1. 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.
- Author
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Hsu EC, Kulp SK, Huang HL, Tu HJ, Salunke SB, Sullivan NJ, Sun D, Wicha MS, Shapiro CL, and Chen CS
- Subjects
- Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid Precursor Protein Secretases genetics, Animals, Blotting, Western, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Caveolae drug effects, Caveolae metabolism, Female, Fluorescent Antibody Technique, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Neoplastic drug effects, Humans, Immunoenzyme Techniques, Immunoprecipitation, Interleukin-6 genetics, Membrane Microdomains drug effects, Membrane Microdomains metabolism, Mice, Mice, Inbred NOD, Mice, SCID, Neoplastic Stem Cells metabolism, Protein Serine-Threonine Kinases antagonists & inhibitors, RNA, Messenger genetics, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Receptors, Notch genetics, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction drug effects, Tumor Cells, Cultured, Amyloid Precursor Protein Secretases metabolism, Breast Neoplasms pathology, Caveolae pathology, Interleukin-6 metabolism, Neoplastic Stem Cells pathology, Protein Serine-Threonine Kinases pharmacology, Receptors, Notch metabolism
- 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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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