expression of serotonin receptors in human hepatocellular ...human cancer biology expression of...

Human Cancer Biology

Expression of Serotonin Receptors in Human HepatocellularCancer

Christopher Soll1, Marc-Oliver Riener2,4, Christian Eugen Oberkofler1, Claus Hellerbrand3, Peter J. Wild2,Michelle L. DeOliveira1, and Pierre-Alain Clavien1

AbstractPurpose: Serotonin is a well-known neurotransmitter and vasoactive substance. Recent research

indicates that serotonin contributes to liver regeneration and promotes tumor growth of human hepato-

cellular cancer. The aim of this study is to investigate the expression of serotonin receptors in hepatocellular

cancer and analyze their potential as a cytotoxic target.

Experimental Design: Using a tissue microarray and immunohistochemistry, we analyzed the expres-

sionof serotonin receptors in the liver from176patientswithhepatocellular carcinoma, ofwhichnontumor

tissue was available in 109 patients. Relevant clinicopathologic parameters were compared with serotonin

receptor expression. Two human hepatocellular cancer cell lines, Huh7 and HepG2, were used to test

serotonin antagonists as a possible cytotoxic drug.

Results: The serotonin receptors 1B and 2B were expressed, respectively, in 32% and 35% of the patients

with hepatocellular cancer. Both receptors were associated with an increased proliferation index, and

receptor 1B correlated with the size of the tumor. Serotonin antagonists of receptors 1B and 2B consistently

decreased viability and proliferation in Huh7 and HepG2 cell lines.

Conclusion: We identified two serotonin receptors that are often overexpressed in human hepatocel-

lular cancer and may serve as a new cytotoxic target. Clin Cancer Res; 18(21); 5902–10. �2012 AACR.

IntroductionHepatocellular carcinoma (HCC) is the 5th most fre-

quent cancer (1) and has become the 3rd leading cause ofcancer-related death worldwide (2). To face this globalhealth problem, the investigation of new molecular targetsis necessary to develop new treatment strategies.

Recent in vitro and in vivo studies have suggested thatserotonin (5HT) contributes to the tumor growth in avariety of cancer including cholangiocarcinoma (3, 4),colon cancer (5), and HCC (6). Within the liver, 5HT wasfound to be implicated in the pathogenesis of liver fibrosis(7, 8), nonalcoholic steatotic hepatitis (9), and viral hep-atitis (10); all these conditions are involved in the tumor-igenesis of HCC. We have recently proposed that 5HT,through its 5HT2B receptor,may represent a potential targetin HCC (6). We observed that the inhibition of 5HT2B

receptors leads to cell death of hepatocellular cancer cells invitro and decreased tumor growth in a subcutaneous tumormodel in mice.

At the cellular level, 5HT acts predominantly via G-protein coupled receptors. The availability of 7 receptorclasses including 14 subtypes of serotonin receptors reflectthe diversity of the serotonergic actions (11). Within thesesubtypes, 5HT receptors (1A, 1B, 2A, 2B, and 7) are widelydistributed in the human gastrointestinal tract and areinvolved in normal liver function and disease (12, 13). Incontrast, 5HT receptors 4 and 5 are not or only poorlyexpressed in the liver (14, 15).

In this study, the expression of different serotoninreceptors was analyzed in specimens obtained from176 patients with HCC using immunohistochemistry(IHC) on a tissue microarray (TMA). Receptor expressionwas compared between nontumoral and tumoral tissue in109 patients, in whom nontumor tissue was available. Wetested whether the expression of serotonin receptors inHCC correlated to the phenotype of the tumor and avariety of clinicopathologic parameters. From these data,we conducted experiments in human cell lines of HCC toinvestigate the cytotoxic effect of antagonizing serotoninreceptors.

Patients and MethodsPatients

One hundred seventy-six patients with HCC who under-went surgery between 1992 and2007 inZ€urich, Switzerland

Authors' Affiliations: Departments of 1Surgery and 2Pathology, SwissHepato-Pancreato-Biliary (HPB) Center, University Hospital Zurich, Zurich,Switzerland; 3Department of Gastroenterology, University of Regensburg,Regensburg, Germany; and 4Institute of Pathology, University HospitalErlangen, Erlangen, Germany

Note: C. Soll and M.-O. Riener contributed equally.

