Differential Proteomic Analysis of Subfractioned Human

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 of Clinical Pathology, University of Udine, 33100 Udine, Italy, Department of Surgery & Transplantation, University of Udine, 33100 Udine, Italy, Azienda Ospedaliero Universitaria, Molinette, 10100 Torino, Italy, and Centro 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 analysis of partially enriched nuclear fractions from liver biopsies of 20 different patients. We obtained a proteomic map of subfractioned liver samples including about 200 common protein spots, among which identified components corresponded to expression products of 52 different genes. A differential analysis of proteins from tumoral and control tissues revealed a significant change in the expression level of 16 proteins associated to cytoskeletal, stress response and metabolic functions. These data may provide novel candidate biomarkers for HCC and useful insights for understanding the mechanisms of HCC pathogenesis and progression. Keywords: hepatocellular carcinoma • cell nucleus • biomarkers

1. Introduction Hepatocellular carcinoma (HCC) is a common malignant tumor, which develops from chronic inflammatory liver diseases 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 most cases. HCC pathogenesis is a multifactorial and multistep process that finally leads to the deregulation of cell homeostasis. It is commonly thought that the occurrence of chronic inflammation and cell damage may provide the proliferative stimuli to promote the hepatocarcinogenetic process.2-4 As the poor survival of HCC patients is largely related to the lack of an early diagnosis, more reliable, sensitive and specific tools for early detection of HCC are urgently needed. An effective marker for diagnosis of HCC is yet to be found, as the utility of R-fetoprotein remains still controversial and the novel tumor † Originally submitted and accepted as part of the “Tissue Proteomics and Metabolomics” special section, published in the April 2009 issue of J. Proteome Res. (Vol 8, No. 4). * Correspondence to: Prof. Gianluca Tell, Department of Biomedical Sciences and Technologies, University of Udine, P.le Kolbe 4, 33100 Udine, Italy.Tel.:++39432494311.Fax:++39432494301.E-mail:[email protected]. Dr. Andrea Scaloni, Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, via Argine 1085, 80147 Naples, Italy. Tel.: ++39 081 5966006. Fax: ++39 081 5965291. E-mail: [email protected]. ‡ Department of Biomedical Sciences and Technologies, University of Udine. § 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

markers proposed (i.e., serum ferritin, γ-glutamyltranspeptidase, alkaline phosphatase, des-γ-carboxy prothrombin, R-1antitrypsin, aldolase A, 5′-nucleotide phosphodiesterase, tissue polypeptide antigen, and R-1-fucosidase) are still inadequate.2,5-7 Proteomic analysis of liver proteins and HCC were predominantly performed using either chemically induced hepatoma in animals (predominantly rat and mouse) or human HCC cell lines, such as HepG2 and Huh7 cells.2,8-11 Several so-called tumor-associated or cancer-related proteins were identified, providing valuable information for the definition of HCC databases.2,8,9,12,13 A major drawback in these studies was the use of total protein extracts, which allowed only the study of the most abundant protein species. To detect low-abundance proteins, the analyzing power of proteomic techniques needs to be improved by using specific subcellular fractionation strategies.14,15 This choice may also reduce the complexity of the protein profile to be analyzed and facilitate the identification of differentially expressed proteins, possibly involved in the tumorigenic process.16-19 Another major limitation that emerged from previous studies was the lack of genetic homogeneity, which hampered a proper comparison of samples from different patients.20,21 Accordingly, the most effective way would be the analysis of both cirrhotic and tumor tissues obtained from the same patient. This would allow to identify distinct and proper HCC proteomic signatures.22,23 Nowadays, it is widely accepted that proteomic technologies may efficiently help the study of biological responses in complex systems in a more global way by the simultaneous evaluation of hundreds of protein components.24,25 In the present study, we used 2-DE and MALDI-TOF-MS for the Journal of Proteome Research 2009, 8, 2273–2284 2273 Published on Web 03/16/2009

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Codarin et al. Table 1. Demographic and Clinical Features of the Patients Studied mean age: 65

Sex Neoplastic Grade Viral Serology

Total no. patients

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

identification of proteins related to hepatocarcinogenesis by differential proteomic analysis on HCC and non-HCC tissue samples enriched for nuclear protein components. To exclude the effect of genetic variation and interindividual variability, the analysis was performed by comparing samples obtained from HCC lesions with those from surrounding liver cirrhosis of the same patient. The deregulated proteins identified may represent valuable biomarker tools in understanding the mechanisms of hepatocarcinogenesis and in aiding HCC diagnosis/ therapy.

