Differential Proteomic Analysis of Subfractioned Human Hepatocellular Carcinoma Tissues

$: Differential Proteomic Analysis of Subfractioned Human Hepatocellular Carcinoma Tissues$
Differential Proteomic Analysis of Subfractioned Human

Hepatocellular Carcinoma Tissues

Erika Codarin,‡ Giovanni Renzone,§ Alessandra Poz,| Claudio Avellini,| Umberto Baccarani,⊥

Francesco Lupo,# Vittorio di Maso,∇ Saveria Lory Croce,∇ Claudio Tiribelli,∇ Simona Arena,§

Franco Quadrifoglio,‡ Andrea Scaloni,*,§ and Gianluca Tell*,‡

Department of Biomedical Sciences and Technologies, University of Udine, 33100 Udine, Italy, Proteomics &Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy, Department ofClinical Pathology, University of Udine, 33100 Udine, Italy, Department of Surgery & Transplantation,

University of Udine, 33100 Udine, Italy, Azienda Ospedaliero Universitaria, Molinette, 10100 Torino, Italy, andCentro Studi Fegato, AREA Science Park, 34012 Trieste, Italy

Received October 30, 2008

To discover new potential biomarkers of HCC, we used 2-DE gel separation and MALDI-TOF-MS analysisof partially enriched nuclear fractions from liver biopsies of 20 different patients. We obtained aproteomic map of subfractioned liver samples including about 200 common protein spots, among whichidentified components corresponded to expression products of 52 different genes. A differential analysisof proteins from tumoral and control tissues revealed a significant change in the expression level of 16proteins associated to cytoskeletal, stress response and metabolic functions. These data may providenovel candidate biomarkers for HCC and useful insights for understanding the mechanisms of HCCpathogenesis and progression.

Keywords: hepatocellular carcinoma • cell nucleus • biomarkers

1. Introduction

Hepatocellular carcinoma (HCC) is a common malignanttumor, which develops from chronic inflammatory liver dis-eases due to hepatitis B virus (HBV) and hepatitis C virus (HCV)infections or exposure to carcinogens, such as aflatoxin B1.1

In addition, cirrhosis is also cause of HCC development in mostcases. HCC pathogenesis is a multifactorial and multistepprocess that finally leads to the deregulation of cell homeo-stasis. It is commonly thought that the occurrence of chronicinflammation and cell damage may provide the proliferativestimuli to promote the hepatocarcinogenetic process.2-4 As thepoor survival of HCC patients is largely related to the lack ofan early diagnosis, more reliable, sensitive and specific toolsfor early detection of HCC are urgently needed. An effectivemarker for diagnosis of HCC is yet to be found, as the utility ofR-fetoprotein remains still controversial and the novel tumor

markers proposed (i.e., serum ferritin, γ-glutamyltranspepti-dase, alkaline phosphatase, des-γ-carboxy prothrombin, R-1-antitrypsin, aldolase A, 5′-nucleotide phosphodiesterase, tissuepolypeptide antigen, and R-1-fucosidase) are still inadequate.2,5-7

Proteomic analysis of liver proteins and HCC were predomi-nantly performed using either chemically induced hepatomain animals (predominantly rat and mouse) or human HCC celllines, such as HepG2 and Huh7 cells.2,8-11 Several so-calledtumor-associated or cancer-related proteins were identified,providing valuable information for the definition of HCCdatabases.2,8,9,12,13 A major drawback in these studies was theuse of total protein extracts, which allowed only the study ofthe most abundant protein species. To detect low-abundanceproteins, the analyzing power of proteomic techniques needsto be improved by using specific subcellular fractionationstrategies.14,15 This choice may also reduce the complexity ofthe protein profile to be analyzed and facilitate the identifica-tion of differentially expressed proteins, possibly involved inthe tumorigenic process.16-19 Another major limitation thatemerged from previous studies was the lack of genetic homo-geneity, which hampered a proper comparison of samples fromdifferent patients.20,21 Accordingly, the most effective waywould be the analysis of both cirrhotic and tumor tissuesobtained from the same patient. This would allow to identifydistinct and proper HCC proteomic signatures.22,23

Nowadays, it is widely accepted that proteomic technologiesmay efficiently help the study of biological responses incomplex systems in a more global way by the simultaneousevaluation of hundreds of protein components.24,25 In thepresent study, we used 2-DE and MALDI-TOF-MS for the

† Originally submitted and accepted as part of the “Tissue Proteomicsand Metabolomics” special section, published in the April 2009 issue of J.Proteome Res. (Vol 8, No. 4).

* Correspondence to: Prof. Gianluca Tell, Department of BiomedicalSciences and Technologies, University of Udine, P.le Kolbe 4, 33100 Udine,Italy.Tel.:++39432494311.Fax:++39432494301.E-mail:[email protected]. Andrea Scaloni, Proteomics and Mass Spectrometry Laboratory, ISPAAM,National Research Council, via Argine 1085, 80147 Naples, Italy. Tel.: ++39081 5966006. Fax: ++39 081 5965291. E-mail: [email protected].

‡ Department of Biomedical Sciences and Technologies, University ofUdine.

§ Proteomics & Mass Spectrometry Laboratory, ISPAAM.| Department of Clinical Pathology, University of Udine.⊥ Department of Surgery & Transplantation, University of Udine.# Azienda Ospedaliero Universitaria.∇ Centro Studi Fegato.

