Atorvastatin Decreases HBx-Induced Phospho- Akt in ...Taken together, we show that statins decrease HBx- or insulin-induced pAkt in hepatocytes. Nuclear translocation of Akt is important

Oncogenes and Tumor Suppressors

Atorvastatin Decreases HBx-Induced Phospho-Akt in Hepatocytes via P2X ReceptorsAram Ghalali, Javier Martin-Renedo, Johan H€ogberg, and Ulla Stenius

Abstract

Hepatocellular carcinoma (HCC) is rated as the fifth mostcommon malignancy and third in cancer-related deathsworldwide. Statins, HMG-CoA reductase inhibitors, arepotent cholesterol-lowering drugs, and recent epidemiologicevidence suggests that statins prevent aggressive HCC devel-opment. Previous experiments revealed that statins down-regulate phosphorylated Akt (pAkt). Here, it is demonstratedthat atorvastatin decreases nuclear pAkt levels in pancreaticand lung cancer cell lines within minutes, and this rapideffect is mediated by the purinergic P2X receptors. Akt isupregulated by hepatitis viruses and has oncogenic activityin HCC; therefore, we tested the possibility that the P2X–Aktpathway is important for the anticipated anticancer effectsof statins in hepatocytes. Atorvastatin decreased hepatitis B

virus X protein- and insulin-induced pAkt and pGsk3b (Ser9)levels. Furthermore, Akt-induced lipogenesis was counter-acted by atorvastatin, and these statin-induced effects weredependent on P2X receptors. Statin also decreased pro-liferation and invasiveness of hepatocytes. These data pro-vide mechanistic evidence for a P2X receptor–dependentsignaling pathway by which statins decrease pAkt, its down-stream phosphorylation target pGsk3b, and lipogenesis inhepatocytes.

Implications: The Akt pathway is deregulated and may act as adriver in HCC development; the P2X–Akt signaling pathwaymay have a role in anticancer effects of statins. Mol Cancer Res;15(6); 714–22. �2017 AACR.

IntroductionHepatocellular carcinoma (HCC) is rated as the fifth most

common malignancy and third in cancer-related deaths world-wide. There are several risk factors for HCC, such as liver cirrhosis,alcohol, aflatoxins, obesity, lipogenesis, and viral infections.Hepatitis B viral (HBV) infection accounts for 50% to 80% ofall HCC cases (1), and recent data show that the relative risk forviral hepatitis increased between 1990 and 2013 and was theseventh leading cause of death worldwide (2).

The viral nonstructural gene hepatitis B virus X protein (HBx)has been shown to activate the PI3K/Akt pathway, which affectscellular and pathologic processes, such as invasion and metas-tasis (3). The molecular mechanism behind the HBx-inducedactivation of Akt pathway is not fully understood, but evidenceindicates that HBx induces activating Akt phosphorylation anddownstream effects of Akt (4, 5). Of perhaps particular inter-est are reports indicating increased activity and expression ofseveral lipogenic enzymes in cancer cells (6, 7) and evidenceidentifying the Akt pathway as a major regulator of the lipo-genic phenotype in liver cancer (8). Data indicate that theAkt pathway induces lipogenesis both via transcriptional and

Gsk3b-dependent posttranscriptional downregulation of ste-rol-regulatory element–binding proteins (SREBP; ref. 8). Acti-vation of the PI3K/Akt signaling pathway has also been seenin HCC, and Akt activation is strongly associated with HCCaggressiveness (9). The PI3K/Akt axis has been suggested as apromising target for HCC treatment (10, 11).

Several epidemiologic studies indicate a chemopreventiveeffect of statins [3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors] against HCC (12, 13), includinghepatitis B virus–related HCC (14). Statins have also beenshown to inhibit early phases of obesity-related experimentalliver tumorigenesis (15). Anticancer effects in HCC and othercancer types are often explained as an on-target effect of statinson HMG-CoA reductase, which leads to depletion of themevalonate pathway and an inhibited prenylation of signalingproteins (16).

Previously, we have found that statins deplete nuclear pAktvia purinergic receptor–dependent signaling in liver, lung,prostate, and pancreatic cells (17–19). We provided evidencefor P2X7 receptor–dependent rapid depletion of nuclear levelsof activated/phosphorylated Akt (pAkt) in cells stimulated byinsulin (17, 20, 21). Akt is expressed mainly in the cytoplasm,but as recently highlighted (22), it is also expressed in thenucleus, and there are data indicating that the nuclear locali-zation of Akt is important for its oncogenic activity (23). Thissuggested that statin-induced purinergic receptor signalingcould be of importance for its cancer-chemopreventive effects.Our interest in statin-induced P2X7 signaling is also stimulatedby recent data showing that P2X7 affects tumor growth (24)and invasion (21).