2. Materials and Methods 2.1. Patient and Sample Collection. Biopsies were obtained from 20 HCC patients with underlying liver cirrhosis. A liver biopsy, called N, was obtained from the tumoral region and a second biopsy, called C2, was collected in the distal region of cirrhotic liver and used as control. Liver tissue was also fixed in 10% formalin for further histological and immunohistochemical examinations (Figure 1A). All biopsies were immediately frozen at -80 °C until processing, through mechanical cell separation. A total of 40 liver biopsies (20 at N and 20 at 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 ethical committee. 2274

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Men Female G1/G2 G3/G4 HBV+ve HCV+ve HBV+ve/HCV+ve HBV-ve/HCV-ve Not reported

no. cases

19 1 14 6 7 5 0 5 3 20

2.2. Patient Demographics. With one exception, all patients were males. The mean age of the patients was 65 (range 53-82) years. Samples were divided into 5 different groups according to the presence of specific serological markers of hepatitis viruses as reported in Table 1. In 3 patients, it was not possible to collect data on viral serology. 2.3. Preparation of Partially Enriched Nuclear Fractions from Liver Biopsies. The partially enriched nuclear fractions from liver sections were obtained with the following experimental procedure. Each liver biopsy (about 100 mg of tissue) was homogenized using a Potter Duall supplied with a PTFE pestle in Lysis Buffer (1 mL of 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT and Protease Inhibitor Cocktail) until more than 90% of the cells was lysed. The disrupted cells were centrifuged at 500g, for 30 min, to separate the cytoplasmic fraction (supernatant). After a rinse with Lysis Buffer, the crude nuclei pellet was then resuspended in Extraction Buffer [about 140 µL of 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 28% (v/v) glycerol, 1 mM DTT and Protease Inhibitor Cocktail], shaken gently for 30 min in ice, and then centrifuged at 18 000g, for 30 min, to obtain the nuclear fraction (supernatant). Protein extracts were precipitated using cold acetone and methanol and subsequently stored at -80 °C. To test for the quality of the partially enriched nuclear extracts, the presence of the TATA binding protein (TBP), cytochrome C, apoptosis inducing factor (AIF) and calreticulin was verified by using anti-TBP, -cytochrome C, -AIF and -calreticulin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Protein quantification was performed by the Bradford 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 (Amersham Biosciences) 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 blue in 0.1 M Tris-HCl, pH 8.8, for other 15 min. The equilibrated strips were applied directly to 12% SDS-polyacrylamide gels and separated at 130 V. Gels were fixed and stained by ammoniacal silver.27 For each bioptic sample, three experimental replicates were subjected to 2-DE; all gels were scanned with an Image Scanner II apparatus (GE Healthcare, Milan, Italy). 2.5. Evaluation of Differentially Represented Spots. Gel images were analyzed by the Image Master 2D Platinum software (GE Healthcare) that allowed performing a compara-

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tive image analysis. Protein spots were detected and matched between the different samples; individual spot volume values were obtained according to the program instruction and normalized using the program volume normalization function. Ratio of the different sample normalized volume values for the candidate protein spots was compared with each others and a mean relative difference in spot intensity was calculated. Differences in protein spot expression levels were considered as significant when Student’s t test gave a P-value 2 were further evaluated by the comparison with their calculated mass and pI using the experimental values obtained from 2-DE. 2.7. Immunoblotting Analysis. Nuclear extracts from HCC liver biopsies were separated on 12% SDS-PAGE. Then, proteins were transferred to nitrocellulose membranes (Schleicher and Schuell Bioscience, Keene, NH) for the detection of ATP synthase, heat shock 60 kDa protein (Hsp60), heat shock 70 kDa protein (Hsp70), heat shock cognate 71 kDa protein (Hsc71), actin-related protein 2 (Arp2), electron transfer flavoprotein β (ETFB) and anti-hnRNP A2/B1. After saturation with 5% (w/v) nonfat dry milk in PBS and 0.1% (w/v) Tween20, the membranes were incubated with one of the following antibodies: anti-ATP synthase polyclonal antibody (homemade, polyclonal antibody against R-β subunit of F1 sector, kindly provided by Prof. G. Lippe, University of Udine), anti-Hsp60 monoclonal antibody (ab8071, Abcam, Cambridge, U.K.), antiHsp70 monoclonal antibody (MA3-007, Affinity Bioreagents, Golden, CO), anti-Hsc71 monoclonal antibody (ab19136, Abcam), anti-Arp2 monoclonal antibody (ab49674, Abcam), antiETFB polyclonal antibody (ab73986, Abcam) and anti-hnRNP A2/B1 monoclonal antibody (sc-32316, Santa Cruz Biotechnology). Filters were incubated overnight, at 4 °C, with each of them. After three washes with PBS, 0.1% (w/v) Tween-20, the membranes were incubated with anti-rabbit, anti-mouse or anti-rat immunoglobulins conjugated with peroxidase (SigmaAldrich, Milan, Italy). After 2 h of incubation at room temperature, the membranes were washed three times with PBS 0.1% (w/v) Tween-20, and the blots were developed by the ECL procedure (Pierce Biotechnology, Rockford, IL). The signals from each protein band were normalized against the actin content by using the polyclonal anti-actin antibody (SigmaAldrich). Blots were quantified by Image Quant (GE Healthcare). Western blot analysis was extended to all the available samples (20 patients), except for hnRNP A2/B1; in this case, the analysis was limited to 11/20 cases, due to sample limitations. 2.8. Immunohistochemical Analysis. Formalin-fixed, paraffin-embedded tumor samples from 5 HCC patients out of 20 subjected to proteomic analysis were used for immunohis-

