histonedeacetylaseinhibitorvalproicacidinhibitscancer … · 2010-03-20 ·...

Histone Deacetylase Inhibitor Valproic Acid Inhibits CancerCell Proliferation via Down-regulation of the AlzheimerAmyloid Precursor Protein*□S

Received for publication, August 20, 2009, and in revised form, February 5, 2010 Published, JBC Papers in Press, February 9, 2010, DOI 10.1074/jbc.M109.057836

Vivek Venkataramani‡, Christian Rossner‡, Lara Iffland‡, Stefan Schweyer§, Irfan Y. Tamboli¶, Jochen Walter¶,Oliver Wirths‡, and Thomas A. Bayer‡1

From the ‡Department of Molecular Psychiatry, Alzheimer Ph.D. Graduate School, and §Department of Pathology, University ofGoettingen, 37075 Goettingen and the ¶Department of Molecular Cell Biology, University of Bonn, 53127 Bonn, Germany

The �-amyloid precursor protein (APP) represents a type Itransmembrane glycoprotein that is ubiquitously expressed. Inthe brain, it is a key player in the molecular pathogenesis ofAlzheimer disease. Its physiological function is however lesswell understood. Previous studies showed that APP is up-regu-lated in prostate, colon, pancreatic tumor, and oral squamouscell carcinoma. In this study, we show that APP has an essentialrole in growth control of pancreatic and colon cancer.AbundantAPP staining was found in human pancreatic adenocarcinomaand colon cancer tissue. Interestingly, treating pancreatic andcolon cancer cells with valproic acid (VPA, 2-propylpentanoicacid), a known histone deacetylase (HDAC) inhibitor, leads toup-regulation of GRP78, an endoplasmic reticulum chaperoneimmunoglobulin-binding protein. GRP78 is involved in APPmaturation and inhibition of tumor cell growth by down-regu-lation of APP and secreted soluble APP�. Trichostatin A, a pan-HDAC inhibitor, also lowered APP and increased GRP78 levels.In contrast, treating cells with valpromide, a VPA derivativelacking HDAC inhibitory properties, had no effect on APP lev-els. VPA did not modify the level of epidermal growth factorreceptor, another type I transmembrane protein, and APLP2, amember of the APP family, demonstrating the specificity of theVPA effect on APP. Small interfering RNA-mediated knock-downofAPPalso resulted in significantly decreased cell growth.Based on these observations, the data suggest that APP down-regulation viaHDAC inhibition provides a novelmechanism forpancreatic and colon cancer therapy.

�-Amyloid precursor protein (APP)2 is a highly conservedsingle transmembrane protein (type I) with a receptor-likestructure and consists of a heterogeneous group of proteins

migrating between 110 and 135 kDa (1, 2). The heterogeneity isdue to alternative splicing, leading to eight distinct isoforms(namelyAPP677, APP695, APP696, APP714, APP733, APP751,APP752, and APP770), as well as by a variety of post-transla-tional modifications, including O- and N-glycosylation, sulfa-tion, and phosphorylation. APP isoforms exist as immature(N-glycosylated) and mature (N- and O-glycosylated, tyrosyl-sulfated) species. Immature APP localizes in the endoplasmicreticulum and cis-Golgi, and the mature APP form preferen-tially localizes in the trans-Golgi network, secretory and endo-cytic vesicles, and at the plasma membrane (3, 4).APP695 is the most common isoform in the central nervous

system, whereas APP751 and APP770 are predominantlyexpressed in non-neuronal cells (5). The key event in the path-ogenic cascade in Alzheimer disease is the amyloidogenic path-way characterized by subsequent cleavage of APP by theenzyme �-secretase and further processing by �-secretase,which finally leads to the generation of A� peptides. However,the predominant route of APP processing consists of successivecleavages by �- and �-secretases in non-neuronal cells (6, 7).The cleavage of APP at Lys16–Leu17 bond by�-secretase withinthe A� sequence liberates the sAPP� and the nonamyloido-genic C-terminal APP fragment (8–10). APP is one of threemembers of a small gene family, which includes amyloid � pre-cursor-like protein 1 (APLP1) and amyloid � precursor-likeprotein 2 (APLP2). All encode type I transmembrane proteinsand share similar domain structures, with a large extracellularN-terminal domain and a short cytoplasmic region thatundergo similar processing. In contrast to APLP1, which pref-erentially is expressed in neuronal tissues, APLP2 is expressedalso in peripheral non-neuronal tissue (11). The biologicalactivity of APP is still not well understood, especially in non-neural and cancer cells. Several studies showed that APP and itssecreted forms promote adhesion, migration, neurite out-growth, and general growth-promoting properties (reviewed inRefs. 2, 11). In a previous study, we demonstrated that SH-SY5Yneuroblastoma cells transfected with APP695 showed anincrease in cell proliferation compared with mock-transfectedcontrols (12).Recent evidence supports the observation of an inverse link

between cancer and Alzheimer disease (13). The authors dem-onstrated that Alzheimer disease was longitudinally associatedwith a reduced risk of cancer, and a history of cancer was asso-ciated with a reduced risk of Alzheimer disease suggesting a

* This work was supported by the Alzheimer Forschung Initiative e.V. (toO. W.) and the European Commission (Marie Curie Early Stage Training,MEST-CT-2005-020013 (NEURAD)).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Materials and Methods” and Figs. 1–3.

1 To whom correspondence should be addressed: Division of Molecular Psy-chiatry, University of Medicine, Gottingen, Von-Siebold-Strasse 5, 37075Gottingen, Germany. Tel.: 49-551-39-22911; Fax: 49-551-39-10291; E-mail:[email protected].