Against this background, we studied the effect of statins onpAkt in hepatocytes further. We here present evidence that

Institute of Environment Medicine. Karolinska Institutet, Stockholm, Sweden.

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

Corresponding Author: Aram Ghalali, Karolinska Institute, Nobels vag 13,Stockholm S-17177, Sweden. Phone: 468-5248-7343; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-16-0373

�2017 American Association for Cancer Research.

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atorvastatin decreases HBx-induced pAkt levels in hepatocytes.We also document downstream events, such as decreasedprotein levels of lipogenic enzymes, and we show an involve-ment of P2X receptors in these effects, indicating an off-targeteffect of atorvastatin.

Materials and MethodsCell culture

The HCC HepG2 and Huh7 cells were purchased from ATCC.TRL-1215 cells were provided as a generous gift from Dr. MichaelP. Waalkes from the NCI (Rockville, MD). This nontumorigeniccell line was originally derived from the livers of 10-day-oldFischer F344 rats (25).

HepG2 cells were grown in MEM, with Earl's salts andL-glutamine supplemented with 1 mmol/L of sodium pyruvate,nonessential amino acids, 10% inactivated FBS, and penicil-lin–streptomycin. The Huh7 cells were grown in DMEM, with10% inactivated FBS, penicillin–streptomycin, and 1 mmol/Lsodium pyruvate. The TRL-1215 cells were grown in William'sEþGlutaMax-I with penicillin/streptomycin and 10% inacti-vated FBS. There are reports that HepG2 and Huh7 cells donot express P2X7 mRNA (26). However, regarding HepG2cells, this is contradicted by other data (27, 28). The lattershows initial low levels but rapid induction of P2X7 mRNA inHepG2 cells.

ReagentsAtorvastatin, insulin, KN-62, and adenosine 50-triphosphate

periodate oxidized sodium salt (o-ATP) were purchased fromSigma-Aldrich. The atorvastatin concentration usedwas1mmol/L.Within 24 hours, this concentration decreased cholesterol esterlevels by 50% in HepG2 cells (29).

Western blottingCells were washed twice with PBS and lysed in IPB-7 with

protein degradation inhibitors (1 mg/mL phenylmethylsulfonylfluoride, 0.1mg/mL trypsin inhibitor, 1mg/mL aprotinin, 1mg/mLleupeptin, 1 mg/mL pepstatin, 1 mmol/L Na3VO4, and 1 mmol/LNaF). The samples were subjected to SDS-PAGE and blotted ontoa PVDF membrane (Bio-Rad). Some samples were subfractio-nated. The protein bands were probed using antibodies againstCdk2, HBx, P2X7, a-tubulin, total Akt, Akt phosphorylated atresidues Ser473 and Thr308 from Santa Cruz Biotechnology,Gsk3b Ser9, ACAC, MMP2, and MMP9 from Cell SignalingTechnology, and FASN from BD Biosciences. Proteins werevisualized with ECL procedure (Amersham Biosciences). TheWestern blotting results were analyzed with NIH Image 1.62software.

Immunocytochemical stainingCells were fixed in 3.7% formaldehyde. After fixation, the

cells were stained with polyclonal antibodies against phos-phorylated Akt at residue Thr308 (Santa Cruz Biotechnology).After incubation with primary antibodies, secondary anti-body conjugated with FITC (Dako) was applied. No stainingwas detected when the primary antibodies were omitted. Thenuclear staining intensity was analyzed with NIH Image 1.62software.

siRNA transfectionCells were transfected with P2X7, or control siRNA (Santa Cruz

Biotechnology) for 40 hours according to the TranIT-TKO pro-tocol (Lipofectamine 2000, Invitrogen).

