nelfinavir induces liposarcoma apoptosis through ... · and extracellular signal-regulated kinase...

Cancer Therapy: Preclinical

Nelfinavir Induces Liposarcoma Apoptosis through Inhibitionof Regulated Intramembrane Proteolysis of SREBP-1 and ATF6

Min Guan1, Kristen Fousek1, Chunling Jiang1, Song Guo1, Tim Synold1, Bixin Xi1,Chu-Chih Shih2, and Warren A. Chow1,3

AbstractPurpose: We previously reported that nelfinavir (NFV) induces G1 cell-cycle block and apoptosis

selectively in liposarcoma cell lines due to increased SREBP-1 (sterol regulatory element binding protein-1)

expression in the absence of increased transcription. We postulate that NFV interferes with regulated

intramembrane proteolysis of SREBP-1 and ATF6 (activating transcription factor 6).

Experimental Design: Time-lapse, confocal microscopic studies show that NFV inhibits the nuclear

translocation of full-length SREBP-1–EGFP and ATF6–EGFP fusion proteins. siRNA-mediated knockdown

of site-1 protease (S1P) and/or site-2 protease (S2P) leads to inhibition of SREBP-1 intracellular trafficking

to the nucleus and reduces liposarcoma cell proliferation. Treatment of LiSa-2 liposarcoma cells with 3,4-

dichloroisocoumarin, a serine protease inhibitor of S1P, did not affect SREBP-1 processing. In contrast,

1,10-phenanthroline, an S2P-specific inhibitor, reproduces the molecular and biological phenotypes

observed in NFV-treated cells, which implicates S2P as a target of NFV. In vivo evaluation of NFV in a

murine liposarcoma xenograft model leads to inhibition of tumor growth without significant toxicity.

Results:NFV-induced upregulation of SREBP-1 and ATF6 results from inhibition of S2P, which together

with S1P mediates regulated intramembrane proteolysis from their precursor to their transcriptionally

active forms. The resulting endoplasmic reticulum (ER) stress and concurrent inhibition of the unfolded

protein response induce caspase-mediated apoptosis.

Conclusions: These results provide new insight into the mechanism of NFV-mediated induction of ER

stress and cell death in liposarcomas and are the first to report targeting S2P for cancer therapy. Clin Cancer

Res; 17(7); 1796–1806. �2011 AACR.

Introduction

The HIV protease inhibitor (PI) nelfinavir (NFV; Vira-cept) has shown promising anticancer activity via theinduction of apoptotic cell death, induction of endoplas-mic reticulum (ER) stress and autophagy, inhibition ofepidermal growth factor receptor– and insulin-like growthfactor receptor–mediated PI3K (phosphoinositide 3-kinase)/Akt activation (1–4), and induction of chemo-and radiosensitization (5). NFV also decreases VEGF/hypoxia-inducible factor-1a expression, tumor hypoxia,and angiogenesis (6). In addition, NFV inhibits STAT3and extracellular signal-regulated kinase 1/2 (7). Such

pleiotropic activities show that NFV has multiple off-targeteffects.

Liposarcomas arise from adipocytes or their precursors(8). Chemotherapy for recurrent or metastatic liposarco-mas is generally palliative (9). We hypothesized that NFVwould inhibit liposarcoma growth because HIV PI use islinked to a clinical syndrome of peripheral lipoatrophyknown as "HIV protease-induced lipodystrophy syn-drome" (10). In this syndrome, adipocyte apoptosis isobserved and associated with alteration of sterol regulatoryelement binding protein-1 (SREBP-1) expression (11–13).

SREBPs are synthesized as inactive, membrane-boundprecursors in the ER, tethered at their carboxy-terminaldomain by SREBP cleavage-activating protein (SCAP; refs.14, 15). In the presence of cholesterol, SCAP binds insulin-induced gene-1 or gene-2 (Insig-1 or Insig-2) in the ER,preventing SCAP-mediated proteolytic processing and acti-vation of SREBPs (16). On cholesterol depletion, SCAPand Insig fail to interact, the SREBP–SCAP complex istransported to the Golgi apparatus, where it is processedin 2 sequential proteolytic cleavage steps by site-1 protease(S1P), a subtilisin-like serine protease, and site-2protease (S2P), a metalloprotease protease, to release thetranscriptionally active amino-terminal fragment to thenucleus in an evolutionarily conserved process, regulated

Authors' Affiliations: 1Department of Molecular Pharmacology, BeckmanResearch Institute of the City of Hope; and Departments of 2Hematology/Hematopoietic Cell Transplantation and 3Medical Oncology and Thera-peutics Research, City of Hope, Duarte, California

