Inhibition of Akt Reverses the Acquired Resistance to Sorafenib by … · Cancer Biology and Signal...

Cancer Biology and Signal Transduction

Inhibition of Akt Reverses the Acquired Resistance toSorafenib by Switching Protective Autophagy to AutophagicCell Death in Hepatocellular Carcinoma

Bo Zhai1, Fengli Hu2, Xian Jiang1, Jun Xu3, Dali Zhao1, Bing Liu1, Shangha Pan1, Xuesong Dong1,Gang Tan1, Zheng Wei1, Haiquan Qiao1, Hongchi Jiang1, and Xueying Sun1

AbstractSorafenib is the standard first-line systemic drug for advanced hepatocellular carcinoma (HCC), but the

acquired resistance to sorafenib results in limited benefits. Activation of Akt is thought to be responsible for

mediating the acquired resistance to sorafenib. The present study aims to examine the underlying mechanism

and seek potential strategies to reverse this resistance. Two sorafenib-resistant HCC cell lines, which had been

established from humanHCCHepG2 andHuh7 cells, were refractory to sorafenib-induced growth inhibition

and apoptosis in vitro and in vivo. Sustained exposure to sorafenib activatedAkt via the feedback loop ofmTOR

but independent of protein phosphatase 2A inHCC cells. Autophagyparticipated in the resistance to sorafenib

as inhibition of autophagy reduced the sensitivity of sorafenib-resistant HCC cells to sorafenib, whereas

activation of autophagy by rapamycin had the opposite effect. However, rapamycin did not show a synergistic

effect with sorafenib to inhibit cell proliferation, while it also activated Akt via a feedback mechanism in

sorafenib-resistant HCC cells. Inhibition of Akt reversed the acquired resistance to sorafenib by switching

autophagy from a cytoprotective role to a death-promoting mechanism in the sorafenib-resistant HCC cells.

Akt inhibition by GDC0068 synergized with sorafenib to suppress the growth of sorafenib-resistant HCC

tumors that possessed the sorafenib-resistant feature in vivo. The results have provided evidence for clinical

investigation of GDC0068, a novel ATP-competitive pan-Akt inhibitor, as the second-line treatment after the

failure of sorafenib-medicated molecular targeted therapy for advancedHCC.Mol Cancer Ther; 13(6); 1589–98.

�2014 AACR.

IntroductionHepatocellular carcinoma (HCC), the second most fre-

quent cause of cancer death in men worldwide (1), isnotoriously resistant to systemic chemotherapy (2). Aftersearching for effective agents to combat HCC for decades,sorafenib has opened a window of hope and presents asthe standard first-line systemic drug for advanced HCC(3). However, the promising treatment has demonstratedlimited survival benefits with very low response rates (3,4), and some patients with HCC initially respond tosorafenib but eventually the disease progresses (4), indi-cating that the resistance to sorafenib is common in HCC.

As a multitargeted kinase inhibitor, sorafenib targetstheRaf/mitogen-activated protein kinase (MAPK)/extra-cellular signaling-regulated kinase (ERK) signaling path-way, and inhibits a number of tyrosine kinase receptors,including VEGF receptor, platelet-derived growth factorreceptor, and c-Kit (3). The phosphoinositide 3-kinase(PI3K)/Akt pathway regulates a large number of mole-cules involved in all aspects of cancer progression (5), andis involved in the development and progression of HCC(6). Sorafenib activates Akt and upregulates its down-stream factors such as ribosomal protein S6 kinase (S6K)and eukaryotic translation initiation factor 4E-bindingprotein 1 (4EBP1) in HCC cells (7, 8). Sorafenib-resistantHCC cells have increased expression of phosphorylatedAkt (p-Akt; ref. 9). There exist cross-talks between thePI3K/Akt and MAPK/ERK pathways (10), indicatingthat the latent compensatory mechanism of the PI3K/Aktpathway may contribute to sorafenib resistance in HCC.However, the mechanisms that underlie the role of Aktactivation in this resistance remain unclear.

