Cancer Therapy: Preclinical

Vorinostat and Sorafenib Synergistically Kill Tumor Cells via FLIP Suppression and CD95 Activation Guo Zhang,1Margaret A. Park,1Clint Mitchell,1Hossein Hamed,1Mohamed Rahmani,1, 2 Aditi Pandya Martin,1David T. Curiel,7 AdlyYacoub,1,4 Martin Graf,3 Ray Lee,2 John D. Roberts,2 Paul B. Fisher,5,6 Steven Grant,1, 2 , 6 and Paul Dent1,4, 6

Abstract Purpose and Design: Mechanism(s) by which the multikinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal, and pancreatic adenocarci- noma cells has been defined. Results: Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal, and pancreatic adenocarcinoma cells in multiple short-term viability (24-96 h) and in long-term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase-8 and, to a lesser extent, by inhibition of caspase-9. Twenty-four hours after exposure, the activities of extracellular signal-regulated kinase 1/2, AKT, and nuclear factor-nB were only modestly modulated by sorafenib and vorinostat treatment. However, 24 h after exposure, sorafenib- and vorinostat-treated cells exhibited markedly diminished expression of c-FLIP-s, full-length BID, BCL-2, BCL-XL, MCL-1, XIAP, increased expression of BIM, and in- creased activation of BAX, BAK, and BAD. Expression of eIF2a S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP-s levels and overexpression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 associ- ation with caspase-8. Knockdown of CD95 or FADD expression significantly reduced sorafenib/ vorinostat-mediated lethality. Conclusions: These data show that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple antiapoptotic proteins and promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the proapoptotic signals from CD95 to promote mitochondrial dysfunction and death.

In the United States, hepatoma is diagnosed in f19,000 diagnosed in f37,000 and f31,000 patients per annum, patients per annum with f17,000 deaths from the disease, respectively, with f34,000 and f13,000 deaths from each with a 5-year survival rate of <10%.Hepatoma is a leading disease every year (1, 2).Pancreatic and renal cancers have cause of diagnosed cancer in Africa and Asia and represents the 5-year survival rates of <5% and 5% to 10%, respectively.These fifth most commonly diagnosed malignancy in the world statistics emphasize the need to develop novel therapies against (1, 2).In the United States, pancreatic and renal cancer are these lethal malignancies. The Raf/mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2) Authors’ Affiliations: Departments of 1Biochemistry, 2Medicine, 3Neurosurgery, pathway is frequently dysregulated in neoplastic transformation 4 5 6 Radiation Oncology, and Human Genetics and The Institute for Molecular (3–5).The MEK1/2-ERK1/2 module comprises, along with Medicine, Virginia Commonwealth University, Richmond, Virginia and 7Division of Human GeneTherapy, Departments of Medicine, Pathology and Surgery, and the c-Jun NH2-terminal kinase (JNK1/2) and p38 MAPK, members GeneTherapy Center, University of Alabama at Birmingham, Birmingham, Alabama of the MAPK superfamily.These kinases are involved in Received 2/20/08; revised 3/24/08; accepted 4/22/08. responses to diverse mitogens and environmental stresses, Grant support: USPHS grants R01-DK52825, P01-CA104177, and R01- including DNA damage, osmotic stress, and hypoxia, and have CA108520 (P. Dent) and USPHS grants R01-CA63753 and R01-CA77141 and Leukemia Society of America grant 6405-97 (S. Grant). These studies were also also been implicated in multiple cellular functions, including funded in part byThe Jimmy V Foundation. A portion of Dr. Yacoub’s funding is from proliferation, differentiation, and cell survival processes. the Department of Radiation Oncology,Virginia Commonwealth University. P. Dent Although exceptions exist, activation of the ERK1/2 pathway is the holder of the Universal, Inc. Professorship in SignalTransduction Research. is generally associated with cell survival, whereas induction of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance JNK1/2 and p38 MAPK pathways generally signals apoptosis. with 18 U.S.C. Section 1734 solely to indicate this fact. There is also evidence that the net balance of signals related to Requests for reprints: Paul Dent, Department of Biochemistry, Virginia the amplitude and duration of the cytoprotective ERK1/2 and Commonwealth University, 401 College Street, Massey Cancer Center, Room the stress-related JNK1/2 and p38 MAPK pathways determines 280a, Box 980035, Richmond,VA 23298-0035. Phone: 804-628-0861;Fax: 804-827-1309; E-mail: [email protected]. whether a cell lives or dies following various insults (3–5). F 2008 American Association for Cancer Research. Although the mechanism(s) by which ERK1/2 activation doi:10.1158/1078-0432.CCR-08-0469 promotes survival is not known with certainty, several

