Circulating Lamin B1 (LMNB1) Biomarker Detects Early Stages of Liver Cancer in Patients Stella Sun,†,‡ Michelle Z. Xu,†,‡ Ronnie T. Poon,†,‡ Philip J. Day,§ and John M. Luk*,†,‡ Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, Center for Cancer Research, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, and Quantitative Molecular Medicine, The Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom Received March 4, 2009
Hepatocellular carcinoma (HCC) is a major liver malignancy possessing a high mortality rate and is particularly prevalent in China and Asia. While surgery is the most effective treatment for liver tumor, about 80% of HCC patients are inoperable at presentation and die early due to late diagnosis. For early cancer detection, we employed a proteomic expression profiling approach to identify biomarkers for early stages of HCC and subsequently assessed the clinical feasibility of a novel marker in plasma. Frozen liver tissues from a retrospective cohort of 75 liver patients (39 HCCs, 20 cirrhosis, and 16 nondiseased subjects) were subjected to proteome-wide expression profiling by 2-DE. MALDI-TOF/ TOF was used to identify differentially expressed proteins, which were further confirmed by immunoblotting, qPCR, and immunohistochemistry. Conventional RT-PCR was employed to further analyze the abundance of selected biomarker at mRNA level in a separate cohort of 63 plasma samples (35 HCCs, 16 liver cirrhosis, 12 healthy individuals). We successfully identified lamin B1 (LMNB1) that was significantly upregulated in HCC tumors and present in patients’ plasma. LMNB1 functions in nuclear envelope lamina and possesses a transcriptional coregulatory activity having an important role in DNA replication, cellular aging, and stress responses. Clinically, the expression level of lamin B1 correlated positively with tumor stages, tumor sizes, and number of nodules. Our findings further showed elevation of circulating LMNB1 marker in plasma could detect early stages of HCC patients, with 76% sensitivity and 82% specificity. In conclusion, lamin B1 is a clinically useful biomarker for early stages of HCC in tumor tissues and plasma, and warrants further clinical investigation. Keywords: circulating mRNA • early HCC • proteomic profiling • tumor marker
Introduction Liver cancer cure remains the enduring millstone worldwide, with the disease accounting for about 500 000-1 000 000 new cases per year and causing 600 000 deaths globally.1 Hepatocellular carcinoma (HCC) shows great geographical variation, particularly in the Asia Pacific region where the incidence of HCC has been static over recent decades at over 20 cases/ 100 000 population.2 About 80% of HCC in this region is attributed to hepatitis (HBV or HCV) infections and patients with chronic hepatitis infections are more likely to develop cirrhosis after 10-30 years and subsequently die of HCC. The prognosis and outcome of HCC remain dismal partly because of the aggressive behavior of the tumor and the current ineffective surveillance programs obliquely leading to the presentation of advanced stages of malignancy at diagnosis * Current and correspondence address: Dr. John Luk, Departments of Pharmacology and Surgery, Yong Loo Lin School of Medicine, National University of Singapore, MD11, Clinical Research Centre, 10 Medical Drive, Singapore 117597, Singapore. Fax: 65-68737690. E-mail:
[email protected]. † Department of Surgery, The University of Hong Kong. ‡ Center for Cancer Research, The University of Hong Kong. § Quantitative Molecular Medicine, University of Manchester.
70 Journal of Proteome Research 2010, 9, 70–78 Published on Web 06/12/2009
which renders treatment ineffective. Besides, only 10%-30% of HCCs are amenable to surgical resection at the time of diagnosis and the overall 5 year survival rate is low at below 60%.3 Prevention of hepatitis infection through vaccination may reduce the incidence of HCC in Asia, but once the malignancy is acquired, surgical resection and liver transplantation provide the best options for effective treatment.4 A surveillance program in cirrhotic patients has the advantage of detecting HCC at earlier stages, when the tumors are amenable to curative treatments. Our group provides evidence that early detection of HCC by screening improves long-term survival by curative treatments, suggesting a prominent role for early detection in HCC.5 However, an effective screening protocol is still under heated debate. Biannual screening with serum alpha-fetoprotein (AFP) and abdominal ultrasonography (US) examination are currently applied as surveillance aids for the early diagnosis of HCC, and the mortality rate has been reduced by about 37%. There is still an urgent need for better surveillance to detect HCC earlier. Patients diagnosed at early stages of HCC should have better clinical outcomes or longer survival time. An effective staging 10.1021/pr9002118
2010 American Chemical Society
LMNB1 Biomarker Detects Early Stages of Liver Cancer Patients Table 1. Clinical and Serological Data of Patients Studied in 2-DE Analysis 2DE clinical features
Male:Female Age (yr)a HBsAg SGOT (u/L)a SGPT (u/L)a Total bilirubin (µmol/L)a Serum AFP level e400 ng/mL >400 ng/mL Tumor Stage (AJCC) Early Stage (stage I and II) Late Stage (stage III and IV) Tumor size (cm)a,b Number of tumor nodules Undiffused (1-2 nodules) Diffused Venous infiltration Absent Present Cellular differentiationc Well differentiated Moderately differentiated Poorly differentiated
normal (n ) 16)
cirrhosis (n ) 20)
HCC (n ) 39)
14:2 46.6 ( 11.2 22.9 ( 11 23.6 ( 16.6 11 ( 6.2
20:0 54 ( 9 + 65 ( 26.6 56.3 ( 44.3 12.8 ( 3.3
39:0 52.2 ( 8.6 + 79.4 ( 59.1 75.5 ( 72.3 15.05 ( 8.7 19 (49%) 20 (51%) 26 (67%) 13 (33%) 8.5 ( 5.9 34 (87%) 5 (13%) 20 (51%) 19 (49%) 11 (28%) 18 (46%) 10 (26%)
a Mean ( SD. b Length of the largest tumor was measured in tumor size. c Well differentiated (Edmonson grade 0-2); moderately differentiated (Edmonson grade 3); poorly differentiated (Edmonson grade 4).