Induction of cell death is the final goal for developinganticancer agents. Autophagy was initially referred as aself-digestion process by which cytoplasmic contents aresequestered in autophagosomes and delivered to lyso-somes for degradation (11), but now autophagy is also

Authors' Affiliations: 1The Hepatosplenic Surgery Center, Department ofGeneral Surgery, the First Affiliated Hospital of Harbin Medical University;and Departments of 2Gastroenterology and 3General Surgery, the FourthAffiliated Hospital of Harbin Medical University, Harbin, China

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Xueying Sun, The Hepatosplenic Surgery Center,Department of General Surgery, the First Affiliated Hospital of HarbinMedical University, Harbin 150001, China. Phone: 86-451-53643628; Fax:86-451-53643628; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-1043

�2014 American Association for Cancer Research.

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considered to be type II programmed cell death (PCD;ref. 12).Apoptosis, the type I PCD, is undoubtedly a tumorsuppressing pathway, whereas autophagy is a double-edged sword depending on the cellular context andnature of the stimuli (13). The autophagic pathway cross-talks with apoptotic and other pathways, and the mutiplemolecular nodes in such pathways offer opportunities fortherapeutic intervention (13). Autophagy-related protein(ATG) 6 (also known as Beclin 1) binds to other compo-nents to form a core complex to allow autophagosomenucleation (14), and interacts with apoptotic molecules(13). The PI3K/Akt pathway inhibits autophagy by acti-vating mTOR (15), which exerts an inhibitory effect onautophagy by dysregulating Beclin 1 and ATG8 (micro-tubule-associated protein 1 light chain 3, LC3; ref. 16).These data indicate that activation of Akt may be respon-sible for mediating the acquired resistance to sorafenib byregulating the autophgic pathway.

Sorafenib promotes autophagic death ofHCC cells (17),but contrary results have also been reported (18). Activa-tion of autophagy is involved in the resistance to cisplatin(19), whereas inhibition of autophagy enhances the effectof sorafenib (20, 21) in HCC cells. However, the role ofautophagy and its cross-talk with other pathways inmediating resistance to sorafenib of HCC have not yetbeen reported. Here, we demonstrate, for the first time,that autophagy switches from a cytoprotective function toa death-promoting role and drives the acquired resistanceto sorafenib, while inhibiting Akt reverses the acquiredresistance to sorafenib by activating the autophagic path-way in sorafenib-resistant HCC cells.

Materials and MethodsCell culture, antibodies, and reagents

Human HCC HepG2 cells were obtained from theAmerican Type Culture Collection, and Huh7 cells fromChineseAcademyof SciencesCell Bank (Shanghai,China)in 2009. Cells were immediately expanded, and multiplealiquots were cryopreserved and used within 3 monthsafter resuscitation. No further authenticationwas done bythe authors. Cells were cultured at 37�C in Dulbecco’sModified Eagle Medium (Gibco BRL) supplementedwith 10% FBS. The antibodies (Abs) against Akt, p-Akt(Ser473), ERK, p-ERK (Thr202/Thyr204), glycogensynthase kinase (GSK)-3b, phosphorylated GSK3b (p-GSK3b; Ser9), mTOR, S6K, phosphorylated S6K (p-S6K;Thr389), 4EBP1, phosphorylated 4EBP1 (p-4EBP1; Ser65),PARP, LC3, Beclin1, ATG5, class III PI3K vascular proteinsorting 34 (Vps34), UV radiation resistance-associatedgene (UVRAG), and p62 were purchased from Cell Sig-nalingTechnology. TheAbs against Bad, caspase-3 and -9,p27, cyclin D1, Bcl-2, and b-actin were from Santa CruzBiotechnology. The anti–Ki-67 Ab was from Abcam. Sor-afenib and perifosine were from Jinan Trio PharmatechCo., Ltd. GDC0068 was from ShangHai BiochempartnerCo., Ltd. Rapamycin (RAP), 3-methyladenine (3-MA),bafilomycin A1 (Baf-A1), okadaic acid (OA), and 1,9-

dideoxyforskolin (Forskolin) were from Sigma-Aldrich.Sorafenib, GDC0068, rapamycin, and Baf-A1 were dis-solved in dimethyl sulfoxide to make a stock solution of100 mmol/L, 100 mmol/L, 1 mmol/L, and 1 mmol/Lfor in vitro assays, respectively. Perifosine was dissolvedin PBS to make a stock solution of 30 mmol/L. 3-MAwasdissolved in PBS at a concentration of 200 mmol/L byheating to 60 to 70�C immediately before use. For animalexperiments, sorafenib was suspended in the vehiclesolution containing Cremophor (Sigma-Aldrich), 95%ethanol and water in a ratio of 1:1:6 (22), and GDC0068was dissolved in 0.5% methylcellulose/0.2% Tween-80(23). The PI (propidium iodide)/Annexin V-FITC apo-ptosis detection kit was from BD Biosciences. The CellCounting Kit-8 (CCK-8) kit was from Dojindo Labora-tories. Terminal deoxynucleotidyl transferase–mediat-ed dUTP nick end labeling agent (TUNEL) was fromRoche.