Corresponding Author: Pierre-Alain Clavien, Department of Surgery,University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland.Phone: 41-44-255-3300; Fax: 41-44-255-4449; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-1813

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(21) November 1, 20125902

on November 13, 2020. © 2012 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 19, 2012; DOI: 10.1158/1078-0432.CCR-11-1813

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(n¼ 94) and Regensburg, Germany (n¼ 82) were includedin this study. The patients’ ages ranged from 20 to 85 years(median 61 years). The underlying liver diseases of theHCCwere: hepatitis B virus (HBV) infection (27 cases, 15.3%),hepatitis C virus (HCV) infection (37 cases, 21%), alcoholabuse (46 cases, 26.1%), hemochromatosis (9 cases, 5.1%),Alagille’s syndrome (1 case, 0.6%), and unknown etiologies(56 cases, 31.8%). Corresponding nonneoplastic liver tis-sue of 109 patients with HCC was available. The medianfollow-up time of all patients was 17 months (range 1–120months). Patients were treated with resection (n ¼ 136) orliver transplantation (n ¼ 40). Median follow-up time ofpatients without disease progression was 17months (range1–120 months). A total of 35% of patients died duringfollow-up after a median time of 14 months (range 1–79months).The study was approved by the local ethics committee

(Kantonale Ethikkommission Zurich, StV 26-2005 and EK-1017). A written informed consent was obtained from thepatients.

TMAFormalin-fixed, paraffin-embedded tissues were used to

construct 5 TMAs with liver tumor tissues. The TMA wasconstructed as described previously (16). Two tissue coresper tumor with a diameter of 0.6 mm were punched out ofthe donor block and transferred to the recipient block.

IHCThe TMA blocks were freshly cut (3 mm) andmounted on

superfrost slides (Menzel Gl€aser). IHC was conducted withthe Ventana Benchmark automated staining system (Ven-tanaMedical Systems) using Ventana reagents for the entireprocedure. The primary antibody was detected using theUltraVIEW DAB detection kit using the benchmarks CC1mheat-induced epitope retrieval. The following primary anti-bodies were used: HTR1A (LS-B970, Lifespan), HTR1B(ab13514, Abcam), HTR2B (ab32994, Abcam), HTR7(AHP1344, AbDSerotec), and Ki67 (M7240, Dako). Slideswere counterstained with hematoxylin, dehydrated, and

mounted. Human tissue from brain, kidneys, smoothmus-cle, and arteries served as positive control.

Evaluation of immunohistochemical stainingThe staining intensity of HTR2B, HTR1A, HTR1B, and

HTR7 was semiquantitatively evaluated with a 4-tieredsystem: 0 (negative), 1þ (weak), 2þþ (moderate), and3þþþ (strong). Weak immunoreactivity was defined asminute granules projecting to the cytoplasm. Moderateimmunoreactivity was diagnosed when a coarser and moreintense stainingwas seen,whereas chunky, dark lumpswerescored as strongly positive. A tumor was scored as positivewhen at least moderate staining (2þþ) was achieved.Because all of the receptors analyzed showed cytoplasmicstaining, only this pattern was used in the evaluation. As apositive control, human brain tissue was used for eachantibody and slide separately. When a tumor was positiveit was homogenously positive in more than 90% of thetumor cells, therefore only the intensity was relevant in theanalysis. For statistical analysis, tumors were grouped intoweakly positive (0 and 1) and strongly positive (2 and 3) forHTR2B, HTR1A, HTR1B, and HTR7. The proliferation rate(Ki-67) was defined as percentage of positive nuclei per 100tumor cells as recently described (17).

Real-time PCRRNA extraction from Huh7, HepG2, and Hep3B was

conducted using theRNeasyMinEluteCleanupkit (Qiagen)following the manufacturer’s instructions. Five microgramsof RNAwere reverse transcribed using ThermoScript reversetranscription PCR System (Invitrogen) yielding the cDNAtemplate. Quantitative real-time PCR amplification anddata analysis were carried out using an ABI Prism 7000Sequence Detector System (PE Applied Biosystems). Taq-Man gene expression assays (PE Applied Biosystems) forHTR1A (Hs00265014_s1), HTR1B (Hs00265286_s1),HTR2A (Hs00167241_m1), HTR2B (Hs00168362_m1),HTR4 (Hs00168380_m1), HTR5 (Hs00168381_m1),HTR7 (Hs00252002_m1), were used to quantify mRNAexpression of the respective genes. Messenger RNA expres-sion levels of each sample were normalized to 18S RNA(TaqMan rRNA control reagents; PE Applied Biosystems).