classification for HCC should incorporate tumor characteristics and the underlying liver disease.6 Traditionally, tumor staging of HCC was classified by tumor/node/metastasis (TNM) staging; however, the system only examines the tumor characteristics but not the liver function conditions. Another tumor staging system was proposed by Okuda et al.7 which includes variables of liver function and was named after by the American Joint Committee on Cancer (AJCC). Consequently, the system was successfully applied in our study to identify biomarkers for the detection of HCC at early stages. Today, the only clinically proven HCC biomarker, alpha fetoprotein (AFP), has been widely applied as part of the surveillance for screening individuals at risk of developing HCC despite the fact that it has very limited sensitivity (39-65%) and specificity (76-94%), in particular for small and early HCC.8 Identification of biomarkers is still in its infancy and researches strive to evaluate the performance of biomarkers either alone or in combination.9 Biomarkers for HCC may be at the levels of genomic, transcriptomics, and proteomics, integrating observations forming an essential step toward the systematic understanding of the disease.10,11 In the present study, we employed two-dimensional gel electrophoresis (2-DE) to compare protein expression profiles between control subjects and HCC tumors of different stages. Lamin B1 was successfully identified by MALDI-TOF/TOF as a potential biomarker candidate for early stages of HCC. Furthermore, its clinical utility was evaluated in plasma samples from a different patient’s cohort.
Patients and Methods Patient Selection and Clinical Specimen Collection. A total of 75 subjects were recruited in this study (Table 1). Resected
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tissues were collected from 20 cirrhosis and 39 HCC patients after hepatectomy at Queen Mary Hospital, Pokfulam, Hong Kong, from 1998 to 2006. In addition, 16 normal liver tissues obtained from residual donor grafts of healthy subjects were included as control for comparison. This study was vetted and approved by the Institutional Ethics Committee, and informed consents were collected from patients. The diagnosis of HCC was confirmed by histopathology and radiological imaging by MRI/CT. HCC tumors were classified into early (Stages I and II) or late/advanced (Stages III and IV) stages based on the American Joint Committee (AJCC) cancer staging system according to the size of primary tumor, presence of the lymph node metastasis and/or distant metastasis.12 The 20 nontumorous cirrhotic tissues were diagnosed to be mildly to severely cirrhotic by histological criteria. Prior to 2DE analysis, histology was confirmed by hematoxylin and eosin staining where morphologies of each tissue showed in excess of 90% of their distinctive nature. A different cohort of plasma samples was recruited from 35 HCC patients and 28 non-neoplastic control subjects for biomarker validation by RT-PCR. Plasma samples were aliquoted and stored at -80 °C prior to use. Cell Lines. Human HCC cell lines MIHA, Huh 7, PLC, Hep G2, Hep 3B, H2P and H2M were obtained and detailed in previous studies.13,14 Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin G, and 50 µg/ mL streptomycin at 37 °C in a 5% CO2 incubator. 2-DE Analysis. Methodology has been previously described.15,16 In brief, approximately 20 mg of tissues was subjected to protein extraction by ReadyPrep Sequential Extraction Kit (Bio-Rad, Hercules, CA) and the concentration was quantified by PlusOne 2-D Quant Kit (GE Biosciences, Buckinghamshire, England). Twenty-five micrograms of tissue lysate was separated by isoelectric focusing using the IPGphor system (GE Biosciences) and the conditions were set as previously described. After equilibrium was established, protein were electrophoresed on a 12.5% precast Ettan DALT Gel (GE Biosciences) for the second-dimension separation. Subsequently, gels were stained with protein silver staining kit (GE Biosciences) according to the manufacturer’s protocol. Gel images were captured by GS-800 Calibrated Densitometer (BioRad), and analyzed using PDQuest 8.0 (Bio-Rad) according to the manufacturer’s guidelines. After spot detection and matching, spot intensities were normalized with total valid spot volume in order to minimize the nonexpression related variations in spot intensity and hence accurately provide semiquantitative information across different gels. Moreover, each sample was performed in duplicate to confirm the reliability of the results. The spots intensities were reported as their % volume. A one-way ANOVA (analysis of variance) test was used to analyze spot intensities in normal, cirrhotic and tumorous samples. Only spots with significant (p < 0.05) expression difference were chosen for mass spectrometry (MS) analysis. MS Identification. Protein spots of interest were excised and the gel pieces were destained followed by trypsin digestion (MS grade, Promega, Madison, WT) as previously described.17 All peptides extracts were pooled to lyophilize, then purified by ZipTip pipet tips (Millipore Corporate, Bedford, MA). Peptides were finally dissolved in 5 mg/mL R-cyano-4-hydroxycinamic acid (CHCA) matrix in 50% acetonitrile and 0.1% trifluoroacetic acid. MS/MS data were determined by an ABI 4800 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, CA) operating in a result-dependent acquisition mode. A maximum of 5 most Journal of Proteome Research • Vol. 9, No. 1, 2010 71
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Figure 1. Lamin B1 protein detects HCC of different tumor stages. (a) A representative 2-DE protein expression profile highlighted a total of 9 protein spots which exhibited significant differential expression between HCC of different tumor stages and the nonmalignant controls (normal and cirrhosis). (b) (i) Overexpression of SSP 3717 (lamin B1) was characterized by histogram of different stages of HCC tumors compared to the nonmalignant controls. Spot intensities are shown in relative % volume. Statistical analysis was performed by Student’s t-test in HCC tumors of different stages relative to nonmalignant control. *p < 0.05, **p < 0.01. (ii) Representative 2-DE gel pictures show SSP 3717 circled in tissues of different liver diagnoses.
