Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in gastrointestinal tumor cells

Abstract

Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95; the present studies have determined how sorafenib and vorinostat individually contribute to CD95 activation. Sorafenib (3-6 micromol/L) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib-induced (3 micromol/L) CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 micromol/L), or sorafenib (3 micromol/L) and vorinostat (500 nmol/L) treatment promoted higher levels of CD95 phosphorylation which correlated with DISC formation, receptor surface localization, and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to localize plasma membrane or induce autophagy. Knockdown/knockout of Src family kinases abolished sorafenib-induced CD95 tyrosine phosphorylation, DISC formation, and the induction of cell death and autophagy. Knockdown of platelet-ived growth factor receptor-beta enhanced Src Y416 and CD95 tyrosine phosphorylation, which correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased reactive oxygen species levels, and in a delayed NF kappa B-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95; however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by approximately 50%. Thus, sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation, whereas vorinostat contributes to CD95 activation via the initial facilitation of reactive oxygen species generation and subsequently of FAS-L expression.

Figure 1. Sorafenib causes a dose-dependent activation of Src, ERBB1 and CD95

Panel A. HEPG2 cells were treated with vehicle (DMSO), sorafenib (3 μM, 6 μM), as indicated. Thirty minutes after treatment cells were isolated and lysed. Portions of lysates were immunoprecipitated (anti-phospho-tyrosine) and IPs probed for PDGFRβ. After SDS PAGE immunoblotting was performed to determine the phosphorylation of the indicated proteins. Representative blots are shown (n = 3). Panel B. Cells were plated in 8 well chamber slides and were treated with vehicle (DMSO), sorafenib (3 μM, 6 μM) or vorinostat (500 nM), as indicated. Cells were fixed 6h after exposure and surface levels of CD95 determined by IHC. The density of CD95 staining was determined in 40 cells (n = 2, +/- SEM). Panel C. HEPG2 cells were treated with vehicle (DMSO), sorafenib (3 μM, 6 μM) or vorinostat (500 nM), as indicated. Cells were isolated 6h after exposure and CD95 immunoprecipitated. The amount of caspase 8 association with CD95 was determined. Representative blots are shown (n = 3). Panel D. HEPG2 cells were treated with vehicle (DMSO), sorafenib (3 μM, 6 μM) or vorinostat (500 nM), as indicated. Thirty minutes after treatment cells were isolated and lysed. Portions of lysates were immunoprecipitated (anti-CD95). After SDS PAGE immunoblotting was performed to determine the phosphorylation of CD95. Representative blots are shown (n = 3).

Figure 2. Sorafenib-induced Src-dependent CD95 tyrosine phosphorylation is due to inhibition of PDGFRβ

Panel A. HuH7 cells were transfected with empty vector plasmid (CMV), a plasmid to express CD95-YFP or a plasmid to express mutant inactive CD95-YFP (Y232F, Y291F). Twenty four h after transfection cells were treated with vehicle (DMSO) or sorafenib (3 μM) and vorinostat (500 nM). Cells were isolated 48h after exposure and viability determined by trypan blue exclusion (n = 3, +/- SEM). Panel B. HuH7 cells were transfected with a plasmid to express CD95-YFP or a plasmid to express mutant inactive CD95-YFP (Y232F, Y291F) and twenty four h later were treated with vehicle (DMSO) or sorafenib (3 μM) and vorinostat (500 nM). Cells were isolated 6h after exposure and CD95 immunoprecipitated. The amount of caspase 8 association with CD95 was determined. Representative blots are shown (n = 3). Panel C. HEPG2 cells were transfected with scrambled siRNA (siSCR) or an siRNA to knock down PDGFRβ. Thirty six h after transfection cells were treated with vehicle (DMSO) or sorafenib (6 μM). Cells were isolated 30 min later and the phosphorylation of Src determined by immunoblotting. Representative blots are shown (n = 3). Panel D. Upper blots: HEPG2 cells were transfected with scrambled siRNA (siSCR) or an siRNA to knock down PDGFRβ. Thirty six h after transfection cells were treated with vehicle (DMSO) or sorafenib (6 μM). Cells were isolated 6 h later and CD95 immunoprecipitated. The association of caspase 8 with CD95 and the tyrosine phosphorylation of CD95 were determined. Representative blots are shown (n = 3). Lower blots: Cells were transfected with scrambled siRNA (siSCR) or an siRNA to knock down PDGFRβ and in parallel were transfected with an empty vector plasmid (CMV) or a plasmid to express dominant negative c-Src. Thirty six h after transfection cells were isolated and CD95 immunoprecipitated. The tyrosine phosphorylation of CD95 was determined. Representative blots are shown (n = 3).

