Carcinogenesis Advance Access published online on June 25, 2008
Carcinogenesis, doi:10.1093/carcin/bgn146
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Targeting lipid rafts inhibits Protein Kinase B by disrupting calcium homeostasis and attenuates malignant properties of melanoma cells.
1 Dept.of Microbiology and Immunology
2 Dept. of Morphology
3 Dept of Physiology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev P.O.B.653, Beer-Sheva 84105, Israel
To whom correspondence should be addressed: Daniel Fishman, Dept. of Morphology, Faculty of Health Sciences, Ben-Gurion University of the Negev POB 653, Beer-Sheva 84105, Israel. Phone: 972-8-6477250 FAX: 972-8-6477626 e-mail: dmitrif{at}bgu.ac.il
Failure of current therapeutic modalities to treat melanoma remains a challenge for clinical and experimental oncology. The aggressive growth and apoptotic resistance of this tumor are mediated, in part, by aberrantly activated protein kinase B/Akt (PKB). In many cells, PKB signaling depends on integrity of cholesterol-enriched membrane micro-domains (rafts). However, it is still unclear if rafts support deregulated PKB activity in melanoma. In this study, ablation of rafts in murine (B16BL6-8, JB/RH1) and human (GA) melanoma lines by cholesterol-chelating methyl-β–cyclodextrin (MβCD) reduced levels of constitutively active PKB in a dose- and time-dependent manner, while reconstitution of micro-domains restored PKB activity. PKB was sensitive to the membrane-permeable Ca2+ chelator BAPTA-AM and to the calmodulin antagonist W7 implying the contribution of Ca2+ signaling to PKB deregulation. Indeed, malignant and apoptosis-resistant clone of B16BL6 melanoma (B16BL6-8) displayed significantly higher [Ca2+]i and store-operated Ca2+ influx (SOC) relative to non-malignant apoptosis-sensitive B16BL6 clone (Kb30) expressing barely-detectable basal levels of active PKB. Raft ablation in B16BL6-8 cells robustly inhibited SOC and decreased [Ca2+]i to levels comparable to those detected in Kb30 cells. Treating cells by PKB-inhibiting doses of MβCD dramatically impaired their apoptotic resistance and capacity to generate tumors. Furthermore, weekly intraperitoneal injections of MβCD to mice grafted with melanoma cells at doses of 300 and 800 mg/kg significantly attenuated tumor development. Our data implicate membrane rafts in enhancing the resistance of melanoma to apoptosis and indicate that targeting raft micro-domains is a potentially effective strategy to cure this frequently fatal form of cancer.
Received December 9, 2007; revised May 19, 2008; accepted June 13, 2008.