intense tandem mass spectra were collected for MS/MS. The NCBI database and MASCOT version 2.1 (Matrix Science) were used for protein identification. Western Blot Analysis. The extracted protein (25 µg) lysates were resolved on 10% polyacrylamide SDS gels and transferred to a nitrocellulose membrane (Millipore Corporate). Membranes were blocked with 5% nonfat dry milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature and incubated with mouse monoclonal anti-lamin B1 antibody (CHEMICON International, MA) (at 1:300 dilution) overnight at 4 °C. The immunoreactive lamin B1 was then detected using a peroxidase-conjugated goat anti-mouse secondary antibody (Invitrogen-Zymed Laboratories, San Francisco, CA). Mouse anti-human β-actin antibody was loaded as internal reference control (Sigma, Santa Cruz, CA) at 1:5 000 dilution. Densitometry data were analyzed by Quantity One (Bio-Rad) for protein quantification of lamin B1 normalized with β-actin.18 Immunohistochemistry. Four micrometers frozen tissue sections were quenched with hydrogen peroxide and blocked with 3% normal goat serum and 1% BSA. The sections were incubated with mouse monoclonal anti-lamin B1 primary antibody (10 µg/mL) (CHEMICON International) overnight at 4 °C. After rinsing with wash buffer, the sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody. Reactivity was detected using diaminobenzidine solution (Invitrogen, Carlsbad, CA), sections were counterstained with hematoxylin (Vector Laboratories, Burlingame, CA), and images were captured as described.19 Purified mouse immunoglobulin (1:500 dilutions, Zymed) was used for negative controls. RNA Extraction. RNA extraction from 100 µL of plasma samples was performed using a commercially available RNA 72
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extraction kit (SV Total RNA Isolation System, Promega, Madison, WI) according to the manufacturer’s instructions. Total RNA was extracted from the liver tissues using TRIZOL reagent (Invitrogen) followed by DNase I treatment (AmbionApplied Biosystems). The final RNA pellets were dissolved in diethylpyrocarbonate-treated water and stored at -80 °C prior to use. Real-Time qPCR. Total RNA extracts from liver tissues were subjected to validation. Primers and probe sequences were designed for lamin B1 using the Universal Probe Library Assay Design Centre [http://www.roche-applied-science.com]. After reverse transcription of 1 µg of total RNA by TaqMan Reverse Transcription Reagents (Applied Biosystems), the Roche LightCycler 480 (Roche, Diagnostic, U.K.) was used for detection and quantification of lamin B1 gene. The geometric mean of two reference genes [ribosomal protein L32 (RPL 32) and β-actin] was used as internal controls for normalization. The amplification was performed in the LightCycler using hot start for 10 min at 95 °C followed by 45 cycles each comprising 95 °C for 10 s and 60 °C for 30 s, and then cooled to 40 °C for 10 s. Standard curves were generated from five 10-fold serial dilutions of template oligonucleotide for quantification. Data were reported as copy numbers of transcript per nanogram of cDNA. RT-PCR of LMNB1 and GAPDH mRNA from Human Plasma. A total of 100 ng of human plasma RNA extracts was reverse-transcribed into cDNA using the TaqMan Reverse Transcription Reagents (Applied Biosystems). PCR amplification was performed in a 20 µL reaction mixture containing 3 µL of cDNA template and 200 nM of each primer [LMNB1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. GAPDH was used as an internal control for PCR quality. The primer sequences for LMNB1 were 5′-TCGCAAAAGC ATGTATGAAGA3′ (sense) and 5′-CTCTACCAAGCGCGTTTCA-3′ (antisense) and
6305
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4610
3309 4111
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0.170 0.002 0.028 0.098 ( 0.088 0.069 ( 0.081 14 100 gil40354192
122
21%
59.0/5.9
45/6
0.033 ( 0.026
0.541 0.002 0.002 0.210 ( 0.153 0.183 ( 0.167 18 100 gil49258360
317
52%
54.9/5.69 55/5.8
0.076 ( 0.079
0.127 0.033 0.009 0.060 ( 0.0760 0.098 ( 0.067 14 100 gil178390
195
30%
56.9/7
55/6.2
0.013 ( 0.024
0.177 0.463 0.003 0.014 0.041 0.031 0.066 ( 0.053 0.012 ( 0.026 10 93.492 gil105294
63
21%
32.3/5.61 26/5.6
0.033 ( 0.027 0.001 ( 0.007
0.095 ( 0.121 0.103 ( 0.021
0.044 0.014 0.087 0.067 0.000 0.000 0.004 0.000 0.015 0.000 0.003 0.018 0.110 ( 0.186 0.115 ( 0.082 0.138 ( 0.108 0.162 ( 0.208 55/5.06 71/5.2 50/4.9 59/5.1 53.7/5.06 66.7/5.11 59.0/5.09 49.7/5.33 32% 31% 40% 27% 16 18 20 12 99.996 99.922 100 99.998 96 82 246 98 gil4507895 gil5031877 gil40354192 gil28193108
Vimentin_HUMAN Lamin B1_HUMAN Keratin 10_HUMAN Heat shock 90 kDa protein_HUMAN Unidentified protein Alternative splicing factor ASF-2-HUMAN Aldehyde dehydrogenase Chain H, Cys302ser mutant of human mitochondrial aldehyde dehydrogenase complexed with Nad+ and Mg2 Keratin 10_HUMAN 1615 3717 1613 2603
protein ID
0.001 ( 0.003 0.016 ( 0.023 0.026 ( 0.062 0.025 ( 0.031
0.053 ( 0.052 0.070 ( 0.060 0.091 ( 0.126 0.093 ( 0.107
early stage late stage early stage vs control vs control vs late stage late stage HCC early stage HCC Nonmalignant control (normal and cirrhosis) gel MW/pl expected MW/pl protein protein peptide sequence score score C.I. % matches coverage accession number spot number
gel spot average intensity
Table 2. Putative Protein Spots Detect Early and Late Stage HCC and the Proteins Characterization by MALDI-TOF/TOF
P-value
LMNB1 Biomarker Detects Early Stages of Liver Cancer Patients
Figure 2. Up-regulation of lamin B1 expression in HCC cell lines. Protein lysates from 7 cell lines were probed by mouse monoclonal anti-human lamin B1 in Western blot analysis. MIHA is an immortalized human primary hepatocytes; Hep G2 is a nontumorigenic HCC. Both showed weak signal of lamin B1. In contrast, the metastatic H2P and H2M revealed strong lamin B1 immunoreactivity. β-Actin was included as loading control in each lane.