In vitro experimentsCell lines were purchased from American Type Culture

Collection and Huh7, HepG2, and Hep3B cultured inDulbecco’sMinimal EssentialMediumwith 4.5 g/L glucose,sodium pyruvate, GlutaMAX (Invitrogen), and 10% fetalcalf serum (FCS; PAA Laboratories), with the addition of100 units/mL of penicillin and 100 mg/mL of streptomycin(Invitrogen). Cells were maintained at 37�C in a 5% CO2

atmosphere.All antagonists were purchased from Tocris. The follow-

ing selective antagonists were used: for HTR1A (S)-WAY100135 dihydrochloride (S-WAY 100), for HTR2B LY272015 hydrochloride (LY272), for HTR7 SB269970(SB269), and for HTR1B SB 216641 hydrochloride(SB216).

Translational RelevanceAccumulating evidence suggests a potential impact of

serotonin in tumor biology, particularly in primary can-cer of the liver. Recently, we have shown that serotoninpromotes tumor growth of human hepatocellular can-cer. In this study, we analyzed the expression of differentserotonin receptors in specimens from a large patientgroup and tested whether specific serotonin receptorantagonists may serve as drugs to reduce tumor growth.As human hepatocellular cancer is a disease with a

poor prognosis and the incidence of this cancer isexpected to increase in the future, the development ofnew treatment strategies is of utmost importance. Ouranalysis suggests serotonin receptors as possible molec-ular targets of human hepatocellular cancer.

Serotonin and Hepatocellular Cancer

www.aacrjournals.org Clin Cancer Res; 18(21) November 1, 2012 5903




Human hepatocellular cell lines, Huh7 and HepG2,were seeded into 24-well plates at a density of approxi-mately 25% corresponding to 2.5 � 104 cells per well andallowed to adhere overnight before the medium waschanged to the specified conditions, containing 100mmol/L 5HT creatinine complex (Sigma Aldrich) andserotonin antagonists. Dosages of 5HT and antagonistswere taken from response curves of previous experiments(6).

Time-dependent experiments were conducted with sub-sequent stimulation after serum withdrawal. In antagonistexperiments, cells were incubatedwith the antagonist for 20minutes before addition of the 5HT or FCS.

The number of viable cells was quantified by theaddition of 25 mL of a 0.5% tetrazolium salt solutionMTT (Sigma Aldrich). After 45 minutes of incubation, theformation of the formazan product was monitored bymeasuring absorbance at 570 nm after solubilization inacidic isopropranol (5% formic acid in isopropranol).Values were calculated as percentage of untreatedcontrols.

Western blottingCells were seeded overnight in 10 cm dishes (1 � 106

/well). After treatment as indicated cells were homogenizedin lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/LNaCl, 5 mmol/L EDTA, 0.5% NP40, containing protease,and phosphatase inhibitor cocktail (Roche Diagnostics)].Cell extracts were diluted in sample buffer [187.5 mmol/LTris-HCl (pH 6.8), 6% SDS, 30% glycerol, 150 mmol/LDTT, and 0.3% bromophenol blue] and heated for 10minutes at 90�C and cooled for 2 minutes on ice. Fortymicrograms of protein were loaded, SDS-PAGE was con-ducted and samples were blotted onto a polyvinylidenedifluoride membrane. Primary antibodies were phospho-p44/42 MAPK(ERK1/2) (#4370, Cell Signaling) and Anti-beta-Tubulin (ab6046; Abcam). Secondary staining anddetection was conducted according to standard protocolswith the enhanced chemiluminescence detection reagent(GE Healthcare Ltd). Results were displayed as fold induc-tion to untreated controls.

StatisticsData are given as mean and SD. Statistical analysis was

conducted using SPSS, version 18.0 (SPSS Inc.) and Prism4.0 (GraphPad, Inc.). Correlations were calculatedaccording to Spearman r. Fisher exact test was appliedto assess the statistical significance of the associationsbetween the expression of 5HT receptors and variousclinicopathologic parameters. Univariate survival analysiswas carried out according to Kaplan–Meier, and differ-ences in survival curves were assessed with the Log-ranktest. Multivariate analyses were calculated according tothe Cox regression model. P values <0.05 were consideredsignificant. For cell culture experiments differencesbetween the groups were assessed by 1-way or 2-wayANOVA using an appropriate posttest, including Dunnettand Bonferroni post hoc test.