Plasmid transfectionThe expression vector for wild-type HBx was provided as a

generous gift from Dr. Vijay Kumar at International Centre forGenetic Engineering and Biotechnology (New Delhi, India). TheHBx plasmid has been described previously (30). P2X7 plasmidand its empty vector were provided as a generous gift fromDr. St�ephane Chevalier (INSERM U1069, University of Tours,Tours, France). pEGFP-N1-mAKT and its empty vector wereprovided as a generous gift from Dr. Arthur Weiss at Universityof California, San Francisco (San Francisco, CA). Cells weretransfected with plasmids according to the Lipofectamine 3000(Invitrogen) protocol. Cells were transfected for 2.5 mg plasmidper 60-mm dish (according to the TranIT-TKO protocol) and for40 hours or for times indicated in the figures.

Cell invasion assayCell invasion assay was performed using 8-mmpore size Trans-

well Biocoat Control inserts (Becton Dickinson) according to themanufacturer's instructions. The cells were fixed with methanoland thereafter stained with Toluidine Blue from Merck. Thenumber of transmembrane cells was counted.

MTT assayCell proliferation was determined by 3-(4,5-dimethylthiazol-

2yl)-2,5-diphenyltetrazolium bromide (MTT) assay detecting thecellularmitochondrial capacity to convertMTT tetrazolium salt toformazan. Cells were incubatedwith themediumcontainingMTT(Sigma-Aldrich) for 4 hours. The cells were then lysed in DMSO.The absorbance was measured at 570 to 620 nm.

Statistical analysisStatistical analysis was conducted using Student t test. The data

were presented as mean � SD. Experiments were performed atleast three times with different batches of cells. Results wereconsidered to be statistically significant at P � 0.05.

ResultsAtorvastatin rapidly decreases HBx-and insulin-inducedpAkt

In line with previously published studies (4, 5), we showedthat overexpression of HBx in HepG2 cells enhanced the levelof pAkt Ser473. Total Akt was not affected. We also found thatthe Akt-induced Ser9-inactivating phosphorylation of Gsk3bwas increased in HepG2 cells (Fig. 1A). We then investigatedthe effect of HBx overexpression on the pAkt level in Huh7 cells.We found that HBx overexpression enhanced the levels ofpAkt Ser473 and pGsk3b Ser9 also in this human hepatocytecell line (Supplementary Fig. S1A).

Our earlier studies show that statins deplete nuclear pAktwithout affecting total Akt levels in many epithelial cell types(17, 18). The effect of atorvastatin on HBx-induced pAkt wasnow studied in HepG2 cells, and we found that atorvastatindecreases HBx-induced pAkt Ser473 (Fig. 1B). The effect ofatorvastatin on the insulin-induced pAkt level was also exam-ined. We found that incubation of cells with atorvastatin

Statin Therapy Inhibits pAkt via P2X Receptors

www.aacrjournals.org Mol Cancer Res; 15(6) June 2017 715




(1 mmol/L) decreased the insulin-induced pAkt Ser473 (signif-icant at 1 and 3 hours) and pGsk3b Ser9 (significant at 3 hours)in HepG2 cells (Fig. 1C). We also used nontransformedTRL-1215 rat liver epithelial cells. We found that atorvastatindecreased the insulin-induced pAkt Ser473 in TRL-1215 cells aswell. The effect on pGsk3b Ser9 was nonsignificant (Fig. 1D).Taken together, we show that statins decrease HBx- or insulin-induced pAkt in hepatocytes.

Nuclear translocation of Akt is important for its activity(22, 23), and we have shown previously that statin decreases thenuclear pAkt within minutes (17). Overexpression of HBx vectorincreased nuclear pAkt and atorvastatin lowered HBx-inducednuclear pAkt in HepG2 cells (Fig. 2A). This effect was confirmedby immunofluorescence microscopy (Fig. 2B). Employing thesame technique, we also found that atorvastatin depleted insu-lin-induced nuclear pAkt in HepG2 and TRL-1215 cells (Fig. 2Cand D). As shown in Fig. 2E and F, fractionation experimentsconfirmed decreased levels of nuclear pAkt by showing decreasedlevels of pAkt Ser437. No concomitant increase of pAkt in thecytoplasmic fraction was seen. The time span used in some ofour experiments (15 minutes, Fig. 2C–F) tended to exclude aninvolvement of mevalonate depletion, as the effects of statinson mevalonate pathway are not detected until 2 to 4 hours (17).In summary, these data indicate that atorvastatin deplete HBx-and insulin-induced nuclear pAkt in hepatocytes via mechanismsthat are not dependent on mevalonate depletion.