tochemical (IHC) analysis. IHC staining was developed on 5 µm thick paraffin sections to detect Hsp60, Hsp70, Hsc71 and hnRNP A2/B1, respectively. After dewaxing, rehydratation, and endogenous peroxidase quenching with 20% H2O2 (v/v) in PBS solution, for 10 min, at room temperature, sections were dipped in 10 mM citrate buffer pH 6.0 for 40 min, at 98 °C, and then cooled 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 antiHsp70 (MA3-007, Affinity Bioreagents) at 1:150 dilution, rat monoclonal anti-Hsc71 (ab19136, Abcam) at 1:50 dilution and mouse monoclonal anti-hnRNP A2/B1 (sc-32316, Santa Cruz Biotechnology) at 1:50 dilution respectively, for 1 h, at room temperature. After a rinse with PBS solution, incubation with the peroxide-based detection system REAL EnVision (Dako A/S, Glostrup, Denmark) for 30 min, at room temperature, and treatment with 3,3′-diaminobenzidine for 10 min followed. Sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted. Cells positive for protein staining were evaluated by a single observer at 20× and 40× of microscope magnification. Negative controls were carried out replacing the primary antibody with nonimmune serum.

3. Results 3.1. Subfractionation of HCC Liver Biopsies. To enrich for low-abundance proteins, a subcellular fractionation of liver biopsies was performed. A well-established protocol32 for partial enrichment of the nuclear fraction was used, thus obtaining about 800 µg of proteins from 100 mg of each tissue sample. To verify for the efficiency and the selectivity of our preparations, Western blot analysis of the nuclear and cytoplasmic extracts was performed. Both fractions were tested for the presence of TATA binding protein (TBP), cytochrome C, apoptosis inducing factor (AIF) and calreticulin (Figure 1B). The first protein is a transcription factor with a well-known nuclear localization;33-35 the second and the third proteins localize within mitochondria,36,37 and the fourth one is a protein usually associated to the endoplasmic reticulum (ER).38 The specific presence of TBP only in the nuclear extract indicated a significant enrichment for the nuclear components, excluding nuclear protein leakage during preparation (Figure 1B), although it did not exclude the co-purification of abundant proteins from other subcellular compartments, such as mitochondria or ER. Despite the limitations of the extraction procedure we used, this protocol can be considered as a good compromise between the amount of proteins fully recovered from the nucleus and the removal of abundant cytoplasmic components on one hand and the limitation on the tissue availability on the other. 3.2. Proteomic Map of Subfractioned HCC Liver Tissue Biopsies. To analyze the proteomic map of the partially enriched nuclear fraction from liver biopsies, samples were subjected to 2-DE analysis in the range of pH 3-10. Staining of 2-DE gels allowed for the simultaneous quantitative evaluation of about 200 common protein spots in each gel (Figure 2A). Protein spots varying among the N and C2 samples plus ones being constant (used as a reference) were excised from the gels and analyzed by MALDI-TOF Peptide Mass Fingerprint (PMF) experiments. Data searching in a nonredundant sequence database allowed the unambiguous identification of 83 protein spots or spot trains, which corresponded to 52 protein species with molecular mass ranging from 13 to 80 kDa. Supporting Information Table 1 reports on the nature of each Journal of Proteome Research • Vol. 8, No. 5, 2009 2275