10.1021/pr8009275 CCC: $40.75 2009 American Chemical Society Journal of Proteome Research 2009, 8, 2273–2284 2273Published on Web 03/16/2009

identification of proteins related to hepatocarcinogenesis bydifferential proteomic analysis on HCC and non-HCC tissuesamples enriched for nuclear protein components. To excludethe effect of genetic variation and interindividual variability,the analysis was performed by comparing samples obtainedfrom HCC lesions with those from surrounding liver cirrhosisof the same patient. The deregulated proteins identified mayrepresent valuable biomarker tools in understanding the mech-anisms of hepatocarcinogenesis and in aiding HCC diagnosis/therapy.

2. Materials and Methods

2.1. Patient and Sample Collection. Biopsies were obtainedfrom 20 HCC patients with underlying liver cirrhosis. A liverbiopsy, called N, was obtained from the tumoral region and asecond biopsy, called C2, was collected in the distal region ofcirrhotic liver and used as control. Liver tissue was also fixedin 10% formalin for further histological and immunohis-tochemical examinations (Figure 1A). All biopsies were im-mediately frozen at -80 °C until processing, through mechan-ical cell separation. A total of 40 liver biopsies (20 at N and 20at C2, from 20 donors) were collected for proteomic analysis.Informed consent was obtained by all patients before surgery.The protocol of the study was approved by the ethicalcommittee.

2.2. Patient Demographics. With one exception, all patientswere males. The mean age of the patients was 65 (range 53-82)years. Samples were divided into 5 different groups accordingto the presence of specific serological markers of hepatitisviruses as reported in Table 1. In 3 patients, it was not possibleto collect data on viral serology.

2.3. Preparation of Partially Enriched Nuclear Fractionsfrom Liver Biopsies. The partially enriched nuclear fractionsfrom liver sections were obtained with the following experi-mental procedure. Each liver biopsy (about 100 mg of tissue)was homogenized using a Potter Duall supplied with a PTFEpestle in Lysis Buffer (1 mL of 10 mM HEPES, pH 7.9, 1.5 mMMgCl2, 10 mM KCl, 1 mM DTT and Protease Inhibitor Cocktail)until more than 90% of the cells was lysed. The disrupted cellswere centrifuged at 500g, for 30 min, to separate the cytoplas-mic fraction (supernatant). After a rinse with Lysis Buffer, thecrude nuclei pellet was then resuspended in Extraction Buffer[about 140 µL of 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420mM NaCl, 0.2 mM EDTA, 28% (v/v) glycerol, 1 mM DTT andProtease Inhibitor Cocktail], shaken gently for 30 min in ice,and then centrifuged at 18 000g, for 30 min, to obtain thenuclear fraction (supernatant). Protein extracts were precipi-tated using cold acetone and methanol and subsequently storedat -80 °C. To test for the quality of the partially enrichednuclear extracts, the presence of the TATA binding protein(TBP), cytochrome C, apoptosis inducing factor (AIF) andcalreticulin was verified by using anti-TBP, -cytochrome C, -AIFand -calreticulin antibodies (Santa Cruz Biotechnology, SantaCruz, CA). Protein quantification was performed by the Brad-ford colorimetric method26 and SDS-PAGE analysis.

2.4. Two-Dimensional Polyacrylamide Gel Electrophoresis.Thirty micrograms of nuclear extracts was loaded onto 13 cm,pH 3-10 L IPG strips (Amersham Biosciences, Milan, Italy).IEF was conducted using an IPGPhor II system (AmershamBiosciences) according to the manufacturer’s instructions.Focused strips were equilibrated with 6 M urea, 26 mM DTT,2% (w/v) SDS, 30% (v/v) glycerol in 0.1 M Tris-HCl (pH 8.8)for 15 min, followed by 6 M urea, 0.38 M iodoacetamide, 2%(w/v) SDS, 30% (v/v) glycerol, and a dash of bromophenol bluein 0.1 M Tris-HCl, pH 8.8, for other 15 min. The equilibratedstrips were applied directly to 12% SDS-polyacrylamide gelsand separated at 130 V. Gels were fixed and stained byammoniacal silver.27 For each bioptic sample, three experi-mental replicates were subjected to 2-DE; all gels were scannedwith an Image Scanner II apparatus (GE Healthcare, Milan,Italy).

2.5. Evaluation of Differentially Represented Spots. Gelimages were analyzed by the Image Master 2D Platinumsoftware (GE Healthcare) that allowed performing a compara-

Figure 1. (A) Hematoxylin and eosin staining of HCC andnontumoral paraffin-embedded tissue sections. N indicates thetissue section from tumoral region. C2 indicates the tissue sectionfar away from cancer site and represents the control sample. (B)Western blotting analysis of nuclear and cytoplasmic enrichedsamples from liver resections. Analysis was performed to evalu-ate the presence of cross-contamination between the extracts.Immunoblot analysis for the evaluation of AIF, cytochrome C,calreticulin and TBP expression in tumoral and nontumoraltissues of a representative patient. Immunoblotting was per-formed as described in Materials and Methods. Actin was usedas a reference. N, tumoral tissue; C2, control tissue.