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Warren A. Chow, Beckman Research Institute ofthe City of Hope, 1500 East Duarte Road, Duarte, CA 91010. Phone: 626-256-4673 (ext. 63712); Fax: 1-626-301-8233. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-3216

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(7) April 1, 20111796

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intramembrane proteolysis (RIP; refs. 16–18). SREBP-1 is akey transcription factor necessary for adipogenesis, choles-terol biosynthesis, and adipocyte differentiation (19). Itstarget genes include genes related to cholesterol synthesissuch as fatty acid (FA) synthase (FASN; ref. 20) and anti-proliferative and proapoptotic genes such as p21WAF1/CIP1,Fas, and Bax (21, 22).Most proteins fold and mature in the ER lumen. An

imbalance between the load of unfolded proteins that enterthe ER (ER stress) and the capacity of the cellular machinerythat handles this load [unfolded protein response (UPR)]eventually triggers apoptosis (23, 24). Activating transcrip-tion factor 6 (ATF6) is a precursor ER-bound transcriptionfactor that activates UPR genes. When unfolded proteinsaccumulate in the ER, ATF6 is processed by RIP by usingS1P and S2P to release its amino-terminal domain analo-gous to SREBPs (18).Our group has shown that NFV induces a G1 cell-cycle

block and apoptosis selectively in liposarcoma cell lines asa consequence of upregulation of SREBP-1 expression (3).In this article, we show that NFV-induced upregulation ofSREBP-1 and ATF6 results from inhibition of S2P, leadingto prevention of intracellular transport of mature SREBP-1and ATF6 to the nucleus. These results provide new insightinto the mechanism for NFV-mediated ER stress in lipo-sarcomas.

Materials and Methods

Cell cultureSW872 dedifferentiated liposarcoma was purchased

from the American Type Culture Collection (25). LiSa-2pleomorphic liposarcoma cell line was a kind gift from Dr.Silke Br€uderlein (University of Ulm, Ulm, Germany; ref.26) and maintained in Iscove’s modified Dulbecco’s med-ium/RPMI in a 4:1 ratio supplemented with 10% FBS, 2mmol/L L-glutamine, and 0.1 mg/mL gentamicin. The cells

were DNA fingerprinted to confirm identity as previouslydescribed (27).

Chemicals and reagentsNFV was obtained from the NIH AIDS Research and

Reference Reagent Program. M8, the primary, active in vivoNFV metabolite was obtained from Pfizer, Inc. Stock solu-tions of NFV and M8 were made in dimethyl sulfoxide(DMSO). 3,4-Dichloroisocoumarin (DCI) and 1,10-phe-nanthroline were purchased from Sigma-Aldrich. SREBP-1,FASN, and GRP78 antibodies were purchased from SantaCruz Biotechnology, Inc., and ATF6 antibody was pur-chased from Abcam, Inc.

Apoptosis, clonogenicity, and proliferation assaysLiSa-2 cells were treated with NFV for 24 hours for

caspase 9/6 assay according to the manufacturer’s protocol(Clontech) or Annexin V detection (Santa Cruz Biotech-nology, Inc.) by flow cytometry. Clonogenic assay wascarried out in NFV-treated LiSa-2 cells, according to theprotocol previously described (3). A fluorescence-baseddigital image microscopy system (DIMSCAN; BioimagingSolutions Inc.) was used to quantify the number of viableNFV- or M8-treated LiSa-2 cells as previously described(28). Briefly, 1,000 cells were seeded in a 96-well plate inmultiples of 10 wells and treated with NFV or M8.

Protein T1/2 assay and Western blot analysisSW872 cells were treated with 10 mmol/L NFV or DMSO

for 4 hours, followed by cycloheximide (100 mmol/L), forthe indicated time. Cell lysates were harvested to detectSREBP-1 or ATF6 by Western blotting. FASN and GRP78were detected in lysates prepared from LiSa-2 cells treatedwith NFV for 24 hours.

Plasmid and quantitative RT-PCRpTK-HSV-BP1a encoding human SREBP-1a and pCGN-

ATF6 encoding human ATF6 were kindly provided by Dr.Guosheng Liang (UT Southwestern Medical School, Dallas,TX) and Dr. Amy Lee (University of Southern California,Los Angeles, CA). cDNA encoding SREBP-1a and ATF6wereamplified from pTK-HSV-BP1a and pCGN-ATF6 by PCR(Supplementary Data). The PCR fragments were insertedinto pEGFP-C2 (Clontech) to obtain fusion EGFP–amino-terminal SREBP-1 and ATF6 plasmids (pSREBP-1–EGFP,pATF6–EGFP). Quantitative reverse transcriptase PCR(qRT-PCR) was carried out in total RNA extracted fromsiRNA-treated LiSa-2 cells with the ABI Prism 7900 HTSequence Detection System (Applied Biosystems; Supple-mentary Data). Relative gene expression quantificationmethod was used to calculate the fold change of mRNAexpression according to the comparative Ct method usingb-actin as an endogenous control.