Establishment of sorafenib-resistant cellsThe IC50 of HCC cells to sorafenib was initially deter-

mined by incubating cells with different concentrations ofsorafenib in 96-well plates, and cell viability was mea-sured 3 days later as described below. The cells werecultured in 6-well plates at 1 � 104 cells/well and incu-bated with sorafenib at a concentration just below theirrespective IC50. The concentration of sorafenibwas slowlyincreased by 0.25 mmol/L per week. After 6 to 7 months,two sorafenib-resistant cell lines were obtained, termedHepG2-SR and Huh7-SR, respectively, and were contin-uously maintained by culturing them in the presence ofsorafenib.

Analysis of autophagy by GFP-LC3 redistributionThe GFP tandem fluorescent-tagged LC3 expression

plasmid was transfected into cells using Lipofectamine2000 (24). LC3 redistribution was detected under a fluo-rescent microscope.

PP2A phosphatase activity assayThe activity of PP2A phosphatase in cell lysates was

measured by using a PP2A immunoprecipitation phos-phatase assay kit (Millipore).

Animal experimentsSix- to 8-week male nude BALB/c-nu/nu mice were

obtained from the Animal Research Center, The FirstAffiliatedHospital ofHarbinMedical University (Harbin,China). The study protocol had been approved by theAnimal Ethic Committee of Harbin Medical University.The animal experiment schedule was designed on thebasis of the preliminary experiments (Fig. 1). Briefly,Huh7-SR cells (5 � 106) were subcutaneously inoculatedinto the back of mice, which received daily oral admin-istration of 15 mg/kg sorafenib. The use of lower dose ofsorafenibwas tomaintain the sorafenib-resistant ability ofHuh7-SR cells, which were kept in the presence of sor-afenib in culture. Fifteen days later, another group ofmice

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received subcutaneous injections of Huh7 cells (5 � 106).Thirty days later, three Huh7 and three Huh7-SR tumorswere harvested. The remaining mice were assigned todifferent treatments. Sorafenibwas orally administered ata dose of 30mg/kg daily, andGDC0068 orally at a dose of25 mg/kg twice per week. The tumor volumes weremeasured every 3 days. The tumors were harvested atthe end of experiments.

Cell viability analysis, in vitro apoptosis assay,visualization of apoptotic cells by laser scanningconfocal microscopy, assessment of Ki-67proliferation index, in situdetection of apoptotic cell,and immunoblottingAll these methods have been described in details pre-

viously (24–26).

Transfection of Akt siRNAAdouble-strand siRNA targeting humanAkt (50- GUG-

GUCAUGUACGAGAUGATT-30 and 50-UCAUCUC-GUACAUGACCACTT-30) encoding nucleotides 1006–1025 of Akt1 (GenBank: NM_001014431.1), nucleotides1210–1229 of Akt2 (GenBank: XM_005336494.1), andnucleotides 955–974 of Akt3 (GenBank: XM_004691046.1)with two introduced thymidine residues at the 30 endwasproduced by GenePharma Co., Ltd. The nonspecificscrambled siRNA (50-UUCUCCGAACGUGUCACGU-30

and 50-ACGUGACACGUUCGGAGAA-30) served as acontrol. Cells were grown to 60% to 70% confluence, andincubated with siRNAs at a final concentration of 0.1mmol/L by using Lipofectamine 2000 (Invitrogen) in aserum-freemedium for 24 hours and then subjected to theassays.

Electronic microscopyCells or tissues were fixed in 2.5% glutaraldehyde

solution for 1 hour, washed twice with PBS, followed byfurther fixation with 1% Osmic acid for 1 hour, dehy-drated with a graded series of ethanol, embedded, andsectioned. Sections were stained with Uranium Acetateand Lead Citrate, and observed under a transmissionelectronic microscope (JEN-M1220, Toshiba; ref. 24).