Table 1. Demographic and Clinical Features of the PatientsStudied

mean age: 65 no. cases

Sex Men 19Female 1

Neoplastic Grade G1/G2 14G3/G4 6

Viral Serology HBV+ve 7HCV+ve 5HBV+ve/HCV+ve 0HBV-ve/HCV-ve 5Not reported 3

Total no. patients 20

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tive image analysis.24,28-30 Protein spots were detected andmatched between the different samples; individual spot volumevalues were obtained according to the program instruction andnormalized using the program volume normalization function.Ratio of the different sample normalized volume values for thecandidate protein spots was compared with each others and amean relative difference in spot intensity was calculated.Differences in protein spot expression levels were consideredas significant when Student’s t test gave a P-value <0.01.

2.6. Mass Spectrometry Analysis. Spots from 2-DE wereexcised from the gel, triturated and washed with water. Proteinswere in-gel reduced, S-alkylated and digested with trypsin aspreviously reported.31 Digest aliquots were removed andsubjected to a desalting/concentration step on µZipTipC18

(Millipore, Bedford, MA) using acetonitrile as eluent beforeMALDI-TOF-MS analysis. Peptide mixtures were loaded on theMALDI target, using the dried droplet technique and R-cyano-4-hydroxycinnamic acid as matrix, and analyzed using Voyager-DE PRO mass spectrometer (Applied Biosystems, Framingham,MA). Internal mass calibration was performed with peptidesderiving from enzyme autoproteolysis. PROWL software pack-age was used to identify spots unambiguously from the NCBInonredundant sequence database. Candidates with ProFound’sEst’d Z-scores >2 were further evaluated by the comparisonwith their calculated mass and pI using the experimental valuesobtained from 2-DE.

2.7. Immunoblotting Analysis. Nuclear extracts from HCCliver biopsies were separated on 12% SDS-PAGE. Then, proteinswere transferred to nitrocellulose membranes (Schleicher andSchuell Bioscience, Keene, NH) for the detection of ATPsynthase, heat shock 60 kDa protein (Hsp60), heat shock 70kDa protein (Hsp70), heat shock cognate 71 kDa protein(Hsc71), actin-related protein 2 (Arp2), electron transfer fla-voprotein � (ETFB) and anti-hnRNP A2/B1. After saturationwith 5% (w/v) nonfat dry milk in PBS and 0.1% (w/v) Tween-20, the membranes were incubated with one of the followingantibodies: anti-ATP synthase polyclonal antibody (homemade,polyclonal antibody against R-� subunit of F1 sector, kindlyprovided by Prof. G. Lippe, University of Udine), anti-Hsp60monoclonal antibody (ab8071, Abcam, Cambridge, U.K.), anti-Hsp70 monoclonal antibody (MA3-007, Affinity Bioreagents,Golden, CO), anti-Hsc71 monoclonal antibody (ab19136, Ab-cam), anti-Arp2 monoclonal antibody (ab49674, Abcam), anti-ETFB polyclonal antibody (ab73986, Abcam) and anti-hnRNPA2/B1 monoclonal antibody (sc-32316, Santa Cruz Biotechnol-ogy). Filters were incubated overnight, at 4 °C, with each ofthem. After three washes with PBS, 0.1% (w/v) Tween-20, themembranes were incubated with anti-rabbit, anti-mouse oranti-rat immunoglobulins conjugated with peroxidase (Sigma-Aldrich, Milan, Italy). After 2 h of incubation at room temper-ature, the membranes were washed three times with PBS 0.1%(w/v) Tween-20, and the blots were developed by the ECLprocedure (Pierce Biotechnology, Rockford, IL). The signalsfrom each protein band were normalized against the actincontent by using the polyclonal anti-actin antibody (Sigma-Aldrich). Blots were quantified by Image Quant (GE Health-care). Western blot analysis was extended to all the availablesamples (20 patients), except for hnRNP A2/B1; in this case,the analysis was limited to 11/20 cases, due to samplelimitations.

2.8. Immunohistochemical Analysis. Formalin-fixed, paraf-fin-embedded tumor samples from 5 HCC patients out of 20subjected to proteomic analysis were used for immunohis-

tochemical (IHC) analysis. IHC staining was developed on 5µm thick paraffin sections to detect Hsp60, Hsp70, Hsc71 andhnRNP A2/B1, respectively. After dewaxing, rehydratation, andendogenous peroxidase quenching with 20% H2O2 (v/v) in PBSsolution, for 10 min, at room temperature, sections were dippedin 10 mM citrate buffer pH 6.0 for 40 min, at 98 °C, and thencooled again at room temperature. After a rinse with PBS,sections were incubated with mouse monoclonal anti-Hsp60(ab8071, Abcam) at 1:100 dilution, mouse monoclonal anti-Hsp70 (MA3-007, Affinity Bioreagents) at 1:150 dilution, ratmonoclonal anti-Hsc71 (ab19136, Abcam) at 1:50 dilution andmouse monoclonal anti-hnRNP A2/B1 (sc-32316, Santa CruzBiotechnology) at 1:50 dilution respectively, for 1 h, at roomtemperature. After a rinse with PBS solution, incubation withthe peroxide-based detection system REAL EnVision (Dako A/S,Glostrup, Denmark) for 30 min, at room temperature, andtreatment with 3,3′-diaminobenzidine for 10 min followed.Sections were counterstained with Mayer’s hematoxylin, de-hydrated and mounted. Cells positive for protein staining wereevaluated by a single observer at 20× and 40× of microscopemagnification. Negative controls were carried out replacing theprimary antibody with nonimmune serum.