S1P, S2P siRNA knockdown assayLiSa-2 cells (5 � 105 per well) were cultured in 6-well

plates overnight prior to transfection. Human S1P and S2Pgene–specific siRNA and scrambled siRNA (Santa Cruz

Translational Relevance

This article describes a novel anticancer pathwayelucidated for the HIV protease inhibitor nelfinavir(NFV). The mechanism involves inhibition of regulatedintramembrane proteolysis through inhibition of site-2protease activity. This results in accumulation of pre-cursor sterol regulatory element binding protein-1(SREBP-1) and activating transcription factor 6(ATF6). The resulting accumulation of unfoldedSREBP-1 and ATF6 results in overwhelming endoplas-mic reticulum stress and an impaired unfolded proteinresponse. The liposarcoma cells respond by undergoingapoptosis. This novel approach to cancer therapeutics isheretofore unreported. A clinical trial for NFV in lipo-sarcoma supported by an FDA Orphan Products Devel-opment grant (R01 FD003006) is currently ongoing,which has shown early activity (NCT00233948: A PhaseI/II Study of Nelfinavir in Liposarcoma; ref. 34).

Nefinavir Inhibits Proteolysis of SREBP-1 and ATF6

www.aacrjournals.org Clin Cancer Res; 17(7) April 1, 2011 1797



Biotechnology, Inc.) were transfected into cells with Lipo-fectamine (Invitrogen Corp.). After 48-hour incubation,the transfected cells were collected for qRT-PCR of S1P andS2P or Western blot of SREBP-1 or subjected to the DIMS-CAN assay.

The DIMSCAN assay was used to quantify the number ofviable siRNA-treated LiSa-2 cells. A total of 2,000 cells wereseeded in a 96-well plate in multiples of 10 wells, followedby transfection with S1P, S2P, or S1P and S2P or scrambledsiRNA. Twenty-four hours later, the cells were transfectedwith pSREBP-1–EGFP. At 48 and 72 hours after siRNAstransfection, cells were stained and scanned.

Confocal microscopy time-lapse imagingOn day 0, LiSa-2 cells were plated in a 2-well chamber

slide at 3 � 105 cells per well. On day 2, cells weretransfected with pSREBP-1–EGFP or pATF6–EGFP. Onday 3, transfected cells were stained with Hoechst 33322(Invitrogen) at final concentration of 1 mg/mL for 5 min-utes and transferred to the Microscopy Core Lab. Cells weremaintained in the live cell chamber at 37�C in 5% CO2

during confocal microscopy. To start the time-lapse experi-ment, DMSO or 10 mmol/L NFV was added to the pre-treated wells. The first image (t ¼ 0 hour) was recorded 5minutes later. The N-terminal EGFP-labeled SREBP-1 orATF6 was visualized with a Zeiss LSM510 META NLOAxiovert microscope, using a 488 argon laser (green)and chameleon 2P laser (blue) and the objective lens(LD-AChroplan 40�/0.6NAwith correction collar). Digitalimages were captured every 2 hours for 12 hours with nosignificant loss of signal.

In the siRNA knockdown study, S1P, S2P, or equalamounts of S1P and S2P siRNA were transfected on day2. On day 3, cells were transfected with pSREBP-1–EGFP.On day 4, nucleus was stained for time-lapse imaging asdescribed. The first image (t ¼ 0 hour) was recorded 24hours after pSREBP-1–EGFP transfection (48 hours aftersiRNA transfection) and recorded every 2 hours for 12hours. Images were generated using Multi-Time Macro inthe LSM 510 software and was analyzed using LSM ImageBrowser and Concatenation Macro software.

In vivo evaluation of NFV in a murine xenograftliposarcoma model

A heterotopic murine liposarcoma model was estab-lished with human LiSa-2 cells in severe-combinedimmunodeficient (SCID) mice. Log-phase growth cellswere harvested and resuspended in PBS at 1 � 107 cells/mL. One hundred microliters of cells was injected sub-cutaneously into the flanks of 6- to 8-week-old femaleSCID mice. Tumors that developed were surgicallyremoved at week 5, cut into 2-mm3 pieces, and placedinto a surgically prepared subcutaneous pocket in therecipient mice. The recipient mice were allowed torecover 7 days before treatment with NFV (n ¼ 7) orcontrol (n ¼ 7). The average starting weight of the micewas 20 gm and tumor volume (V) was determined by theequation: V ¼ 1/2(l � w2).