for GAPDH were 5′-AGCCACATCGCTCAGACAC-3′ (sense) and 5′-GCCCAATACGACCAAATCC-3′ (antisense). Each primer set was optimized so the absolute difference in samples could be detected with known amount of LMNB1 transcripts. The RTPCR conditions were programmed and started at initial incubation of 94 °C for 5 min to heat-activate the Taq DNA polymerase followed by 35 cycles comprising 94 °C for 45 s, 58 °C for 45 s, 72 °C for 40 s and a final extension at 72 °C for 10 min. The RT-PCR assay was repeated twice and PCR products were separated by electrophoresis on 2% agarose gel and visualized under UV light after ethidium bromide staining.20 The results were considered positive or negative if PCR products were reproducibly present or absent, respectively. To confirm the reaction specificity, PCR products were isolated, cloned, and sequenced using an ABI3700 genetic analyzer (Applied Biosystems, Inc.). Statistical Analysis. Parametric analyses were performed for all log-transformed 2-DE data using both SPSS for Windows (version 16.0, SPSS, Chicago, IL) and Prism 4 software (GraphPad, Inc., San Diego, CA). Student’s t-test and ANOVA analysis were used to compare the differences between two groups or more than 2 groups, respectively. P-values of less than 0.05 were considered statistically significant.
Results Proteomic Identification of Lamin B1 in Early Stage HCC. A total of 75 liver tissues were successfully profiled by 2-DE analysis followed by steps which included spot detection, matching and normalization. Nine protein spots were calculated by ANOVA to be significantly overexpressed in both the early and late stages of HCCs when compared to the nonmalignant controls (Figure 1A) and only 8 spots had been successfully identified by MALDI-TOF/TOF (Table 2). Among the 9 spot candidates, SSP 3717 was found to be constantly overexpressed in different tumor stages of HCC when compared to cirrhotic and normal liver tissues (Figure 1B). Protein spot SSP3717 was identified as lamin B1 [Homo sapiens] locus NP_005564 by MALDI-TOF/TOF (Supplementary Figure 1), which exhibited the highest significance (p < 0.0001) with a high protein score, C.I. value (%) and sequence coverage among the identified proteins, with the most significant p-value in discriminating between early and late stage tumors (Table 2). Furthermore, lamin B1 has been reported to be expressed on Journal of Proteome Research • Vol. 9, No. 1, 2010 73
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Figure 3. Verification of lamin B1 overexpressed in HCC of different tumor stages. (A) Representative Western blot analysis demonstrates up-regulation of lamin B1 in different stages of HCC tumors compared to cirrhosis samples. Numbers represent percentages of lamin B1 signal in each fraction from one representative experiment. (B) Overexpression of lamin B1 transcripts was confirmed using qPCR. Student’s t-test was performed to demonstrate the significant elevated expression of lamin B1 in HCC of different tumor stages versus the malignant control. *P < 0.05, **P < 0.01. Data presented in normalized copies per nanogram of RNA. (C) Immunohistochemical staining of liver tissues. Lamin B1 stain was found to localize at the nuclear envelope of HCC tumor hepatocytes but no staining was found in the cirrhotic tissues. Original magnification × 200 and × 400.