3. Results

3.1. Subfractionation of HCC Liver Biopsies. To enrich forlow-abundance proteins, a subcellular fractionation of liverbiopsies was performed. A well-established protocol32 forpartial enrichment of the nuclear fraction was used, thusobtaining about 800 µg of proteins from 100 mg of each tissuesample. To verify for the efficiency and the selectivity of ourpreparations, Western blot analysis of the nuclear and cyto-plasmic extracts was performed. Both fractions were tested forthe presence of TATA binding protein (TBP), cytochrome C,apoptosis inducing factor (AIF) and calreticulin (Figure 1B). Thefirst protein is a transcription factor with a well-known nuclearlocalization;33-35 the second and the third proteins localizewithin mitochondria,36,37 and the fourth one is a protein usuallyassociated to the endoplasmic reticulum (ER).38 The specificpresence of TBP only in the nuclear extract indicated asignificant enrichment for the nuclear components, excludingnuclear protein leakage during preparation (Figure 1B), al-though it did not exclude the co-purification of abundantproteins from other subcellular compartments, such as mito-chondria or ER. Despite the limitations of the extractionprocedure we used, this protocol can be considered as a goodcompromise between the amount of proteins fully recoveredfrom the nucleus and the removal of abundant cytoplasmiccomponents on one hand and the limitation on the tissueavailability on the other.

3.2. Proteomic Map of Subfractioned HCC Liver TissueBiopsies. To analyze the proteomic map of the partiallyenriched nuclear fraction from liver biopsies, samples weresubjected to 2-DE analysis in the range of pH 3-10. Stainingof 2-DE gels allowed for the simultaneous quantitative evalu-ation of about 200 common protein spots in each gel (Figure2A). Protein spots varying among the N and C2 samples plusones being constant (used as a reference) were excised fromthe gels and analyzed by MALDI-TOF Peptide Mass Fingerprint(PMF) experiments. Data searching in a nonredundant se-quence database allowed the unambiguous identification of 83protein spots or spot trains, which corresponded to 52 proteinspecies with molecular mass ranging from 13 to 80 kDa.Supporting Information Table 1 reports on the nature of each

HCC Proteomics research articles

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identified spot, the measured 2-DE coordinates, the relativesequence coverage, together with the known functional proper-ties. As expected, the distribution of spots across the gel wasnot homogeneous, with a prevalence of focalized spots presentin the mass range 20-70 kDa and the pI range 4-7. Thisproteomic map was also characterized by the presence ofmultiple horizontal spot trains with different pI values, associ-ated to the same protein entry, as shown for spots 26-31(THIL), 35-37 (ARGI1) and 53-55 (ECH1), which were likelyderived from the occurrence of post-translational modifica-tions. In addition, some spots associated to the same proteinspecies were also distributed over the whole map, showing amarked difference in their apparent mass; their presence wasassociated to protein processing events.

Identified proteins were divided into clusters according totheir functional properties (Figure 2B). All the major functionalgroups are represented including proteins involved in binding,catalysis, structure, redox regulation or detoxification, metabo-lism/transport regulation, signal transduction and transcrip-tional regulation. Among the identified enzymes, those involved

in lipid, energy and protein or amino acid metabolism werethe most abundant.

3.3. Changes in Liver Proteomic Profile betweenTumoral and Distal Regions. Bioinformatic analysis of 2-DEgel images allowed for the simultaneous quantitative evaluationof almost 200 protein spots within each gel. Thirty-three proteinspots, each of them representative of the different conditionsexamined, displayed a statistically significant change in abun-dance when comparing samples from the neoplastic (N) to thedistal regions (C2) (Figure 3). PMF analysis allowed theunambiguous identification of 31 of them, which were associ-ated to 16 ORFs. Tables 2 and 3 report on the nature of eachidentified spot, as well as the gel coordinates, the relative N/C2abundance ratio and the known functional properties. Identi-fied proteins were classified into two groups according to theirassociation to HCC. While Table 2 includes proteins that havenever been associated to this type of cancer (electron transferflavoprotein �, hnRNP A2/B1, UTP-glucose-1-phosphate uridyl-transferase 2A, hCG2001950 scaffold protein, hydroxymethyl-glutaryl-CoA synthase, actin-related protein 2 and methylma-lonate-semialdehyde dehydrogenase), thus representing possiblenovel candidate biomarkers, Table 3 lists proteins alreadydescribed as deregulated in HCC (Hsc71, Hsp70, Hsp60,R-enolase, cathepsin D, 3-ketoacyl-CoA thiolase, ATP synthaseR, fructose-bisphosphate aldolase B and peptidyl-prolylcis-trans isomerase A),39-41 thus reinforcing the reliability ofthe procedure used. In addition, two of the differentiallyexpressed proteins, namely, ATP synthase R and Hsc71, wereidentified both as full-length and truncated proteins, possiblyas the consequence of proteolytic events.

All quantitative variations for proteins already described asassociated to HCC reflected expression data42-44 previouslyreported. The majority (8/16) of the changes observed consistedin a decreased protein expression level in tumoral tissue. Infact, a reduced abundance was observed for cathepsin D, ATPsynthase R, electron transfer flavoprotein �, fructose-bispho-sphate aldolase B, 3-ketoacyl-CoA thiolase, UTP-glucose-1-phosphate uridyltransferase 2A, hCG2001950 scaffold proteinand methylmalonate-semialdehyde dehydrogenase. One pro-tein, namely, peptidyl-prolyl cis-trans isomerase A, exhibiteda different behavior being up- or down-regulated in differentpatients. All the other proteins (7/16) were characterized byan increased abundance in the tumoral tissue. They wereidentified as Hsc71, Hsp60, Hsp70, R-enolase, hydroxymeth-ylglutaryl-CoA synthase, hnRNP A2/B1 and actin-related pro-tein 2.