2 The abbreviations used are: APP, amyloid precursor protein; sAPP�, solubleAPP�; EGFR, epidermal growth factor receptor; H4Ac, hyperacetylated his-tone H4; HDAC, histone deacetylase; siRNA, small interfering RNA; TSA,trichostatin A; VPA, valproic acid; VPM, valpromide; ANOVA, analysis ofvariance; MAPK, mitogen-activated protein kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10678 –10689, April 2, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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common mechanism linking both diseases. In this study, wecould demonstrate that APP is selectively overexpressed inpancreatic and colon carcinoma, but not in healthy tissues.Because APP seems to have an important function as a growthfactor, it has received considerable attention in the oncologyfield. Studies showed that patients with increased APP levelshave a significantly lower survival rate and has therefore beensuggested as a potential biomarker to evaluate cancer prognosis(14–17).For decades, valproic acid (VPA, 2-propylpentanoic acid) has

been the drug of choice for the treatment of epilepsy and bipo-lar disorder and represents one of the most important thera-peutic agents in psychiatry (18, 19), although the underlyingmechanisms for brain activity are controversial. Furthermore,VPA is a well established histone deacetylase (HDAC) inhibitorand affects cell growth in different types of cancer in vitro and invivo (19–24). We examined VPA-induced alterations in theprocessing of endogenous APP. We further focused on themolecular mechanism responsible for the highly specificimpairment in the maturation of APP and the reduction ofsecreted sAPP� caused by VPA in the cancer cell lines. Thebinding immunoglobulin protein (BiP) (also called glucose-reg-ulated protein 78, GRP78) is a molecular chaperone that usesATP/ADP cycling to regulate protein folding. GRP78 is a78-kDa heat shock protein induced by VPA (25), and it isinvolved in maturation of APP (26). The aim of this report wasto study the potential impact of APP on prominent gastrointes-tinal tumor growth and to elucidate the underlying molecularmechanism.

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—The following antibodies wereused: monoclonal APP/A� antibodyW0-2 (1:5000, TheGenet-ics Co.), APP (1:250 ofmonoclonal antibody 22C11, Chemicon;1:500 of polyclonal antibody 23850, generous gift from GerdMulthaup), polyclonal APP antibody 5313 (27), anti-acetyl his-tone H4 (1:2000, Millipore), EGFR (1:200, Santa Cruz Biotech-nology), APLP2 (1:5000, Calbiochem), GRP78 (1:1000, Cell Sig-naling Technology), andmonoclonal mouse anti-actin (1:5000,Sigma). VPA (Sigma) was prepared in sterile water as concen-trated stock solution and added to the final concentrations asindicated. Trichostatin A stock solution (5 mM in DMSO) waspurchased from Sigma. Valpromide (VPM), a kind gift fromKatwijk Chemie B.V., was dissolved in DMSO and added tofinal concentrations as indicated.Human Specimens—Histological classification (tumor type,

grade of malignancy) was carried out according to the currentWorld Health Organization and International Union AgainstCancer criteria. All slides were re-evaluated again, and diagno-sis was approved by an experienced pathologist. All tumorspecimens (n � 3 of each tumor type) were obtained from theDepartment of Pathology, University Medicine, Goettingen,Germany.Cell Culture and Transfection—Stably expressing cell lines

were obtained by transfecting the mammalian expression vec-tor pCEP4 (Invitrogen) alone (mock) or with the APP695wt orSPA4CT constructs into SH-SY5Y cells using Lipofectin 2000(Invitrogen). 300 �g/ml hygromycin (Invitrogen) was added to

maintain stable integration of the constructs in the transfectedcells. APP695-transfected and mock-transfected SH-SY5Ycontrol cells have been in culture for an identical period of timewith a similar number of passages. All transfected cell lineswere cultured in Dulbecco’s modified Eagle’s medium/F-12(Pan Biotech GmbH), supplemented with 10% fetal calf serum,2 mM L-glutamine, and 1% nonessential amino acids. Threepancreatic cancer cell lines (BxPC3, PANC-I, and CFAPC-1)and four colon cancer cell lines (SW480, LoVo, CaCo-2, andT84) were used in this study (kindly provided by Prof. Ghadimi,University of Gottingen) and were cultured in RPMI 1640medium (Pan Biotech GmbH) containing 10% fetal calf serumand 2mM L-glutamine. All cell cultures were incubated at 37 °Cin a humidified atmosphere of 5%CO2. Data are presented onlywith the BxPC3 and SW480 cell lines.Immunohistochemistry on Paraffin Sections—Paraffin-em-

bedded colon and pancreas tissue sections (4 �m) were depar-affinized in xylene and rehydrated in a series of ethanol concen-trations. Primary antibodies 22C11 and 23850 were incubatedovernight in a humid chamber at room temperature. Sectionswere subsequently incubated with a horseradish peroxidase-conjugated polymer, which carries antibodies to rabbit andmouse immunoglobins (EnVision/HRPTM, Dako, Hamburg,Germany), and signals were visualized with 3,3�-diaminobenzi-dine (Dako). Counterstaining was carried out with Meyer’shematoxylin, mounted in SuperMount medium, and equippedwith an Olympus DP-50 camera using the software ViewfinderLite version 1.0.134 (Pixera Co.). All tissue sections were ana-lyzedwith themonoclonal antibody 22C11 and polyclonal anti-body 23850. For negative controls, blocking solution was usedin place of the primary antibody.Cell Growth Assay—Cell growth was measured by a colori-

metric cell proliferation assay (CellTiter 96AQassay, Promega)using the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (Owens’s rea-gent) to formazan according to the protocol of the supplier asreported previously (12). In brief, BxPC3 andSW480 cancer celllines were seeded at 5 � 103 cells/well in 96-well culture platesand incubated for 24 h with RPMI 1640 medium containing10% fetal bovine serum. The cells were than treated withmedium containing VPA, TSA, and VPH for 24 h. After 24 h,20 �l of dye solution was added to each well, and the platewas incubated for 2 h at 37 °C, 5% CO2. The absorbance wasmeasured at 490 nm using an enzyme-linked immunosor-bent assay microplate reader. Each assay was performed atleast six times.Western Blot Analysis—For treatment experiments, 1 � 106