the cell surfaces and present in culture supernatant,21 having a diagnostic potential for noninvasive test. Up-Regulation of Lamin B1 Expression in HCC Cell Lines. The 2-DE expression data was confirmed by Western blotting analysis against a panel of normal liver and HCC cell lines. As shown in Figure 2, the lamin B1 level was generally higher in the HCC cell lines (PLC/PRF/5, Huh7, Hep3B, H2P and H2M) compared to the MIHA (immortalized hepatocytes) and nontumorigenic Hep G2 (Figure 2). Validation of Lamin B1 Overexpression in HCC Tissues. The aberrant expression of lamin B1 in HCC was confirmed in an independent set of 10 liver samples from each group of 74
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cirrhosis, early stage HCC, late stage HCC and their adjacent nontumorous tissues. Cirrhotic liver and adjacent nontumorous livers were grouped as the nonmalignant controls and then compared with HCCs of different tumor stages by Western blot analysis, qPCR, and immunohistochemical staining. In agreement with the 2-DE data, lamin B1 was most abundant in both early and late stage HCCs when compared to the cirrhosis group by Western blot analysis (Figure 3A). Furthermore, the expression of lamin B1 was approximately 2-fold higher in early stage HCCs (71%) and more than 2-fold in the late stage HCCs (83%) when compared to the cirrhosis samples (33%). Likewise, real-time qPCR demonstrated a similar expression pattern, in
LMNB1 Biomarker Detects Early Stages of Liver Cancer Patients Table 3. Clinical Correlation of Lamin B1 (SSP3717) in HCC
clinicopathological features
Age (mean ( SD) Number of tumor nodules Undiffused (1-2 nodules) Diffused Tumor sizea e2 cm >2 cm Cellular differentiationb,d Well differentiated Moderately differentiated Poorly differentiated Serum AFP level e400 ng/mL >400 ng/mL Venous infiltrationc,d Absent Present Tumor stage (AJCC) Early stage (stage I&II) Late stage (stage III&IV)
total patients (n ) 39)
lamin B 1 (SSP 3717) intensity
52.2 ( 8.6
P-value
0.871
35 4
0.075 ( 0.063 0.153 ( 0.072
0.029
9 30
0.059 ( 0.016 0.091 ( 0.076
0.039
11 17 7
0.117 ( 0.080 0.070 ( 0.061 0.073 ( 0.067
0.192
19 20
0.086 ( 0.066 0.081 ( 0.071
0.829
20 19
0.075 ( 0.062 0.093 ( 0.074
0.412
26 13
0.068 ( 0.055 0.114 ( 0.082
0.040
a Length of the largest tumor was measured in tumor size. b Well differentiated (Edmonson grade 0-2); moderately differentiated (Edmonson grade 3); poorly differentiated (Edmonson grade 4). c Venous infiltration was defined by findings on microscopic and major pathologic examination. d Statistic was based on available data.
that the lamin B1 transcript level was significantly elevated in early stage HCCs (134 normalized copies/ng) and late stage HCCs (105 normalized copies/ng) when compared to the nonmalignant controls (25 normalized copies/ng) (Figure 3B). Heavily localized immunoreactivity signal of lamin B1 was revealed at the nuclear envelope of HCC tumor cells, but limited immunoreactivity was found in cirrhotic tissues (Figure 3C). Clinical Relevance of Lamin B1 Expression in HCC. Clinicopathological correlation with lamin B1 expression was analyzed in a total of 39 human HCCs (Table 3). Overexpression of lamin B1 was showed to be significantly associated with the increased number of tumor nodules (p ) 0.029), the size of tumors (p ) 0.039), and advanced characteristics of tumor stage (p ) 0.04). There was no apparent correlation with other clinical features, including cellular differentiation, serum APF level, and venous infiltration. Circulating Lamin B1 (LMNB1) mRNA Detects HCC Tumors of Different Stages. A separate cohort of 63 individuals was tested for the presence of circulating LMNB1 mRNA in their plasma. A representative RT-PCR gel image was shown in Figure 4A where faint or no signals were estimated as negative and GAPDH was used as PCR and mRNA quality control mainly because its concentration was considered to reflect total plasma mRNA. Lamin B1 (LMNB1) mRNA was detected in 16 of 21 (76%) plasma from patients with early stage HCC and 12 of 14 (86%) from patients with late stage HCC, whereas only 19% and 17% in cirrhosis and normal subjects demonstrated LMNB1, respectively [Figure 4B (i)]. The positivity rate of circulating LMNB1 mRNA gradually increased with tumor stages progression and it was significantly higher in HCC than in cirrhosis and normal individuals (p < 0.05 and p < 0.01, respectively). The overall detection sensitivity of LMNB1 mRNA for HCC was 80% and the specificity was 82%, where in particular for the detection of early stage and late stage HCCs the sensitivity is 76% and 86%, respectively [Figure 4B (ii)].