3.4. Validation of Differential Protein Expression UsingWestern Blot Analysis. To check for the reliability of thequantitative data obtained, seven differentially expressed pro-teins found by 2-DE (Hsc71, Hsp60, Hsp70, hnRNP A2/B1, Arp2,ETFB and ATP synthase R) were also validated by Westernblotting analysis (Figure 4A). In all cases, Western blotting datawere in good agreement with 2-DE, thus confirming a higherexpression for Hsc71, Hsp60, Hsp70, Arp2 and hnRNP A2/B1,and a lower expression for ATP synthase R and ETFB in thetumor samples, respectively (Figure 4B).

3.5. Immunohistochemistry Analysis of DifferentiallyExpressed Proteins in HCC Liver Biopsies. To further validateour data, we performed IHC analysis of the most relevantprotein species in tumor samples from 5 randomly selectedHCC patients. The expression of Hsp60 protein, which has anexclusive cytoplasmic localization, was decreased in patientswith poorly differentiated HCC (Edmondson grade III and IV

Figure 2. (A) 2-DE proteomic map obtained from enriched HCCliver samples. 2-DE was performed on immobilized pH 3-10strips, followed by the second-dimensional separation on 12%polyacrylamide gels. Resolved proteins were visualized by am-moniacal silver staining. Identification numbers of the spotscorrespond to numbering in Supporting Information Table 1. Allprotein species were identified by MALDI-TOF PMF experiments.(B) Functional clustering of the identified proteins shows therelative representation (indicated as percentage of all the identi-fied proteins) for each protein family.



HCC) as compared to those with a better differentiatedphenotype (Edmondson grade I or II HCC). Moreover, inagreement with present proteomic analysis data, Hsp60 expres-sion level was higher in HCC than in cirrhotic tissue (Figure5A). Similarly, Hsp70 showed a proper cytoplasmic localization;immunohistochemical analysis also denoted a lower reactivityfor Hsp70 in cirrhotic parenchyma (C2) than in the tumoralregion (N) (Figure 5B). Furthermore, a correlation similar tothat observed for Hsp60 was maintained also in associationwith neoplastic grading, so that Hsp70 had lower expressionlevels in poorly differentiated HCC with respect to less malig-nant neoplasias. This trend was maintained in all the casesobserved.

On the other hand, Hsc71 had both nuclear and cytoplas-mic localization, depending on the tumoral grading. The IHCstaining revealed a more intense reaction in better differenti-ated neoplasias (G1 and G2) compared to the more aggres-sive forms of the tumor. In particular, G3 and G4 neoplasiashad almost an exclusive Hsc71 cytoplasmic expression andsuch expression levels were higher in the neoplastic tissue(N) with respect to the cirrhotic parenchyma (C2) (Figure5C).

The expression of hnRNP A2/B1 was almost totally nuclear(Figure 5D) without any significant change in the percentagevalues between neoplastic and cirrhotic tissues and withoutany kind of correlation with the tumor grading.

4. Discussion

Previous reports have demonstrated that proteomic analysisof tumor tissues, by combined 2-DE and MALDI-TOF-MStechnologies, may provide useful information for cancer clas-sification, identification of diagnostic markers and selection oftherapeutic target candidates.45-47 Such approaches have beenapplied to the analysis of specimen or cell line samples derivedfrom liver cancer.8,12,13,48 In this way, several so-called livertumor-associated or cancer-related proteins have been identi-fied, which provided a valuable contribution to the establish-ment of HCC databases.2,8,9,12,13 However, previous proteomicstudiesonHCChavebeenoftenrestrictedtoafewpatients,20,22,23,49,50

limiting a wider applicability of the identified HCC biomarkers,or have shown the limitation due to the reduced dynamic rangeof 2-DE analysis, which allowed only visualization of the mostabundant proteins.20,22,23,49,50 Thus, enrichment strategies fordetection of poorly represented proteins51,52 have been veryrecently applied also to the study of HCC-related componentsfrom subcellular compartments, such as Golgi, ER, plasmamembrane and mitochondria.53-57 At present, however, thereare no examples in the literature for differential investigationson nuclear proteins from HCC versus non-HCC specimens.

In this study, we report the first partially enriched nuclearproteomic map from liver tissues, which describes the expres-sion products of 52 genes, including those involved in signaltransduction, transcriptional regulation, structure, stress re-sponse and various metabolic pathways. This study wasperformed by comparing the protein fingerprint from tumoraland nontumoral, cirrhotic tissues of the same patient, thusremoving genetic and environmental factors possibly affectingthe results. Despite contaminations from other subcellularcompartments, the enriched nuclear preparations allowed usto detect low-abundance proteins and reduced the complexityof the resulting protein patterns profile, facilitating the iden-tification of novel deregulated proteins in HCC. This compara-tive analysis performed on 20 patients allowed the identification

Figure 3. Differential proteomic analysis of nuclear enriched HCC andcontrol samples. Representative gel regions comprising some of thestatistically significant changes in proteome repertoire were cropped.(A) Up-regulated protein species found in N with respect to C2; (B)down-regulated protein species found in N with respect to C2.



of 31 differentially expressed spots in HCC tissue, which wereassociated to 16 ORFs. Possible functional implications oncarcinogenesis of their quantitative changes and previousreports on their expression in HCC are reported below.