BxPC3 and SW480 cells were seeded per 25-cm2 flask. Cellswere treated for 24 h with serum-freemedia followed by 24 h oftreatment with the compounds. Protein concentration wasmeasured using a commercially available kit (Roti-Quant,Roth). Cells were harvested using lysis buffer (50 mM Tris, 150mMNaCl, 1%Nonidet P-40, 1% Triton X-100, 2 mM EDTA, pH7.5) supplemented with 1� complete protease inhibitor mix-ture (Roche Applied Science) for 30min at 4 °C. Cellular debriswas removed by centrifugation (5000 � g, 10 min), and equalamounts of soluble proteins (usually 10–15 �g) were separatedby electrophoresis on 4–12% Vario-Gels (Anamed) and trans-

�-Amyloid Precursor Protein Is a Tumor Growth Factor

APRIL 2, 2010 • VOLUME 285 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 10679


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ferred to nitrocellulose membranes (GE Healthcare). Afterwashing in Tris-buffered saline, including Tween 20 (TBS-T),the blotswere incubatedwith the primary antibody, followedbyhorseradish peroxidase-conjugated secondary mouse/rabbitantibodies (1:4000/1:3000, Dako). For detection of sAPP�, con-ditioned culture media were collected and centrifuged at14,000� g for 2min at 4 °C to remove cell debris. Subsequently,supernatant (2 ml) was concentrated by centrifugal filterdevices (Amicon Ultra-4 50K, Millipore) at 4000 � g for 5 min.Protein levels in the supernatant were normalized to the pro-tein concentration in the corresponding cell lysate. For quanti-fication of relative protein levels, x-ray films (Hyperfilm EC,AmershamBiosciences) were scanned, and densitometric anal-ysis was carried out using ImageJ software (version 1.41o,National Institutes of Health). Each immunoblot was donethree to six times.Knockdown of APP Using siRNA—BxPC3 and SW480 cells

were grown in 6-well plates to �30–50% confluence in antibi-otic-free medium and transfected with siRNA duplexes(Ambion, Silencer� Select validated siRNA, s1500) targeted toAPP as follows: sense sequence, CAAGGAUCAGUUACG-GAAATT; antisense sequence, UUCCGUAACUGAUCCU-UGGT. Transfection of the cells was carried out usingLipofectamine 2000 and Opti-MEM I medium (Invitrogen)according to the manufacturer’s protocol at a final concentra-tion of 50 nM. Random siRNA (Ambion, Silencer� negativecontrol siRNA) served as negative control targeted to a scram-bled sequence. According to the manufacturer, the specificityof the Silencer� select siRNAs was validated by TaqMan geneexpression assays. Specific knockdown of APP by Silencer�select siRNA has been shown previously (28). After 48 h, celllysates and media were collected and analyzed using the proto-col for cell lysis described above.Statistical Analysis—Statistical differences were evaluated

using one-wayANOVA followed by Bonferroni post hoc test orunpaired t test as indicated. All data are given as means � S.E.All statistics were calculated using GraphPad Prism version5.00 software.

RESULTS

Expression of APP in Colon and Pancreas Carcinoma—Us-ing immunohistochemistry, the expression of APP in neoplas-tic tissue was studied in colon carcinoma and pancreas carci-noma. Immunohistochemistry revealed that colon carcinomacells showed a strong expression ofAPP,whereas no expressionwas seen in normal epithelial cells of the colon (Fig. 1, A–D). Inpancreas carcinoma, an intense expression of APP could befound in tumor cells (Fig. 1,G andH). Interestingly, islet cells ofthe endocrine pancreas presented a strong expression of APP(Fig. 1,E andF), whereas acinar cells showed a slight staining forAPP. In contrast, APPwas not detected in the normal epithelialcells of the salivary duct system aswell as the exocrine pancreas,respectively (Fig. 1, E and F). To detect APP, we used themono-clonal antibody 22C11, which recognizes residues 66–81 (29)and the polyclonal antibody 23850 which detects residues18–491 of human APP (30, 31).

APP Knockdown Results in Inhibition of Tumor CellGrowth—To further validate the functional relevance of APPin pancreatic and colon cancer, a loss-of-function study usingsiRNA was performed. Detection of APP in SW480 cells byWestern immunoblotting revealed two bands at 110 and 130kDa. To test whether these two variants represent immatureand mature APP, respectively, we performed pulse-chaseexperiments. After pulse labeling, a major band of 110 kDa wasdetected in cell lysates. During the chase period an additionalband was detected at 130 kDa, indicating that immature APPwas converted to its mature form byO-glycosylation. Immuno-precipitation of APP from conditioned media revealed secre-tion of soluble APP after 20 min that accumulated during thechase. These data indicate that the two bands observed byWestern immunoblotting represent immature and maturevariants of a single splice variant (supplemental Fig. S1).The expression of APP was efficiently suppressed by treat-

ment of SW480 cells with APP siRNA but not with randomsiRNAs (Fig. 2). Cellular and secreted APP were reduced by56 � 3.9 and 67 � 5.7%, respectively. In BxPC3 cells, treatmentwith APP siRNA also resulted in significantly reduced levels ofcellular (62 � 2.7%) and secreted sAPP� (69 � 7.7%). Impor-tantly, siRNA-mediated down-regulation of APP significantlyreduced cell growth in both cell types. siRNA treatment did notaffect cellular morphology either in pancreatic or in colon can-cer cells (data not shown). APP siRNA treatment had no effecton APLP2 levels (supplemental Fig. S2).VPA Treatment Inhibits Cell Growth, Decreases APP Lev-