www.aacrjournals.org 5385 Clin Cancer Res 2008;14(17) September 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical downstream antiapoptotic effector proteins have been identi- in a highly synergistic manner to induce cell death in fied, including direct inactivation of proapoptotic proteins, hepatoma, pancreatic, and renal cell carcinoma cells through such as caspase-9, BAD, and BIM, and increased expression multiple interacting mechanisms, most notably enhanced of antiapoptotic proteins, such as BCL-XL, MCL-1, and c-FLIP activation of the extrinsic apoptotic pathway. (6–11).In view of the importance of the Raf-MEK1/2-ERK1/2 pathway in neoplastic cell survival, clinically relevant inhibitors Materials and Methods have been developed and have now entered clinical trials, including sorafenib (Bay 43-9006, Nexavar; a Raf kinase Materials inhibitor) and AZD6244 (a MEK1/2 inhibitor; refs.12, 13). Sorafenib and sorafenib tosylate (Bay 43-9006 and BAY 54-9085; Sorafenib is a multikinase inhibitor that was originally Bayer) as well as vorinostat (Merck) were provided by the Cancer developed as an inhibitor of Raf-1 but which was subsequently Treatment and Evaluation Program, National Cancer Institute/NIH shown to inhibit multiple other kinases, including platelet- (Bethesda, MD).Phosphorylated/total (ERK1/2, JNK1/2, and p38 derived growth factor, vascular endothelial growth factor MAPK) antibodies, phosphorylated/total AKT (T308; S473), and the receptors 1 and 2, c-Kit, and FLT3 (14).Antitumor effects of total and cleaved caspase-3 antibodies were purchased from Cell sorafenib in renal cell carcinoma and in hepatoma have been Signaling Technology.Anti-BID and anti-cathepsin B antibodies were ascribed to antiangiogenic actions of this agent through purchased from Cell Signaling Technology.All the secondary anti- bodies (anti-rabbit horseradish peroxidase, anti-mouse horseradish inhibition of multiple growth factor receptors (15–17). peroxidase, and anti-goat horseradish peroxidase) were purchased from However, several groups, including our own, have shown Santa Cruz Biotechnology.Enhanced chemiluminescence and terminal in vitro that sorafenib kills human leukemia cells at concen- deoxynucleotidyl transferase–mediated dUTP nick end labeling trations below those achievable in the plasma (e.g., a Cmax (TUNEL) kits were purchased from NEN Life Science Products and of 15-20 Amol/L) through a mechanism involving down- Boehringer Mannheim, respectively.Trypsin-EDTA, DMEM, RPMI regulation of the antiapoptotic BCL-2 family member MCL-1 1640, and penicillin-streptomycin were purchased from Life Technol- (18, 19).In these studies, sorafenib-mediated MCL-1 down- ogies.HEPG2, HEP3B, HuH7, A498, CAKI-1, MiaPaCa2, and PANC1 regulation occurred through a translational rather than a cells were purchased from the American Type Culture Collection. UOK121LN cells were kindly provided by Dr.Lineham Marston transcriptional or posttranslational process that was mediated -/- -/- -/- by endoplasmic reticulum (ER) stress signaling (20, 21).This (NIH).BAK , BAK , and BID fibroblasts were kindly provided by Dr.S.Korsmeyer (Harvard University, Boston, MA).Commercially suggests that the previously observed antitumor effects of available validated short hairpin RNA molecules to knock down RNA/ sorafenib are partially mediated by a complex combination protein levels were from Qiagen: CD95 (SI02654463; SI03118255) of inhibition of Raf family kinases and the ERK1/2 pathway, and FADD (SI00300223; SI03648911).Reagents and performance receptor tyrosine kinases that signal angiogenesis, and the of experimental procedures were in general as described in refs.20, 21, induction of ER stress signaling. 29–33. Histone deacetylase inhibitors (HDACI) represent a class of Methods agents that act by blocking histone deacetylation, thereby in vitro modifying chromatin structure and gene transcription.HDA- Culture and exposure of cells to drugs. All established cell j CIs, along with histone acetyltransferases, reciprocally regulate lines were cultured at 37 C [5% (v/v) CO2] in vitro using RPMI 1640 supplemented with 5% (v/v) FCS and 10% (v/v) nonessential amino the acetylation status of the positively charged NH -terminal 2 acids.For short-term cell killing assays, immunoblotting, and apoptosis- histone tails of nucleosomes.In general, HDACIs promote inducing factor/cathepsin release studies, cells were plated at a density histone acetylation and neutralization of positively charged of 3 Â 103/cm2 (f2 Â 105 per well of a 12-well plate) and, 48 h after lysine residues on histone tails, allowing chromatin to assume a plating, treated with various drugs.Unless otherwise indicated, cells more relaxed, open conformation, which favors gene transcrip- were plated in triplicate and treated with vehicle (DMSO), sorafenib tion (22).However, HDACIs also induce acetylation of (3.0 Amol/L), vorinostat (500 nmol/L), or both sorafenib and many other nonhistone targets, actions that may have vorinostat. In vitro vorinostat and sorafenib treatments were from pleiotropic biological consequences, including inhibition of 100 mmol/L stock solutions of each drug and the maximal concen- HSP90 function, induction of oxidative injury, disruption of tration of vehicle (DMSO) in medium was 0.02% (v/v). Cells were not checkpoint control, and up-regulation of death receptor cultured in reduced serum medium during any study in this article. In vitro cell treatments, microscopy, SDS-PAGE, and Western blot expression (23–25).With respect to combinatorial drug studies analysis. For in vitro analyses of short-term cell death effects, cells were with a multikinase inhibitor such as sorafenib, HDACIs are of treated with vehicle or vorinostat/sorafenib for the indicated times in particular interest in that they also down-regulate/inactivate the figure legends.For apoptosis assays where indicated, cells were multiple oncogenic kinases by interfering with HSP90 function, pretreated with vehicle (DMSO), zVAD (50 Amol/L), calpain inhibitor leading to proteasomal degradation of these proteins.Vorino- [acetyl-calpastatin (amino acids 184-210); 5 Amol/L], or cathepsin B stat (suberoylanilide hydroxamic acid, Zolinza) is a hydroxamic inhibitor ([L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl- acid HDACI that has shown preliminary preclinical evidence L-proline methyl ester; 1 Amol/L); isolated at the indicated times; either subjected to trypan blue cell viability assay by counting in a of activity in hepatoma and other malignancies with a Cmax of f9 Amol/L (26–28).Based on the single-agent activity of light microscope or fixed to slides; and stained using a commercially sorafenib in hepatoma patients, as well as its approval for available Diff-Quick (Giemsa) assay kit.Alternatively, the Annexin V/propidium iodide assay was carried to determine cell viability out as treatment of patients with renal cell carcinoma and hepatocel- per the manufacturer’s instructions (BD PharMingen) using a Becton lular carcinoma, and the fact that sorafenib and vorinostat Dickinson FACScan flow cytometer.Vorinostat/sorafenib lethality, as target multiple overlapping pathways implicated in tumor cell judged by Annexin V-propidium iodide, was first evident f24 h after survival, the possibility arose that a combined approach might drug exposure (data not shown). be more effective than either agent administered individually. For immunohistochemistry of cells fixed in situ, fixed cells were The present studies reveal that sorafenib and vorinostat interact blocked 1 h with antibody dilution buffer [2% (v/v) rat serum, 1%