Among deregulated proteins here associated to HCC, ninespecies have been already reported as involved in cancer onsetand progression. In fact, up-regulation of molecular chaperonesHsc71, Hsp60 and Hsp70 has been already reported in HCC,colorectal adenomas/cancer, breast cancer or high-grade pro-static lesions.9,20,22,39,58-66 The overexpression of these proteinshas been interpreted as a response to the stressful cancerousenvironment for cyto-protective functions. In the case of Hsp60,its up-regulation may contribute to the tumor cell adaptiveresponse to low oxygen levels.67 Since, in cancer cells, expres-sion of chaperone Hsp70 has been implicated in regulation ofapoptosis, immune response against tumors and multidrugresistance,68-70 understanding its role in carcinogenesis mayhave important implications regarding tumor behavior/prog-nosis.71 Thus, these findings suggest that Hsps are of specialrelevance in human cancer, representing candidate biomarkersfor oncological management.

Up-regulation of R-enolase has also been described as acommon feature of HCC8,72,73 and the expression of thisglycolytic enzyme increased with tumor dedifferentiation (asshown in biopsies from HCV-related HCC).73 Expression ofR-enolase positively correlates also with tumor size and venousinvasion,73 thus suggesting this protein as candidate biomarkerfor liver tumor progression.

Different mechanisms associated with tumor invasion/metastasis,23,74-77 also involving deregulation of cellular pro-teolytic activities,23,78 could explain the down-regulation ofcathepsin D. In HCC, this acid protease has been proposed as

a biochemical marker of malignant progression of liver cir-rhosis.74 Similarly, down-regulation of mitochondrial ATPsynthase R, observed in HCC or other cancers,79-83 could berelated to the mitochondrial bioenergetic deregulation typicalof the tumoral condition. A metabolic derangement peculiarof the neoplastic process can be hypothesized also for thedown-regulation of mitochondrial 3-ketoacyl-CoA thiolaseobserved in neoplastic tissues.84 In fact, a bioenergetic dys-function of mitochondria, affecting lipid/fatty acid metabolism,has been reported as a hallmark of many types of cancers.85,86

In addition, a specific down-regulation of fructose-bisphos-phate aldolase B in tumoral tissues has been described forHCC.87 Cancer cells with a high glycolytic rate may have anadvantage in tumor growth. HCC often exhibits an aberrantexpression of glycolytic enzymes, particularly hexokinases andaldolases.88 In particular, a decreased expression of fructose-bisphosphate aldolase B has been associated with advanceddisease, early tumor recurrence and has been used as apredictive marker of poor prognosis.87,88

A peculiar behavior was observed for peptidyl-prolylcis-trans isomerase A (PPIA), which exhibited up/down-regulated expression levels depending on the patient. ThePPIase family of proteins regulates the activities of matureproteins by promoting their assembly/intracellular transport,89

due to the association with the dynein/dynactin motor com-plex. Recent findings suggest that PPIA may be involved in theexecution stage of the p53-mediated apoptosis44,90 by interact-ing with p53 and participating in the retrograde movement ofthe p53 complex to the nucleus.90 Furthermore, PPIA also playsan essential role in the G phase of the cell cycle.91 Nowadays,this protein has been proposed as a marker candidate fornonsmall cell lung, pancreatic and liver tumors.22,92-94

Table 2. Proteins Identified in This Work As Differentially Expressed in HCC and Never Associated to Liver Tumoral Tissuesa

spotno. protein identity

gi/Swiss-Protentry MW (kDa) pI

relative intensityN/C2 ratio function

referenceno.

9 Methylmalonate-semialdehydedehydrogenase[acylating] (MMSA)

Q02252 56 (58) 6.7 (8.7) 0.71 Plays a role in valine andpyrimidine metabolismand binds fatty acyl-CoA.

106, 107

12 UTP-glucose-1-phosphteuridylyltransferase 2isoform A (UGPA)

Q16851 53 (57) 6.8 (8.1) 0.52 Plays a central role as aglucosyl donor in cellularmetabolic pathways.

108, 109

38 Actin-related protein2 (ARP2)

P61160 41 (44) 5.9 (6.3) 3.50 Functions as ATP-bindingcomponent of theArp2/3 complex which isinvolved in regulation ofActin polimerization.

110

45 Hydroxymethylglutaryl-CoA synthase (HMCS2)

P54868 36 (56) 7.2 (8.4) 4.06 It condenses acetyl-CoAwith acetoacetyl-CoAto form HMG-CoA.

95, 111

42; 44;46; 47

hnRNP A2/B1 protein(ROA2)

P22626 36, 36, 36, 36 (37) 6.9, 7.7, 8.4,8.6 (8.9)

1.45, 1.50,1.47, 1.55

Heterogeneous nuclearribonucleoprotein.

99, 112

63 Electron transferflavoprotein subunitbeta (ETFB)

P38117 28 (28) 7.6 (8.2) 0.50 It transfers theelectrons to the mainmitochondrial respiratorychain viaETF-ubiquinoneoxidoreductase(ETF dehydrogenase).