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Discussion Alpha-fetoprotein (AFP) is a widely used HCC serological marker together with the ultrasonography (US) to monitor the detection of early HCC. Because of its known limitations, several other biomarkers have been identified to be employed either alone or in combination with AFP in an attempt to increase the detection sensitivity of early HCC.22–24 However, none of these markers have been validated sufficiently for clinical use; thus, novel biomarkers are urgently needed for the detection of early HCC. Recently, an exciting finding in the biomarker field suggests that circulating nucleic acids detected in the human plasma/ serum may provide a very promising assay for the early detection of malignancies.25 The discovery supplements the fact that current proteomic methods are insufficient for the detection of subtle changes of circulating oncoprotein that underlie the key cancer signaling processes.26 Therefore, different new technical platforms have recently been developed to increase the detection sensitivity of biomarkers whose expression exhibited slight changes in response to tumorigenesis. For instance, the development of a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in nanoliter volumes26 and the microfluidics digital PCR based platform for detection of mRNA in the circulation.27 Indeed, correlation of protein and mRNA abundance may be poor mainly because of the possible post-translational modification and/or degradation of these molecules, and varied losses during extraction. Nevertheless, if an association exists, this could advocate better insight on the mechanism of secretory pathways. We employed 2-DE proteomic analysis of 75 individuals to identify proteins that enabled early detection of HCC. Lamin B1 was revealed to be one of the most promising candidates for subsequent validation mainly because of its remarkable expression level (p < 0.0001) in HCC compared to the nonmalignant controls. In fact, lamin B1 has been previously suggested as a potential tumor tissue marker; however, the validity has not been clinically proven nor correlated with the clinicopathological characteristics.28 Clinical application of biomarker requires a simple serological test. Lamin B1 is a potential diagnostic biomarker candidate because of its overexpression in HCC and presence in the circulation. In fact, our 2-DE data showed that lamin B1 was not only a potential marker for the detection of early HCC, but the expression of this protein was positively correlated with the increased number of tumor nodules, the size of the tumors and the advanced stage of the tumor characteristics. This suggests that lamin B1 overexpression may relate to active tumor development. Validation of aberrant expression of lamin B1 in HCC tissues and cell lines further reinforced the use of lamin B1 as a potential tumor marker. However, the nonavailability of commercial ELISA assays limits our ability to detect the level of soluble lamin B1 in the circulation. Recently, a new nonprotein candidate appears to be an alternative modality to detect and monitor HCC. The tumor-related cell-free circulating mRNAs in the plasma are most likely tyrosine kinase mRNA,29 telomerase components30 and mRNAs that are encoded by different tumorrelated genes.31 Lamin B1 is the major component of nuclear lamina which provides structural support of the nuclear envelope and is involved in controlling gene expression by managing organization of chromatin, DNA replication, repair and transcription.32–34 Lamin B1 was also found to control oxidative stress via a Journal of Proteome Research • Vol. 9, No. 1, 2010 75
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Figure 4. Circulating LMNB1 mRNA expression in human plasma by RT-PCR. (A) Representative DNA gels of LMNB1 and GAPDH RT-PCR products. The images demonstrate results from the plasma of 8 patients in different analyzed groups. (B) (i) Histogram shows the percentage of individuals with circulating LMNB1 mRNA present in each liver diagnosis. (ii) Summary of the diagnostic performance of LMNB1 mRNA for the detection of HCCs at different tumor stages.
transcription factor Oct-1.35 The phosphorylation of lamin B1 mediated by the inositide-specific phospholipase C β1 (PLCβ1) signaling results in cell proliferation via G2/M cell cycle progression.36 Lamin B1 also appears to have many other functions as reflected in the array of diseases caused by lamin mutations.37 This intermediate filament protein that forms a network lining the inner nuclear membrane works as transcriptional coregulator whose dyregulation may initiate some key cancer signaling processes. In fact, this protein was also found to be overexpressed in HCC tumors compared to the control subjects.28 More importantly, proteolytic cleavage and subsequent degradation of lamin B1 have been previously reported during radiation induced apoptosis.38 Nucleic acid in 76
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plasma may come from lysis of circulating cancer cells caused by tumor necrosis, apoptosis, or active release.39 On the basis of the above evidence, we hypothesize that at least some of the apoptotic cells would release lamin B1 mRNA into the circulation and that such lamin B1 mRNA might be detectable in the patients’ plasma.40,41 Therefore, the present study explored the possibility of using RT-PCR in measuring plasma lamin B1 mRNA in the HCC patients. The finding of a detectable quantity of lamin B1 mRNA in the plasma of HCC patients is an exciting development. Lamin B1 was identified to detect HCCs with sensitivity of 80% and specificity of 82%, where in particular for the detection of early stage and late stage HCCs, the sensitivity is 76% and 86%,
LMNB1 Biomarker Detects Early Stages of Liver Cancer Patients respectively. In conclusion, lamin B1 is a potential HCC marker that acts intracellularly or extracellularly in the form of protein and mRNA, respectively. The expression of this marker could be detected in the earliest stage of HCC. Additionally, overexpression of lamin B1 is positively correlated with the tumor development. Finally, the development of a TaqMan-probe quantitative PCR approach for large-scale studies in multiple centers is underway to investigate whether plasma LMNB1 mRNA detection would have a role in population screening for early stage HCC among the at-risk subjects.