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(w/v) bovine serum albumin in Dulbecco’s PBS] and then incubated 2 mmol/L MgCl2, 200 mmol/L h-mercaptoethanol, 1.34 mg/mL overnight at 4jC in antibody dilution buffer with addition of anti- O-nitrophenyl-h-D-galactopyranoside] were added to 50 AL cell lysate CD95 antibody (2 Ag/mL; Abcam).Cells were then washed and and incubated at 37jC for 10 min.The product of the assay was incubated for 1 h with a 488 nm–tagged secondary antibody measured at an absorbance of 405 nm.( c) Transfection with siRNA: (Invitrogen).Cells were washed, coverslipped, and analyzed on a Cells were plated in 60-mm dishes from a fresh culture growing in log fluorescent microscope (Â100 magnification). phase as described above and, 24 h after plating, transfected.Before For SDS-PAGE and immunoblotting, cells were plated at 5 Â transfection, the medium was aspirated and 1 mL serum-free medium 105/cm2, treated with drugs at the indicated concentrations and after was added to each plate.For transfection, 10 nmol/L of the annealed the indicated time of treatment, and lysed in whole-cell lysis buffer siRNA, the positive sense control double-stranded siRNA targeting [0.5 mol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1% h- glyceraldehyde-3-phosphate dehydrogenase or the negative control mercaptoethanol, 0.02% bromphenol blue], and the samples were (a ‘‘scrambled’’ sequence with no significant homology to any known boiled for 30 min.The boiled samples were loaded onto 10% to 14% gene sequences from mouse, rat, or human cell lines), were used.siRNA SDS-PAGE and electrophoresis was run overnight.Proteins were (10 nmol/L; scrambled or experimental) was diluted in serum-free electrophoretically transferred onto 0.22-Am nitrocellulose and immu- medium.HiperFect (4 AL; Qiagen) was added to this mixture and the noblotted with various primary antibodies against different proteins. solution was mixed by pipetting up and down several times.This All immunoblots were visualized by enhanced chemiluminescence. solution was incubated at room temperature for 10 min and then added For presentation, immunoblots were digitally scanned at 600 dpi using dropwise to each dish.The medium in each dish was swirled gently to Adobe Photoshop CS2, and their color was removed and figures were mix and then incubated at 37jC for 2 h.One milliliter of 10% (v/v) generated in Microsoft PowerPoint. serum-containing medium was added to each plate, and cells were Infection of cells with recombinant adenoviruses. Cells were plated incubated at 37jC for 48 h before replating (50 Â 103 each) onto at 3 Â 103/cm2 in each well of a 12-well, 6-well, or 60-mm plate.After 12-well plates.Cells were allowed to attach overnight and then treated plating (24 h), cells were infected (hepatoma and pancreatic carcinoma; with vorinostat/sorafenib (0-48 h).Trypan blue exclusion/TUNEL/flow at a multiplicity of infection of 50; UOK121LN renal carcinoma at a cytometry assays and SDS-PAGE/immunoblotting analyses were done multiplicity of infection of 400) with a control empty vector virus at the indicated time points. [cytomegalovirus (CMV)] and adenoviruses to express CrmA, c-FLIP-s, Isolation of a crude cytosolic fraction. A crude membrane fraction BCL-XL, and XIAP or to express dominant-negative AKT/MEK1/InB/ was prepared from treated cells.Briefly, cells were washed twice in ice- caspase-9 or activated MEK1/AKT (Vector BioLabs).Twenty-four hours cold isotonic HEPES buffer [10 mmol/L HEPES (pH 7.5), 200 mmol/L after infection, cells were treated with the indicated concentrations of mannitol, 70 mmol/L sucrose, 1 Amol/L EGTA, 10 Amol/L protease vorinostat/sorafenib and/or other drugs, and cell survival or changes inhibitor cocktail (Sigma)].Cells on ice were scraped into isotonic in expression/protein phosphorylation were determined 0 to 96 h HEPES buffer and lysed by passing 20 times through a 25-gauge needle. after drug treatment by trypan blue/TUNEL/flow cytometry assays and Large membrane pieces, organelles, and unlysed cells were removed immunoblotting, respectively. from the suspension by centrifugation for 5 min at 120 Â g.The crude Transfection of cells with small interfering RNA or with plasmids. (a) granular fraction and cytosolic fraction were obtained by centrifugation For plasmids: Cells were plated as described above and, 24 h after for 30 min at 10,000 Â g, leaving the cytosol as supernatant. plating, transfected with a variety of constructs.For mouse embryonic In vivo exposure of carcinoma tumors to drugs. Athymic female fibroblasts (MEF; 2-5 Ag) or other cell types (0.5 Ag), plasmids NCr-nu/nu mice were obtained from The Jackson Laboratory.Mice were expressing a specific mRNA [or small interfering RNA (siRNA)] or maintained under pathogen-free conditions in facilities approved by appropriate vector control plasmid DNA was diluted in 50 AL serum- the American Association for Accreditation of Laboratory Animal Care free and antibiotic-free medium (one portion for each sample). and in accordance with current regulations and standards of the U.S. Concurrently, 2 AL Lipofectamine 2000 (Invitrogen) was diluted into Department of Agriculture (Washington, DC), the U.S. Department of 50 AL of serum-free and antibiotic-free medium (one portion for each Health and Human Services (Washington, DC), and the NIH.HEP3B sample).Diluted DNA was added to the diluted Lipofectamine 2000 for cells were cultured and isolated by trypsinization followed by cell each sample and incubated at room temperature for 30 min.This number determination using a hemacytometer.Cells were resuspended mixture was added to each well/dish of cells containing 200 AL serum- in PBS and 10 million tumor cells per 100 AL PBS were injected into the free and antibiotic-free medium for a total volume of 300 AL, and the right rear flank of each mouse, and tumors were permitted for form to cells were incubated for 4 h at 37jC.An equal volume of 2 Â medium a volume of f150 mm3 over the following 6 wk.The tumor take rate was then added to each well.Cells were incubated for 48 h, then treated for HEP3B tumors was f20%.Vials of vorinostat (stored in a -20 jC with vorinostat/sorafenib, and subsequently analyzed.( b) For nuclear cold room under vacuum and protected from light; f30 mg/vial) were factor-nB (NF-nB) promoter-luciferase assays: Cells or MEFs were plated first dissolved in 30 AL DMSO, diluted in sterile saline, and heated to as described above and, 24 h after plating, transfected with either a boiling for complete dissolution before injection.Mice were adminis- control luciferase plasmid (1 Ag) + h-galactosidase plasmid (15 ng) or tered 25 mg/kg vorinostat by oral gavage based on body mass (0.2 mL luciferase plasmid with 4Â nuclear factor-nBconsensusbinding total volume per 30 g mouse).Animals received two more admin- sequences (1 Ag) + h-galactosidase plasmid (15 ng), incubated for istrations of vorinostat, 24 h apart, for an additional 2 d.Sorafenib was 5 min in serum-free medium, then added to GeneJuice (2 AL per administered 30 min before the first vorinostat administration each condition; EMD Biosciences), and incubated for 15 min together at day.Sorafenib tosylate (BAY 54-9085) was dissolved fresh each day. room temperature.This mixture was added to cells and incubated at The dosing volume used is 0.3 mL/30 g body weight. The compound 37jC for 24 h, after which cells were treated with vorinostat/sorafenib was dissolved in a 50% Cremophor EL/50% ethanol mixture.The for 0 to 24 h, then washed twice with PBS, and harvested in cell lysis mixture was heated to 60jC and sonicated for 20 min to solubilize. buffer [25 mmol/L Tris phosphate (pH 7.8), 2 mmol/L DTT, 2 mmol/L Once the BAY 54-9085 entered solution, the aqueous component was CDTA, 10% glycerol, 1% (v/v) Triton X-100].The lysate was centrifuged added gradually and with mixing to generate the 1Â dosing solution. for 5 min at 13,000 Â g at 4jC to pellet debris.The luciferase assay was Animals were administered with BAY 54-9085 for a final concentration done according to the manufacturer’s instructions (Promega).Briefly, of 45 mg/kg.Animals received two more administrations of vorinostat, luciferase substrate was brought to room temperature, then added to 24 h apart, for an additional 2 d.Each animal not receiving a dose of 20 AL lysate, and measured immediately on a Perkin-Elmer lumin- sorafenib or vorinostat at the time of drug treatment was administered ometer.The luciferase measurement was normalized to h-galactosidase diluent alone in a volume equal to the amount given with the drug. Â measurement to control for transfection efficiency; 50 ALof2 Ex vivo manipulation of tumors. Animals were euthanized by CO2 h-galactosidase reagent [200 mmol/L Na2HPO4/NaH2PO4 (pH 7.4), and placed in a BL2 cell culture hood on a sterile barrier mat.The

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Fig. 1. Sorafenib and vorinostat interact in a synergistic fashion to kill pancreatic, liver, and kidney tumor cells in colony formation assays. A, pancreatic (PANC-1) and hepatoma (HEP3B) cells were plated as single cells (250-1,500 per well) in sextuplicate and, 12 h after plating, treated with vehicle (VEH ;DMSO),sorafenib (Sor.; 3.0-6.0 Amol/L), or vorinostat (Vor. ; 250-500 nmol/L) or with both drugs combined, as indicated at a fixed concentration ratio to do median dose effect analyses for the determination of synergy. After drug exposure (48 h), the medium was changed and cells were cultured in drug-free medium for an additional 10 to 14 d.Cellswere fixed and stained with crystal violet, and colonies of >50 cells/colony were counted. Columns, true percentage inhibition of colony formation plotted from the means of sextuplicate plates from two separate experiments (n = 3 total studies); bars, SE. *, P < 0.05, greater cell killing than compared with any other treatment condition. Colony formation data were also entered into the CalcuSyn program and CI values were determined. A CI value of <1.00 indicates synergy: CI values for PANC-1and HEP3B were all below 0.70. B, HEPG2 cells were plated in triplicate and treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. After drug exposure (48 h), cells were isolated, treated with AnnexinVand propidium iodide according to the manufacturer’s instructions, and stained using these established methods. Cells were subjected to flow cytometry to determine the numbers of AnnexinV ^ positive and propidium iodide ^ positive cells F SE. Data shown are from a representative of three independent studies. *, P < 0.05, greater cell killing than compared with any other treatment condition. C, UOK121LN and HEPG2 cells were plated in triplicate and treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. After drug exposure (48 and 96 h), cells were isolated and stained using trypan blue indicative of later stages of cell death/plasma membrane disruption as described in Materials and Methods. Columns, percentage of trypan blue ^ positive cells; bars, SE. Data are from a representative of three independent studies. *, P < 0.05, greater cell killing than compared with any other treatment condition.