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

identified spot, the measured 2-DE coordinates, the relative sequence coverage, together with the known functional properties. As expected, the distribution of spots across the gel was not homogeneous, with a prevalence of focalized spots present in the mass range 20-70 kDa and the pI range 4-7. This proteomic map was also characterized by the presence of multiple horizontal spot trains with different pI values, associated to the same protein entry, as shown for spots 26-31 (THIL), 35-37 (ARGI1) and 53-55 (ECH1), which were likely derived from the occurrence of post-translational modifications. In addition, some spots associated to the same protein species were also distributed over the whole map, showing a marked difference in their apparent mass; their presence was associated to protein processing events. Identified proteins were divided into clusters according to their functional properties (Figure 2B). All the major functional groups are represented including proteins involved in binding, catalysis, structure, redox regulation or detoxification, metabolism/transport regulation, signal transduction and transcriptional regulation. Among the identified enzymes, those involved 2276


Codarin et al. in lipid, energy and protein or amino acid metabolism were the most abundant. 3.3. Changes in Liver Proteomic Profile between Tumoral and Distal Regions. Bioinformatic analysis of 2-DE gel images allowed for the simultaneous quantitative evaluation of almost 200 protein spots within each gel. Thirty-three protein spots, each of them representative of the different conditions examined, displayed a statistically significant change in abundance when comparing samples from the neoplastic (N) to the distal regions (C2) (Figure 3). PMF analysis allowed the unambiguous identification of 31 of them, which were associated to 16 ORFs. Tables 2 and 3 report on the nature of each identified spot, as well as the gel coordinates, the relative N/C2 abundance ratio and the known functional properties. Identified proteins were classified into two groups according to their association to HCC. While Table 2 includes proteins that have never been associated to this type of cancer (electron transfer flavoprotein β, hnRNP A2/B1, UTP-glucose-1-phosphate uridyltransferase 2A, hCG2001950 scaffold protein, hydroxymethylglutaryl-CoA synthase, actin-related protein 2 and methylmalonate-semialdehyde dehydrogenase), thus representing possible novel candidate biomarkers, Table 3 lists proteins already described as deregulated in HCC (Hsc71, Hsp70, Hsp60, R-enolase, cathepsin D, 3-ketoacyl-CoA thiolase, ATP synthase R, fructose-bisphosphate aldolase B and peptidyl-prolyl cis-trans isomerase A),39-41 thus reinforcing the reliability of the procedure used. In addition, two of the differentially expressed proteins, namely, ATP synthase R and Hsc71, were identified both as full-length and truncated proteins, possibly as the consequence of proteolytic events. All quantitative variations for proteins already described as associated to HCC reflected expression data42-44 previously reported. The majority (8/16) of the changes observed consisted in a decreased protein expression level in tumoral tissue. In fact, a reduced abundance was observed for cathepsin D, ATP synthase R, electron transfer flavoprotein β, fructose-bisphosphate aldolase B, 3-ketoacyl-CoA thiolase, UTP-glucose-1phosphate uridyltransferase 2A, hCG2001950 scaffold protein and methylmalonate-semialdehyde dehydrogenase. One protein, namely, peptidyl-prolyl cis-trans isomerase A, exhibited a different behavior being up- or down-regulated in different patients. All the other proteins (7/16) were characterized by an increased abundance in the tumoral tissue. They were identified as Hsc71, Hsp60, Hsp70, R-enolase, hydroxymethylglutaryl-CoA synthase, hnRNP A2/B1 and actin-related protein 2. 3.4. Validation of Differential Protein Expression Using Western Blot Analysis. To check for the reliability of the quantitative data obtained, seven differentially expressed proteins found by 2-DE (Hsc71, Hsp60, Hsp70, hnRNP A2/B1, Arp2, ETFB and ATP synthase R) were also validated by Western blotting analysis (Figure 4A). In all cases, Western blotting data were in good agreement with 2-DE, thus confirming a higher expression for Hsc71, Hsp60, Hsp70, Arp2 and hnRNP A2/B1, and a lower expression for ATP synthase R and ETFB in the tumor samples, respectively (Figure 4B). 3.5. Immunohistochemistry Analysis of Differentially Expressed Proteins in HCC Liver Biopsies. To further validate our data, we performed IHC analysis of the most relevant protein species in tumor samples from 5 randomly selected HCC patients. The expression of Hsp60 protein, which has an exclusive cytoplasmic localization, was decreased in patients with poorly differentiated HCC (Edmondson grade III and IV