Acknowledgment. We thank the technical support from the Surgical Tissue Bank staff (Jasmine Yu, S. Y. Yik, Noel Kwong) and insightful advice and comments by Professor S. T. Fan (Queen Mary Hospital, HK). The work was supported by the Research Grants Council of Hong Kong (N_HKU718/03) and Sun C.Y. Research Foundation for Hepatobiliary and Pancreatic Surgery. Supporting Information Available: Supplementary Figure 1, MALDI-TOF/TOF of putative lamin B1 spots, which explicitly confirmed the identify of lamin B1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Jemal, A.; Siegel, R.; Ward, E.; Murray, T.; Xu, J.; Thun, M. J. Cancer statistics, 2007. CA Cancer J. Clin. 2007, 57 (1), 43–66. (2) Kamangar, F.; Dores, G. M.; Anderson, W. F. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J. Clin. Oncol. 2006, 24 (14), 2137–50. (3) Poon, R. T.; Fan, S. T. Hepatectomy for hepatocellular carcinoma: patient selection and postoperative outcome. Liver Transplant. 2004, 10 (2 Suppl. 1), S39–45. (4) Lai, E. C.; Lau, W. Y. The continuing challenge of hepatic cancer in Asia. Surgeon 2005, 3 (3), 210–5. (5) Chan, A. C.; Poon, R. T.; Ng, K. K.; Lo, C. M.; Fan, S. T.; Wong, J. Changing paradigm in the management of hepatocellular carcinoma improves the survival benefit of early detection by screening. Ann. Surg. 2008, 247 (4), 666–73. (6) Lau, W. Y.; Lai, E. C. Hepatocellular carcinoma: current management and recent advances. Hepatobiliary Pancreatic Dis. Int. 2008, 7 (3), 237–57. (7) Okuda, K.; Ohtsuki, T.; Obata, H.; Tomimatsu, M.; Okazaki, N.; Hasegawa, H.; Nakajima, Y.; Ohnishi, K. Natural history of hepatocellular carcinoma and prognosis in relation to treatment. Study of 850 patients. Cancer 1985, 56 (4), 918–28. (8) Collier, J.; Sherman, M. Screening for hepatocellular carcinoma. Hepatology 1998, 27 (1), 273–8. (9) Sun, S.; Lee, N. P.; Poon, R. T.; Fan, S. T.; He, Q. Y.; Lau, G. K.; Luk, J. M. Oncoproteomics of hepatocellular carcinoma: from cancer markers’ discovery to functional pathways. Liver Int. 2007, 27 (8), 1021–38. (10) Aravalli, R. N.; Steer, C. J.; Cressman, E. N. Molecular mechanisms of hepatocellular carcinoma. Hepatology 2008, 48 (6), 2047–63. (11) Pang, R. W.; Joh, J. W.; Johnson, P. J.; Monden, M.; Pawlik, T. M.; Poon, R. T. Biology of hepatocellular carcinoma. Ann. Surg. Oncol. 2008, 15 (4), 962–71. (12) Greene, F. L. P. D.; Fleming, I. D.; Fritz, A. G.; Balch, C. M.; Haller, D. G. AJCC Cancer Staging Manual, 6th ed.; Springer: Chicago, IL, 2002; pp 131-144. (13) Wong, B. W.; Luk, J. M.; Ng, I. O.; Hu, M. Y.; Liu, K. D.; Fan, S. T. Identificationofliver-intestinecadherininhepatocellularcarcinomasa potential disease marker. Biochem. Biophys. Res. Commun. 2003, 311 (3), 618–24. (14) Lee, N. P.; Leung, K. W.; Cheung, N.; Lam, B. Y.; Xu, M. Z.; Sham, P. C.; Lau, G. K.; Poon, R. T.; Fan, S. T.; Luk, J. M. Comparative proteomic analysis of mouse livers from embryo to adult reveals an association with progression of hepatocellular carcinoma. Proteomics 2008, 8 (10), 2136–49. (15) Luk, J. M.; Lam, C. T.; Siu, A. F.; Lam, B. Y.; Ng, I. O.; Hu, M. Y.; Che, C. M.; Fan, S. T. Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values. Proteomics 2006, 6 (3), 1049–57.
research articles
(16) Lee, N. P.; Chen, L.; Lin, M.; Tsang, F.; Yeung, C.; Poon, R.; Peng, J.; Leng, X.; Beretta, L.; Sun, S.; Day, P.; Luk, J. M. Proteomic Expression Signature Distinguishes Cancerous and Non-Malignant Tissues in Hepatocellular Carcinoma. J. Proteome Res. 2009, 8 (3), 1293–1303. (17) Yi, X.; Luk, J. M.; Lee, N. P.; Peng, J.; Leng, X.; Guan, X. Y.; Lau, G. K.; Beretta, L.; Fan, S. T. Association of mortalin (HSPA9) with liver cancer metastasis and prediction for early tumor recurrence. Mol. Cell. Proteomics 2008, 7 (2), 315–25. (18) Wang, Y. Q.; Luk, J. M.; Ikeda, K.; Man, K.; Chu, A. C.; Kaneda, K.; Fan, S. T. Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells. Biochem. Biophys. Res. Commun. 2004, 317 (2), 358–62. (19) Mok, B. W.; Yeung, W. S.; Luk, J. M. Differential expression of gapjunction gene connexin 31 in seminiferous epithelium of rat testes. FEBS Lett. 1999, 453 (3), 243–8. (20) Wang, X. Q.; Luk, J. M.; Leung, P. P.; Wong, B. W.; Stanbridge, E. J.; Fan, S. T. Alternative mRNA splicing of liver intestine-cadherin in hepatocellular carcinoma. Clin. Cancer Res. 2005, 11 (2 Pt 1), 483– 9. (21) Moisan, E.; Girard, D. Cell surface expression of intermediate filament proteins vimentin and lamin B1 in human neutrophil spontaneous apoptosis. J. Leukocyte Biol. 2006, 79 (3), 489–98. (22) Ikoma, J.; Kaito, M.; Ishihara, T.; Nakagawa, N.; Kamei, A.; Fujita, N.; Iwasa, M.; Tamaki, S.; Watanabe, S.; Adachi, Y. Early diagnosis of hepatocellular carcinoma