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Vorinostat and Sorafenib bodies of the mice were soaked with 70% (v/v) ethanol and the skin the remainder of the tumor was placed in 5 mL of Streck tissue fixative around the tumor was removed using small scissors, forceps, and a (Fisher Scientific) in a 50 mL conical tube for H&E fixation.The tumor disposable scalpel.These implements were flame sterilized between sample that had been placed in RPMI 1640 was minced with a sterile removal of the outer and inner layers of skin.A piece of the tumor disposable scalpel into the smallest possible pieces and then placed in (f50% by volume) was removed and placed in a 10-cm dish a sterile disposable flask.The dish was rinsed with 6.5mL of RPMI containing 5 mL of RPMI 1640 cell culture medium on ice.In parallel, 1640, which was then added to the flask.A 10 Â solution of collagenase (2.5 mL, 28 units/mL final concentration; Sigma) and 10Â of enzyme mixture containing DNase (308 units/mL final concentration; Sigma) and Pronase (22,500 units/mL final concentration; EMD Sciences) in a volume of 1 mL was added to the flask.The flasks were placed into an orbital shaking incubator at 37jC for 1.5 h at 150 rpm. Following digestion, the solution was passed through a 0.4-Am filter into a 50 mL conical tube.After mixing, a sample was removed for viable and total cell counting using a hemacytometer.Cells were centrifuged at 500 Â g for 4 min, the supernatant was removed, and fresh RPMI 1640 containing 10% (v/v) FCS was added to give a final resuspended cell concentration of 1 Â 106 cells/mL.Cells were diluted and plated in 10-cm dishes in triplicate at a concentration of 2 Â 103 to 6 Â 103 per dish for control, sorafenib, and vorinostat treatments and 4 Â 103 to 10 Â 103 per dish for combined sorafenib and vorinostat exposure. Immunohistochemistry and staining of fixed tumor sections. After sacrifice, tumors were fixed in OCT compound (Tissue-Tek) and cryostat sectioned (Leica) as 12-Am sections.Nonspecific binding was blocked with a 2% (v/v) rat sera, 1% (v/v) bovine sera, 0.1% (v/v) Triton X-100, and 0.05% (v/v) Tween 20 solution and then sections were stained for cell signaling pathway markers: cleaved caspase-3 (rabbit IgG, 1:100; Cell Signaling Technology), phosphorylated AKT1/ 2/3 (S473; mouse IgG, 1:100; Santa Cruz Biotechnology), and phosphorylated ERK1/2 (mouse IgG, 1:100; Santa Cruz Biotechnolo- gy).For staining of sectioned tumors, primary antibodies were applied overnight, sections were washed with phosphate buffer solution, and secondary antibodies were applied for detection [as indicated in the figure; goat anti-rat Alexa Fluor 488/647 (1:500; Invitrogen) and goat anti-mouse Alexa Fluor 488/647 (1:500; Invitrogen) secondary anti- body as per the primary antibody used] or detected by way of 3,3¶- diaminobenzidine substrate peroxidase detection kit (Biogenex) as per the manufacturer’s instructions.Sections were then dehydrated, cleared, and mounted with coverslips using 4¶,6-diamidino-2-phenylindole mounting medium (Vectastain).Apoptotic cells with double-stranded DNA breaks were detected using the Upstate TUNEL Apoptotic Detection kit according to the manufacturer’s instructions.Slides were applied to high-powered light/confocal microscopes (Zeiss LSM 510 Meta confocal scanning microscope; Zeiss HBO 100 microscope with AxioCam MRm camera) at the indicated magnification in the figure/ figure legend.The proliferation zone that included both tumor and normal tissue was usually selected as the site of interest within 2 mm of, or juxtaposed to, the leading edge of the tumor. Data analysis. Comparison of the effects of various treatments was done using ANOVA and the Student’s t test.Differences with a P value of <0.05 were considered statistically significant. Experiments shown are the means of multiple individual points (FSE).Median dose effect isobologram analyses to determine synergism of drug interaction were done according to the methods of Chou and Talalay using the CalcuSyn program for Windows (Biosoft).Cells are treated with agents at a fixed Fig. 2. Inhibition of caspase-8 and cathepsin function suppresses sorafenib and concentration dose.A combination index (CI) value of <1.00indicates vorinostat lethality in gastrointestinal tumor cells. A, HEPG2 cells plated in triplicate, synergy of interaction between the drugs, a value of 1.00 indicates as indicated, were infected to express empty vector (CMV ), dominant-negative additivity, and a value of >1.00 equates to antagonism of action caspase-9 (dn casp. 9), caspase-8 inhibitor CrmA, or dominant-negative caspase-9 and CrmA. Cells were treated with vehicle, sorafenib, vorinostat, or both sorafenib between the agents. and vorinostat. Ninety-six hours after drug exposure, cells were isolated and viability was determined by trypan blue assay. Columns, percentage of trypan blue ^ positive cells; bars, SE. Data are from the mean of three independent studies. Results #, P < 0.05, less cell killing than compared with parallel condition in vehicle-treated cells. B, SV40 largeTantigen ^ transformed MEFs lacking expression of various proapoptotic genes were plated in triplicate and treated with vehicle, sorafenib, Treatment of human liver, pancreatic, and kidney tumor cell vorinostat, or both sorafenib and vorinostat. Forty-eight hours after drug exposure, lines with increasing low relatively concentrations of vorinostat cells were isolated and viability was determined by trypan blue assay. Columns, and sorafenib at a fixed dose ratio resulted in a synergistic percentage of trypan blue ^ positive cells; bars, SE. Data are from the mean of three independent studies. #, P < 0.05, less cell killing than compared with parallel enhancement in tumor cell killing as measured by median condition in vehicle-treated cells. dose effect long-term colony formation assays (Fig.1A;

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Supplementary Figs.S1 and S2).Reexpression of the VHL killing assays, examined the interaction between vorinostat and protein in 786 renal carcinoma cells did not significantly alter sorafenib.In HEPG2 and UOK121LN cells, TUNEL, Annexin V- the synergistic interaction of vorinostat and sorafenib (data not propidium iodide, and trypan blue exclusion viability assays shown).Additional studies, using a variety of short-term cell generated quantitatively and qualitatively similar data (i.e.,

Fig. 3. Sorafenib and vorinostat treatment modulates the expression of c-FLIP-s, BCL-XL, and MCL-1in tumor cells. A, HEPG2 cells were plated and treated 24 h after plating with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Six and 24 h after drug exposure, cells were isolated and subjected to SDS-PAGEfollowedby immunoblotting to determine the expression of BID, procaspase-8, procaspase-3, XIAP, BCL-2, BCL-XL, MCL-1, c-FLIP-s, phosphorylated eIF2a S51 (P-eIF2a S51), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ERK2. Data are from a representative study (n =3).B, HEPG2 cells infected to express empty vector (CMV), BCL-XL, XIAP, or c-FLIP-s. Cells were treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Ninety-six hours after drug exposure, cells were isolated and viability was determined by trypan blue assay. Columns, percentage of trypan blue ^ positive cells; bars, SE. Data are from the mean of three independent studies. #, P < 0.05, less cell killing than compared with parallel condition in vehicle-treated cells. C, HEPG2 and UOK121LN cells were plated and treated 24 h after plating with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Six and 24 h after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS-PAGE followe d by immunoblotting to determine the expression of BIM, procaspase-8, BAD S112 phosphorylation, BAX activation, BAK activation, and glyceraldehyde-3-phosphate dehydrogenase. Data are from a representative study (n =3).