els, and Reduces Secreted sAPP� in Pancreatic and ColonCell Lines—A range of studies have shown that VPA and otherHDAC inhibitors alter cellular proliferation and induce pro-grammed cell death in pancreatic and colon cancer in vitro andin vivo (23, 32). Based on these observations, we performed cellproliferation assays on BxPC3 (pancreatic cancer) and SW480(colon cancer) treated with VPA (Fig. 3), as well as using addi-tional pancreatic (PANC-I and CFAPC-1) and colon (LoVo,CaCo-2, and T84) cancer cell lines (data not shown). All cellswere treated with different concentrations of VPA rangingfrom 0 to 100 mM and showed similar growth inhibition. After24 h of incubation, we performed a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium assay to evaluate drug effect on cellular growth. Resultsare presented as percentage inhibition compared withuntreated control (Fig. 3, A and D). VPA treatment resulted indose-dependent inhibition in proliferation with both cell lines.To further evaluate the effect of VPA on APP expression,BxPC3 and SW480 were exposed to 0, 1, 2.5, 5, or 10 mM VPAfor 24 h. To analyze total APP and the processing productsAPP�, we used the APP/A�-specific antibodyW0-2 recogniz-ing a region between amino acid 5 and 8 of humanA� (33). Thisantibody detects full-length APP and C99 in cell lysates andsAPP� in culturemedia. All blots were re-probedwith a�-actinantibody to ensure equal protein load. Significant down-regu-lation was seen in BxPC3 cells at 1 mM and in SW480 at 2.5 mM

VPA treatment. APP decreased with higher doses of VPA inBxPC3 as follows: 1 mM, 30 � 2.3%; 2.5 mM, 40 � 8.9%; 5 mM,43 � 6.2%, and 10 mM 80 � 5.4% (Fig. 3C). APP expressionfurther decreased with higher doses of VPA in SW480




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(means � S.E. in % reduction of APP levels versus untreatedcontrol) as follows: 1mM, 6� 10%; 2mM, 43� 8.1%; 5mM, 55�1.7%; 10 mM, 82 � 3.3% (Fig. 3E). Next, we evaluated APP

metabolism by measuring sAPP� inthe supernatant of conditioned cells.Exposure to VPA markedly reducedsAPP� corresponding to full-lengthAPP levels. Significant down-regula-tion of sAPP� was seen in BxPC3cells at 1 mM and in SW480 at 2.5mM VPA treatment. sAPP� de-creased with higher doses of VPA inBxPC3 (means � S.E. in percentreduction of sAPP� levels versusuntreated control) as follows: 1 mM,33� 2.7%; 2.5mM, 27� 3.5%; 5mM,45 � 1.7%; 10 mM, 87 � 2.3% (Fig.3E). sAPP� decreased with higherdoses of VPA in SW480 (means �S.E. in % reduction of sAPP� levelsversus untreated control) as follows:1 mM, 0 � 2.7%; 2.5 mM, 40 � 7.1%;5 mM, 67 � 2.1%; 10 mM, 72 � 4.8%(Fig. 3F).To evaluate the effect of VPA on

mature and immature APP, levelswere quantified and indicated as aratio of mature to immature APP.The quantification of the ratioimmature APP to �-actin showedno significant changes (Fig. 4).These results indicate that VPAimpairsmaturation ofAPP and con-secutively leads to reduced secretedsAPP�. In SW480 cells, mature APPdecreased in a concentration-de-pendent fashion (means � S.E. in %reduction of mature APP levels ver-sus untreated control) as follows: 1mM, 30.7 � 10.7%; 2.5 mM, 52.1 �8.9%, 5 mM 73 � 5.9%. Similarresults were obtained with BxPC3cell lines (data not shown).HDAC Inhibitory Properties of

VPA Are Specific for APP Reductionand Responsible for the Suppres-sion of Tumor Growth—Consistentwith previous studies, VPA inhibitsHDAC isoenzymes in several celltypes (19, 22, 23) causing hyper-acetylation of N-terminal tails(lysine residues) of histone H4. VPAtreatment of SW480 resulted inhyperacetylated histone H4 in adose-dependent manner using anantibody specific to acetylated his-tone H4 (H4Ac) (Fig. 5B). The sameresult was obtained using BxPC3

(data not shown). To determine whether the APP reducingeffect of VPA is specific, epidermal growth factor receptor(EGFR) and the highly homologous family member protein,

FIGURE 1. Immunohistochemical staining of APP in gastrointestinal tumors. Representative sections dem-onstrating expression of APP in formalin-fixed, paraffin-embedded pancreatic and colon tissues. A–D, APPstaining in colon tissue stained with polyclonal antiserum 23850 recognizing N-terminal APP antibody (1:500).A and B, low level of APP expression in healthy nonmalignant human colonic mucosa. C and D, abundantcytoplasmic APP staining in the tumor glands. E–H, APP staining in pancreatic tissue stained with the mono-clonal APP antibody recognizing N-terminal APP (22C11; 1:250). E and F, normal human pancreas having weakAPP staining in pancreatic acini (star). Pancreatic duct epithelia show no staining for APP (arrowhead). Arrowsshow positive staining in adjacent islet cells. G and H, human pancreatic adenocarcinoma with intenseAPP staining in ductal carcinoma cells (stars). Arrows show positive staining in adjacent islet cells. Scale bars,A, C, E, and G, 100 �m; B, D, F, and H, 50 �m.