Effects of atorvastatin on Akt and lipogenic enzymes aremediated by P2X receptors

We have previously shown an involvement of the purinergicP2X7 receptor in pAkt depletion induced by statins (21) andthat P2X7 colocalizes with P2X4 upon the addition of xeno-biotics (31). The role of P2X in HBV-induced pathogenesis islargely unknown, but significant increase of purinergic receptorlevels has been reported in hepatic cells stably expressing hep-atitis C virus (HCV) structural proteins (32). We now investi-gated the role of the P2X receptor in statin-induced effects inHepG2 cells. The P2X antagonist, oxidized-ATP (o-ATP), and aselective inhibitor of P2X7 receptor, KN-62, were tested. Wefound that atorvastatin-induced depletion of pAkt and pGsk3bSer9 was inhibited by P2X7 inhibitors (Fig. 3A). Next, weinvestigated the involvement of P2X7 receptor in insulin-induced pAkt. As shown in Fig. 3B and C, preincubation witho-ATP or KN62 abrogated the atorvastatin-induced effect onpAkt Ser473 in insulin-treated HepG2 and TRL-1215 cells. o-ATP or KN62 alone did not have any effect on pAkt (Fig. 3B andC). In addition, the siRNA against P2X7 was tested in TRL-1215cells. As shown in Fig. 3D, the atorvastatin-induced depletion ofinsulin-induced pAkt Ser473 was completely blocked by siRNAP2X7. To further investigate the role of P2X7 receptor, we testedthe effect of statin on P2X7-negative Huh7 cells (Fig. 3E; ref. 32).We found that atorvastatin had no effect on insulin-inducedpAkt Ser473 in Huh7 cells, but that atorvastatin depleted pAkt

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Atorvastatin decreases HBx- and insulin-induced pAkt levels in hepatocytes. A, HepG2 cells were transfected with HBx-expressing plasmid for 40 hours.B, HepG2 cells were transfected with HBx-expressing plasmid for 40 hours, serum starved (24 hours), and thereafter treated for atorvastatin (1 mmol/L) foradditional 24 hours. C, Serum-starved HepG2 cells were treated with insulin (1 mg/mL) for 15 minutes and thereafter with atorvastatin for 1 or 3 hours.D, Serum-starved rat primary liver TRL-1215 cells were treated with 1 mg/mL insulin for 15 minutes and thereafter with atorvastatin (1 mmol/L) for 1 or 3 hours.A–D, Cell lysates were analyzed for Akt1, pAkt Ser473, pGsk3b Ser9, and HBx by Western blotting. Cdk2 was used as loading control. Bars represent themean� SD from three independent experiments. �, Significantly different from control cells; ¤, significantly different from HBx-overexpressed cells; #, significantlydifferent from insulin-treated cells, P < 0.05.

Ghalali et al.

Mol Cancer Res; 15(6) June 2017 Molecular Cancer Research716




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Atorvastatin decreases nuclear pAkt in hepatocytes. A, HepG2 cells were transfected with HBx vector for 40 hours, serum-starved for 24 hours, andthereafter incubated with 1 mmol/L atorvastatin for 3 hours. In B, HepG2 cells were transfected with HBx vector for 40 hours followed by starvation for 24 hoursand thereafter treated with 1 mmol/L atorvastatin for 3 hours, and the cells were stained for pAkt Thr308. C–F, Serum-starved cells were incubated with1 mg/mL insulin for 15 minutes and thereafter with 1 mmol/L atorvastatin for 15 minutes. C and D, Cells were stained for pAkt Thr308. E and F, Western blotsand densitometric analysis of nuclear and cytoplasmic fractions are shown. A–F, Bars represent the mean � SD from three independent experiments. Cdk2was used as a loading control. �, Significantly different from control cells; #, significantly different from insulin-treated cells; ¤, significantly different fromHBx vector–transfected cells, P < 0.05.