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Vorinostat and Sorafenib greater than additive induction of cell killing following low- had just begun to display signs of cell death at this interval (e.g., dose vorinostat and sorafenib exposure compared with either time course data in Fig.1; data not shown).Thus, the observed agent individually; Fig.1B and C; Supplementary Figs.S3 reductions in prosurvival protein expression occurred before and S4).Substratum attachment did not alter the sensitivity significant manifestations of cell killing. of A498 cells to vorinostat and sorafenib exposure (data not The ability of overexpression of proteins whose levels were shown).Based on the encouraging findings in Fig.1, the reduced in Fig.3A and in Supplementary Figs.S8 and S9 molecular mechanisms by which vorinostat and sorafenib to prevent cell killing was then tested.Overexpression of interacted to kill cells were then investigated. BCL-XL, XIAP, or c-FLIP-s significantly reduced vorinostat and In HEPG2 and MiaPaCa2 cells, pan-inhibition of caspase sorafenib lethality (Fig.3B; Supplementary Fig.S10).Whether function using zVAD significantly reduced vorinostat and the expression and/or activity of additional proapoptotic sorafenib lethality, as did inhibition of cathepsin protease proteins correlated with increased cell killing was also function; in UOK121LN cells, the relative role of caspases in the determined.Treatment of cells with vorinostat and sorafenib death process seemed to be greater than that of cathepsins increased the expression of BIM, including promotion of BIM (Supplementary Figs.S5-S7).Inhibition of caspase-8 function dephosphorylation as well as the dephosphorylation of BAD (e.g., by ectopic expression of CrmA) significantly reduced, to a S112 and the activation of BAX and BAK (Fig.3C).Based on greater extent than inhibition of caspase-9 (e.g., by ectopic data showing that c-FLIP-s overexpression largely abolished expression of dominant-negative caspase-9), the lethality of low-dose vorinostat and sorafenib lethality, regardless of the vorinostat and sorafenib in our panel of tumor cells (Fig.2A; fact that in parallel drug exposure also suppressed expression of Supplementary Figs.S5-S7).Similar cell killing data after multiple downstream prosurvival proteins and activated BAX vorinostat and sorafenib treatment were obtained using the and BAK, the possibility that vorinostat- and sorafenib-induced caspase-8 and caspase-9 inhibitors IETD and LEHD, respective- killing was death receptor dependent, specifically CD95 (FAS ly (data not shown).Based on data showing that inhibition receptor) dependent, was explored. of caspase-8 and caspase-9 significantly reduced vorinostat and Notably, knockdown of FADD or CD95 expression signifi- sorafenib lethality, genetically modified transformed MEFs cantly reduced the lethality of low-dose combined vorinostat lacking expression of various cell survival modulator genes and sorafenib exposure in HEPG2 and UOK121LN cells were used.Combined loss of BAX and BAK function or loss of (Fig.4A; Supplementary Fig.S11).Vorinostat and sorafenib BID function significantly reduced vorinostat and sorafenib exposure in HEPG2 and UOK121LN cells, in a cell type– lethality in transformed fibroblasts (Fig.2B).These findings dependent fashion, also enhanced expression of FAS-L and/or suggest that lysosomal dysfunction plays a role in the killing CD95 proteins as well (Supplementary Fig.S12).In addition, process and that caspase-8 signaling and the extrinsic pathway combined treatment of HEPG2 and UOK121LN cells with are also involved in vorinostat- and sorafenib-induced trans- vorinostat and sorafenib promoted the rapid association of formed cell lethality. procaspase-8 with CD95 (i.e., DISC complex formation; To further define the processes of cell death, immunoblotting Fig.4C).Knockdown of CD95 abolished drug-induced analyses in vorinostat- and sorafenib-treated HEPG2 and procaspase-8 and BID cleavage in total cell lysates (data not UOK121LN cells were done (Fig.3; Supplementary Fig.S8). shown).Studies in primary hepatocytes treated with death- In UOK121LN cells, within 6 h of combined, but not indivi- inducing natural compounds that act via CD95, such as toxic dual, drug exposure, the expression of the proforms of BID, bile acids, have shown that these agents cause plasma caspase-8, and caspase-3 (Supplementary Fig.S8) declined membrane localization and clustering of CD95 as part of the relative to vehicle control–treated cells.These observations receptor activation/hepatocyte killing process (34, 35).Treat- correlated with decreased expression of BCL-2 and c-FLIP-s ment of HEPG2 cells with vorinostat and sorafenib caused (Supplementary Fig.S8).The cleaved form of BID was poorly increased plasma membrane localization of CD95 and the visualized by immunoblotting in all studies using this drug appearance of intense-staining punctate bodies containing combination (data not shown).Twenty-four hours after drug CD95, demonstrative of CD95 clustering and its activation exposure, the expression of BCL-2, BCL-XL, MCL-1, c-FLIP-s, (Fig.4D).Note that in HEPG2 cells, CD95 activation and DISC BID, procaspase-8, and procaspase-3 had further declined after formation occurred at time points (i.e., 6 h) without alteration combined, but not individual, drug exposure. in either total protein levels of CD95 or FAS-L/FAS-L cleavage, In HEPG2 cells, little obvious change in the expression of any arguing that CD95 activation was ligand independent.Over- protein was observed 6 h after combined drug exposure with expression of c-FLIP-s significantly suppressed vorinostat and the exception of c-FLIP-s and procaspase-3, whereas 24 h after sorafenib lethality as measured in TUNEL assays and markedly combined, but not individual, vorinostat and sorafenib diminished cytochrome c release into the cytosol of HEPG2 exposure, expression of BID, procaspase-8, procaspase-3, XIAP, cells (Supplementary Fig.S13). BCL-2, BCL-XL, MCL-1, and c-FLIP-s was all reduced (Fig.3A). We next defined how combined exposure to vorinostat and In both HEPG2 and UOK121LN cells, decreased expression of sorafenib could rapidly suppress the expression of multiple prosurvival proteins correlated with increased phosphorylation prosurvival proteins.High doses of sorafenib, >3 Amol/L as of eIF2a S51; increased phosphorylation of eIF2a S51 is known a single agent, have been shown by our laboratories to cause to correlate with increased activity of this protein and with ER stress, translational inhibition, and reduced expression of suppression of translation/initiation in cells (Fig.3A; ref.21). MCL-1 that correlated with eIF2a phosphorylation (19, 21); in Similar immunoblotting data to that obtained in HEPG2 and the present studies, lower doses of sorafenib (f3 Amol/L) do UOK121LN cells in Fig.3A was obtained in MiaPaca2 cells not enhance eIF2a phosphorylation but did synergize with (Supplementary Fig.S9).Of note, cell viability data obtained vorinostat to cause eIF2a phosphorylation.Expression of 24 h after drug exposure argued that drug-treated tumor cells dominant-negative eIF2a S51A abolished low-dose combined

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Fig. 4. Sorafenib and vorinostat interact to kill tumor cells via activation of CD95 and suppression of c-FLIP-s expression. A, HEPG2 cells were transfected with a nonspecific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24 h after transfection with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Ninety-six hours after drug exposure, cells were isolated and viability was determined by trypan blue assay. Columns, percentage of trypan blue ^ positive cells (n = 3); bars, SE. #, P < 0.05, less cell killing than compared with parallel condition in vehicle-treated cells. B, HEPG2 cells were plated and treated 24 h after plating with vehicle or with sorafenib and vorinostat. Cells were isolated at the indicated time points after sorafenib and vorinostat exposure and CD95 was immunoprecipitated from the cell lysate. SDS-PAGE followed by immunoblotting of CD95 immunoprecipitates was done to determine the association of procaspase-8 and caspase-8 with CD95 (n =3).C, HEPG2 and UOK121LN cells were plated on glass slides and treated 24 h after plating with vehicle (DMSO) or with sorafenib and vorinostat. Six hours after drug exposure, cells were fixed in situ. Fixed cells were blocked, incubated overnight with anti-CD95 antibody, and then incubated with a 488 nm ^ tagged fluorescent secondary antibody. Cells were analyzed on a fluorescent microscope. Magnification, Â100.The intensity of CD95 staining was determined at 50 random points per cell for a total of five cells F SE (n = 3 separate studies).