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amyloid � (A4) precursor-like pro-tein 2 (APLP2) (34), were examinedby Western blot. There was noeffect of even 5 mM VPA treatmenton EGFR or APLP2 protein levels.The same was found in BxPC3 cells(data not shown). GRP78 proteinlevels, an APP-binding protein,were elevated after VPA treatment(Fig. 5B) suggesting that the VPA-induced impairment of APP matu-ration is due to up-regulation ofGRP78.VPM (19, 35), in which the car-

boxyl group ismodified to an amide,lacking HDAC inhibitory activity.VPM had no effects on GRP78,H4Ac, EGFR, or APLP2 protein lev-els. Consistent with our hypothe-ses, APP protein levels were notaffected. VPM is an even morepotent anti-convulsive drug thanVPA having no anti-neoplasticpotency (Fig. 6) (36, 37).Treatment of SW480 cells with

TSA, a potent but to VPA structur-ally unrelated pan-HDAC inhibitor(38, 39), resulted in a similarly sig-nificant reduction of APP andincreased hyperacetylation of his-tone H4. APLP2 and EGFR levelswere again unchanged. GRP78 lev-els were also elevated after TSAtreatment (Fig. 7).As expected, VPA treatment trig-

gered GRP78 mRNA levels and hadno effect on APPmRNA levels indi-cating that the HDAC inhibitoryeffect was mediated via GRP78transcriptional regulation (supple-mental Fig. S3). Taken together,these findings suggest that thereduction of APP levels by inhibi-tion of class I HDAC enzymes(HDAC1–3 and 8) is highly specificand mediated by GRP78 elevation.N-terminal Domain of APP/

sAPP� Is Essential for Growth-pro-moting Effects—To further charac-terize the growth promotingactivity of APP, we stably trans-fected SH-SY5Y neuroblastomacells with APP695, SPA4CT (N-ter-minal domain deleted), or the emp-ty vector pCEP4 construct alone(mock) (Fig. 8A). APP695-trans-fected cells presented a 43 � 4.8%higher proliferation rate compared




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withmock-transfected control cells (Fig. 8B).We hypothesizedthat the growth regulating activity is localized to theN-terminalpart of APP/sAPP�. To test this hypothesis, we used the shorter

construct lacking the N-terminaldomain (SPA4CT). The SPA4CTconstruct contains the signal pep-tide of APP followed by leucine andglutamic acid and the C-terminalfragment (C99) of APP (40). In con-trast, the SPA4CT construct thatlacks the ectodomain of APP had noeffect on cellular growth comparedwith mock-transfected neuroblas-toma cells. This observation revealsthat the N-terminal domain isessential for the growth-promotingeffect of APP (Fig. 8C).We next sought to determine

whether addition of conditionedmedia derived from APP-overex-pressing cells would increase cellularproliferation. Importantly, condi-tionedmedia fromAPP-overexpress-ing cells significantly increased prolif-eration of both mock-transfectedcontrol and the SPA4CT-transfectedcell.Mock-transfectedcellspresenteda 76 � 13.3% increase in cell growth,whereas SPA4CT-transfected cellsshowed a 40 � 7.5% increase in cellgrowth (Fig. 8D).In addition, a 24-h treatment of

SW480 cells with conditioned mediafrom APP-overexpressing SY5Y cellsincreased proliferation by 55%. Thetreatment with 5 mM VPA was com-pletely rescued by co-treatment withconditionedmedia (Fig. 9).

DISCUSSION

Growth factors and their recep-tors make significant and well doc-umented contributions to the proc-ess of tumorigenesis and representan important hallmark of cancer.Their contribution to tumorigene-sis is in part due to their ability tocontrol cell cycle entry, to conferresistance to apoptotic stimuli, andto increase cellular invasion andmetastasis (41).This study shows that the class I

HDAC inhibitor VPA is an effectiveand highly specific repressor of APP maturation and secretion.This pharmacological strategy might therefore have therapeu-

FIGURE 2. siRNA-mediated knockdown of APP decreased cell growth. siRNA treatment targeting endogenous APP decreases both full-length APP andsecreted sAPP� levels in human BxPC3 pancreatic (A–C) and human SW480 colon cancer (E–G) cell lines. Cells were transiently transfected with equimolarrandom siRNA showing no effect. APP knockdown resulted in a significant reduction of cell growth of BxPC3 (D) and SW480 (H) cells. Results are from anaverage of six experiments. Data were expressed as means � S.E. Differences were calculated using one-way ANOVA followed by Bonferroni post hoc analyses(*, p � 0.05; ***, p � 0.001).

FIGURE 3. VPA inhibits tumor cell growth and reduces APP and sAPP� levels. VPA inhibits proliferation ina concentration-dependent manner in pancreatic (BxPC3) and colon cancer (SW480) cell lines. BxPC3 andSW480 cells were exposed to VPA in different concentrations up to 100 mM. The inhibition of cell growth wasdetermined 48 h after plating using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium assay (A and D). VPA down-regulates full-length APP (B and E) and secreted sAPP� (C andF) levels in a concentration-dependent manner in both cell lines. Results are from an average of at least threeexperiments. Data were expressed as mean � S.E. Differences were calculated using one-way ANOVA followedby Bonferroni post-hoc analyses (*, p � 0.05; **, p � 0.01; ***, p � 0.001).




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tic implication for the treatment of different carcinomas. It is ofinterest that both pancreatic adenocarcinoma, a highly invasivetumor with poor prognosis, and colon carcinoma, one of themost abundant tumors, overexpress APP and that this featuremight serve as a potent diagnostic tumor marker. The majorityof patients are diagnosed at an advanced state of gastrointesti-nal cancer development with a bad prognosis and a short sur-vival time, even after surgical resection. Therefore, there is aneed for newmolecular targets for additional adjuvant and neo-adjuvant pharmacological treatment options.Very little is known concerning the role of APP in carcino-

genesis. Gain-of-function studies showed that APP overexpres-sion leads to increased cellular proliferation (12, 17, 42). Loss-of-function studies either by APP knockdown (14, 16, 17) orblockage of sAPP function by antibody application (15) demon-strated regression of carcinoma growth in vitro and in vivo.Such a functional relationship between APP expression andproliferation demonstrates the importance of APP in thepathogenesis of cancer. APP-overexpressing mice are prone todevelop brain carcinomas (43). However, the precise mecha-nism underlying the tumor growth-promoting effect of APPremains to be clarified. The large N-terminal domain of APP