Statistical analysisThe data were expressed as mean values � SD. Com-

parisons were made using one-way ANOVA followed byDunnet t test. P < 0.05 was considered significant.

ResultsSorafenib-resistant HCC cells are refractory tosorafenib-induced growth inhibition and apoptosis

After incubation with 10 mmol/L of sorafenib for 48hours, the viability of HepG2-SR and Huh7-SR cells were78.8% and 77.2%, respectively, which were significantlyhigher than that of HepG2 and Huh7 cells (18.4% and31.9%, respectively; Fig. 2A). Evenwhen the concentrationof sorafenib reached 20mmol/L, the viability ofHepG2-SRand Huh7-SR still remained at 38.5% and 46.2%, respec-tively, whereas HepG2 and Huh7 cells were almostcompletely dead (Fig. 2A). The in vitro findings weresupported by the in vivo data (Supplementary Fig. S1).Apoptotic rates of HepG2 cells were 4.1- and 3.9-foldhigher than that of HepG2-SR cells, after exposure to 5and 10 mmol/L of sorafenib, respectively (Fig. 2B and C).Apoptotic rates ofHuh7 cellswere 2.8- and 4.0-fold higherthan that of Huh7-SR cells, after exposure to 5 and

Figure 1. Animal experimental schedule. Two groups of mice (n ¼ 5 and n ¼ 35) underwent subcutaneous injections of Huh7 or Huh7-SR cells (5 � 106),respectively, and received daily oral sorafenib at 15 mg/kg. Fifteen days later, another group of mice (n ¼ 15) underwent subcutaneous injection ofHuh7 cells (5� 106). At day 30, none of the mice (0/5) receiving inoculation of Huh7 cells and sorafenib administration had palpable tumors, whereas 31mice(31/35) receiving inoculation of Huh7-SR cells and sorafenib administration, and 14 mice (14/15) receiving inoculation of Huh7 cells, had tumors ofapproximately 100 mm3 in volume. The mice without tumors were killed. Three Huh7 and 3 Huh7-SR tumors were harvested at day 30. The remaining micewere assigned to different treatments. Sorafenibwas orally administered at a dose of 30mg/kg daily, andGDC-0068 orally administered at a dose of 25mg/kgtwice per week. The frames with solid lines indicate start of the experiment, and frames with dotted lines indicate end of the experiment.

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10 mmol/L of sorafenib, respectively (Fig. 2B and C). Theapoptotic resultswere supported by the expression of twokey apoptotic proteins, caspase-3 and PARP (Fig. 2D andE), indicating that sorafenib-resistant cells are refractoryto sorafenib-induced apoptosis through caspase-depen-dent and -independent ways.

Autophagy participates in the mechanisms for theresistance to sorafenib

After incubation for 48 hours in the presence andabsence of sorafenib (10 mmol/L), sorafenib-resistantcells expressed higher levels of p-Akt and differentlevels of its downstream factors, including p-GSK3b,mTOR, p-S6K and p-4EBP1, Bad, p27, and cyclin D1,

compared with their respective parental cells (Supple-mentary Fig. S2A and Supplementary Table S1). Theresults indicate that sustained exposure to sorafenibactivates the Akt pathway. In addition, sorafenib-induced phosphorylation of Akt was independent ofprotein phosphatase inactivation (Supplementary Fig.S2B and S2C). Given that mTOR is a gatekeeper ofautophagy by dysregulating ATGs (16), we hypothe-sized that autophagy may participate in the acquiredresistance to sorafenib. Accordingly, sorafenib-resistantcells expressed lower levels of LC3-II, ATG5, Vps34 andBeclin 1, and expressed higher levels of P62 and Bcl-2,than their respective parental cells in either the presenceor absence of sorafenib (Fig. 3A). Sorafenib upregulated

Figure 2. Sorafenib-resistant HCCcells are refractory to sorafenib-induced growth inhibition andapoptosis. A, the sorafenib-resistant cells HepG2-SR andHuh7-SR and parental HepG2 andHuh7 cells were incubated withincreasing concentrations ofsorafenib for 48 hours. Cell viability(%) was compared with thecorresponding untreated cells.B–E, the above cells wereincubated with 0, 5 or 10 mmol/L ofsorafenib for 48 hours. The cellswere analyzed cytometrically todetect apoptosis (B) and therates of apoptosis weredetermined (C), and cell lysateswere immunoblotted (D). E, banddensities were normalized tob-actin. Data represent threeindependent experiments.��, P < 0.001; †, P < 0.05; z, P <0.001 versus the correspondinguntreated cells.