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P2X receptors mediate the atorvastatin-induced effect on HBx-and insulin-induced pAkt in hepatocytes. A, HepG2 cells were transfected with HBx vector for40 hours, followed by 24-hour starvation and thereafter incubated with o-ATP (250 mmol/L) or KN-62 (100 mmol/L) for 10 minutes, and thereafter withatorvastatin (1 mmol/L) for 3 hours.B andC, Serum-starved cells were treatedwith insulin (1 mg/mL) for 15minutes followed by treatment with P2X inhibitors (o-ATP,250 mmol/L or KN-62, 100 mmol/L) for 10minutes, and thereafter with atorvastatin (1mmol/L) for 3 hours.D, TRL-1215 cellswere transfectedwith siRNA against P2�7(50 nmol/L) for 40 hours and thereafter serum starved (24 hours) and incubated with insulin (1 mg/mL) for 15 minutes then with atorvastatin (1 mmol/L) for 3 hours.E, Huh7 cells were transfected with P2�7-expressing plasmid for 40 hours and thereafter serum starved (24 hours) and treated with insulin (1 mg/mL) for15minutes then atorvastatin 1mmol/L for 3 hours.A–E,Westernblots anddensitometric analysis of total fractions are shown. Bars represent themean�SD from threeindependent experiments. Cdk2 was used as a loading control. �, Significantly different from control cells; #, significantly different from insulin-treated cells;P < 0.05.

Ghalali et al.





Ser473 after transfection of P2X7 (Fig. 3E), confirming a dys-functional P2X7 in this cell line as well as a role for P2X7 inatorvastatin-induced effects.

Gsk3bmaydownregulate SREBPs and lipogenesis (33, 34), andAkt-induced Gsk3b Ser9 phosphorylation inhibits this down-regulation. Thus, Akt overexpression enhances the levels of severalproteins involved in lipogenesis (8). We studied the effect ofatorvastatin on lipogenic enzymes inducedbyAkt overexpression.We employed HepG2 cells transfected with HBx. However, thisdid not give clear enough effects on lipogenic enzymes. Instead,we transfected HepG2 cells with Akt, which resulted in clearlyincreased FASN and ACAC levels (Fig. 4A). Similar effects wereseen in TRL-1215 cells (data not shown). We also treated the Akt-overexpressing HepG2 cells with atorvastatin and found that Akt-induced FASN was decreased significantly by atorvastatin. Thiseffect was seen after 24 hours of treatment (Fig. 4A) and also after3 hours of atorvastatin treatment (data not shown). No effect ofstatin on ACACwas seen.We then investigated the levels ofHMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis,which is targeted by statin therapy (35). We found that Akttransfection induced HMG-CoA reductase protein levels inHepG2 cells and that atorvastatin reduced these levels. We alsofound that KN62 counteracted this effect of atorvastatin (Fig. 4B),indicating the involvement of P2X7. A role for P2X receptors wasconfirmed by using Huh7 cells and the P2X4 inhibitor TNP-ATP(18). As shown in Supplementary Fig. S1B, also this inhibitorcounteracted the statin-induced effect on HMG-CoA reductaseprotein levels.

It was recently shown that FASN deletion or overexpressionmay influence Akt activation via Rictor in mouse liver (36). Incontrol experiments employing HBx-transfected cells (data notshown), we observed no effects of atorvastatin on Rictor levels,indicating that atorvastatin did not affect pAkt levels via down-regulating Rictor.

Atorvastatin reduces HBx-induced cellular invasiveness andproliferation.

We have earlier shown that statin-induced pAkt depletion isassociated with decreased invasiveness and proliferation of dif-

ferent cancer cell lines (21). We have also shown that P2X7 isinvolved in invasive growth and that atorvastatin inhibits inva-siveness through a P2X7–Akt axis (21). The expression of matrixmetalloproteinase (MMP) family has been shown tohave a role incellular invasion andmetastasis. Two particular genes of theMMPfamily,MMP-9 andMMP-2, have been reported to be increased byHBV infection (37). MMP-9 is activated through activation ofERKs and PI3K–Akt pathways (38). We found that HBx over-expression enhanced MMP-9 and MMP-2 protein levels in theHepG2 cells. Treatment of cells by atorvastatin for 24 hoursdecreased HBx-induced MMP-9 and MMP-2 levels significantly(Fig. 5A). Furthermore, we found that HBx transfection inducedinvasiveness and that atorvastatin counteracted this effect. Theinhibiting effect shown in Fig. 5B correlated with decreases inMMP levels (Fig. 5A) and with inhibition of cell proliferation(Fig. 5C). As shown in Supplementary Fig. S2A and S2B, atorvas-tatin also induced a significant inhibition of hepatic cell prolif-eration. Atorvastatin have even a stronger antiproliferative effectonHBx-overexpressing cells than on untransfected cells (Fig. 5C).Taken together, we found that atorvastatin decreased HBx-induced cellular invasiveness and proliferation in HepG2 cells,and that was correlated to decrease in MMP2 and MMP9 levels.