(residues 28–123) contains cys-teine-rich regions and a heparin-binding site and shows similaritiesto well known growth factors andcan therefore be classified as amember of the cysteine-rich growthfactor superfamily (44). The extra-cellular domain interacts withmatrix proteins and heparan sulfateproteoglycans reflecting its role inmigration, adhesion, and cell-ma-trix and cell-cell interactions (45–48). Recently, it has been shown thatAPP is up-regulated in several can-cer species, including pancreatic(15), colon (14),melanoma (49), andprostate cancer (17), as well as oralsquamous cell carcinoma (15), andhas growth-promoting features.Different kinds of stimuli can

enhance APP expression. A recentstudy has revealed that APP tran-scription is androgen-inducible inprostate cancer (17). Under onco-genic and pro-inflammatory condi-tions (50), activation of Ras-MAPKcan induce APP expression (51).Moreover, a variety of ligands ofgrowth factor receptors withintrinsic tyrosine kinase activity,like nerve growth factor (51),fibroblast growth factor, and epi-dermal growth factor, were shownto increase APP secretion (52–54).Several studies have evaluated APPas a potential tumor marker in tis-

FIGURE 4. VPA effect on mature and immature APP levels. A, SW480 cellswere treated with increasing concentrations of VPA for 24 h. Cell lysates wereanalyzed by Western blot for full-length APP. B and C, signals correspondingto the different bands were quantified by densitometric analysis, and theratios of the mature (130 kDa) to the immature (110 kDa) APP levels werecalculated. Results are from an average of at least three experiments. Datawere expressed as mean � S.E. Differences were calculated using one-wayANOVA followed by Bonferroni post hoc analyses (**, p � 0.01; ***, p � 0.001).

FIGURE 5. HDAC inhibitor VPA specifically up-regulates GRP78 and has no effect on APLP2 and EGFRlevels. A, molecular structure of VPA. B, SW480 cells were treated with increasing concentrations of VPA for24 h. Cell lysates were analyzed by Western blot with antibodies against EGFR, GRP78, and APLP2, H4Ac, and�-actin. Like H4Ac, GRP78 protein levels clearly showed dose-response characteristics. C, quantification ofEGFR levels; D, quantification of GRP78; E, quantification of APLP2. Results were from an average of at leastthree experiments. Data were expressed as mean � S.E. Differences were calculated using one-way ANOVAfollowed by Bonferroni post hoc analyses (*, p � 0.05; ***, p � 0.001).




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sue, mainly using immunohisto-chemistry. Interestingly, in prostateand oral squamous cell carcinoma,the rate of cancer-specific survivalfor patients with APP-positivetumors was significantly lower thanthose with APP-negative tumors(16, 17). This study is in good agree-ment with the previous reports aswe have demonstrated that APP ishighly expressed in only pancreaticand colon carcinoma but not inhealthy control tissue.Moreover, APP is expressed very

early during embryogenesis in new-born vessels and could have a role inangiogenesis (55). Interestingly, arecent study using short hairpinRNA silencing in utero could clearlydemonstrate that the regulation andamount of full-length APP is essen-tial for the proper migration of neu-rons in the developing cerebral cor-tex suggesting that dysregulationmight have an impact on corticaldevelopment (56). In good agree-ment with previous studies, wecould demonstrate that overexpres-sion of APP induces increased cellu-lar proliferation compared withmock-transfected controls. To de-termine the growth promotingactivity of APP, we used the shorterSPA4CT construct, lacking the N-terminal domain of APP. SPA4CT-transfected cells (40) did not pro-mote increased proliferation rate ascompared with mock-transfectedcontrols. Furthermore, we showedthat addition of conditioned me-dium from APP-overproducingcells could stimulate proliferation ofSPA4CT- and mock-transfectedcells. These results clearly indicatethat the secreted N-terminal do-main of APP plays an essential reg-ulatory role in promoting cellulargrowth. Several lines of evidenceshowed that sAPP� fulfills criteriaof a trophic growth factor (42, 45,57–59) and that the growth regulat-ing activity of APP is specified to theN-terminal domain of sAPP con-taining the 5-amino acid sequenceRERMS (APP-(328–332)). Peptidescontaining this pentapeptide se-quence could increase cellular pro-liferation in fibroblasts and induce

FIGURE 6. Valpromide has no effect on APP and GRP78 levels. A, molecular structure of VPM. B, SW480 cellswere treated for 24 h with 5 mM VPA or equimolar concentrations of VPM. VPM is a carboxamide derivative ofVPA lacking HDAC inhibitory activity. Although VPM had no effect, VPA significantly inhibited cell growth.C, SW480 cells were treated with increasing concentrations of VPM for 24 h. Cell lysates were analyzed byWestern blot with antibodies against APP, EGFR, GRP78, APLP2, and H4Ac (prolonged exposure), and �-actin,respectively. Representative Western blots show no changes on any protein levels. Results were from anaverage of at least six experiments. Data were expressed as means � S.E. Differences were calculated usingone-way ANOVA followed by Bonferroni post hoc analyses (***, p � 0.001).

FIGURE 7. Structurally unrelated HDAC inhibitor trichostatin A has the same effects as VPA. A, molecularstructure of (TSA. B, SW480 cells were treated for 24 h with 250 nM TSA having a potent inhibitory effect on cellgrowth. C, cell lysates were analyzed by Western blot with antibodies against APP, EGFR, GRP78, APLP2, H4Ac,and �-actin. TSA specifically down-regulates APP and up-regulates GRP78 protein levels. No effect was seen onEGFR and APLP2 levels. Results were from an average of at least six (B) and three (D and E) experiments. Datawere expressed as means � S.E. Differences were calculated using unpaired t test (**, p � 0.01; ***, p � 0.001).