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HCC) as compared to those with a better differentiated phenotype (Edmondson grade I or II HCC). Moreover, in agreement with present proteomic analysis data, Hsp60 expression level was higher in HCC than in cirrhotic tissue (Figure 5A). Similarly, Hsp70 showed a proper cytoplasmic localization; immunohistochemical analysis also denoted a lower reactivity for Hsp70 in cirrhotic parenchyma (C2) than in the tumoral region (N) (Figure 5B). Furthermore, a correlation similar to that observed for Hsp60 was maintained also in association with neoplastic grading, so that Hsp70 had lower expression levels in poorly differentiated HCC with respect to less malignant neoplasias. This trend was maintained in all the cases observed. On the other hand, Hsc71 had both nuclear and cytoplasmic localization, depending on the tumoral grading. The IHC staining revealed a more intense reaction in better differentiated neoplasias (G1 and G2) compared to the more aggressive forms of the tumor. In particular, G3 and G4 neoplasias had almost an exclusive Hsc71 cytoplasmic expression and such expression levels were higher in the neoplastic tissue (N) with respect to the cirrhotic parenchyma (C2) (Figure 5C). The expression of hnRNP A2/B1 was almost totally nuclear (Figure 5D) without any significant change in the percentage values between neoplastic and cirrhotic tissues and without any kind of correlation with the tumor grading.

4. Discussion

Figure 3. Differential proteomic analysis of nuclear enriched HCC and control samples. Representative gel regions comprising some of the statistically 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.

Previous reports have demonstrated that proteomic analysis of tumor tissues, by combined 2-DE and MALDI-TOF-MS technologies, may provide useful information for cancer classification, identification of diagnostic markers and selection of therapeutic target candidates.45-47 Such approaches have been applied to the analysis of specimen or cell line samples derived from liver cancer.8,12,13,48 In this way, several so-called liver tumor-associated or cancer-related proteins have been identified, which provided a valuable contribution to the establishment of HCC databases.2,8,9,12,13 However, previous proteomic studiesonHCChavebeenoftenrestrictedtoafewpatients,20,22,23,49,50 limiting a wider applicability of the identified HCC biomarkers, or have shown the limitation due to the reduced dynamic range of 2-DE analysis, which allowed only visualization of the most abundant proteins.20,22,23,49,50 Thus, enrichment strategies for detection of poorly represented proteins51,52 have been very recently applied also to the study of HCC-related components from subcellular compartments, such as Golgi, ER, plasma membrane and mitochondria.53-57 At present, however, there are no examples in the literature for differential investigations on nuclear proteins from HCC versus non-HCC specimens. In this study, we report the first partially enriched nuclear proteomic map from liver tissues, which describes the expression products of 52 genes, including those involved in signal transduction, transcriptional regulation, structure, stress response and various metabolic pathways. This study was performed by comparing the protein fingerprint from tumoral and nontumoral, cirrhotic tissues of the same patient, thus removing genetic and environmental factors possibly affecting the results. Despite contaminations from other subcellular compartments, the enriched nuclear preparations allowed us to detect low-abundance proteins and reduced the complexity of the resulting protein patterns profile, facilitating the identification of novel deregulated proteins in HCC. This comparative analysis performed on 20 patients allowed the identification Journal of Proteome Research • Vol. 8, No. 5, 2009 2277

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Table 2. Proteins Identified in This Work As Differentially Expressed in HCC and Never Associated to Liver Tumoral Tissuesa spot no.

9

12

38

45

protein identity

gi/Swiss-Prot entry

MethylmalonateQ02252 semialdehyde dehydrogenase [acylating] (MMSA) UTP-glucose-1-phosphte Q16851 uridylyltransferase 2 isoform A (UGPA) Actin-related protein P61160 2 (ARP2)

Hydroxymethylglutaryl- P54868 CoA synthase (HMCS2)

pI

relative intensity N/C2 ratio

function

reference no.

56 (58)

6.7 (8.7)

0.71

Plays a role in valine and 106, 107 pyrimidine metabolism and binds fatty acyl-CoA.