DiscussionIn this study, we show that atorvastatin decreases nuclear pAkt

levels in hepatocytes. We employed insulin, the oncogenic virusprotein HBx, and Akt transfection to increase pAkt levels, and ageneralized ability of atorvastatin to abrogate Akt activation inhepatocytes was indicated. A mitigated inhibition of Gsk3b,decreased levels of lipogenic enzymes, decreased invasive growth,and decreased cell proliferation, all possible downstream effectsof decreased pAkt levels, were also documented. Typically, weused 1 mmol/L concentration of atorvastatin in our experiments,but previously, we also reported pAkt depletion at 50 nmol/L(20, 21), so an effect on pAkt by statin therapy in humans can beexpected (39).

The time frame for many of our experiments indicates thatdecreased pAkt levels are not caused by an inhibited

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Atorvastatin decreases Akt-induced lipogenesis enzymes. A, HepG2 cells were transfected with Akt-expressing plasmid for 40 hours, serum starved (24 hours),and thereafter treated with atorvastatin (1 mmol/L) for additional 24 hours. B, HepG2 cells were transfected with Akt vector for 24 hours followed by serumstarvation (24 hours) and treatment with KN-62 100 mmol/L for 10 minutes and then by treatment with atorvastatin (1 mmol/L) for 3 hours. A and B, Westernblots and densitometric analysis of total fractions are shown. Bars represent the mean � SD from three independent experiments. Cdk2 was used as aloading control. �, Significantly different from control cells; #, significantly different from Akt-overexpressed cells; P < 0.05.






mevalonate pathway, as components in this pathway are notaffected until 2 to 4 hours after statin addition (40). Purinergicreceptors, on the other hand, induce rapid effects on cellsignaling (17), and our data showing an involvement of P2Xreceptors are consistent with a rapid effect on pAkt levels. Ourdata thus indicate that the pAkt effects shown here can bedelineated as an off-target effect of atorvastatin, and as we arguebelow, statin-induced pAkt depletion may lead to effects medi-ated by Gsk3b and SREBP2 as well as to other effects discussedin our previous studies (17–21).

There is ample evidence that PI3K/Akt regulates SREBPs viaAkt phosphorylation of Gsk3b, and also that PI3K/Akt inhibi-tors downregulate the mevalonate pathway (41–43). We foundthat Akt overexpression increased FASN and HMG-CoA reduc-tase protein levels, and we found that atorvastatin counteractedthe Akt-induced effects via a P2X-dependent mechanism. Theeffect of Akt overexpression is in line with earlier studiesshowing that Akt increases the expression of HMG-CoA reduc-tase and FASN genes (44–46). Atorvastatin may thus not onlyact as a competitive inhibitor of HMG-CoA reductase, but alsodecreased its protein levels by downregulating Akt via an off-target effect. The regulation of HMG-CoA reductase is verycomplex, and there are reports that statins upregulate unin-duced HMG-CoA reductase expression (see e.g., ref. 29). How-

ever, we have found no literature data on how statins affect Akt-induced HMG-CoA reductase levels.

In experimental studies of statins and cancer-chemopreven-tive effects, a role for depleted metabolites in the mevalonatepathway is often demonstrated by adding mevalonate in highconcentrations (see e.g., ref. 16). Our data suggest that meva-lonate may counteract not only the competitive inhibition ofHMG-CoA reductase (16), but also the off-target effect onlipogenic enzyme expression, including a decreased HMG-CoAreductase expression as shown here. Consequently, counter-acting effects of mevalonate addition may not exclude aninvolvement of an off-target effect on pAkt by statins.

In our previous work on statins and their effect on pAktlevels, we discussed anticancer effects other than a decreasedHMG-CoA reductase expression and an inhibited mevalonatepathway (17–19, 21). For example and as mentioned above,we recently suggested a statin-induced pAkt-dependent effecton MMP-9 activity (21). Of further interest is that in a well-characterized three-dimensional (3D) model of mammaryglands, mutant p53 caused malignant phenotypes by upregu-lating SREBP2 and the mevalonate pathway (i.e., a mechanismsimilar to the one induced by pAkt) and that simvastatin veryeffectively inhibited this effect (16). In fact, the authors com-ment that simvastatin in some aspects had more dramatic

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Figure 5.