Results5HT receptors in human hepatocellular cancer celllines

First, we screened 3 human hepatocellular cancer celllines (Huh7, HepG2, and Hep3B) for transcription of7 different 5HT receptors (HTR1A,HTR1B, HTR2A,HTR2B,HTR4, HTR5, and HTR7). In all 3 cell lines, PCR productswere identified for HTR1A, HTR1B, HTR2B, and HTR7(Fig. 1). Therefore, we focused on the expression of thesefour 5HT receptors using TMA in the available specimens ofHCC.

5HT-receptor expression in HCC and correspondingnontumor liver tissue

The expression of HTR1A, HTR1B, HTR2B, and HTR7was compared on 109 TMA tissue cores of HCC’s andcorresponding nontumor liver tissue of the same patient(Fig. 2). A similar expression rate was found for HTR1Aand HTR7 in nontumoral tissue and in tumoral tissue. Incontrast, the expression of HTR1B and HTR2B weresignificantly increased in HCC (HTR1B: P < 0.0001;HTR2B: P ¼ 0.001). A total of 32 (29.3%) and 38(34.9%) of the 109 HCC specimens evaluated displayeda strong expression for HTR1B and HTR2B, respectively(Table 1).

Comparison of 5HT-receptor expression withclinicopathologic parameters

First, we asked whether HTR1B and HTR2B were con-comitant expressed to assure that statistical analyses are notconfounded by coexpression of these receptors. We com-pared all specimens of 176 patients and only 21 HCCspecimens were double-positive for HTR1B and HTR2B(Fig. 3).

Next, we analyzed a potential association of 5HTreceptors expression with a number of clinicopathologicparameters including age and gender of the patient,tumor stage, histology grade, size, proliferation rate, type

Figure 1. Detection of 5HT-receptor mRNA expression in humanhepatocellular cancer cells HuH7, HepG2, and Hep3B. The reciprocalcycle threshold in relation to the housekeeping gene is plotted to displaymRNA expression qualitatively (n ¼ 4). ct, cycle threshold.

Soll et al.

Clin Cancer Res; 18(21) November 1, 2012 Clinical Cancer Research5904




of underlying liver disease, and patient survival (Table 2).Significant associations were identified for HTR1Band tumor size above 5 cm (P ¼ 0.002). Interestingly,the expression of HTR1B was also associated with ahigher proliferation rate, as assessed by Ki-67 staining

(P ¼ 0.036). HTR1B correlated with higher proliferationand size in HCC (Spearman r; Fig. 4). Consistent with ourprevious observation, we found that HTR2B correlated toa higher proliferation rate (Spearman r: n ¼ 176, r ¼0.214, P ¼ 0.004; ref. 6). HTR1A and HTR7 were notassociated with any of the evaluated clinicopathologicparameters.

To test whether the expression of 5HT receptors mayimpact on patient’s outcome, we evaluated a possible cor-relation between receptor expression and survival. An asso-ciation could not be identified for any of the receptorsstudied (Table 2). These findings were similar even byexcluding the patients who received transplantation or whohad anoncurative resection. Aunivariate survival analysis of171 patients showed that transplantation and R0-resectionwas significantly associated with prolonged overall survival(data not shown).

Effect of 5HT antagonists on proliferationIn the light of the significant correlation between the

expression of HTR1B and HTR2B markers of tumorcell proliferation, we asked whether 5HT-receptorantagonists might block proliferation in vitro. There-fore, we quantified the proliferation rate, using MTT

Nontumor tissue HCC

HT

R2B

H

TR

1A

HT

R1B

H

TR

7

Figure 2. Representative IHCof four 5HT receptors (HTR2B, 1A, 1B, and7)in human hepatocellular cancer compared with corresponding nontumortissue of the same liver (40 � magnification).