using a sensitive assay for serum desgamma-carboxy prothrombin: a prospective study. Hepatogastroenterology 2002, 49 (43), 235–8. (23) Henry, L.; Lavabre-Bertrand, T.; Vercambre, L.; Ramos, J.; Carillo, S.; Guiraud, I.; Pouderoux, P.; Bismuth, M.; Valats, J. C.; Demattei, C.; Duny, Y.; Chaze, I.; Funakoshi, N.; Bureau, J. P.; Daures, J. P.; Blanc, P. Plasma proteasome level is a reliable early marker of malignant transformation of liver cirrhosis. Gut 2009, 58 (6), 833– 8. (24) Zinkin, N. T.; Grall, F.; Bhaskar, K.; Otu, H. H.; Spentzos, D.; Kalmowitz, B.; Wells, M.; Guerrero, M.; Asara, J. M.; Libermann, T. A.; Afdhal, N. H. Serum proteomics and biomarkers in hepatocellular carcinoma and chronic liver disease. Clin. Cancer Res. 2008, 14 (2), 470–7. (25) Miura, N.; Maruyama, S.; Oyama, K.; Horie, Y.; Kohno, M.; Noma, E.; Sakaguchi, S.; Nagashima, M.; Kudo, M.; Kishimoto, Y.; Kawasaki, H.; Hasegawa, J.; Shiota, G. Development of a novel assay to quantify serum human telomerase reverse transcriptase messenger RNA and its significance as a tumor marker for hepatocellular carcinoma. Oncology 2007, 72 Suppl 1, 45–51. (26) Fan, A. C.; Deb-Basu, D.; Orban, M. W.; Gotlib, J. R.; Natkunam, Y.; O’Neill, R.; Padua, R. A.; Xu, L.; Taketa, D.; Shirer, A. E.; Beer, S.; Yee, A. X.; Voehringer, D. W.; Felsher, D. W. Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nat. Med. 2009, 15 (5), 566–71. (27) Yung, T. K.; Chan, K. C.; Mok, T. S.; Tong, J.; To, K. F.; Lo, Y. M. Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients. Clin. Cancer Res. 2009, 15 (6), 2076–84. (28) Lim, S. O.; Park, S. J.; Kim, W.; Park, S. G.; Kim, H. J.; Kim, Y. I.; Sohn, T. S.; Noh, J. H.; Jung, G. Proteome analysis of hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 2002, 291 (4), 1031– 7. (29) Kopreski, M. S.; Benko, F. A.; Kwak, L. W.; Gocke, C. D. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin. Cancer Res. 1999, 5 (8), 1961–5. (30) Chen, X. Q.; Bonnefoi, H.; Pelte, M. F.; Lyautey, J.; Lederrey, C.; Movarekhi, S.; Schaeffer, P.; Mulcahy, H. E.; Meyer, P.; Stroun, M.; Anker, P. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin. Cancer Res. 2000, 6 (10), 3823–6. (31) Cheung, S. T.; Fan, S. T.; Lee, Y. T.; Chow, J. P.; Ng, I. O.; Fong, D. Y.; Lo, C. M. Albumin mRNA in plasma predicts post-transplant recurrence of patients with hepatocellular carcinoma. Transplantation 2008, 85 (1), 81–7. (32) Dechat, T.; Pfleghaar, K.; Sengupta, K.; Shimi, T.; Shumaker, D. K.; Solimando, L.; Goldman, R. D. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 2008, 22 (7), 832–53. (33) Tang, C. W.; Maya-Mendoza, A.; Martin, C.; Zeng, K.; Chen, S.; Feret, D.; Wilson, S. A.; Jackson, D. A. The integrity of a laminB1-dependent nucleoskeleton is a fundamental determinant of RNA synthesis in human cells. J. Cell Sci. 2008, 121 (Pt 7), 1014– 24. (34) Chiarini, A.; Whitfield, J. F.; Pacchiana, R.; Armato, U.; Dal Pra, I. Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - a novel mechanism of action
Journal of Proteome Research • Vol. 9, No. 1, 2010 77
research articles (35) (36)
(37) (38)
78
of a photodynamic tumor therapy agent. Biochim. Biophys. Acta 2008, 1783 (9), 1642–53. Malhas, A. N.; Lee, C. F.; Vaux, D. J. Lamin B1 controls oxidative stress responses via Oct-1. J. Cell Biol. 2009, 184 (1), 45–55. Fiume, R.; Ramazzotti, G.; Teti, G.; Chiarini, F.; Faenza, I.; Mazzotti, G.; Billi, A. M.; Cocco, L. Involvement of nuclear PLCbeta1 in lamin B1 phosphorylation and G2/M cell cycle progression. FASEB J. 2009, 23 (3), 957–66. Goldberg, M. W.; Fiserova, J.; Huttenlauch, I.; Stick, R. A new model for nuclear lamina organization. Biochem. Soc. Trans. 2008, 36 (Pt 6), 1339–43. Dynlacht, J. R.; Earles, M.; Henthorn, J.; Seno, J. D. Different patterns of DNA fragmentation and degradation of nuclear matrix proteins during apoptosis induced by radiation, hyperthermia or etoposide. Radiat. Res. 2000, 154 (5), 515–30.
Journal of Proteome Research • Vol. 9, No. 1, 2010
Sun et al. (39) Silva, J. M.; Rodriguez, R.; Garcia, J. M.; Munoz, C.; Silva, J.; Dominguez, G.; Provencio, M.; Espana, P.; Bonilla, F. Detection of epithelial tumour RNA in the plasma of colon cancer patients is associated with advanced stages and circulating tumour cells. Gut 2002, 50 (4), 530–4. (40) Wong, S. C.; Lo, S. F.; Cheung, M. T.; Ng, K. O.; Tse, C. W.; Lai, B. S.; Lee, K. C.; Lo, Y. M. Quantification of plasma beta-catenin mRNA in colorectal cancer and adenoma patients. Clin. Cancer Res. 2004, 10 (5), 1613–7. (41) Salway, F.; Day, P. J.; Ollier, W. E.; Peakman, T. C. Levels of 5′ RNA tags in plasma and buffy coat from EDTA blood increase with time. Int. J. Epidemiol. 2008, 37 (Suppl. 1), i11–5.
PR9002118