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Table 1. The synergy of killing by sorafenib and vorinostat is dependent on loss of c-FLIP-s expression

MiaPaca2: CMV Hep3B: CMV Sorafenib Vorinostat Fraction CI Sorafenib Vorinostat Fraction CI (Mmol/L) (Mmol/L) affected (Mmol/L) (Mmol/L) affected

3.00 0.250 0.34 0.32 3.0 0.250 0.43 0.47 4.50 0.375 0.42 0.40 4.5 0.375 0.56 0.58 6.00 0.500 0.50 0.45 6.0 0.500 0.74 0.58

MiaPaca2: c-FLIP-s Hep3B: c-FLIP-s

Sorafenib Vorinostat Fraction CI Sorafenib Vorinostat Fraction CI (Amol/L) (Amol/L) affected (Amol/L) (Amol/L) affected

3.00 0.250 0.16 0.98 3.0 0.250 0.26 0.90 4.50 0.375 0.21 1.23 4.5 0.375 0.40 0.97 6.00 0.500 0.26 1.42 6.0 0.500 0.54 1.08

NOTE: Pancreatic (MiaPaca2) and hepatoma (HEP3B) cells were infected to express empty vector (CMV) or c-FLIP-s. Twenty-four hours after infection, cells were plated as single cells (250-1,500 per well) in sextuplicate and, 12 h after plating, treated with vehicle (DMSO), sorafenib (3.0-6.0 Amol/L), or vorinostat (250-500 nmol/L) or with both drugs combined, as indicated at a fixed concentration ratio to do median dose effect analyses for the determination of synergy. Forty-eight hours after drug exposure, the medium was changed and cells were cultured in drug-free medium for an additional 10 to 14d. Cells were fixed and stained with crystal violet, and colonies of >50 cells/colony were counted. Colony formation data were also entered into the CalcuSyn program and CI and fraction affected values were determined. A CI value of <0.90 to 1.00 indicates synergy, a CI value of 0.90 to 1.10 approximates to additive interactions between the drugs, and a CI value of >1.10 indicates antagonism (n = 2 independent studies).

sorafenib- and vorinostat-induced suppression of c-FLIP-s and Supplementary Fig.S14).Overexpression of c-FLIP-s in hepa- MCL-1 expression in HEP3B cells and expression of eIF2a toma and pancreatic cancer cells suppressed the synergistic S51A in transformed fibroblasts and in HEP3B cells suppressed lethality of vorinostat and sorafenib in median dose effect the toxic interaction between sorafenib and vorinostat (Fig.4D; colony formation assays, reducing the CI value from f0.45 to

Fig. 4 Continued. D, blotting section: HEP3B cells were transfected either with an empty vector plasmid (CMV) or to express dominant-negative eIF2a S51A.Twenty-four hours after plating, cells were treated with vehicle (DMSO) or with sorafenib (S;3.0Amol/L) and vorinostat (V; 500 nmol/L). Cells were isolated 6 or 24 h, as indicated, after drug exposure and the expression of c-FLIP-s and glyceraldehyde-3-phosphate dehydrogenase was determined at each time point. A representative study (n = 2) is shown. Graphical section: transformed MEFs [wild-type (WT); expressing eIF2a S51A] were plated in triplicate and treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Forty-eight hours after drug exposure, cells were isolated and viability was determined by trypan blue assay. Columns, percentage of trypan blue ^ positive cells; bars, SE. Data are from the mean of three independent studies. #, P < 0.05, less cell killing than compared with parallel condition in vehicle-treated cells.

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Fig. 5. Delayed inactivation of ERK1/2 and AKTcorrelates with profound and long-term loss of c-FLIP-s expression: delayed activation of NF-nB is a toxic signal following sorafenib and vorinostat exposure. A, HEPG2 cells, 24 h after plating, were treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Cells were isolated 24 to 96 h after drug exposure and subjected to SDS-PAGE and immunoblotting to determine the phosphorylation of ERK1/2, JNK1/2, p38 MAPK, and AKT(S473 )aswellas total ERK2. A representative of three separate studies is shown. B, top, HEPG2 cells, 24 h after plating, were infected with empty vector control virus (CMV) or viruses to express constitutively active AKT( caAKT) and constitutively active MEK1EE (caMEK1EE).Twenty-four hours after infection, cells were treated with vehicle or with sorafenib and vorinostat. Forty-eight hours after drug exposure, cells were isolated and subjected to SDS-PAGE and immunoblotting to determine the expressionofc-FLIP-saswell as total ERK2 levels. Bottom, HEPG2 cells, 24 h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1EE, or both the active AKTand active MEK1EE viruses.Twenty-four hours after infection, cells were treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. Ninety-six hours after drug exposure, cells were isolated and viability was determined by trypan blue assay. Columns, percentage of trypan blue ^ positive cells; bars, SE. Data are from the mean of three independent studies. #, P < 0.05, less cell killing than compared with parallel condition in vehicle-treated cells; ##, P < 0.05, less cell killing than compared with parallel condition in constitutively active AKT^ treated or constitutively active MEK1EE ^ treated cell s. C, left, HEPG2 cells, 24 h after plating in triplicate, were transfected with NF-nB-luciferase (NF-jB-luc)andh-galactosidase (b-gal) constitutive reporter constructs. Parallel control plates of cells (data not shown) were transfected with NF-nB-luciferase and h-galactosidase constitutive reporter constructs and cotransfected with a plasmid to express dominant-negative InB or with a plasmid to express a constitutively activated form of NF-nB as internal negative and positive controls. Thirty-six hours after transfection, cells were treated with vehicle, sorafenib, vorinostat, or both sorafenib and vorinostat. At the indicated times, cells were isolated and the amount of luciferase per cell and the amount of h-galactosidase per cell were determined as described in Materials and Methods. Columns, mean from two separate studies; bars, SE. Control studies showed that overexpression of dominant-negative InB blocked vorinostat-induced activation of NF-nB-luciferase activity (data not shown). Right, HEPG2 cells, 24 h after plating in triplicate, were infected with control empty vector virus (CMV) or a recombinant virus to express dominant-negative InBS32AS36A(dn IjB). Twenty-four hours after infection, cells were treated with vehicle, sorafenib, vorinostat, 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 Materials and Methods. Columns, percentage ofTUNEL-positive cells; bars, SE. Data are from a representative of two independent studies. #, P < 0.05, less cell killing than compared with the same condition in CMV-infected cells.

Clin Cancer Res 2008;14(17) September1,2008 5394 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Vorinostat and Sorafenib f1.10 (Table 1). These findings argue that low doses of MAPK pathway signaling (23–25, 36–39).Furthermore, vorinostat and sorafenib induce activation of the extrinsic changes in NF-nB, ERK1/2, AKT, JNK1/2, and p38 MAPK apoptotic pathway at the level of the CD95 death receptor and signaling have been linked to the modulation of CD95 that the initial rapid loss of c-FLIP-s expression within 24 function and that of c-FLIP-s, expression, also in a cell h occurs via activation of eIF2a and plays a critical role in type–dependent manner.These considerations prompted transmitting death signals from the plasma membrane to the further examination of whether significant alterations in cytosol, resulting in a pleiotropic activation of multiple signaling pathway function occurred in vorinostat/sorafenib- downstream apoptotic processes, including mitochondrial treated cells and whether these changes could be related to dysfunction. CD95 activation, changes in apoptosis-regulatory protein Sorafenib was originally developed as an inhibitor of Raf expression, and overall tumor cell survival. family protein kinases but subsequently shown to be an Twenty-four hours after drug exposure, a time at which inhibitor of several receptor tyrosine kinases and an inducer CD95 activation had occurred, eIF2a phosphorylation had of ER stress signaling (12, 14, 21).Vorinostat has been shown occurred, and the expression of multiple prosurvival proteins to modulate in a concentration-dependent and cell type– such as c-FLIP-s had already declined; no profound change in dependent manner ERK1/2, NF-nB, AKT, JNK1/2, and p38 the basal activities of ERK1/2, AKT, JNK1/2, or p38 MAPK was

Fig. 5 Continued. D, HEP3B cells were injected (107) into the flanks of athymic mice and tumors were permitted to form. The tumor take rate was f20%.Tumors were permitted to grow to f15 0 mm 3, after which time tumors were segregated based on volume into relatively normalized groups. Animals/tumors in triplicate were subjected to vehicle/drug administration by oral gavage once daily for 3 consecutive days. BAY 54-9085 was administered at a dose of 45 mg/kg.Vorinostat was administered at a dose of 25 mg/kg. Twelve hours after the final gavage dosing, the animals were humanely sacrificed and the tumors were removed and sectioned. One portion of thetumorwas subjected to immunohistochemical analyses (left) to determine eIF2a phosphorylation, ERK1/2 phosphorylation, AKT(S473) phosphorylation, c-FLIP-s expression, MCL-1 expression, caspase-3 cleavage status,TUNEL positivity, and tumor morphology by H&E staining. Another portion of the tumor was macerated and digested to obtain i ndividual tumor cells. Individual viable isolated tumor cells were plated at 2,000 to 10,000 per 10-cm radius dish and colonies were permitted to form over the following 10 d. Cells were fixed and stained with crystal violet, and colonies of >50 cells/colony were counted. The colony formation for vorinostat-treated cells was 0.78 F 0.04 and the colony formation for vorinostat- and sorafenib-treated cells corrected for the toxicity of sorafenib was 0.58 F 0.06. #, P < 0.05, colony formation for vorinostat- and sorafenib-treated cells corrected for the toxicity of sorafenib less than colony formation for vorinostat-treated cells.