Given our previous experience, prolonged twice-dailyoral gavage was not considered feasible. NFV 625 mgtablets were purchased from the City of Hope Pharmacyand crushed into a fine powder. NFV powder was admixedwith Transgenic Dough Diet (Bio-Serv) to dose at 500 mg/kg/d. Mouse feed was replaced daily with 4 g of TransgenicDough Diet alone (control) or admixed with NFV. Tumorswere bidimensionally measured, and the mice wereweighed 3 times weekly.

NFV pharmacokinetics in SCID miceNFV powder was resuspended in 1% carboxymethylcel-

lulose to 100 mg/mL. Groups of three 8- to 10-week-oldfemale SCIDmice underwent oral gavagewith 500mg/kg ofNFV in carboxymethylcellulose and then euthanized at 0, 1,2, 4, 8, and 24 hours after oral gavage. Blood was collectedand plasmawas separated for analysis ofNFV concentrationwith anUPLC-tandemmass spectrometric assay, developed,and validated in the Analytical Pharmacology Core Facilityat the City of Hope (Supplementary Data).

Measurement of ER stressS1P, S2P, or equal amounts of S1P and S2P siRNA were

transfected on day 1. Cell lysates for Western blot detectionof GRP78were prepared from the transfected cells on day 3.NFV-treated LiSa-2 cells were collected at 24 hours forWestern blot detection of GRP78. Thapsigargin (TG; 0.5mmol/L) served as a positive control for ER stress induction.

Statistical analysisData are presented as the mean � SD of 3 independent

experiments. Group comparisons for continuous data weredone with Student’s t test for independent means or 2-wayANOVA.

Results

NFV induces caspase-dependent apoptosisCaspase activation was analyzed in NFV-treated LiSa-2

and SW872 cells. As shown in Figure 1A, activity level ofcaspase 9/6 increases in a dose-dependent fashion andreaches a maximum of 4 and 5 fold over baseline activityat the highest dose (20 mmol/L). Clonogenic assays in LiSa-2 cells (Fig. 1B) confirm NFV inhibits clonogenicity in adose-dependent manner.

M8, the primary NFV metabolite, induces dose-independent cell death

NFV is metabolized in vivo to its hydroxyl-t-butylamidemetabolite, M8, which possesses similar in vitro antiviralactivity as its parent compound. Figure 1C reveals similar(day 2) or slightly higher survival (day 6) in M8-treatedLiSa-2 cells thanNFV at the same doses. This shows that M8possesses comparable antitumor activity as NFV.

NFV increases SREBP-1 protein half-lifeOur previous data showed NFV leads to significantly

elevated expression of precursor SREBP-1 (125 kDa) in

Guan et al.

Clin Cancer Res; 17(7) April 1, 2011 Clinical Cancer Research1798



the absence of increased transcription (3). Consequently,protein stability of SREBP-1 was evaluated in the presenceof NFV. DMSO-treated SW872 cells show degradation ofprecursor SREBP-1 (125 kDa) by 1 hour and its completeabsence by 4 hours (Fig. 2A), along with increased detec-tion of processed SREBP-1 (68 kDa) up to 24 hours. Incontrast, NFV leads to accumulation of precursor SREBP-1 and minimal detection of its processed form through 24hours (Fig. 2A). The results are shown quantitatively inFigure 2A, with the half-life (T1/2) of 2 hours for controland 13 hours for NFV-treated cells. Similarly, ATF6(Fig. 2B) was also tested in LiSa-2 cells and shows asimilar dose-dependent increase of precursor ATF6 (90kDa) by NFV and prolonged T1/2 from 6 to 9 hours(Supplementary Data). Importantly, NFV did not alterSREBP-1 ubiquitination or acetylation (SupplementaryData).

NFV inhibits intracellular trafficking of SREBP-1 andATF6To explore whether the increased T1/2 of precursor

SREBP-1 or ATF6 results from NFV-mediated inhibitionof RIP, plasmids encoding the full-length SREBP-1 or ATF6gene fused in frame at their amino terminus with EGFP

were constructed and transfected into LiSa-2 cells to moni-tor the trafficking of their mature forms from the cytoplasm(ER-Golgi) to the nucleus. As shown in Figure 2C and D,control cells show intense green fluorescence throughoutthe nucleus by 12 hours, which reflects normal RIP of theprecursor SREBP-1–EGFP fusion protein to its transcrip-tionally active form. In contrast, the NFV-treated cells showretention of the fusion protein in the cytoplasm andminimal movement into the nucleus at 12 hours. Similarly,inhibition of RIP of the precursor ATF6 was observed, asshown in Figure 2D. These data show that NFV inhibitsintracellular trafficking of SREBP-1 and ATF6 by inhibitingRIP.