Figure 3. Src family kinase signaling is essential for sorafenib-induced CD95 activation and drug toxicity

Panel A. Wild type or Src / Fyn / Yes null SV40 Large T transformed MEFs plated in 8 well chamber slides and were treated with vehicle (DMSO), sorafenib (3 μM, 6 μM) or vorinostat (500 nM), as indicated. Cells were fixed 6h after exposure and surface levels of CD95 determined by IHC. The density of CD95 staining was determined in 40 cells (n = 2, +/- SEM). Panel B. Wild type or Src / Fyn / Yes null SV40 Large T transformed MEFs were transfected with a plasmid to express CD95-YFP or a plasmid to express mutant inactive CD95-YFP (Y232F, Y291F) and twenty four h later were treated with vehicle (DMSO) or sorafenib (6 μM). Cells were isolated 6 h later and CD95 immunoprecipitated via the YFP tag. The association of caspase 8 with CD95 and the tyrosine phosphorylation of CD95 were determined. Representative blots are shown (n = 3). Panel C. Wild type or Src / Fyn / Yes null SV40 Large T transformed MEFs were treated with vehicle (DMSO) or increasing concentrations of sorafenib (0.1-6.0 μM). Cells were isolated 48 h after drug exposure and viability determined by trypan blue exclusion (n = 3, +/- SEM). Panel D. HEPG2 cells were transfected with empty vector plasmid (CMV) or a plasmid to express dominant negative Src. Twenty four h after transfection cells were treated with sorafenib (3 μM, 6 μM), as indicated. Upper Panel: Cells were isolated 6h after drug exposure, CD95 immunoprecipitated and the levels of CD95 tyrosine phosphorylation and the association of pro-caspase 8 with CD95 determined. Representative blots are shown (n = 3). Lower Panel: Cells were isolated 48h after treatment and viability determined by trypan blue exclusion (n = 3, +/- SEM).

Figure 4. PDGFRβ regulates CD95-dependent sorafenib –induced autophagy

Panel A. HEPG2 cells plated in 8 well chamber slides and were transfected with scrambled siRNA (siSCR) or an siRNA to knock down PDGFRβ. Thirty six h after transfection cells were treated with vehicle (DMSO) or sorafenib (6 μM). Cells were fixed 6h after exposure and surface levels of CD95 determined by IHC. The density of CD95 staining was determined in 40 cells (n = 2, +/- SEM). Panel B. HEPG2 cells plated in 8 well chamber slides and were transfected with a plasmid to express LC3-GFP and in parallel with scrambled siRNA (siSCR) or siRNA molecules to knock down PDGFRβ or CD95. Thirty six h after transfection cells were treated with vehicle (DMSO) or sorafenib (6 μM). The number of intense punctate GFP-LC3 staining vesicles was determined in 40 cells (n = 2, +/- SEM). Panel C. HEPG2 cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down PDGFRβ or CD95. Thirty six h after transfection cells were treated with vehicle (DMSO) or sorafenib (6 μM). Forty eight h later cells were isolated and viability determined by trypan blue exclusion (n = 3, +/- SEM). Panel D. HEPG2 cells plated in 8 well chamber slides and were transfected with a plasmid to express LC3-GFP and in parallel with a plasmid to express dominant negative Src. Thirty six h after transfection cells were treated with vehicle (DMSO) or sorafenib (6 μM). The number of intense punctate GFPLC3 staining vesicles was determined in 40 cells (n = 2, +/- SEM).