Table 1. 5HT-receptor expression in HCC livertissue and corresponding nontumor tissue

5-HT receptorexpression (%)

Nontumor HCC P valuea

5-HT1A 94.9 94.9 ns5-HT1B 11.1 29.3 <0.00015-HT2B 22.9 34.9 0.0015-HT7 17.2 18.2 ns

Abbreviation: ns, not significant.aMcNemar Test, n ¼ 109.

Figure 3. Distribution of HTR1B and HTR2B on 176 HCC specimens illustrated as a map. Gray squares are positive for the indicated receptor, white squaresare negative.






assays, of 2 human HCC cell lines (Huh7 and HepG2)exposed to specific antagonists S-WAY (HTR1A), SB216(HTR1B), LY272 (HTR2B), and SB269 (HTR7). SB216and LY216 significantly decreased proliferation in the

presence of FCS, whereas antagonists against HTR1Aattenuated proliferation only under serum-free con-ditions, and HTR7 had no impact on proliferation(Fig. 5).

Table 2. 5HT-receptor expression in HCC

HTR1A HTR1B HTR2B HTR7

Variable Category Totala Positive % P value Totala Positive % P value Totala Positive % P value Totala Positive % P value

Age at <60 years 76 74 97.4 1 78 20 25.6 1 78 21 26.9 1 75 14 18.7 0.704

diagnosis �60 years 97 95 97.9 97 24 24.7 98 28 28.6 97 21 21.6

Gender Female 40 39 97.5 1 41 8 19.5 0.414 41 13 31.7 0.849 41 7 17.1 0.66

Male 133 132 99.2 134 36 26.9 135 41 30.4 131 28 21.4

Tumor stage pT1 42 40 95.2 0.703 42 5 11.9 0.068 42 10 23.8 0.17 42 13 31.0 0.165

pT2 68 67 98.5 69 18 26.1 70 16 22.9 69 15 21.7

pT3 53 52 98.1 54 18 33.3 54 20 37.0 52 7 13.5

pT4 4 4 100.0 4 2 50.0 4 1 25.0 3 0 0.0

Histologic G1 41 38 92.7 0.05 42 12 28.6 0.523 42 13 31.0 0.391 41 9 22.0 0.166

grade G2 115 114 99.1 115 26 22.6 116 33 28.4 115 20 17.4

G3 17 15 88.2 18 6 33.3 18 8 44.4 16 6 37.5

Size �5 cm 93 91 97.8 1 96 16 16.7 0.002 93 23 24.7 0.205 95 20 21.1 1

>5 cm 62 60 96.8 61 24 39.3 62 21 33.9 59 13 22.0

Etiology HBV 27 25 92.6 0.229 27 9 33.3 0.148 27 7 25.9 0.304 25 9 36.0 0.111

HCV 35 35 100.0 37 5 13.5 37 9 24.3 36 10 27.8

Alcohol 45 45 100.0 46 11 23.9 46 13 28.3 46 6 13.0

Others 10 10 100.0 10 1 10.0 10 2 20.0 10 2 20.0

Unknown 55 53 96.4 54 18 33.3 55 23 41.8 54 9 16.7

Proliferation �5% 73 72 98.6 0.639 76 13 17.1 0.036 76 16 21.1 0.021 74 17 23.0 0.567

rate >5% 100 97 97.0 99 31 31.3 100 38 38.0 98 18 18.4

Overall Alive 108 104 96.3 0.298 109 27 24.8 1 110 25 22.7 0.116 108 19 17.6 0.316

survival Deceased 61 60 98.4 61 15 24.6 61 22 36.1 60 15 25.0

aTMA cores of some carcinomas were not evaluable because of insufficient number of carcinoma cells.

A100

80

60

40

20

0

20.0

15.0

10.0

5.0

0.0

Pro

lifer

atio

n r

ate

% (

Ki6

7)

HTR1B

Negative Positive

HTR1B

Negative Positive

Tu

mo

r si

ze (

cm)

B

Figure 4. Correlation of HTR1Bwith (A) Ki67 (n ¼ 157, r ¼ 0.279,P < 0.0001) and (B) tumor size(n ¼ 175, r ¼ 0.214, P < 0.004).

Soll et al.





