Pranlukast Antagonizes CD49f and Reduces Stemness in Triple-Negative Breast Cancer Cells

Abstract

Introduction

Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists.

Materials and Methods

We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation.

Results

Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f⁺ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC.

Conclusion

Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

Keywords:

• CD49f
• alpha6 integrin
• breast cancer stem cells
• pranlukast
• drug repositioning
• triple-negative breast cancer cells

Acknowledgments

This work was partially carried out with the equipment of the National Laboratory for Specialized Services of Investigation, Development and Innovation (I+D+i) for Pharma Chemicals and Biotechnological products, LANSEIDI-FarBiotec-CONACyT, which is part of the Research and Development in Bioprocess Unit (UDIBI)-IPN. We thank Andrea Rodríguez-Moreno, Sandra Guerrero-Rodríguez, Mireya Velázquez-Paniagua, Homero Gómez-Velasco, Luis A. Valencia-Flores, and Israel Castro-Cruz for technical assistance and Ms. Josefina Bolado, Head of the Scientific Paper Translation Department from the Research Division, School of Medicine, UNAM, for language editing. We also thank the support of “Programa de Becas Posdoctorales, DGAPA-UNAM”. Charmina Aguirre-Alvarado is currently affiliated with the Unit for Research in Immunology and Infectology, CMN “La Raza”, Instituto Mexicano del Seguro Social, Mexico City, Mexico.

Abbreviations

CSC, cancer stem cell; CysLTR, cysteinyl leukotriene receptor 1; DMSO, dimethyl sulfoxide; FAK, focal adhesion kinase; FDA, Food and Drug Administration, USA; IC₅₀, half-maximal inhibitory concentration; MD, molecular dynamics; NCI, National Cancer Institute, USA; PI3K, phosphatidylinositol 3-kinase; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation; AU, arbitrary units.

Ethics Approval

Animal procedures were performed per Mexican guidelines for the production, care, and use of laboratory animals (NOM-062-ZOO-1999) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1978). The animal experiments were approved by the IACUC of “Facultad de Medicina, UNAM” (FMED/CI/JMO/102/2012).

Author Contributions

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

This paper was presented at the 2018 NCRI Conference as a poster presentation with interim findings. The poster’s abstract was published in “Selected Abstracts from the 2018 NCRI Cancer Conference of National Cancer Research Institute” in British Journal of Cancer; 2018;119:46; https://doi.org/10.1038/s41416-018-0299-z. Dr Inés Velázquez-Quesada reports a patent for pranlukast as an antagonist of CD49f and derived uses pending. Dr Aliesha González-Arenas reports a patent pending: Mx/a/2018/014001. Dr Marco A Velasco-Velázquez reports grants from CONACYT, and PAPIIT UNAM and Dr. Diana Casique-Aguirre reports personal fees from POSDOC DGAPA-UNAM during the conduct of the study; in addition, they have a patent pending: MX/a/2018/014001. Sonia M Pérez-Tapia was employed by the National Laboratory for Specialized Services of Investigation, Development and Innovation (I+D+i) for Pharma Chemicals and Biotechnological Products, LANSEIDI-FarBiotec-CONACyT, during the study. The authors report no other conflicts of interest in this work.