 com- PCR and western blot analyses confirmed the overexpression of pared with the control cells (Figure 3c, left). When MDR1 was Pygo2 in all three resistant cell lines (Figure 1c). When Pygo2 was overexpressed in the Pygo2 knockdown cells to a level compar- knocked down in MCF-7/ADR cells, the TopFlash assay showed able with that of the control cells (Figure 3c, right), the knocking significantly reduced TCF transcriptional activity (Figure 1d). As down of Pygo2 no longer increased the intracellular accumulation negative controls, NF-κB and FOXO1/3a signaling pathways, two of rhodamine-123 (Figure 3c, left). Finally, we explored the other key MDR1 (see below) transcription regulators, were not mechanisms involved in drug sensitization after Pygo2 inhibition. affected upon Pygo2 downregulation, when we performed NF-κB- A TUNEL assay showed that Pygo2 knockdown in the MCF-7/ADR and FOXO-luciferase assays in MCF-7/ADR cells (Supplementary cells resulted in apoptotic cells in response to chemotherapy Figures S1A-C). Moreover, the nucleus/cytoplasm ratio of β- (Figure 3d). Consistent with this result, cleaved poly ADP-ribose catenin was found to be reduced in MCF-7/ADR cells in which polymerase, an apoptotic protein, was detected in the TUNEL- Pygo2 was knocked down (Figure 1e). By contrast, the nucleus/ positive cells as revealed by a western blot analysis (Figure 3e). cytoplasm ratio of β-catenin was increased when Pygo2 was The induction of caspase-dependent cell death was also measured overexpressed (Figure 1f). Taken together, these results suggest using a caspase-3-like activity assay. As shown in Figures 3f and g, that the Wnt/β-catenin pathway is activated in drug-resistant cells Pygo2 knockdown in the MCF-7/ADR cells resulted in a higher and that this activity is likely mediated by Pygo2.

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