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the expression of LC3-II, ATG5, Beclin 1, and Vps34,downregulated the expression of P62, and had littleeffects on UVRAG and Bcl-2, in either parental orsorafenib-resistant cells (Fig. 3A). The main in vitrofindings were further supported by the in vivo results(Supplementary Fig. S3).We next showed that inhibition of autophagy by

3-MA and Baf-A1 protected HepG2-SR and Huh7-SRcells against sorafenib-induced reduction in cell viabil-

ity (Fig. 3B and C). In contrast, rapamycin, an inhibitorof mTOR and an inducer of autophagy (27), enhancedsorafenib-induced growth inhibition in a dose-depen-dent manner (Fig. 3D). However, the synergistic effectsof rapamycin and sorafenib were not significant, as thevalues for the coefficient of drug interaction (CDI;refs. 25, 28) for HepG2-SR and Huh7-SR cells treatedwith 1, 10, or 100 nmol/L of rapamycin in the presenceof sorafenib (10 mmol/L) were all more than 0.7.

Figure 3. Autophagy affects thesensitivity of sorafenib-resistantHCC cells to sorafenib. A, lysatesof HepG2, HepG2-SR, Huh7, andHuh7-SR cells incubated with 0, 5or 10 mmol/L of sorafenib for 48hours were immunoblotted todetect expression of autophagy-associated proteins. B–D, theabove cells were incubated for 48hours in the presence or absenceof sorafenib (10 mmol/L in B and Dor 15 or 20 mmol/L in C), and/or 3-MA (3-methyladenine; 10 mmol/L),and/or Baf-A1 (bafilomycin A1, 50nmol/L), or with differentconcentrations of rapamycin (D).Cell viability (%) was comparedwith the corresponding untreatedcells. E, immunoblot analyses oflysates of Huh7-SR cells from B. F,immunoblot analyses of lysates ofHuh7-SR cells from D. The banddensity of LC3-II was normalizedto LC3-I, and that of other proteinswas normalized to b-actin. Datarepresent three independentexperiments. The statisticalcomparison was performed forsorafenib-resistant cells only.�, P < 0.05 and ��, P < 0.001 versusrespective untreated cells; †,P < 0.05 and z, P < 0.001 versussorafenib alone; #, P < 0.05 and ##,P < 0.001 versus rapamycin alone.øø, P < 0.001.






3-MA downregulated the expression of LC3-II andBeclin 1, whereas Baf-A1 upregulated LC3-II and down-regulated Beclin 1, in Huh7-SR cells (Fig. 3E). Both 3-MAand Baf-A1 increased the expression of pro–caspase-3 buthad little effect on cleaved-PARP expression (Fig. 3E).Rapamycinupregulated the expression of LC3-II, Beclin 1,p-Akt, and cleaved-PARP, and increased the activation ofcaspase-3 (Fig. 3F), indicating that induction of autophagyby rapamycin promotes apoptosis, but also results inactivation of Akt, in sorafenib-resistant cells.

Inhibition of Akt synergizes with sorafenib to inhibitcell viability, promote apoptosis, and induceautophagic cell death in sorafenib-resistant cells

Inhibition of Akt by GDC0068 synergized with sorafe-nib to reduce the viability ofHepG2-SR andHuh7-SR cellsin dose- and time-dependent manners (Fig. 4A and Sup-plementary Fig. S4A). The CDIs for HepG2-SR cells trea-ted with 1, 5, or 10 mmol/L of GDC0068 in the presence ofsorafenib (10 mmol/L) were 0.67, 0.63, or 0.65, respective-

ly; and that for Huh7-SR were 0.79, 0.59, or 0.61, respec-tively, indicating that the synergistic effects were signif-icant. The effects of Akt inhibition on cell viability werefurther supported by application of perifosine, anotherAkt inhibitor (Supplementary Fig. S4B). GDC0068 alsoshowed a significantly synergistic effect with sorafenib ininducing cell apoptosis with CDIs of 0.48 and 0.53, inHepG2-SR and Huh7-SR cells, respectively (Fig. 4Band C).