Atorvastatin decreases HBx-induced invasiveness and proliferation in HepG2 cells.A andB,HepG2 cells were transfectedwith HBx-expressing plasmid for 24 hours,serum starved for 40 hours, and thereafter treated with atorvastatin (1 mmol/L) for additional 24 hours. B, Cells analyzed by invasion assay. C, HepG2 cellswere transfected with HBx-expressing plasmid for 40 hours. The cells were treated during the transfection with atorvastatin (1 mmol/L) for 24 hours and the cellproliferation was measured by MTT assay. A–C, Bars represent the mean � SD from three independent experiments. Cdk2 was used as a loading control.�, Significantly different from control cells; #, significantly different from HBx-overexpressing cells; *, significantly different from empty vector–overexpressing cells;¤, significantly different from empty vector–overexpressing cells treated with atorvastatin; P < 0.05.

Ghalali et al.





effects than deleting mutant p53 (16). This latter observationleads us to speculate that simvastatin might have decreasedpAkt levels and that additional targets (9), besides SREBP2and the mevalonate pathway, were affected by pAkt depletion.This 3D model might thus be suitable for further characteri-zation of off-target effects of statins.

Epidemiologic studies indicate that statins prevent HBV-induced HCC (14) as well as perhaps any type of HCC(13, 47). Our data suggest that statin-induced downregulationof pAkt might be part of an explanation of this chemopreven-tive effect. Of perhaps particular interest is the question whetherdownregulation of lipogenic enzymes via pAkt depletion isof relevance for a chemopreventive effect on NASH andNASH-related HCC. Further studies are warranted, as FASN hasbeen implicated in this type of hepatocellular carcinogenesis(8). For example, recent work indicates that FASN ablationprevents HCC development in Akt-overexpressing mice (36).

Our data indicate a role for P2X receptors in atorvastatin-induced effect in hepatocytes. P2X receptors are mainly studiedin inflammatory and mesenchymal cells, and less is knownabout their function in epithelial cells even though P2X recep-tors are expressed in these cells (48). In HCC, high P2X7expression in peritumoral hepatocytes, but not in tumor tissue,correlates with poor prognosis for HCC patients (49). It corre-lated to aggressive clinicopathologic features, especially highrates of vascular invasion, a marker of HCC invasiveness (49).The role of P2X receptors in HBV-induced pathogenesis isunknown, but significant increase of purinergic receptor levelsin hepatic cells stably expressing HCV structural proteins hasbeen reported (32).

Invasion andmetastasis are crucial events in HCC progression,and our findings complement growing evidence that HBxoverexpression is associated with increased levels of MMPs and

contributes to cell invasion. HBx-induced invasiveness correlatesto the PI3K/Akt pathway activity (3), and our finding that HBxtransfection induced invasiveness supports a role for Akt inHBx-induced HCC.

In summary, our data show that atorvastatin counteractsHBx-induced pAkt levels in human hepatocytes. In line with this,inhibiting effects on cellular invasiveness and proliferation wereseen in HBx-overexpressing cells, and on lipogenic enzymeexpression in Akt-overexpressing cells. We also provide evidencethat the atorvastatin-induced downregulation of pAkt was medi-ated by P2X receptors, and our data suggest that this effect mightwork in parallel with a statin-induced competitive inhibition ofHMG-CoA reductase to counteract HCC development as docu-mented in epidemiologic studies.

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

Authors' ContributionsConception and design: A. Ghalali, J. Martin-Renedo, J. H€ogberg, U. SteniusDevelopment of methodology: A. Ghalali, J. Martin-RenedoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Ghalali, J. Martin-Renedo, J. H€ogbergWriting, review, and/or revision of the manuscript: A. Ghalali, J. Martin-Renedo,J. H€ogberg, U. SteniusAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Ghalali, J. Martin-RenedoStudy supervision: A. Ghalali, U. Stenius

The costs of publication of this articlewere defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 25, 2016; revised January 24, 2017; accepted January 25,2017; published OnlineFirst February 16, 2017.

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Ghalali et al.




2017;15:714-722. Published OnlineFirst February 16, 2017.Mol Cancer Res Aram Ghalali, Javier Martin-Renedo, Johan Högberg, et al. via P2X ReceptorsAtorvastatin Decreases HBx-Induced Phospho-Akt in Hepatocytes

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Atorvastatin Decreases HBx-Induced Phospho- Akt in ...Taken together, we show that statins decrease HBx- or insulin-induced pAkt in hepatocytes. Nuclear translocation of Akt is important