title: Amplified lipid rafts of malignant cells constitute a target for inhibition of aberrantly active NFAT and melanoma tumor growth by the aminobisphosphonate zoledronic acid.
abstract: Nuclear factors of activated T cells (NFAT) are critical modulators of cancer cell growth and survival. However, the mechanisms of their oncogenic dysregulation and strategies for targeting in tumors remain elusive. Here, we report coupling of anti- apoptotic NFAT (NFAT2) activation to cholesterol-enriched lipid raft microdomains of malignant melanoma cells and interruption of this pathway by the aminobisphosphonate zoledronic acid (Zol). The pathway was indicated by capability of Zol to promote apoptosis and to retard in vivo outgrowth of tumorigenic melanoma cell variants through inhibition of permanently active NFAT2. NFAT2 inhibition resulted from disintegration of cholesterol-enriched rafts due to reduction of cellular cholesterol by Zol. Mechanistically, raft disruption abolished raft-localized robust store-operated Ca(2+) (SOC) entry, blocking constitutive activation of protein kinase B/Akt (PKB) and thereby reactivating the NFAT repressor glycogen synthase kinase 3β (GSK3β). Pro-apoptotic inactivation of NFAT2 also followed reactivation of GSK3β by direct inhibition of PKB or SOC, whereas GSK3β blockade prevented Zol-induced NFAT2 inhibition and cell death. The rescuing effect of GSK3β blockade was reproduced by recovery of entire SOC/PKB/GSK3β cascade after reconstitution of rafts by cholesterol replenishment of Zol-treated tumorigenic cells. Remarkably, these malignant cells displayed higher cholesterol and lipid raft content than non-tumorigenic cells, which expressed weak SOC, PKB and NFAT2 activities and resisted raft-ablating action of Zol. Together, the results underscore the functional relevance of amplified melanoma rafts for tumor-promoting NFAT2 signaling and reveal these distinctive microdomains as a target for in vitro and in vivo demise of tumorigenic cells through NFAT2 inhibition by the clinical agent Zol.
authors.affiliation: Department of Physiology and Cell Biology and The Shraga Segal Department of Microbiology, Immunology and Virology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84015, Israel.
authors.forename: Uliana
authors.initials: U
authors.lastname: Levin-Gromiko
authors.affiliation: Department of Physiology and Cell Biology and The Shraga Segal Department of Microbiology, Immunology and Virology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84015, Israel.
authors.forename: Valeria
authors.initials: V
authors.lastname: Koshelev
authors.affiliation: Department of Physiology and Cell Biology and The Shraga Segal Department of Microbiology, Immunology and Virology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84015, Israel.
authors.forename: Paz
authors.initials: P
authors.lastname: Kushnir
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Virology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84015, Israel.
authors.forename: Shlomit
authors.initials: S
authors.lastname: Fedida-Metula
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Virology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84015, Israel.
authors.forename: Elena
authors.initials: E
authors.lastname: Voronov
authors.affiliation: Department of Physiology and Cell Biology and The Shraga Segal Department of Microbiology, Immunology and Virology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84015, Israel dmitrif@bgu.ac.il.
authors.forename: Daniel
authors.initials: D
authors.lastname: Fishman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism; D004164:Diphosphonates / Q000008:administration & dosage; D005434:Flow Cytometry; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000077211:Zoledronic Acid
chemical_list: D004164:Diphosphonates; D007093:Imidazoles; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D000077211:Zoledronic Acid; D002784:Cholesterol