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neurite outgrowth in neuronal cells; moreover, infusion intobrain ventricles led to increased synaptogenesis and improvedmemory retention in rat brains (60–62).To evaluate whether the proliferative effect of APP/sAPP� is

of pathophysiological relevance, we used siRNA to selectivelyknock down APP gene expression. siRNA treatment led todecreased APP and sAPP� protein levels and significantlyinhibited cellular growth in both cancer cell lines. This obser-vation is in good agreement with earlier work in N2a cells (63).An important finding of our study is that APP levels and

sAPP� secretion are down-regulated upon HDAC inhibition.Other type I transmembrane proteins like EGFR and the APPfamily member APLP2 were not affected, demonstrating thespecificity of HDAC activity on APP metabolism. VPA, a welltolerated drug with an extensively characterized toxicity pro-file, emerged as one of the leading candidates for epilepsy andnumerous neuropsychiatric disorders, including mania,migraine prophylaxis, and neuropathic pain (18). However, theexact molecular pathways affected are not well understood.During the last 2 decades, it has become evident that VPA alsopossesses a variety of anti-neoplastic effects, including suppres-sion of tumor growth, metastasis, and induction of differentia-tion in vitro and in vivo (23). It has been shown that VPA exhib-its potent anti-tumor effects in a variety of human cancer cell

lines, including pancreatic (32),colon adenocarcinoma (64), malig-nantmelanoma (65), cervical cancer(64), sarcoma (64), breast adenocar-cinoma (64), malignant glioma (66),prostate (24), bladder cancer (67),and neuroblastoma (68). Severaltargets like extracellular signal-reg-ulated kinase (ERK)-signaling path-way, peroxisome proliferator-acti-vated receptor � and peroxisomeproliferator-activated receptor �activation, protein kinase C down-regulation, or inhibition of GSK-3�(23) have been discussed as beinginvolved in the inhibition of tumorgrowth. However, binding studiesrevealed that VPA can inhibitHDACenzymes by blocking the cat-alytic center of the enzymes (22). Incontrast to other potent HDACinhibitors like TSA, VPA preferen-tially inhibits class IHDACenzymesthat include HDAC1, -2, and -3 iso-formswith IC50 values ranging from0.7 to 1 mM (22, 69). Both pan-HDAC and class I-specific inhibi-tors bring out similar changes in lev-els of H4Ac.In this study, we demonstrated

for the first time that VPA selec-tively decreases APP expression in aconcentration-dependent manner.Interestingly, although VPA down-

regulated APP protein levels, transcriptional inhibition of APPmRNA was not affected in pancreatic (BxPC3) and colon ade-nocarcinoma (SW480) cell lines as demonstrated previously(70).We and others have shown that APP overexpression leads to

increased proliferation and that APP is pathophysiologicallyup-regulated in various cancer types (12, 14, 16, 17). On theother side, VPA treatment leads to growth inhibition by phar-macological down-regulation specifically of APP. Accordingly,VPM, an amide analogue of VPA, with no HDAC inhibitoryactivity did not affect APP levels, whereas the pan-HDACinhibitor TSA had the same effects as VPA.To investigate whether HDAC inhibition alters the level of

other membrane-anchored proteins, EGFR protein levels werestudied, because of its involvement in tumor pathogenesis.EGFR is a member of the HER family of receptor tyrosinekinases and is overexpressed in a broad range of tumors, includ-ing pancreatic and colon cancer. Like APP, this cell surfacereceptor plays important roles in growth control, and increasedexpression has been associated with increased cellular prolifer-ation (71, 72). Our results demonstrate that EGFR levels werenot altered by treatment with HDAC inhibitors.Moreover, VPA treatment also did not affect amyloid � (A4)

precursor-like protein 2 (APLP2) protein levels.With 71% sim-

FIGURE 8. N-terminal domain is essential for the growth-promoting properties of APP. A, schematicillustration of APP695 and the N-terminal truncated SPA4CT construct used in the study. The A� domainis shown in black. SH-SY5Y cells were transfected with plasmids coding for APP695, SPA4CT, or with theempty vector pCEP4 alone. For all constructs the signal peptide sequence (SP) of APP was introducedin-frame with the remaining domains. The SPA4CT lacking the entire N-terminal ectodomain precedes theA� domain. AICD, APP intracellular domain. B, SH-SY5Y cells were stably transfected with APP695, SPA4CT,and empty vector pCEP4 construct alone (mock). Cell lysates were analyzed by Western blotting forfull-length APP and the different APP cleavage products C99 and A�. C, proliferation analysis showing asignificant increased cell growth rate only in APP696-transfected cells. SPA4CT did not stimulate cellgrowth. D, to study the influence of exogenous sAPP�, media were replaced with conditioned media(cond. Medium) from SH-SY5Y APP695-overexpressing cells (hatched bars) or treated with serum-freemedia (solid bars). Cellular proliferation was significantly elevated in mock-transfected as well as inSPA4CT-transfected cell lines. Results were from an average of at least six experiments. Data wereexpressed as means � S.E. Differences were calculated using one-way ANOVA followed by Bonferroni posthoc analyses (C) and unpaired t test (D) (**, p � 0.01; ***, p � 0.001).




