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Published on: December 26, 2016
Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2
Wing-Kee Lee1, Michelle Maaß2, Amy Quach3
1Institute for Physiology, Pathophysiology and Toxicology, ZBAF, Witten/Herdecke University, Witten, Germany; Physiology & Pathophysiology of Cells and Membranes, Medical School OWL, Bielefeld University, Bielefeld, Germany.
Abstract:
Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.
Insights
Ceramide synthases (CerS) regulate multidrug resistance in renal cancer by controlling ABCB1 transporter activity. Targeting CerS2 and CerS6 can reverse chemoresistance by altering lipid composition and ABCB1 expression.
Area of Science:
- Oncology
- Biochemistry
- Molecular Biology
Background:
- Multidrug resistance in renal cancer is often intrinsic, driven by ABCB1 transporters.
- ABCB1 transporter activity is influenced by its lipid microenvironment, specifically cholesterol- and sphingomyelin (SM)-rich domains.
- Ceramides are precursors to SMs, suggesting a role for ceramide synthases (CerS) in regulating ABCB1 activity.
Purpose of the Study:
- To investigate the hypothesis that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity in renal cancer.
- To explore the potential of targeting ceramide synthesis to overcome chemotherapy resistance.
Main Methods:
- Analysis of RNA-Seq data from patient kidney tumors to assess CerS mRNA expression.
- Cellular experiments using chemoresistant and chemosensitive renal cancer cell lines to compare CerS activity and lipid profiles.
- Lipidomics analysis using HPLC-MS/MS to determine ceramide and SM composition.
- Inhibition of de novo ceramide synthesis and CerS activity using specific inhibitors.
- Downregulation of CerS expression via siRNA and assessment of ABCB1 levels, localization, and transport activity.
- Treatment with inhibitors of endoplasmic reticulum-associated degradation and proteasome.
Main Results:
- Kidney tumors showed increased CerS2 mRNA, inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas.
- Chemoresistant cells exhibited higher CerS2 to CerS5/6 activity, with a bias toward longer-chain ceramides and altered SM composition.
- Inhibiting ceramide synthesis or CerS activity partially reversed doxorubicin resistance.
- Downregulation of CerS2/6 reduced ABCB1 expression, plasma membrane localization, efflux activity, and doxorubicin resistance.
- Proteasome and ER-associated degradation inhibitors prevented ABCB1 loss upon CerS2/6 downregulation.
Conclusions:
- A specific balance of ceramide and SM species is critical for ABCB1 expression and function.
- Targeting ceramide metabolism, particularly CerS2 and CerS6, offers a potential strategy to reverse multidrug resistance in renal cancers.
- The findings highlight the intricate link between lipid metabolism and drug transporter regulation in cancer.
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