Amplified lipid rafts of malignant cells constitute a target for inhibition of aberrantly active NFAT and melanoma tumor growth by the aminobisphosphonate zoledronic acid.

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 3ß (GSK3ß). Pro-apoptotic inactivation of NFAT2 also followed reactivation of GSK3ß by direct inhibition of PKB or SOC, whereas GSK3ß blockade prevented Zol-induced NFAT2 inhibition and cell death. The rescuing effect of GSK3ß blockade was reproduced by recovery of entire SOC/PKB/GSK3ß 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.

Lipid rafts couple store-operated Ca2+ entry to constitutive activation of PKB/Akt in a Ca2+/calmodulin-, Src- and PP2A-mediated pathway and promote melanoma tumor growth.

Central role of constitutively active protein kinase B/Akt (PKB) in melanoma drives the search for new targets to abolish its deranged signaling. PKB activation is promoted by cholesterol-enriched lipid rafts and is Ca(2+)-dependent, but the pathway linking rafts and Ca(2+) to deregulation of this enzyme remains poorly understood. Here employing B16BL6 melanoma model, we show that ablation of rafts with methyl-ß-cyclodextrin (MßCD) inactivated PKB by inhibiting Src kinase and reactivating the negative PKB modulator, PP2A phosphatase. Blockade of PP2A with okadaic acid rescued PKB, indicating that raft ablation reactivated PP2A through inhibiting Src. Indeed, direct Src blockade with the Src kinase inhibitor-1 or the dominant-negative Src-mutant was sufficient for PP2A reactivation and downregulation of PKB, whereas reconstitution of rafts in MßCD-treated cells restored PKB, PP2A and Src activities to their basal levels. This pathway was also interrupted by inhibition of the Ca(2+) sensor calmodulin, either by its antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide or the Ca(2+)-insensitive calmodulin-mutant or the intracellular Ca(2+)-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N#N#-tetraacetic acid tetra-(acetocymethyl)-ester or by diminishing the store-operated Ca(2+) entry with 2-aminoethoxydiphenyl borate or small hairpin RNA against Stim1. Ablation of rafts prevented Stim1-mediated store-operated Ca(2+) entry, aborted Ca(2+) stimulation of raft-residing calmodulin and disrupted its Ca(2+)-dependent binding to Src, abolishing Src activity and entire Src/PP2A/PKB cascade. Most importantly, blockade of this cascade in the tumor site by raft-ablating MßCD, administered to melanoma-bearing mice, robustly retarded tumor growth and extended animal survival. Together, our data suggest that lipid rafts couple store-operated Ca(2+) entry to sustained activation of major tumor-promoting signaling elements in melanoma cells and underscore the potential of raft-targeting agents as effective anticancer drugs.