NFV inhibits FASN expressionFASN is a transcriptional target of SREBP-1 (29). Accord-

ingly, FASN was evaluated in NFV-treated LiSa-2 cells toevaluate the effect of inhibition of SREBP-1 production ondownstream targets. Figure 2B shows NFV dose depen-dently inhibits FASN expression (confirmed by FASNqRT-PCR gene expression; Supplementary Data), consis-tent with the hypothesis that NFV interferes with SREBP-1processing.

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Figure 1. Nelfinavir and its metabolite M8 induce caspase-dependent apoptosis and dose-dependent cell death in liposarcoma. A, caspase9/6 activation was analyzed in LiSa-2 (left) and SW872 (right) cells treated with 0, 2.5, 10, or 20 mmol/L nelfinavir (NFV). After 24 hours, cells wereharvested for caspase analysis. B, clonogenic assay in pretreated LiSa-2 cells with NFV. C, DIMSCAN assay was performed in NFV- or M8-treatedLiSa-2 cells. A total of 1,000 cells seeded in a 96-well plate for overnight incubation, followed by treatment with NFV or M8 (0, 2.5, 10, or 20 mmol/L) for 2 or 6days. Each experiment was done in triplicate. Results are presented as mean � SD. *, P < 0.05; **, P < 0.01 compared with DMSO control (0 mmol/L).





siRNA-mediated inhibition of S1P or S2P blocksintracellular trafficking of SREBP-1 and reducesliposarcoma proliferation

Both SREBP-1 and ATF6 are cleaved by S1P and S2Pduring RIP. To further explore the mechanism of NFV-induced accumulation of unprocessed SREBP-1 and ATF6,siRNAs targeting S1P, S2P, or S1P plus S2P were transfectedinto LiSa-2 cells. siRNA reduced target S1P or S2P RNA levelby more than 90% (Fig. 3A). Western blot confirms anaccumulation of precursor SREBP-1 (Fig. 3A) in S1P and/orS2P siRNA–transfected cells. Notably, time-lapse micro-scopy shows the absence of nuclear green fluorescent

protein in both S1P and S2P siRNA–transfected cellswhereas control cells show diffuse nuclear fluorescence(Fig. 3B and C). These results indicate that inhibition ofS1P and S2P results in potent inhibition of SREBP-1trafficking to the nucleus. Significantly, combined S1Pand S2P knockdown leads to the greatest inhibition ofSREBP-1 movement, as shown by nearly complete exclu-sion of fluorescence in the nucleus (Fig. 3D). To determinethe consequence of S1P and S2P knockdown on cell pro-liferation, a fluorescence-based assay, DIMSCAN, was car-ried out. Figure 4A shows that S1P or S2P siRNA reducescell survival to 85% of control at 72 hours. Notably, both

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Figure 2.Nelfinavir increases SREBP-1 protein half-life (T1/2) and inhibits intracellular trafficking of SREBP-1 and ATF6. A, SREBP-1 T1/2 determination. Lysatefrom SW872 cells pretreated with 10 mmol/L NFV or DMSO for 4 hours followed by cycloheximide (100 mmol/L) for the indicated time points was analyzed byWestern blot. Relative SREBP-1 expression was quantitated by Image Quant (Molecular Dynamics). B, dose-dependent induction of precursor ATF6 andFASN reduction byNFV. LiSa-2 cell was treated with NFV at the indicated dose for 24 hours and lysate was harvested forWestern blot analysis. C, visualizationof SREBP-EGFP transport from cytoplasm to nucleus in NFV-treated cells. A total of 3� 105 LiSa-2 cells/well were seeded in glass-bottomed 2-well chamberslide for overnight incubation and transfected with pSREBP-1-EGFP, cells were then cultured 24 hours to allow transgene expression before staining withHoechst 33322 1 mg/ml for 5 minutes. The live cell image was captured and recorded at indicated time points using a confocal microscopy (t = 0 hour,5 minutes after addition of 10 mmol/L nelfinavir or DMSO). D, visualization of transport of ATF6-EGFP from cytoplasm to nucleus in NFV-treated LiSa-2 cells.Cells were transfected with pATF6-EGFP for time-lapse imaging. Bar, 20 mm.

Guan et al.




siRNAs reduce survival by 50%. These results show thatinhibition of S1P- and/or S2P-mediated RIP of SREBP-1leads to cell death, which supports the notion that S1P and/or S2P may be potential targets of NFV.