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MAPK pathways did not correlate strongly with cell death Finally, we did in vivo analyses using established f150 mm3 induction.In contrast to signaling pathways that promote HEP3B flank tumors to determine whether sorafenib and death, activities of pathways that protect against cell death vorinostat interacted in a toxic manner in vivo.We noted that began to decline in cells treated with vorinostat and sorafenib unselected clones of HEP3B and HEPG2 cells are poorly 24 to 96 h after drug exposure.Within 48 h of exposure, tumorigenic in the flanks of athymic mice and form tumors ERK1/2 was inactivated in combined drug-exposed cells; that rapidly become necrotic on growth beyond >200 mm3, within 72 h of exposure, AKT became inactivated.Inactivation potentially due to a relatively low CD31 staining (data not of the ERK1/2 pathway was not due to the prior activation of shown).As such, we chose an in vivo treatment, ex vivo colony CD95 or FADD, in view of the findings that knockdown of formation assay approach, to assess tumor cell killing and long- CD95 or FADD suppressed cell killing but did not maintain term survival.A 3-day treatment of animals with either ERK1/2 phosphorylation (Supplementary Fig.S15).Thus, vorinostat or sorafenib caused little alteration in the cleavage inactivation of ERK1/2 and AKT were relatively late events in status of caspase-3 or the TUNEL positivity of flank tumor cell death induction after sorafenib and vorinostat exposure sections (Fig.5D, left).Combined exposure of animals/tumors but were not causally dependent on the primary CD95- to vorinostat and sorafenib caused a large increase in both the dependent apoptotic signal. number of apoptotic TUNEL-positive cells and a large increase Based on the findings in Fig.5A, attempts were made to in immunoreactivity for the cleaved form of caspase-3. determine whether expression of constitutively active MEK1 Combined, but not individual, exposure of animals/tumors and/or AKT protected cells from vorinostat and sorafenib to vorinostat and sorafenib caused a large increase in eIF2a exposure.Expression of constitutively active MEK1 maintained phosphorylation and large decreases in the expression of ERK1/2 phosphorylation in HEPG2 cells treated with vorinostat c-FLIP-s and MCL-1 and phosphorylation of AKT (S473).Of and sorafenib, as did expression of constitutively active AKT particular note, at the in vivo concentrations of sorafenib used in maintaining levels of AKT S473 phosphorylation (Supplemen- as a single agent in our study, we observed near total inhibition tary Fig.S16).Expression of either activated MEK1 or activated of ERK1/2 phosphorylation by the drug, but this inhibition of AKT almost abolished the toxicity of the individual drugs and ERK1/2 phosphorylation did not correlate with the cleavage significantly suppressed the toxicity of the drug combination of caspase-3, strong enhancement of TUNEL positivity, or (Fig.5B, bottom).These findings correlated with maintenance of profoundly lower c-FLIP-s or MCL-1 levels.In ex vivo colony c-FLIP-s expression in tumor cells expressing activated MEK1 formation assays using viable cells isolated from treated tumors and activated AKT and treated with vorinostat and sorafenib after cessation of drug treatment, and with cells cultured in the (Fig.5B, top).Collectively, these data further argue that absence of any drug in vitro, combined exposure of animals/ maintained c-FLIP-s expression prevents CD95 signaling from tumors to sorafenib and vorinostat caused a greater reduction activating the caspase-8-BID pathway to induce mitochondrial in cell survival of the explanted tumor cells growing ex vivo than dysfunction and death.These findings also argue that primary was observed in the cells that had been exposed to either drug activation of eIF2a followed by the secondary inhibition of individually (Fig.5D, right).Collectively, these findings argue ERK1/2 and AKT represents the likely sequence of events by that our molecularly defined markers for sorafenib and which low doses of sorafenib and vorinostat suppress c-FLIP-s vorinostat lethality are observed in vitro and also in drug- levels and subsequently maintain suppression of c-FLIP-s treated tumors and that these effects correlate with an increase expression in transformed cells. in both short-term and long-term tumor cell killing. Prior studies using vorinostat have shown that this agent activates the transcription factor NF-nB and that this can act Discussion against the lethal actions of this drug (29, 36).Whether NF- nB function plays any role in cell survival after low-dose vorinostat Previous studies from our laboratories have shown that and sorafenib treatment in our cell system was then examined. sorafenib and vorinostat interact in vitro in a greater than Treatment of HEPG2 and UOK121LN cells with vorinostat additive fashion to kill malignant hematologic cells.In chronic caused a late post-24 h exposure-induced activation of NF-nB, myelogenous leukemia cells, sorafenib- and vorinostat-induced which was not significantly altered by incubation of cells with cell killing correlated with decreased expression of the low doses of sorafenib (Fig.5C; Supplementary Fig.S17).As mitochondrial protective protein MCL-1 and the cyclin kinase noted above, NF-nB activation following vorinostat treatment inhibitor p21Cip-1/WAF1/mda6 (20).The present studies attemp- has been shown as a protective signal in malignant hematologic ted to determine whether sorafenib and vorinostat also cells, we determined whether genetic inhibition of NF-nB interacted to kill malignant epithelial cells attached to a function via expression of the super-repressor InB S32A S36A substratum and, if so, to elucidate the mechanism responsible altered the survival response of drug-treated carcinoma cells. for this phenomenon. Expression of InB S32A S36A–inhibited vorinostat-induced The results of the present study indicate that low clinically activation of NF-nB and did not significantly alter the lethality relevant concentrations of sorafenib and vorinostat interact in of vorinostat as a single agent (Fig.5C; Supplementary Fig.S17; a synergistic manner to kill liver, kidney, and pancreatic tumor data not shown).However, inhibition of NF- nB function cells in vitro. In vitro, the enhanced lethality of the regimen significantly suppressed the death of cells treated with sorafenib toward transformed cells was blocked by inhibition of and vorinostat.Our findings argue that hyperactivation of CD95 or FADD function and abolished by overexpression of ERK1/2 and AKT can suppress killing that may be due to c-FLIP-s.Significantly, the protective effect of c-FLIP-s maintained expression of c-FLIP-s and that the observed late- overexpression was observed despite the fact that sorafenib