53 (57)

6.8 (8.1)

0.52

41 (44)

5.9 (6.3)

3.50

36 (56)

7.2 (8.4)

4.06

Plays a central role as a glucosyl donor in cellular metabolic pathways. Functions as ATP-binding component of the Arp2/3 complex which is involved in regulation of Actin polimerization. It condenses acetyl-CoA with acetoacetyl-CoA to form HMG-CoA. Heterogeneous nuclear ribonucleoprotein. It transfers the electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF dehydrogenase). Genomic scaffold

42; 44; hnRNP A2/B1 protein 46; 47 (ROA2) 63 Electron transfer flavoprotein subunit beta (ETFB)

P38117

36, 36, 36, 36 (37) 6.9, 7.7, 8.4, 1.45, 1.50, 8.6 (8.9) 1.47, 1.55 28 (28) 7.6 (8.2) 0.50

72

119623362

26

Scaffold protein hCG2001950

P22626

MW (kDa)

8.5 (8.6)

0.30

108, 109

110

95, 111

99, 112 101, 113

114

a Spot number, protein name, accession number (gi/Swiss-Prot entry), experimental (theoretical) mass and pI value, and relative protein abundance (as compared 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 ratio was calculated for matched pair samples from each patient.

of 31 differentially expressed spots in HCC tissue, which were associated to 16 ORFs. Possible functional implications on carcinogenesis of their quantitative changes and previous reports on their expression in HCC are reported below. Among deregulated proteins here associated to HCC, nine species have been already reported as involved in cancer onset and progression. In fact, up-regulation of molecular chaperones Hsc71, Hsp60 and Hsp70 has been already reported in HCC, colorectal adenomas/cancer, breast cancer or high-grade prostatic lesions.9,20,22,39,58-66 The overexpression of these proteins has been interpreted as a response to the stressful cancerous environment for cyto-protective functions. In the case of Hsp60, its up-regulation may contribute to the tumor cell adaptive response to low oxygen levels.67 Since, in cancer cells, expression of chaperone Hsp70 has been implicated in regulation of apoptosis, immune response against tumors and multidrug resistance,68-70 understanding its role in carcinogenesis may have important implications regarding tumor behavior/prognosis.71 Thus, these findings suggest that Hsps are of special relevance in human cancer, representing candidate biomarkers for oncological management. Up-regulation of R-enolase has also been described as a common feature of HCC8,72,73 and the expression of this glycolytic enzyme increased with tumor dedifferentiation (as shown in biopsies from HCV-related HCC).73 Expression of R-enolase positively correlates also with tumor size and venous invasion,73 thus suggesting this protein as candidate biomarker for liver tumor progression. Different mechanisms associated with tumor invasion/ metastasis,23,74-77 also involving deregulation of cellular proteolytic activities,23,78 could explain the down-regulation of cathepsin D. In HCC, this acid protease has been proposed as 2278


a biochemical marker of malignant progression of liver cirrhosis.74 Similarly, down-regulation of mitochondrial ATP synthase R, observed in HCC or other cancers,79-83 could be related to the mitochondrial bioenergetic deregulation typical of the tumoral condition. A metabolic derangement peculiar of the neoplastic process can be hypothesized also for the down-regulation of mitochondrial 3-ketoacyl-CoA thiolase observed in neoplastic tissues.84 In fact, a bioenergetic dysfunction 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-bisphosphate aldolase B in tumoral tissues has been described for HCC.87 Cancer cells with a high glycolytic rate may have an advantage in tumor growth. HCC often exhibits an aberrant expression of glycolytic enzymes, particularly hexokinases and aldolases.88 In particular, a decreased expression of fructosebisphosphate aldolase B has been associated with advanced disease, early tumor recurrence and has been used as a predictive marker of poor prognosis.87,88 A peculiar behavior was observed for peptidyl-prolyl cis-trans isomerase A (PPIA), which exhibited up/downregulated expression levels depending on the patient. The PPIase family of proteins regulates the activities of mature proteins by promoting their assembly/intracellular transport,89 due to the association with the dynein/dynactin motor complex. Recent findings suggest that PPIA may be involved in the execution stage of the p53-mediated apoptosis44,90 by interacting with p53 and participating in the retrograde movement of the p53 complex to the nucleus.90 Furthermore, PPIA also plays an essential role in the G phase of the cell cycle.91 Nowadays, this protein has been proposed as a marker candidate for nonsmall cell lung, pancreatic and liver tumors.22,92-94

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Table 3. Proteins Identified in This Work As Differentially Expressed in HCC and Already Associated to Liver Tumoral Tissuesa spot no.

protein identity

gi/Swiss-Prot entry

2a, 2b

Heat shock cognate 71 P11142 kDa protein (HSP7C)

3

Heat shock 70 kDa protein 1 (HSP71)