101, 113

72 Scaffold proteinhCG2001950

119623362 26 8.5 (8.6) 0.30 Genomic scaffold 114

a Spot number, protein name, accession number (gi/Swiss-Prot entry), experimental (theoretical) mass and pI value, and relative protein abundance (ascompared to the values for the C2 samples) are listed (quotients of N vs C2: ratio above 1, up-regulation; ratio below 1, down-regulation in C2). The ratiowas calculated for matched pair samples from each patient.



Among the deregulated proteins we found, seven polypeptidespecies (electron transfer flavoprotein �, hnRNP A2/B1, UTP-glucose-1-phosphate uridyltransferase 2A, hCG2001950 scaffoldprotein, hydroxymethylglutaryl-CoA synthase, actin-related

protein 2 and methylmalonate-semialdehyde dehydrogenase)have never been associated with HCC before. Hydroxymeth-ylglutaryl-CoA synthase, which regulates ketone body produc-tion, is expressed in liver and other several extra-hepatic tissues,

Table 3. Proteins Identified in This Work As Differentially Expressed in HCC and Already Associated to Liver Tumoral Tissuesa

spotno. protein identity

gi/Swiss-Protentry MW (kDa) pI

relative intensityN/C2 ratio function

referenceno.

2a, 2b Heat shock cognate 71kDa protein (HSP7C)

P11142 77, 77 (71) 5.5,5.6 (5.4)

2.20, 2.00 Chaperone. Translocatesrapidly from thecytoplasm to the nuclei andespecially to thenucleoli upon heatshock.

39, 40

3 Heat shock 70 kDaprotein 1 (HSP71)

P08107 70 (70) 5.7 (5.5) 3.46 Stabilizes preexistentproteins againstaggregation and mediate thefolding of newlytranslated polypeptidesin thecytosol as well aswithin organelles.

21, 66

4a, 4b, 4c,4d, 4e

60 kDa heatshock protein,mitochondrial(CH60)

P10809 58, 58, 58,58, 58 (61)

4.9, 5.0, 5.1,5.1, 5.2 (5.7)

1.37, 1.20, 1.10,1.25, 1.15

Implicated inmitochondrial proteinimport and macromolecularassembly.

21, 39

10 ATP synthasealpha chain,mitochondrial(ATPA)

P25705 50 (60) 8.7 (9.1) 0.48 Produces ATP from ADPin the presence of aproton gradient acrossthe membrane. The alphachain isa regulatory subunit.

115, 116

17 Alpha-enolase(ENOA)

P06733 48 (47) 6.6 (7.0) 1.36 Multifunctional enzymethat plays a part invarious processes such asgrowth control, hypoxiatolerance and allergicresponses.

42, 117

22a, 22b, 22c 3-ketoacyl-CoAthiolase(THIM)

P42765 47 (42) 7.3, 7.4,7.6 (8.3)

0.61, 0.80, 0.67 It has catalytic activityin lipid and fattyacid metabolism.

84

23a, 23b 3-ketoacyl-CoAthiolase(THIM)

P42765 47 (42) 8.3, 8.4 (8.3) 0.70, 0.57 It has catalytic activityin lipid and fattyacid metabolism.

84

39 Aldolase B,fructose-biphosphate(ALDOB)

Q5T7D5 41 (24) 7.5 (6.6) 0.70 It has catalytic activityin carbohydratedegradation and glycolysis.

87, 118

52 Heat shock cognate71 kDa protein(fragment)(HSP7C)

P11142 34 (71) 7.3 (5.4) 2.20 Chaperone. Translocatesrapidly from thecytoplasm to the nuclei andespecially to thenucleoli upon heatshock.

39, 40

56 Cathepsin Dheavy chain(CATD)

P07339 32 (44) 5.6 (6.1) 0.45 Acid protease active inintracellular proteinbreakdown. Involved inthe pathogenesis ofseveral diseases suchas breast cancer and possiblyAlzheimer disease.

43, 119

73 ATP synthasealpha chain,mitochondrial(fragment)(ATPA)

P25705 23 (60) 7.0 (9.1) 0.48 Produces ATP from ADPin the presence of aproton gradient acrossthe membrane.The alphachain is a regulatorysubunit

115, 116

80; 81 Peptidyl-prolylcis-transisomeraseA (PPIA)

P62937 18, 18 (18) 7.5, 7.7 (7.7) 2.35, 0.66 PPIases acceleratethefolding of proteins bycatalyzing the cis-trans isomerizationof prolineimidic peptidebonds inoligopeptides.

44, 90, 120

a Spot number, protein name, accession number (gi/Swiss-Prot entry), experimental (theoretical) mass and pI value, and relative protein abundance (ascompared to the values for the C2 samples) are listed (quotients of N vs C2: ratio above 1, up-regulation; ratio below 1, down-regulation in C2). The ratiowas calculated for matched pair samples from each patient.



such as colon. In CaCo-2 colonic epithelial cells, the expressionof this protein increases with cell differentiation.95 Moreover,its expression was found altered in moderately/poorly dif-ferentiated colon and rectal carcinomas as well as in smallintestine Myc-independent tumors.95 Our finding is in agree-ment with a deregulation of this protein, as found in HBVtransgenic mice livers at early stages of fibrosis.96