Figure 5. Vorinostat –induced ROS plays a key role in promoting PTPase inactivation and CD95 tyrosine phosphorylation

Panel A. HEPG2 cells were transfected with empty vector plasmid (CMV) or a plasmid to express thioredoxin (TRX). Twenty four h after transfection cells were treated with vehicle (DMSO) or sorafenib (3 μM) and vorinostat (500 nM). Cells were isolated 48h after exposure and viability determined by trypan blue exclusion (n = 2, +/- SEM). Panel B. HEPG2 cells were plated in 96 well plates and 24h after plating were transfected with empty vector plasmid (CMV) or a plasmid to express thioredoxin (TRX). Twenty four h after transfection cells were treated with vehicle (DMSO) or sorafenib (3 μM) and vorinostat (500 nM). PTPase activity was measured using a commercial kit as described in the Methods (n = 2, +/- SEM). Panel C. Left Section: HuH7 cells transfected with plasmids to express CD95-YFP or CD95-YFP FF, 24h after plating in 8 well chamber slides were treated with vehicle (DMSO) or sorafenib (3 μM), and vorinostat (500 nM) in combination. Cells were fixed after 6h and cell surface CD95 levels determined. Right Section: HEPG2 cells were transfected with empty vector (CMV) or to express either wild type Thioredoxin (TRX). Twenty-four h after transfection cells were treated with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Cells were isolated after 6h and CD95 immunoprecipitated to determine DISC formation and CD95 tyrosine phosphorylation (n = 3, +/- SEM).

Figure 6. Vorinostat promotes sorafenib toxicity by increasing expression of FAS-L

Panel A. Upper IHC: HEPG2 cells plated in 8 well chamber slides were pre-treated with control IgG or an IgG to neutralize FAS-L (1 μg/ml) and 30 min later treated with vehicle (DMSO) or sorafenib (3 μM), and vorinostat (500 nM) in combination. Cells were fixed 6h after exposure and surface levels of CD95 determined by IHC. The density of CD95 staining was determined in 40 cells (n = 2, +/- SEM). Lower Graph: HEPG2 cells were pre-treated with control IgG or an IgG to neutralize FAS-L (1 μg/ml) and 30 min later treated with vehicle (DMSO) or sorafenib (3 μM), and vorinostat (500 nM) in combination. Forty eight h later cells were isolated and viability determined by trypan blue exclusion (n = 3, +/- SEM). Panel B. Left Graph: HEPG2 cells 24h after plating in 96 well plates were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs. Thirty six hours after transfection cells were treated with vehicle (DMSO), sorafenib (3.0 μM), vorinostat (500 nM) or both sorafenib and vorinostat. Cells were assayed for NFκB-luciferase and β-galactosidase activity 24h after treatment (± SEM, a representative from 2 separate studies). Control studies demonstrated that over-expression of dominant negative IκB blocked vorinostat-induced activation of NFκB-luciferase activity (not shown). Right Graph: HEPG2 cells 24h after plating were infected with control empty vector virus (CMV) or a recombinant virus to express dominant negative IκB S32A S36A (dn IκB). Twenty four hours after infection, cells were treated with vehicle (DMSO), sorafenib (3.0 μM), vorinostat (500 nM) or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated, spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods (n = 2, +/- SEM). Panel C. HEPG2 and HEP3B cells 24h after plating were infected with control empty vector virus (CMV) or a recombinant virus to express dominant negative IκB S32A S36A (dn IκB). Twenty four hours after infection, cells were treated with vehicle (DMSO) or vorinostat (500 nM). Cells were isolated 12h and 24h after treatment. SDS PAGE and immunoblotting were performed to determine changes in the expression of CD95 and FAS-L, as indicated. A representative is shown of 3 separate studies.