Sorafenib and GDC0068 upregulated, and their combi-nation showed an even stronger effect in increasing theexpression of LC3-II and Beclin 1 in Huh7-SR cells (Fig.4D). Inhibition of Akt by perifosine or depletion of Akt bysiRNAs had a similar effect on the expression of autop-hagic proteins (Supplementary Fig. S4C and S4D). Con-sequently, the number of GFP-LC3–positive dots in cellstreated with sorafenib or GDC0068 were significantlyhigher than untreated cells, and their combinationresulted in the highest number of GFP-LC3–positive dots(Fig. 4E). The CDI for GDC0068 and sorafenib to induce

Figure 4. Inhibition of Akt enhances the sensitivity of sorafenib-resistant HCC cells to sorafenib by inducing autophagy. A, HepG2-SR andHuh7-SR cells wereexposed for 48 hours to different concentrations of GDC0068, or 5 mmol/L of GDC0068 for different periods, in the presence or absence of sorafenib(10 mmol/L). Cell viability (%) was compared with the corresponding untreated cells. B–G, HepG2-SR and Huh7-SR cells were incubated for 48 hourswith sorafenib (10 mmol/L), GDC0068 (5 mmol/L), or in combination. Untreated cells served as controls. B, the apoptosis rates were measured by cytometry.C, representative images were taken from Huh7-SR cells stained with Annexin V/propidium iodide, and viewed by microscopy. D, cell lysates wereimmunoblotted. E and F, the above cells were transfected with GFP-LC3 expression vectors, GFP-LC3–positive dots were counted in 200 randomly selectedcells (E), and viewed under fluorescent microscopy (F). Arrows point to colocalized particles representing autophagosomes. G, electron microscopy;arrows point to autophagosomes (magnification, �10,000). ø, P < 0.05 and øø, P < 0.001. �, P < 0.05 and ��, P < 0.001 versus untreated control; z, P < 0.001versus sorafenib alone; #, P < 0.05 and ##, P < 0.001 versus GDC0068 alone.

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autophagy of Huh7-SR cells was 0.51, indicating that thesynergistic effectwas significant. The treated cells showedthat GFP-LC3 signals shifted from a diffuse cytoplasmicpattern to a punctate membrane pattern, suggesting theformation of autophagic vacuoles (Fig. 4F). Electronmicroscopy revealed abundant autophagic vacuoles insorafenib or GDC0068-treated cells, but scarcely inuntreated cells; and the combinational therapy resultedin the most autophagosomes (Fig. 4G).

GDC0068 synergizes with sorafenib to suppresssorafenib-resistant tumorsAdministration of sorafenib andGDC0068 into themice

reduced the size of Huh7-SR tumors by 15.8% and 37.5%,respectively, and the combination therapy furtherreduced the size by 71.3%, comparedwith vehicle-treatedtumors at day 15 (Fig. 5A).Immunoblotting analysis of tumor lysates demonstrat-

ed the similar alteration of expression of p-Akt, LC3-II,pro–caspase-3, and PARP (Fig. 5B), as shown in vitro(Supplementary Fig. S5A–S5C). Electron microscopy alsorevealed abundant autophagosomes in cells from tumors

treated by sorafenib plus GDC0068, but scarcely in vehi-cle-treated tumors (Fig. 5C). There were fewer Ki-67–positive cells in tumors treated with sorafenib orGDC0068, compared with vehicle-treated tumors; andthe combination therapy resulted in even fewer Ki-67–positive cells (Fig. 5D and E). Tumors treated with sor-afenib and GDC0068 had a greater number of TUNEL-positive cell than vehicle-treated tumors, and the combi-nation therapy resulted in even more TUNEL-positivecells (Fig. 5D and F).

DiscussionConsidering the dilemma that no effective systemic

therapy for HCC is available so far after failure of sor-afenib therapy (29), sorafenib remains in auniquepositionfor the treatment of HCC. The present study has demon-strated that activation ofAkt induced by chronic exposureof sorafenib regulates the acquired resistance to sorafenibby switching autophagy from a cytoprotective role to adeath-promoting mechanism in HCC cells.

