title: Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6-Abundant Breast Cancer Cells by Regulating γ-Secretase-Mediated Notch1 Activation in Caveolae.
abstract: Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6-driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6-driven Notch1 activation by ILK in IL-6-producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7(IL-6) cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase-mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6-induced breast CSCs.
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: En-Chi
authors.initials: EC
authors.lastname: Hsu
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Kulp
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Han-Li
authors.initials: HL
authors.lastname: Huang
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Huang-Ju
authors.initials: HJ
authors.lastname: Tu
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Santosh B
authors.initials: SB
authors.lastname: Salunke
authors.affiliation: Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Sullivan
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA.
authors.forename: Duxin
authors.initials: D
authors.lastname: Sun
authors.affiliation: Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA.
authors.forename: Max S
authors.initials: MS
authors.lastname: Wicha
authors.affiliation: Division of Medical Oncology, Department of Internal Medicine, College of Medicine, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Shapiro
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Electronic address: chen.844@osu.edu.
authors.forename: Ching-Shih
authors.initials: CS
authors.lastname: Chen
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D000099200:Scaffold Protein ILK
chemical_list: D053829:Amyloid Precursor Protein Secretases; D015850:Interleukin-6; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051880:Receptors, Notch; D000099200:Scaffold Protein ILK; C508600:IL6 protein, human