5HT activates the mitogen-activated protein kinasespathway (MAPK) or extracellular signal-regulatedkinases (ERK) through the stimulation of HTR1B andHTR2B (8, 18–21). The MAPK/ERK pathway is involvedin the activation of transcription factors that regulateproliferation and cell-cycle progression (22). To testwhether 5HT and specific receptor antagonists changesthe activation of the MAPK/ERK pathway, we measuredthe phosphorylation of ERK (pERK) with immuno-blots. Stimulation with 5HT of HepG2 cells lead to atemporary phosphorylation after 5 minutes, whichdecreased again after 30 minutes. In the presence ofthe HTR1B-antagonist SB216 and the HTR2B-antago-nist LY272, no phosphorylation of ERK could bedetected (Fig. 6). These findings suggest (1) that pro-liferation of HCC cell lines is mediated by 5HT recep-tors and that (2) specific antagonists inhibit HCC cellgrowth in vitro.

DiscussionIn this study, we identified that serotonin receptors

1A, 1B, 2B, and 7 are expressed in human HCC tissue aswell as in corresponding nontumoral liver tissue.HTR1B and HTR2B are overexpressed in tumor tissue,however, compared with the adjacent liver, and theexpression of these receptors correlated with a higherproliferation index. Specific antagonists against HTR1Band HTR2B inhibited the proliferation of cells in 2human HCC cell lines suggesting a new putative targetfor therapy.

These findings are consistent with our previous obser-vation, that almost one-third of HCCs are positive forHTR2B, whereas 20 normal livers used an internal con-trol, were negative (6). In the current study, we extendedour analysis in nontumoral tissue, as control of thecorresponding HCC tumoral tissue. The expression rate

Figure 5. Viability and proliferation of Huh7 and HepG2 in the presence of specific 5HT-receptor antagonists. Cells were exposed to 4 differentculture conditions: standard growth conditions with 10% FCS, FCS in the combination with an antagonist (100 mmol/L), serum-free media with100 mmol/L 5HT, and 5HT in combination with an antagonist (without FCS). A, LY272, an HTR2B-antagonist, inhibited growth in the presence of FCSand 5HT (n ¼ 6, FCS vs. FCS/LY272: P < 0.001; 5HT vs. 5HT/LY272: P < 0.001). B, S-WAY100, an HTR1A-antagonist, attenuated growth onlyin serum-free media (n ¼ 6, 5HT vs. 5HT/S-WAY100: P < 0.001). C, SB216, an HTR1B-antagonist, inhibited growth in the presence of FCS and5HT (n ¼ 6, FCS vs. FCS/SB216: P < 0.001; 5HT vs. 5HT/SB216: P < 0.001). D, SB269 revealed no effect on growth of Huh7 and HepG2 (n ¼ 6). OD,optical density.






of HTR1B was 29.3% and 34.9% for HTR2B. Comparedwith nontumoral tissue the expression increased by18.2% and 12% for HTR1B and HTR2B, respectively.These data suggest an impact in a subset of patients withHCC.

An interesting new finding was the significant corre-lation identified between the tumor size and HTR1B, aswell as proliferation rates with both HTR1B and HTR2B.Such correlation suggests a role for these 2 receptors inthe cancer biology of HCC. These receptors have alreadybeen implicated in a variety of liver conditions, inter-estingly all those conditions may lead to cancer. There-by, HTR2B increasingly triggers some interest to betterunderstand the mechanisms of a variety of liver diseases.HTR2B is involved in hepatocellular regeneration andproliferation (23, 24). It could be shown in animalmodels that HTR2B agonists improved liver regenera-tion after extended hepatectomy (25) and also improvedhepatosinusoidal blood flow after hepatectomy by aVEGF-driven pathway (26). In regards to HCC, ourgroup identified HTR2B-mediated signaling down-stream of the mammalian target of rapamycin (mTOR),where mTOR signaling pathway may serve as a newmolecular target against HCC in approximately half ofthe patients (6, 27). We found that one-third of thepatients with HCC exhibit an activation of the mTORpathway, as assessed by immunohistochemical analysisof phosphorylated p70S6K, a direct downstream targetof mTOR (6, 27).

The involvement of HTR1B in HCC is novel and needsfurther investigation.Within the liver, HTR1B is involved inthe growth of the biliary tree by autocrine and paracrineregulation of 5HT (3, 13). In relation to cancer the role ofHTR1B is less defined. A few in vitro studies suggest apo-ptotic and antiproliferative properties of HTR1B antago-nists (28, 29).