Although it is not a direct target of sorafenib, the PI3K/Akt pathway plays an important role in sorafenib

Figure 5. Inhibition of Akt overcomes sorafenib resistance in vivo. A, subcutaneous tumors were established in mice that received different treatments for 15days as described inMaterials andMethods and Fig. 1. The size (mm3) of tumorswas recorded. B, immunoblot analyses of lysates of tumors harvested fromAat the end of experiments. C, sections of tumors treated with vehicle or sorafenibþGDC0068 were viewed under electron microscopy. Arrows point toautophagosomes (magnification, �10,000). D, representative images from tumor sections stained with an anti–Ki-67 Ab (top) or TUNEL (bottom;magnification,�200). Proliferation index (E) and apoptosis index (F) were quantified. n, the number of samples examined. �, P < 0.05 and ��, P < 0.001 versusvehicle-treated tumors; z, P < 0.001 versus sorafenib alone; ##, P < 0.001 versus GDC0068 alone.






resistance because it crosstalks with the MAPK/ERKpathway (10). Many lines of studies have shown thatsorafenib activates the PI3K/Akt pathway (7, 8, 18, 30),and blockage of this pathway enhances the efficacy ofsorafenib (7, 30), but it has also been reported that sor-afenib enhances proteasome inhibitor-induced cell deathby inactivating Akt in HCC (31), indicating that thecomplicated role of the PI3K/Akt pathway in sorafenibresistance is far from clear. Here, we have demonstratedthat Akt was activated in sorafenib-resistant cells, inaccordance with a previous report (9). Although phos-phorylation ofAkt can be induced byprotein phosphataseinactivation (32), the present study showed that the sor-afenib-induced Akt phosphorylation was independent ofprotein phosphatase 2A (PP2A), amajor serine/threoninephosphatase in eukaryotic cells (33). In accord, the syn-ergistic interaction between sorafenib and bortezomib,but not sorafenib alone, involved PP2A-dependent Aktinactivation in HCC cells (34). Akt regulates the expres-sion and/or phosphorylation of mTOR, GSk3b, Badand p27; mTOR regulates the apoptotic proteins S6Kand 4EBP1, and the autophagic proteins LC3 and Beclin 1;and GSk3b regulates cyclin D1 and caspase-9 (refs.5, 13, 29, 35; Fig. 6). Therefore, specific inhibition of Aktcould downregulate the above downstream factors, thusinducing apoptosis and inhibiting proliferation of HCCcells.

Among the over hundred molecules in the PI3K/Aktpathway, mTOR interacts with theMARK, apoptotic, andautophagic pathways and has been most extensivelystudied, and several mTOR inhibitors are under evalua-tion in clinical trials (36). Inhibition ofmTOR enhances thechemosensitivity (37), yields antiproliferative effects inboth parental and sorafenib-resistant HCC cells (35), andaugments the effects of sorafenib in a syngeneic orthotopicmodel of HCC (32). However, here we found that rapa-mycin did not show a synergistic effect with sorafenib ininhibiting the growth of sorafenib-resistant HCC cellsdespite the higher basal expression of mTOR (8). Theunhampered feedback loop driving PI3K/Akt activationby mTOR inhibition has been recently reported, andinhibition of Akt interrupted this rapamycin-inducedfeedback loop, thereby enhancing the antiproliferativeeffects of mTOR inhibition (38). Accordingly, here wehave shown that chronic exposure of sorafenib inhibitedactivation of ERK (9), sequentially inhibited mTOR inde-pendent of the PI3K/Akt pathway (39), resulting in acti-vation of Akt via the feedback loop in sorafenib-resistantHCC cells (Fig. 6). This may explain that the effect ofmTOR inhibition by rapamycin is not ideal to reversesorafenib resistance.

The role of autophagy as a target in cancer therapyremains controversial (40). It has been reported that sor-afenib induced autophagic death of HCC cells (17).

Figure 6. Proposedmechanisms bywhich activation of AKT contribute to acquired resistance to sorafenib via regulating autophagy, and the actions of agentsin parental and sorafenib-resistant cells. Blank area indicates aparental cell, and shadowed area a sorafenib-resistant cell.!, positive regulation or activation;?, negative regulation or blockade; þ P, regulation by phosphorylation.