Blocking S2P function with a small molecule inhibitorreproduces the NFV-treated phenotypeTo more closely examine whether the activity observed

for NFV was attributable to the inhibition of S1P or S2P,small molecule inhibitors were utilized. DCI is a potentserine PI of recombinant S1P (30). It does not alter theprecursor SREBP-1 or ATF6 accumulation (Fig. 4B). In

contrast, treatment of LiSa-2 cells with 1,10-phenanthro-line, a metalloprotease-specific S2P inhibitor, leads to bothdose- and time-dependent accumulation of precursorSREBP-1 and ATF6 (Fig. 4C), similar to the phenotypeobserved for NFV (31). Likewise, 1,10-phenanthrolineleads to dose-dependent apoptosis (Fig. 4D), which highlysuggests that NFV inhibits the proteolytic activity of S2P.

NFV inhibits liposarcoma growth in a murine modelTo determine whether NFV possesses in vivo anticancer

activity, a heterotopic SCID murine liposarcoma modelwas established and the mice were treated with NFV 500

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Figure 3. siRNA-mediated inhibition of S1P or S2P blocks intracellular trafficking of SREBP-1. A, siRNA knockdown of S1P (top) and S2P (bottom).LiSa-2 cells were transfected with S1P, S2P or scrambled siRNA by Lipofectamine RNAiMAX. The transfected cells were cultured for 48 hours and collectedfor S1P or S2P qRT-PCR and Western blot for SREBP-1 detection. **, P < 0.01 compared to control siRNA. B, visualization of transport of SREBP-EGFP inS1P siRNA treated LiSa-2 cells. A total of 3� 105 cells were seeded on day 1 and S1P siRNA were transfected on day 2. On day 3, cells were transfected withpSREBP-1-EGFP. On day 4, the nucleus wasHoechst-stained for time-lapse imaging. t¼ 0 hour, 24 hours after pSREBP-1-EGFP transfection. C, visualizationof transport of SREBP-EGFP in S2P siRNA treated LiSa-2 cells. D, visualization of transport of SREBP-EGFP in S1P and S2P siRNA treated LiSa-2 cells. Equalamounts of S1P and S2P siRNA were transfected and follow the treatment as (B). Bar, 20 mm.





mg/kg/d orally for 6 weeks. The results shown in Figure 5Aindicate that by day 24, the tumor growth curve of the NFV-treated mice begin to diverge from the control mice andcontinue to do so throughout the entire experimentalperiod (P < 0.05), which suggest that the in vitro observa-tions for NFV may be equally applicable in vivo.

Clinical use of NFV in the treatment of HIV infection iscommonly associated with diarrhea but rarely leads tohepatotoxicity and/or myelosuppression (32). To evaluatetoxicities associated with NFV in SCID mice, after 2 weeksof continuous twice-daily dosing with vehicle only (car-boxymethylcellulose), NFV 1 gm/kg/d, or 2 gm/kg/d byoral gavage, the mice were euthanized and subjected tonecropsy. Figure 5B shows minimal or no weight lossassociated with NFV with either dose level (P < 0.05). Dailycage examination failed to show diarrhea or its predeter-mined surrogate, 10% weight loss. Histologic evaluation ofliver and bone marrow showed no microscopic difference

in liver architecture or levels of myeloid or erythroid pre-cursors between control and NFV-treated mice (data notshown).

To determine the pharmacokinetics of oral NFV in vivo,cohorts of 3 mice underwent a single, oral gavage with 500mg/kg of NFV and the mice were euthanized for cardiacpuncture at the indicated time points. The results show thatat a dose of 500 mg/kg, a peak plasma concentration ofNFV that approximates the in vitro biologically active dose(�10 mmol/L) is reached in 1 hour and remains steady for 8hours (Fig. 5C). These data suggest that twice-daily dosingis feasible for long-term NFV dosing.

NFV and S2P siRNA induce ER stressNFV induces ER stress (4). To determine whether S2P

inhibition also induces ER stress, siRNAs targeting S1P,S2P, or S1P plus S2P were transfected into LiSa-2 cells andexpression of the ER stress response protein GRP78 was

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Figure 4. A, siRNA-mediated inhibition of S1P or S2P reduces liposarcoma proliferation. A total of 2,000 LiSa-2 cells were seeded in a 96-well plate,followed by transfection with S1P, S2P, or S1P and S2P, or scrambled siRNA. Twenty-four hours later, the cells were transfected with pSREBP-1-EGFP. At 48and 72 hours after siRNA transfection, cells proliferation was determined by DIMSCAN assay. *, P < 0.05, **, P < 0.01 compared with control siRNA. B, S1Pinhibitor does not alter the expression of SREBP-1 and ATF6. LiSa-2 cells were treated at the indicated DCI dose for 4 hours then lysed for Western blotanalysis of SREBP-1. pATF6-EGFP (or mock) was transfected 24 hours prior to the treatment of DCI for 4 hours for analysis of ATF6. C, S2P inhibitor alters theexpression of SREBP-1 and ATF6. Western blot analysis of SREBP-1 and ATF6 as (B) for LiSa-2 cells treated with 1,10-phenanthroline for 4 hours. D, S2Pinhibitor induces a dose-dependent apoptosis. LiSa-2 cells were treated with indicated dose of DCI, 1,10-phenanthroline, or NFV for 4 hours and collected forAnnexin V detection by flow cytometry.