P08107

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

60 kDa heat shock protein, mitochondrial (CH60) ATP synthase alpha chain, mitochondrial (ATPA)

P10809

10

17

Alpha-enolase (ENOA)

22a, 22b, 22c 3-ketoacyl-CoA thiolase (THIM) 23a, 23b 3-ketoacyl-CoA thiolase (THIM) 39 Aldolase B, fructosebiphosphate (ALDOB) 52 Heat shock cognate 71 kDa protein (fragment) (HSP7C)

P25705

P06733

P42765 P42765 Q5T7D5

MW (kDa)

77, 77 (71)

relative intensity N/C2 ratio

pI

5.5, 5.6 (5.4)

function

2.20, 2.00

Chaperone. Translocates rapidly from the cytoplasm to the nuclei and especially to the nucleoli upon heat shock. 70 (70) 5.7 (5.5) 3.46 Stabilizes preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. 58, 58, 58, 4.9, 5.0, 5.1, 1.37, 1.20, 1.10, Implicated in 58, 58 (61) 5.1, 5.2 (5.7) 1.25, 1.15 mitochondrial protein import and macromolecular assembly. 50 (60) 8.7 (9.1) 0.48 Produces ATP from ADP in the presence of a proton gradient across the membrane. The alpha chain is a regulatory subunit. 48 (47) 6.6 (7.0) 1.36 Multifunctional enzyme that plays a part in various processes such as growth control, hypoxia tolerance and allergic responses. 47 (42) 7.3, 7.4, 0.61, 0.80, 0.67 It has catalytic activity 7.6 (8.3) in lipid and fatty acid metabolism. 47 (42) 8.3, 8.4 (8.3) 0.70, 0.57 It has catalytic activity in lipid and fatty acid metabolism. 41 (24) 7.5 (6.6) 0.70 It has catalytic activity in carbohydrate degradation and glycolysis.

P11142

34 (71)

7.3 (5.4)

2.20

56

Cathepsin D heavy chain (CATD)

P07339

32 (44)

5.6 (6.1)

0.45

73

ATP synthase alpha chain, mitochondrial (fragment) (ATPA)

P25705

23 (60)

7.0 (9.1)

0.48

80; 81

Peptidyl-prolyl cis-trans isomerase A (PPIA)

P62937

18, 18 (18)

7.5, 7.7 (7.7)

2.35, 0.66

Chaperone. Translocates rapidly from the cytoplasm to the nuclei and especially to the nucleoli upon heat shock. Acid protease active in intracellular protein breakdown. Involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer disease. Produces ATP from ADP in the presence of a proton gradient across the membrane. The alpha chain is a regulatory subunit PPIases accelerate the folding of proteins by catalyzing the cis -trans isomerization of proline imidic peptide bonds in oligopeptides.

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39, 40

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115, 116

44, 90, 120

a Spot number, protein name, accession number (gi/Swiss-Prot entry), experimental (theoretical) mass and pI value, and relative protein abundance (as compared 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 ratio was calculated for matched pair samples from each patient.

Among the deregulated proteins we found, seven polypeptide species (electron transfer flavoprotein β, hnRNP A2/B1, UTPglucose-1-phosphate uridyltransferase 2A, hCG2001950 scaffold protein, hydroxymethylglutaryl-CoA synthase, actin-related

protein 2 and methylmalonate-semialdehyde dehydrogenase) have never been associated with HCC before. Hydroxymethylglutaryl-CoA synthase, which regulates ketone body production, is expressed in liver and other several extra-hepatic tissues, Journal of Proteome Research • Vol. 8, No. 5, 2009 2279

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Figure 4. Expression levels of some of the tumor-associated proteins identified by 2-DE were evaluated on the whole casistic through Western blot analysis. (A) Representative immunoblot analysis for the evaluation of Hsc71, Hsp60, Hsp70, hnRNP A2/B1, Arp2, ETFB and 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 loaded onto a 12% SDS-PAGE. For Hsp60, 10 µg of nuclear enriched samples was loaded onto a 12% SDS-PAGE. β-Actin was always measured as 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 β-actin signal were plotted as intensity ratio. N, tumoral tissue; C2, nontumoral tissue.