Our study also revealed an up-regulation of the actin-relatedprotein 2 in HCC. In mammalian cells, the actin-related protein2 and 3 (Arp2/3) complex is important for various cell functionsinvolving cytoskeletal actin dynamics, such as cell polarity, celllocomotion, and intracellular motility.97 Highly invasive/metastatic carcinomas are characterized by the formation ofprotrusions due to the assembly of branched actin filamentnetworks.98 Thus, it is tempting to speculate that higherexpression levels of this protein may be associated to apromoted assembling of these cancer-related macromolecularstructures and considered as a manifestation of the intrinsicmigratory ability of malignant cancer cells. In this sense, Arp2may be tentatively proposed as a marker predicting enhancedinvasive/metastatic potential of HCC.

hnRNP A2/B1 is a protein with a nucleic acid bindingfunction, whose involvement in HCC pathogenesis and pro-gression has never been documented. Our experiments re-vealed an up-regulation of this protein in HCC. A recent reportsuggests that the gene encoding for this protein is a newly

identified fusion partner during chromosomal rearrangementsassociated to prostate neoplasia.99 Since the role played by thishousekeeping gene in cancer seems to demonstrate thatdormant oncogenes may be activated during neoplastic trans-formation by juxtaposition to ubiquitously active genomicloci,99 hnRNP A2/B1 protein should participate in the earlymolecular events leading to neoplastic transformation of livercells. For this reason, we propose hnRNP A2/B1 as a newmarker for early detection of human liver cancer.

A genomic scaffold protein, namely hCG2001950, was alsofound deregulated in HCC by our proteomic analysis. Its rolein HCC pathogenesis is unknown at present. Nonetheless, dueto its important structural and regulatory features, genomicscaffold proteins may play a key role in controlling structuralchanges of the genome and in preparing genomic regions fortranscription. Furthermore, alterations in the expression of keygenes through aberrant epigenetic regulation in breast cellshave been recently described as related to initiation, promotionand maintenance of carcinogenesis.100 Epigenetic regulationmodulates chromatin structure to activate or silence geneexpression. Thus, the observed deregulation of hCG2001950 inHCC may have a major role in chromatin remodeling, influ-encingcancerriskandplayinganimportantroleintumorigenesis.

A number of metabolic enzymes were also observed asdown-regulated in HCC lesions, although clear evidence on therelationship between their deregulation and neoplastic pro-

Figure 4. Expression levels of some of the tumor-associated proteins identified by 2-DE were evaluated on the whole casistic throughWestern blot analysis. (A) Representative immunoblot analysis for the evaluation of Hsc71, Hsp60, Hsp70, hnRNP A2/B1, Arp2, ETFBand ATP synthase R expression in tumoral and nontumoral tissues of three representative patients. The expression levels of Hsc71,Hsp60, Hsp70, hnRNP A2/B1, Arp2, ETFB and ATP synthase R were analyzed using the antibodies described in Materials and Methods.For evaluation of Hsc71, Hsp70, hnRNP A2/B1, Arp2, ETFB and ATP synthase alpha, 20 µg of nuclear enriched samples was loadedonto a 12% SDS-PAGE. For Hsp60, 10 µg of nuclear enriched samples was loaded onto a 12% SDS-PAGE. �-Actin was always measuredas loading control and was used for data normalization. (B) The quantification of the signal has been obtained by densitometric scanning;data obtained after normalization for �-actin signal were plotted as intensity ratio. N, tumoral tissue; C2, nontumoral tissue.



gression are actually lacking. They included (i) electron transferflavoprotein �, a specific electron acceptor that transferselectrons to the main mitochondrial respiratory chain. Thisprotein may represent a new interesting candidate biomarkerof neoplasia as its deregulated expression has been docu-mented in brains of p53-deficient mice;101 (ii) UTP-glucose-1-phosphate uridylyltransferase 2 isoform A, a protein involvedin glucose metabolism;102-104 (iii) methylmalonate-semialde-hyde dehydrogenase that, located in the mitochondrial matrixspace, catalyzes some crucial reactions in the distal portionsof the valine and pyrimidine catabolic pathways.105,106 Althougha distinct role for these proteins cannot be rationalized at themoment, their expression data provide valuable informationto supplement the present knowledge on the processes in-volved in the development of HCC.

Despite the rather limited number of patients, the compara-tive analysis reported in this study has revealed a significantlyaltered expression level for various proteins. Some of them havebeen already proposed as candidate biomarkers of HCC;39-44

others have been originally proposed here. Future experimentswill investigate the involvement of these proteins in HCC,further exploring the possibility that tumor development maybe directly related to their deregulated expression. We hopethese results may provide positive insights for further studiesdirected to a better understanding of hepatocarcinogenesis aswell as to the identification of biomarkers useful for HCCdiagnosis and treatment.

Abbreviations: AIF, apoptosis inducing factor; HCC, hepa-tocellular carcinoma; Hsc71, heat shock cognate 71 kDa

protein; Hsp 60, heat shock 60 kDa protein; Hsp 70, heat shock70 kDa protein; IHC, immunohistochemistry; TBP, TATA bind-ing protein.

Acknowledgment. This work was supported by grantsfrom MIUR (FIRB Prot. RBRN07BMCT and RBNE08YFN3),Regional FVG AIRC and National Research Council (RSTL862) to G.T., A.S. and C.T.

Supporting Information Available: List of spots/protein species identified in partially enriched nuclear HCCproteome 2-DE map. This material is available free of chargevia the Internet at http://pubs.acs.org.

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Differential Proteomic Analysis of Subfractioned Human Hepatocellular Carcinoma Tissues

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