Many 5HT-receptor agonist and antagonists are avail-able for experimental research. Most of them are notapproved for human use. In the current study, we useda new chemical antagonist against HTR2B [LY272 insteadof SB204741, used in recent experiments (6)]. This water-soluble antagonist avoids complicated solubilization pro-tocols such as SB204741. Interestingly, LY272 exhibitedthe same effect on survival in Huh7 and HepG2 asSB204741, excluding a general toxic effect of SB204741and suggesting that specific inhibition of HTR2B impairsgrowth of HCC cells.

Proliferation assays were conducted without (serum-freemedium) and with FCS, as standard growth medium. 5HTstimulated HCC cell growth as strong as FCS. Only antago-nists against HTR2B and HTR1B inhibited proliferation inthe presence of FCS. These experiments suggest 5HT as aserum factor needed for optimal growth of HCC cellsmediated by 2 different types of 5HT receptors. The prolif-erative effect of 5HT is cell-type specific because 5HT didnot stimulate proliferation in non-HCC cell lines, asshown in our previous publication (6). 5HT-mediated cellgrowth may be at least partially explained by the activationof classical MAP kinases such as ERK1/2, a pathway that isfrequently activated in cancer including HCC (30–32).Interestingly, either inhibition of HTR1B or HTR2B sup-pressed the phosphorylation of ERK1/2. This finding maybe explained with cross-talks between 5HT-receptor sub-types or 5HT receptor and other cell-surface receptors. Across-talk means a transactivation of a distinct cellulartarget by different signal transduction pathways. As a resultthe activation of a target can be blocked or attenuated ifone of several activating pathways is inhibited. Cross-talksbetween different 5HT receptors (33, 34) and HTR2Band the platelet growth factor receptor (18) have beendescribed.

The first, and still only clinically available success of amolecular targeted therapeutic strategy was the use of themultikinase inhibitor sorafenib, leading to survival benefitin patients with advanced HCC (35). This successencourages a molecular classification of HCC to promotea personalized treatment (36). The detection of 5HT recep-tors in HCCmay have a clinical relevance, as a considerablenumber of patients overexpress HTR1B and HTR2B andmany different specific 5HT antagonists are available on themarket.

A large amount of work has been done to identifyprognostic molecular markers in HCC, for example, topredict survival or help in deciding a specific therapy. Aparticular difficulty is the presence of a variety of under-lying diseases, such as various etiologies for the livercirrhosis, which may exert differential effects on involved

pERK1/2

ββ-Tubulin

5HT

5HT + SB216

5HT + LY272

0′ 5′ 15′ 30′

pERK1/2

β-Tubulin

0′ 5′ 15′ 30′

pERK1/2

β-Tubulin

0′ 5′ 15′ 30′

C

A

B

Figure 6. Immunblot detection of pERK1/2 in HEPG2. A, stimulationwith 5HT led to a phosphorylation of ERK1/2, whereas antagonistagainst (B) HTR1B (SB216) and (C) HTR2B (LY272) inhibited anactivation of ERK1/2.

Soll et al.





molecular pathways leading to HCC. For example, amolecular marker for HBV-related HCC may not berelevant for HCV-related disease. In addition, the major-ity of patients are likely to have been exposed to acombination of etiologic factors. Therefore, our cohortof 176 patients may still be too small to reveal 5HT-receptors as a predictor of survival or an association witha distinct etiology. It is also possible that not the expres-sion of 5HT receptors itself serves as a marker, for exam-ple, predicting survival, but downstream molecules acti-vated by 5HT receptors.In conclusion, the 5HT receptor 1B and 2B are involved

in tumor growth of human HCC. Therefore, 5HT-medi-ated signaling pathways in the liver may represent a target

for new molecular treatment strategies in a subset ofpatients.

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

Grant SupportKrebsliga Zurich (to C. Soll), Sassella Foundation (to C. Soll and C.E.

Oberkofler), and Swiss National Science Foundation Grant 3200B0-109906(to P.-A. Clavien).

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

Received July 13, 2011; revised July 29, 2012; accepted August 27, 2012;published OnlineFirst October 19, 2012.

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Soll et al.





2012;18:5902-5910. Published OnlineFirst October 19, 2012.Clin Cancer Res Christopher Soll, Marc-Oliver Riener, Christian Eugen Oberkofler, et al. CancerExpression of Serotonin Receptors in Human Hepatocellular

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