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Vorinostat and Sorafenib and vorinostat, but not the drugs individually, suppressed mechanism by which vorinostat lethality is enhanced by expression of multiple downstream mitochondrial protective sorafenib in carcinoma cells. proteins, including BCL-2, BCL-XL, and MCL-1, and increased Prior studies from this group have shown that sorafenib in the expression/activity of multiple downstream mitochondrial the f10 to 15 Amol/L range, which are within pharmacolog- toxic proteins, including BAX, BAK, BIM, BID, and BAD, ically achievable concentrations, induced cell death in human events thought to act independently of CD95 and c-FLIP-s leukemic cells by promoting ER stress and diminishing to also lower cell viability.These findings suggest that one expression of MCL-1 rather than by inhibiting either Raf family central mechanism of sorafenib and vorinostat lethality in kinases or receptor tyrosine kinases (14–16, 20, 21).The epithelial tumor cells involves primary activation of the present results in epithelial cancer cells showed that much extrinsic apoptosis pathway, in concert with diminished lower concentrations of sorafenib (f3 Amol/L) as a single c-FLIP-s levels, which in turn facilitate secondary activation agent did not significantly alter MCL-1 levels or increase the of a destabilized intrinsic apoptosis pathway (Supplementary phosphorylation of eIF2a, as was also the case for vorinostat Fig.S18). exposure.However, treatment of carcinoma cells with both Inhibition of cathepsin B protease function also suppressed sorafenib and vorinostat resulted in a large increase in the the toxicity of sorafenib and vorinostat.Cathepsin proteases are phosphorylation of PERK8 as well as its substrate eIF2a. secreted enzymes as well as localized in the cell within acidic Collectively, these findings are suggestive of ER stress signaling. endosomes, and in some studies, cathepsins have been shown Findings in malignant hematologic cells argued strongly that to promote cell death by cleaving the caspase-8 substrate BID down-regulation of the short-lived protein (half-life, 2-4 h) independently of caspase-8, thereby causing mitochondrial MCL-1 by translational inhibition played an important dysfunction (e.g., ref. 31). In the present studies, overexpression functional role in the lethality of the sorafenib and vorinostat of the caspase-8–specific inhibitor c-FLIP-s abolished drug- drug combination.MCL-1 is a multidomain antiapoptotic induced cell killing in liver, kidney, and pancreatic tumor cells, member of the BCL-2 family that acts through several arguing that for cathepsin proteases to act as apoptotic mechanisms, including cooperation with BCL-XL, to block mediators in sorafenib- and vorinostat-treated cells first mitochondrial outer membrane permeabilization by BAK and requires activation of caspase-8.The expression and activity of BAX and to prevent apoptosis (18).Translation inhibition of cathepsin proteases is frequently increased in malignant cells MCL-1 expression was independent of ERK1/2 inactivation. and the secreted forms of these enzymes have been shown to Notably, MCL-1 down-regulation was also observed in sorafe- a play a key role in promoting invasion and angiogenesis, two nib/vorinostat-treated epithelial tumor cells in an eIF2 - critical characteristic features of the malignant phenotype dependent fashion.Additional studies will be required to (40, 41).Death receptor signaling (the extrinsic apoptosis define the functional roles of ER stress signaling and the pathway) is generally viewed as a pathway that proceeds via unfolded protein response in sorafenib and vorinostat lethality and relationship between these events, the activation of CD95 caspase-8 signaling to induce both BID cleavage and mito- and enhanced CD95/FAS-L expression that occurred shortly chondrial dysfunction or directly to procaspase-3 cleavage in following CD95 activation/DISC formation, and the rapid both cases culminating in cell death (4–6).However, cathepsin suppression of prosurvival protein expression (e.g., MCL-1 and proteases have also been shown to play a dynamic role in c-FLIP-s) in malignant epithelial cells. tumor necrosis factor-a and FAS-stimulated cell death processes HDACIs, such as vorinostat, kill tumor cells through highly wherein cathepsin proteases cooperate with caspase-8 to cause diverse mechanisms, including blocking histone deacetylation, mitochondrial dysfunction and cell death (40, 41).Palacios and have been proposed to down-regulate/inactivate multiple et al.(42) recently showed that the cyclin-dependent kinase oncogenic kinases by interfering with HSP90 function, leading inhibitor flavopiridol (100 nmol/L) potentiated the lethality of to proteasomal degradation of these proteins.Prior studies in tumor necrosis factor–related apoptosis-inducing ligand in breast cancer cells have established that low (e.g., 500 nmol/L) MDA-MB-231 mammary tumor cells and that this effect was concentrations of vorinostat, which are substantially below the due to the suppression of c-FLIP-s/l expression by flavopiridol. 9 Amol/L Cmax of this agent, promoted activation of caspase- Analogously, interactions between much higher concentrations 8 and that cleavage of BID was involved in drug lethality, in of sorafenib and tumor necrosis factor–related apoptosis- agreement with the present data involving liver, kidney, and inducing ligand in malignant hematopoietic cells have been pancreatic tumor cells (29).Disruption of signal transduction related to inhibition of c-FLIP protein translation (43).Others, pathway function, reducing the activities within the Raf-MEK- also using much higher concentrations of sorafenib, have ERK and phosphatidylinositol 3-kinase–phosphoinositide- observed c-FLIP and MCL-1 expression suppression by this dependent protein kinase 1–AKT pathways, will exert pleiotro- agent, which is also likely due to profound suppression of pic actions in tumor cells, including destabilization of c-FLIP-s transcription and translation (44).Inasmuch as cathepsins play and BCL family member proteins.Inhibition of these path- a proapoptotic role in sorafenib- and vorinostat-treated cancer ways was not readily measurable by immunoblotting over the cells, such findings raise the possibility that tumor cells that initial 24 h after drug exposure but could, nevertheless, survive exposure to these agents may do so by down-regulating significantly contribute at later times to diminished expression cathepsin expression, which may result in diminished angio- of prosurvival molecules such as BCL-XL and MCL-1 and to genesis and a less invasive tumor.It also argues that inhibition increased expression of prodeath molecules such as BIM of cyclin-dependent kinase 9 function or suppression of protein (3–6).Hence, although the initial insult of vorinostat and translation by sorafenib (compare our data showing increased eIF2a phosphorylation; see also refs.18, 19, 21), events that induce c-FLIP-s/l down-regulation, may represent a common 8 G. Zhang, M.A. Park, and P. Dent, unpublished observation.

www.aacrjournals.org 5397 Clin Cancer Res 2008;14(17) September 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical sorafenib exposure caused CD95 activation and translational of vorinostat increase tumor necrosis factor-a expression in repression, shifting the apoptotic rheostat of a cell toward cell epithelial tumor cells and whether this represents a portion death and away from cell survival, the secondary destabiliza- of its toxic biology via activation of the extrinsic pathway tion of Raf-MEK-ERK and phosphatidylinositol 3-kinase- independently of CD95 are beyond the scope of the present PDK1-AKT pathway function, whether by changes in HSP90 article. activity or other unknown factors, likely prevented any form In conclusion, our data indicate that sorafenib and vorinostat of compensatory survival response being mounted by the interact in a highly synergistic manner to kill liver, kidney, and tumor cell resulting in the death response becoming ‘‘locked pancreatic tumor cells in vitro.They also show that this effect in place.’’ translates into an in vivo model system using hepatoma cells Studies in leukemia and lymphoma cells have shown that growing in the flanks of athymic mice.Ongoing in vitro and vorinostat can enhance cell killing in these cells particularly animal studies and phase I studies in renal and liver cancer with when vorinostat-induced NF-nB activation has been suppressed these agents are presently defining in greater detail the via expression of a dominant-negative InB protein (e.g., refs. mechanism(s) of action of these drugs and their putative clinical 45–47).In leukemic cells, the actions of the extrinsic pathway relevance. in vorinostat lethality were not noted as a primary effector in the actions of vorinostat, although they could possibly play a Disclosure of Potential Conflicts of Interest role.We were surprised to discover, however, in hepatoma, renal, and pancreatic tumor cells that low-dose vorinostat- No potential conflicts of interest were disclosed. induced activation of NF-nB had little effect on cell survival, as previously observed in breast cancer cells, and that when Acknowledgments combined with sorafenib, NF-nB activation facilitated the cell killing process (29).Activation of NF- nB in some cell systems P. Dent thanks Dr. S. Lin for assisting Dr. G. Zhang in these studies and Dr. O. n Korchinsky for assistance with NF-KB-luciferase assays and the training of Dr. M.A. has been linked to cell death processes, and NF- B can increase Park for these assays. the expression of death receptor ligands, such as tumor necrosis This manuscript is dedicated to the corresponding author’s Great AuntVera in her factor-a, at a transcriptional level (48, 49).Whether low doses continued fight against renal cell carcinoma.

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Guo Zhang, Margaret A. Park, Clint Mitchell, et al.

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