such as colon. In CaCo-2 colonic epithelial cells, the expression of this protein increases with cell differentiation.95 Moreover, its expression was found altered in moderately/poorly differentiated colon and rectal carcinomas as well as in small intestine Myc-independent tumors.95 Our finding is in agreement with a deregulation of this protein, as found in HBV transgenic mice livers at early stages of fibrosis.96 Our study also revealed an up-regulation of the actin-related protein 2 in HCC. In mammalian cells, the actin-related protein 2 and 3 (Arp2/3) complex is important for various cell functions involving cytoskeletal actin dynamics, such as cell polarity, cell locomotion, and intracellular motility.97 Highly invasive/ metastatic carcinomas are characterized by the formation of protrusions due to the assembly of branched actin filament networks.98 Thus, it is tempting to speculate that higher expression levels of this protein may be associated to a promoted assembling of these cancer-related macromolecular structures and considered as a manifestation of the intrinsic migratory ability of malignant cancer cells. In this sense, Arp2 may be tentatively proposed as a marker predicting enhanced invasive/metastatic potential of HCC. hnRNP A2/B1 is a protein with a nucleic acid binding function, whose involvement in HCC pathogenesis and progression has never been documented. Our experiments revealed an up-regulation of this protein in HCC. A recent report suggests that the gene encoding for this protein is a newly 2280


identified fusion partner during chromosomal rearrangements associated to prostate neoplasia.99 Since the role played by this housekeeping gene in cancer seems to demonstrate that dormant oncogenes may be activated during neoplastic transformation by juxtaposition to ubiquitously active genomic loci,99 hnRNP A2/B1 protein should participate in the early molecular events leading to neoplastic transformation of liver cells. For this reason, we propose hnRNP A2/B1 as a new marker for early detection of human liver cancer. A genomic scaffold protein, namely hCG2001950, was also found deregulated in HCC by our proteomic analysis. Its role in HCC pathogenesis is unknown at present. Nonetheless, due to its important structural and regulatory features, genomic scaffold proteins may play a key role in controlling structural changes of the genome and in preparing genomic regions for transcription. Furthermore, alterations in the expression of key genes through aberrant epigenetic regulation in breast cells have been recently described as related to initiation, promotion and maintenance of carcinogenesis.100 Epigenetic regulation modulates chromatin structure to activate or silence gene expression. Thus, the observed deregulation of hCG2001950 in HCC may have a major role in chromatin remodeling, influencing cancer risk and playing an important role in tumorigenesis. A number of metabolic enzymes were also observed as down-regulated in HCC lesions, although clear evidence on the relationship between their deregulation and neoplastic pro-

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HCC Proteomics

protein; Hsp 60, heat shock 60 kDa protein; Hsp 70, heat shock 70 kDa protein; IHC, immunohistochemistry; TBP, TATA binding protein.

Acknowledgment. This work was supported by grants from MIUR (FIRB Prot. RBRN07BMCT and RBNE08YFN3), Regional FVG AIRC and National Research Council (RSTL 862) to G.T., A.S. and C.T. Supporting Information Available: List of spots/ protein species identified in partially enriched nuclear HCC proteome 2-DE map. This material is available free of charge via the Internet at http://pubs.acs.org. References

Figure 5. Immunohistochemistry of Hsp60, Hsp70, Hsc71 and hnRNP A2/B1 in nontumoral (C2) and tumoral tissues (N) of a representative patient. Magnification 20×. N section of Hsp70, magnification 10×.

gression are actually lacking. They included (i) electron transfer flavoprotein β, a specific electron acceptor that transfers electrons to the main mitochondrial respiratory chain. This protein may represent a new interesting candidate biomarker of neoplasia as its deregulated expression has been documented in brains of p53-deficient mice;101 (ii) UTP-glucose-1phosphate uridylyltransferase 2 isoform A, a protein involved in glucose metabolism;102-104 (iii) methylmalonate-semialdehyde dehydrogenase that, located in the mitochondrial matrix space, catalyzes some crucial reactions in the distal portions of the valine and pyrimidine catabolic pathways.105,106 Although a distinct role for these proteins cannot be rationalized at the moment, their expression data provide valuable information to supplement the present knowledge on the processes involved in the development of HCC. Despite the rather limited number of patients, the comparative analysis reported in this study has revealed a significantly altered expression level for various proteins. Some of them have been already proposed as candidate biomarkers of HCC;39-44 others have been originally proposed here. Future experiments will investigate the involvement of these proteins in HCC, further exploring the possibility that tumor development may be directly related to their deregulated expression. We hope these results may provide positive insights for further studies directed to a better understanding of hepatocarcinogenesis as well as to the identification of biomarkers useful for HCC diagnosis and treatment. Abbreviations: AIF, apoptosis inducing factor; HCC, hepatocellular carcinoma; Hsc71, heat shock cognate 71 kDa

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