Guan et al.




evaluated. TG served as a positive control for ER stressinduction. Figure 6A shows that TG, high-dose NFV, andS2P or S1P plus S2P siRNA strongly induce expression ofGRP78, whereas control and S1P siRNA alone does not.These results are consistent with the hypothesis that NFVinhibits S2P activity, which induces ER stress.

Discussion

NFV is a PI that was originally developed for therapyagainst HIV. NFV may inhibit cancer growth via multiplepathways (1–6). Recently, a study in ovarian cancersuggested a novel additional mechanism that NFV maytarget cancer stem cells (33). These reports have ledsupport for the use of NFV alone or in combination withradiation and/or chemotherapy for advanced cancers inseveral clinical trials, including our ongoing trial in lipo-sarcomas (5, 34).Liposarcomas are one of the most common soft tissue

sarcomas, and outcomes for recurrence remains poor. They

may be particularly sensitive to NFV because it interfereswith maturation of SREBP-1, an adipocytic transcriptionfactor (19). Our data strongly suggest that NFV inhibitsliposarcoma proliferation and promotes apoptosis by inhi-biting RIP of SREBP-1 and ATF6. This results in accumula-tion of unfolded SREBP-1 and ATF6 precursor proteins (ERstress) and inhibition of the UPR because ER stress-inducedactivation of ATF6 is neutralized.

Regulation of SREBP-1 and ATF6 is similar (Fig. 6B).Both transcription factors are synthesized as ER transmem-brane proteins (precursor) and transported to the Golgiwhere RIP occurs. Luminal S1P cleavage occurs first, fol-lowed by intramembrane S2P cleavage, which liberates thetranscriptionally active amino-terminal segments ofSREBP-1 and ATF6 to migrate to the nucleus in order totransactivate sterol biosynthesis or UPR activation genes.

Our previous data showed that precursor and processedforms of SREBP-1 were consistently upregulated by NFV invarious liposarcoma cell lines (3). Our present results showthat NFV induces similar upregulation of ATF6 (Fig. 2).

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Figure 5. A, nelfinavir inhibits liposarcoma growth in a murine model. LiSa-2 liposarcoma xenografts were established in female SCID mice andthe mice were treated by daily oral feeding of NFV at 500 mg/kg/d. Size of tumors was measured and presented as mean � SE (n ¼ 7 mice/group).*, P < 0.05 compared with the control animals. B, no significant toxicity associated with NFV treatment. The mice were weighed 3 times weekly.Weight was measured and presented as mean � SE. #, P < 0.05 compared with the control animals. C, pharmacokinetics determination of NFV inSCID mice. A single oral dose of NFV at 500 mg/kg was given to the SCID mice. Blood samples of the treated mice at 0, 1, 2, 4, 8, and 24 hours after oralgavage were analyzed to determine the plasma concentration of NFV using an UPLC-tandem mass spectrometric assay.





Confocal, time-lapse microscopy (Fig. 3) shows thatSREBP-1 and ATF6 processing is inhibited by NFV, withretention of precursor SREBP-1 and ATF6 in the cytoplasm.This results in SREBP-1–mediated downregulation ofFASN. Accumulation of precursor SREBP-1 and ATF6induces ER stress, activates the UPR, and induces apoptosis.Our results indicate that inhibition of RIP of SREBP-1 andATF6 results from NFV-mediated inhibition of S2P activity,which is supported by the demonstration that siRNA-mediated knockdown of S2P, but not S1P, induces ERstress (Fig. 6A). Interestingly, HIV PIs inhibit proteolyticactivation of zinc metalloproteinase, ZMPSTE24, andmatrix metalloproteinase-2 (35, 36). S2P is also a metal-loprotease and likely shares a similar domain with otherfamily members recognized by a PI.

Our current model illustrated in Figure 6B shows thatNFV-induced upregulation of precursor SREBP-1 and ATF6is mediated through inhibition of S2P, which leads toinhibition of RIP. Confirmatory support remains pendingour current efforts to purify S2P protein for enzymatic

cleavage assays. The precursor proteins are retained inthe cytoplasm, their transport to the nucleus is halted,and transcriptional activation of target genes is blocked.Accumulation of unprocessed SREBP and ATF6 induces ERstress, and a deficient UPR leads to caspase-dependentapoptosis.