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However, the authors used a high concentration of sor-afenib at 20 mmol/L (18). Even with the same concentra-tion of sorafenib, another study showed that autophagyprotected against sorafenib-induced cell death (20). Here,we have demonstrated that sorafenib at 10 mmol/L acti-vated autophagy, which played a protective role againstcell death of parental HCC cells, and is in agreement witha previous report (21); but exhibited a death-promotingrole in sorafenib-resistant HCC cells. Autophagy inducedbyvorinostat, a histonedeacetylase inhibitor that has beenapproved for treating cutaneousT-cell lymphoma in clinic(41), protected against cell death independent ofmTOR invorinostat-resistant lymphoma cells (42). The autophagicpathway crosstalks with both the caspase-dependent and-independent apoptotic pathways (13, 21, 43). Sustaineddrug exposure could induce imbalanced apoptotic path-ways, leading to the resistance to apoptosis (44).When theapoptotic pathway is suppressed, autophagy as an adap-tive responsewill switch from a protective role to a death-promoting function through the existing cross-talk withthe apoptotic pathway (13, 45). This explanation is sup-ported by a recent study that paclitaxel resistance isassociated with switch from apoptotic to autophagic celldeath in breast cancer cells (46).Themechanisms accounting for the acquired resistance

to sorafenib in HCC remain complex (47). The detailedcellular signaling pathways and their interactions inparental and sorafenib-resistant HCC cells, and the inter-ventions with agents tested in the present study aresummarized in Fig. 6. Briefly, autophagy plays a protec-tive role against sorafenib-induced cell death in the paren-tal cells. In sorafenib-resistant cells, sustained exposure tosorafenib activates Akt directly or via the feedback loop ofmTOR, which is inhibited by sorafenib through the ERKpathway (Fig. 6). Sorafenib induces autophagy indepen-dent of the ERKpathway (20). Activation of autophagy byrapamycin partially reverses the acquired resistance tosorafenib as rapamycin-mediated mTOR inhibition inturn activates Akt via the feedback loop. However, inhi-bition of Akt by GDC0068 significantly enhances the

efficacy of sorafenib to suppress the growth of sorafe-nib-resistant HCC cells by inducing autophagic celldeath. As a novel ATP-competitive pan-Akt inhibitor,GDC0068 binds to the active site of Akt and protects itfrom phosphatases, leading to increased Akt phosphor-ylation (48, 49). It has been reported that GDC0068 hasless dose-limiting toxicity and is more effective in inhibit-ing the function of Akt than allosteric Akt inhibitors (48,49). GDC0068 has displayed anticancer activities againstseveral types of cancers (49). The results presented war-rant clinical investigation of GDC0068 as the second-linetreatment after the failure of sorafenib to treat advancedHCC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H. Qiao, H. Jiang, X. SunDevelopment of methodology: B. Zhai, F. Hu, X. SunAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Zhai, X. Jiang, J. Xu, D. Zhao, X. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): B. Zhai, S. Pan, H. Qiao, H. Jiang, X. SunWriting, review, and/or revision of the manuscript: B. Zhai, X. Dong,X. SunAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G. Tan, Z. Wei, H. Jiang, X. SunStudy supervision: J. Xu, B. Liu, H. Qiao, H. Jiang, X. Sun

AcknowledgmentsThe authors thank Dr. Shiva Reddy for reviewing this article.

Grant SupportThisworkwas supported by theNationalNatural Scientific Foundation

of China (30973474, to X. Sun and H. Qiao; 81172331, to X. Sun), theHeilongjiang Provincial Scientific Foundation (GB07C32405, to B. Liu;H201307, to G. Tan), and The Youth Scientific Fund of HeilongjiangProvince (QC2013C103, to B. Zhai; QC2013C098, to X. Jiang), China.

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.

ReceivedDecember 17, 2013; revisedMarch 18, 2014; acceptedMarch 25,2014; published OnlineFirst April 4, 2014.

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2014;13:1589-1598. Published OnlineFirst April 4, 2014.Mol Cancer Ther Bo Zhai, Fengli Hu, Xian Jiang, et al. Hepatocellular CarcinomaSwitching Protective Autophagy to Autophagic Cell Death in Inhibition of Akt Reverses the Acquired Resistance to Sorafenib by

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