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ilarity in the amino acid sequence, APLP2 is the closest homo-logue to APP (73). Because APP and APLP2 are highly homol-ogous proteins with similar protein domain organization inperipheral tissue (73), it is of interest that VPA specificallydown-regulated APP and did not affect APLP2 levels. Asexpected, the levels of soluble APLP2 (sAPLP2�) were notchanged(datanotshown).Therefore,wesuggestthattheHDAC-dependent APP down-regulation underlies a highly specificmechanism.The metabolism and trafficking of APP are highly coordi-

nated processes and include essential post-translational modi-fications to be transported in the secretory pathway. Glycosy-lation is an important factor in APP maturation and transportfunction to the cell surface. Several lines of evidence haveshown that predominantly O-linked oligosaccharides play animportant role in intracellular sorting, processing, and secre-tion of APP (4).Differential display PCR has been used to identify genes reg-

ulated byVPA in rat cerebral cortex. GRP78was found to be thegene product with the highest level of up-regulation after VPAtreatment (74). Previously, it has been demonstrated that VPAand other potent HDAC inhibitors specifically up-regulateGRP78, without affecting other targets of the unfolded proteinresponse like CHOP, XBP-1, and HSP70. Furthermore, theHDAC inhibitor response elements in the Grp78 promotercould be identified, revealing that HDAC inhibitors exclusivelyup-regulate GRP78 (25, 75). Consistent with these studies, wedemonstrate that HDAC inhibition promotes GRP78 levelscorrelating with the induction of histone H4 hyperacetylation(H4Ac). GRP78, a 78-kDa glucose-regulated protein alsoknown as BiP or immunoglobulin heavy chain binding protein,is an endoplasmic reticulum chaperone acting as a molecular

chaperone and is essential for the proper glycosylation, folding,and assembly of many membrane-bound and -secreted pro-teins (76). In contrast to other chaperones, GRP78 seems tobind APP in the endoplasmic reticulum, and this interactionhas functional consequences on APPmetabolism. Overexpres-sion of GRP78 leads to impairment in APP maturation andsecretion of sAPP. Furthermore, co-precipitation studiesshowed that GRP78 selectively binds to the immature form ofAPP. These results could suggest that interaction of GRP78withAPP inhibits the translocation from the endoplasmic retic-ulum lumen to the cis-Golgi network, where maturation iscompleted (26, 77, 78). Moreover, semiquantitative compari-son of interactomes of APP, APLP1, andAPLP2 confirms selec-tive binding of GRP78 to APP but not to APLP1 or APLP2 (79).Taken together, these results are consistent with our observa-tion and underline that HDAC inhibition led to a selectivedecrease of mature APP.It is well known that HDAC family members control hall-

marks of cancer cell biology. HDAC inhibitors exert a plethoraof effects on cancer cells, including induction of apoptosis, dif-ferentiation, cell cycle arrest, and inhibition of neo-angiogene-sis.ModulatingHDACexpression by siRNAknockdown impli-cates that class I HDAC enzymes are essential for proliferationand survival. In particular, HDAC1 andHDAC3 are believed tobe crucial in controlling cellular proliferation in vitro andin vivo (80–83). Moreover, HDAC inhibition causes rapidchanges in gene expression of a distinct set of genes, mediatedby modulation of the chromatin structure. Besides these epige-netic mechanisms, HDAC inhibitors also modulate the acety-lation state of numerous cytoplasmic proteins, clearly in a non-epigenetic manner (84, 85). VPA has shown potent anti-tumoreffects in a variety of in vitro and in vivo systems. Encouragingresults were obtained in early clinical trials either with VPAalone or in combination with demethylating and cytotoxicagents. In addition, transcriptomemicroarray analysis from theprimary tumors of patients treated with VPA showed signifi-cant up-regulation of many genes belonging to multiple path-ways, including ribosomal proteins, oxidative phosphorylation,MAPK signaling, focal adhesion, cell cycle, antigen processingand presentation, proteasome, apoptosis, phosphatidylinositol3-kinase, Wnt signaling, calcium signaling, transforminggrowth factor-� signaling, and ubiquitin-mediated proteolysisamong others (36, 86).Overall, our data indicate that HDAC inhibitors increase

GRP78 levels correlating with a decrease in mature APP andreduced secretion of sAPP�. The possibility that HDAC inhib-itors specifically down-regulate APP, but not its homologueAPLP2or EGFR,might open additional therapeutic approachesfor the treatment of cancer. The down-regulation of APP maythereby inhibit tumor growth, tumor invasion, and neoangio-genesis in various cancer species. Moreover, selective down-regulation of APP by HDAC inhibitors might also be an expla-nation for the teratogenic side effect, leading to neural tubedefects (87).In conclusion, our results support previous observations that

APP is a crucial mediator of tumor growth in general. The anti-epileptic and well tolerated HDAC inhibitor VPA can be usedto modulate endogenous growth-promoting APP levels in gas-

FIGURE 9. Secreted APP rescues inhibitory effect of VPA treatment.SW480 treatment with conditioned media from SH-SY5Y APP695-overex-pressing cells increased the proliferation rate significantly (�55%) comparedwith treatment with serum-free media. The VPA-induced inhibitory effect(�29%) was completely rescued by treatment with conditioned media.Results were from an average of at least six experiments. Data were expressedas means � S.E. Differences were calculated using one-way ANOVA followedby Bonferroni post hoc analyses (**, p � 0.01; ***, p � 0.001).




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trointestinal carcinomas and might therefore provide a molec-ular tool for therapeutic intervention.

Acknowledgments—We thank B. Gunnawan (University of Gottin-gen) for the generous gift of pancreatic and colon tissues, J. Schmidt(University of Gottingen) for the APP siRNAs, andM. Ghadimi (Uni-versity of Gottingen) for the pancreatic and colon cancer cell lines.Wealso thank Katwijk Chemie BV for the generous gift of valpromide.

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Tamboli, Jochen Walter, Oliver Wirths and Thomas A. BayerVivek Venkataramani, Christian Rossner, Lara Iffland, Stefan Schweyer, Irfan Y.

Down-regulation of the Alzheimer Amyloid Precursor ProteinHistone Deacetylase Inhibitor Valproic Acid Inhibits Cancer Cell Proliferation via

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