Although our data indicate that NFV inhibits S2P activ-ity, partial S1P inhibition cannot be excluded. RIP ofSREBP-1 and ATF6 requires sequential S1P and S2P clea-vage. Prywes and colleagues previously noted that thebulky ATF6 luminal domain blocks S2P cleavage and theprimary role of S1P is to reduce the size of the luminaldomain to prepare ATF6 to be an optimal S2P substrate(37). An analogousmodel for SREBP-1 exists, with removalof its second transmembrane domain by S1P, which allowsfor the movement of the substrate site within the mem-brane (38). This may explain why knockdown of S1P bysiRNA still leads to accumulation of unprocessed precursorSREBP-1 in the cytoplasm in the absence of precursorSREBP-1 accumulation in DCI-treated cells. However,

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ApoptosisUPR target genesLipogenic target genes:

FASN etc. ( )

S1Pand S2P

Figure 6. A, nelfinavir induces ER stress. siRNA transfected (48 hours) or NFV treated (24 hours) LiSa-2 cells were lysed for Western blot detection of GRP78.TG (0.5 mmol/L) was served as a positive control. B, current model of nelfinavir in liposarcoma. SREBP and ATF6 are synthesized as ER transmembraneproteins and transported to the Golgi upon appropriate stimulus. Luminal S1P cleaves first, followed by intramembrane S2P to liberate the transcriptionallyactive amino-terminal segments of nSREBP and nATF6 from the Golgi membrane to migrate to the nucleus to activate transcription of lipogenic target genessuch as FASN or UPR targets genes (solid arrow); NFV inhibits liposarcoma proliferation, and promotes apoptosis through inhibition of RIP processing ofSREBP and ATF6. Inhibition of S2P cleavage by NFV leads to retention of precursor SREBP and ATF6 proteins in the ER or Golgi and block of transcriptionalactivation of target genes (FASN). Accumulation of unprocessed SREBP and ATF6 induces ER stress, which leads to caspase-dependent apoptosis (dottedarrow). NFV, nelfinavir; ER, endoplasmic reticulum; nSREBP-1, nuclear active SREBP; nATF6, nuclear active ATF6; FASN, fatty acid synthase; UPR, unfoldedprotein response; S1P, site-1 protease; S2P, site-2 protease; RIP, regulated intramembrane proteolysis.

Guan et al.




knockdown of S2P by siRNA or inhibition of S2P catalyticactivity by 1,10-phenanthroline leads to a phenotype char-acterized by NFV treatment.Our previous data showed that conditional expression

of SREBP-1 in SW872 liposarcoma cells reduces cellularproliferation and induces antiproliferative and proapop-totic genes such as p21WAF1/CIP1, Fas, and Bax. SREBP-1acts as a proapoptotic gene in pancreatic b-islet cells, andcaspases 2 and 7 are activated by SREBP-1 and SREBP-2 instatin-induced apoptotic gastric cancer cells (39, 40).ATF6-mediated apoptosis resulting from ER stress hasbeen elucidated (41). Accumulation of misfolded pro-teins in the ER induces the UPR to promote cell survivalby adjusting ER protein–folding capacity. However, ifhomeostasis cannot be reestablished, apoptosis isinduced through ATF6-mediated activation of CHOP/GADD153 (42).A notable target gene of SREBP is FASN. One of the

hallmarks of cancer is increased de novo FA synthesis,referred to as the "lipogenic phenotype" (43, 44). FAsare essential constituents of all biological membrane lipidsand are important substrates for energy metabolism (44).FASN catalyzes the terminal steps of long-chain FA synth-esis (44). FASN is overexpressed in many cancers (43, 44).

Inhibition of FASN by RNA interference or small moleculesinhibits growth and survival of tumors (44–46). We alsoobserved NFV-dependent reduction in FASN expression,which was mediated through depletion of transcriptionallyactive SREBP-1. This provocative result suggests that NFVmay possess anticancer activity in other cancers whichexhibit a "lipogenic phenotype," an avenue that we arecurrently actively pursuing.

In summary, this study shows that NFV induces lipo-sarcoma apoptosis through ER stress induction and defi-cient UPR. These biological effects arise from accumulationof precursor SREBP-1 and ATF6 as a result of S2P-regulatedRIP cleavage inhibition. To our knowledge, this is the firstreport of a novel strategy targeting S2P and RIP for cancertherapeutics.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 3, 2010; revised February 9, 2011; accepted February10, 2011; published OnlineFirst February 25, 2011.

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