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Inhibition of HER2 Increases JAGGED1-Dependent Breast Cancer Stem Cells: Role for Membrane JAGGED1 Deep Shah1, Debra Wyatt2, Andrew T

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Inhibition of HER2 Increases JAGGED1-Dependent Breast Cancer Stem Cells: Role for Membrane JAGGED1 Deep Shah1, Debra Wyatt2, Andrew T Published OnlineFirst June 12, 2018; DOI: 10.1158/1078-0432.CCR-17-1952 Cancer Therapy: Preclinical Clinical Cancer Research Inhibition of HER2 Increases JAGGED1-dependent Breast Cancer Stem Cells: Role for Membrane JAGGED1 Deep Shah1, Debra Wyatt2, Andrew T. Baker3, Patricia Simms4, Daniel S. Peiffer3,5, Michelle Fernandez6, Emad Rakha7, Andrew Green7, Alexandra Filipovic8, Lucio Miele9, and Clodia Osipo2,6 Abstract Purpose: HER2-positive breast cancer is driven by cells tivity to anti-HER2 treatment within the bulk cell popula- possessing stem-like properties of self-renewal and differen- tion. This increase in membrane JAGGED1 is associated tiation, referred to as cancer stem cells (CSC). CSCs are with higher NOTCH receptor expression, activation, and implicated in radiotherapy, chemotherapy resistance, and enrichment of CSCs in vitro and in vivo. Importantly, lapa- tumor recurrence. NOTCH promotes breast CSC survival and tinib treatment results in growth arrest and cell death of self-renewal, and overexpression of NOTCH1 and the NOTCH JAGGED1 low-expressing cells while the JAGGED1 high- ligand JAGGED1 predict poor outcome. Resistance to anti- expressing cells continue to cycle. High membrane þ HER2 therapy in HER2 breast cancer requires NOTCH1, and JAGGED1 protein expression predicts poor overall cumu- þ that combination of trastuzumab and a gamma secretase lative survival in women with HER2 breast cancer. inhibitor (GSI) prevents tumor relapse in xenograft models. Conclusions: These results indicate that higher membrane Experimental Design: The current study investigates JAGGED1 expression may be used to either predict response to mechanisms by which HER2 tyrosine kinase activity regulates anti-HER2 therapy or for detection of NOTCH-sensitive CSCs NOTCH-dependent CSC survival and tumor initiation. posttherapy. Sequential blockade of HER2 followed by Results: Lapatinib-mediated HER2 inhibition shifts the JAGGED1 or NOTCH could be more effective than simulta- þ population of HER2 breast cancer cells from low membrane neous blockade to prevent drug resistance and tumor progres- JAGGED1 expression to higher levels, independent of sensi- sion. Clin Cancer Res; 1–13. Ó2018 AACR. Introduction approximately 4,571,210 breast cancer survivors in the United States. Gene expression profiling has revealed distinct Breast cancer remains the most common form of cancer among intrinsic subtypes of breast cancer. There are four major classes— women worldwide. By 2026, it is estimated that there will be þ Luminal A, Luminal B, HER2 enriched (HER2 ), and basal-like þ (1–3). Approximately, 15%–20% of breast cancers are HER2 (4, 5). These cancers contain amplification for a section of chro- 1Molecular Pharmacology and Therapeutics Program, Loyola University Chicago: mosome 17q containing the ERBB2 gene locus and neighboring Health Sciences Division, Maywood, Illinois. 2Oncology Research Institute, genes GRB7 and MIEN1, accompanied by protein overexpression 3 Loyola University Chicago: Health Sciences Division, Maywood, Illinois. Inte- of HER2 detectable as 3þ staining by IHC (6). Treatment options grated Cell Biology Program, Loyola University Chicago: Health Sciences Divi- include the use of biologics, trastuzumab and pertuzumab, along 4 fi sion, Maywood, Illinois. FACS Core Facility, Of ce of Research Services, Loyola – University Chicago: Health Sciences Division, Maywood, Illinois. 5MD/PhD Pro- with taxane-based chemotherapy, use of the antibody drug con- gram, Loyola University Chicago: Health Sciences Division, Maywood, Illinois. jugate TDM1 (trastuzumab emtansine) or dual EGF receptor 6Department of Microbiology and Immunology, Loyola University Chicago: (EGFR)/HER2 tyrosine kinase inhibitor lapatinib (6). þ Health Sciences Division, Maywood, Illinois. 7Departments of Histopathology Despite improved prognosis for women with HER2 breast and Medicine, University of Nottingham and University Hospital NHS Trust, cancer, drug resistance, disease progression, and tumor recurrence 8 Nottingham, United Kingdom. Emperial College of London, London, United remain major challenges preventing a cure (6). It is estimated that Kingdom. 9Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Los Angeles. less than 35% of the patients initially respond to trastuzumab. This could be deemed as primary or intrinsic resistance (7, 8). Out Note: Supplementary data for this article are available at Clinical Cancer of those who respond, about 70% exhibit metastatic progression Research Online (http://clincancerres.aacrjournals.org/). within one year, suggesting secondary or acquired drug resistance D. Shah and D. Wyatt contributed equally to this article. (9). Drug resistance could be due to multiple factors including Corresponding Author: Clodia Osipo, Loyola University Chicago, 2160 S. First survival of resistant cells referred to as cancer stem cells (CSC). The Ave, Bldg 112, Room 238, Maywood, IL 60153. Phone: 708-327-2372; Fax: 708- CSC hypothesis suggests that a tumor is comprised of a hetero- 327-2245; E-mail: [email protected] geneous population of cells including small subsets or clones with doi: 10.1158/1078-0432.CCR-17-1952 stem-like properties. These stem-like properties can be intrinsic to Ó2018 American Association for Cancer Research. cell clones or acquired through epigenetic remodeling by hypoxia, www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst June 12, 2018; DOI: 10.1158/1078-0432.CCR-17-1952 Shah et al. Here, we show that overexpression, and importantly, hyperac- Translational Relevance tivity of HER2 restricts JAGGED1 cell surface expression. When þ Critical concerns for women with HER2 breast cancer are HER2 is inactivated by lapatinib, there is an increase in cell surface drug resistance, tumor recurrence, and disease progression. JAGGED1, NOTCH target gene expression, percentage of cells in The current study describes a novel role for membrane the S-phase of the cell cycle, and increased survival of breast CSCs. þ JAGGED1 in enrichment of breast CSCs and resistance to In HER2 breast cancer tissue, higher JAGGED1 membrane anti-HER2 therapy. The clinical significance of this work is staining, but not cytoplasmic or perinuclear JAGGED1 expression, that higher membrane JAGGED1 is a biomarker for higher predicts poor overall survival for women with primary, invasive þ NOTCH activation, and it predicts poor overall cumulative HER2 breast cancer. Overall, results of the study demonstrate survival. Sequential blockade of HER2 followed by JAGGED1 that HER2 expression and activity status specifically restricts high or NOTCH could be more effective than simultaneous block- membrane JAGGED1-enriched breast CSCs. The consequence of ade to prevent drug resistance and tumor progression. HER2 inactivation is a shift from low JAGGED1 membrane expression to high JAGGED1 membrane expression responsible for breast CSCs that are resistant to lapatinib. EMT, or treatment-induced cell stress. These small subsets of stem- Materials and Methods like cells (CSC) are thought to be selected by treatment that kills the majority of nonstem-like cells, while the surviving CSCs are Cell culture responsible for drug-resistant recurrences (10). NOTCH signaling HCC1954, MDA-MB-453, and MCF-7 cells were purchased has been implicated in mammary stem cell self-renewal as well as from ATCC. MCF-7 cells stably overexpressing HER2, designated in survival and self-renewal of breast CSCs (11, 12). as MCF-7-HER2, were generously provided by Dr. Mien-Chie NOTCH signaling is a developmentally conserved signaling Hung from The University of Texas MD Anderson Cancer Center pathway mediating communications between cells. Typically, (Houston, TX). HCC1954 and MDA-MB-453 cells were grown in fi the ligand is expressed on the signal sending cell and the DMEM (Thermo Fisher Scienti c), whereas MCF7 and MCF7- fi receptor is expressed on the signal receiving cell. Mammals HER2 cells were cultured in RPMI1640 (Thermo Fisher Scienti c). express five NOTCH ligands (JAGGED1, JAGGED2, DLL 1, 3, The cell culture media were supplemented with 10% FBS (Gemini and 4), and four NOTCH receptors (NOTCH1–4). Delta-like 3 Bio-Products), 1% L-glutamine (2 mmol/L, Thermo Fisher Scien- fi is thought to be an inhibitory ligand, while Delta-like 1 and 4 ti c), and 1% nonessential amino acids (100 mmol/L, Invitrogen). and JAGGED1 and 2 are stimulatory ligands. The relative Cell lines were authenticated using short tandem repeat allelic fi affinity of NOTCH receptors for different ligand classes is pro ling (DCC Medical) in 2015 and maintained at low passage numbers (below 20). All cell lines were maintained by incubating modulated by glycosylation of the extracellular subunits of NOTCH receptors mediated by Fringe family N-acetyl-glucos- at 37 C with 95% humidity and 5% CO2. aminidyltransferases, POFUT fucosyltransferase, and RUMI glucosyl transferase (13). When a ligand engages a receptor, it Drugs and chemicals is ubiquitinylated by an E3 ligase, Neuralized or Mindbomb A g-secretase inhibitor (GSI), MRK-003, was provided by Merck (14). This allows the ligand to be endocytosed into ligand- Oncology & Co. For in vitro studies, 5 mmol/L MRK-003 GSI stock expressing cells with the extracellular subunit of the NOTCH solution was prepared by dissolving in DMSO. The working receptor, separating the latter from the transmembrane subunit. concentration was 5 mmol/L and the prepared
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