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Novel Identification of Zyxin Upregulations in the Motile Phenotype of Hepatocellular Carcinoma

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Novel Identification of Zyxin Upregulations in the Motile Phenotype of Hepatocellular Carcinoma Modern Pathology (2006) 19, 1108–1116 & 2006 USCAP, Inc All rights reserved 0893-3952/06 $30.00 www.modernpathology.org Novel identification of zyxin upregulations in the motile phenotype of hepatocellular carcinoma Shirley M-H Sy1, Paul B-S Lai2, Etonia Pang1, Navy L-Y Wong1, Ka-Fai To1, Philip J Johnson3 and Nathalie Wong1 1Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Shatin, N.T., SAR Hong Kong, China; 2Department of Surgery, Chinese University of Hong Kong, Shatin, N.T., SAR Hong Kong, China and 3Institute for Cancer Studies, University of Birmingham, Birmingham, UK Genome-wide copy number aberrations are common in hepatocellular carcinoma, although the precise genetic events underlying disease progression remain poorly defined. Previous work from our group has indicated several regional chromosomal gains such as chromosome 7q34–q36 that are associated with advanced metastatic tumors. Although the distal chromosome 7q gains have also been implicated in the progression of other malignancies, information on underlying targeted genes is limited. In this study, we have examined the chromosome 7q34–q36 region for involved gene(s) (or genes of interest). An integrated array-based comparative genomic hybridization and transcriptional mapping analyses has enabled us to identify a single candidate, zyxin on chromosome 7q34–q36. This array-derived finding was supported by quantitative reverse transcription-polymerase chain reaction, which also indicated common upregulations of zyxin in hepatocellular carcinoma tumors compared to their corresponding nonmalignant liver tissue (17/52 cases; 33%). Although there was no correlation between zyxin expression and tumor stagings, there was a significant increase in messenger RNA levels in hepatocellular carcinoma cases that presented with multifocal disease (211.57936.9- fold) compared to those with solitary lesions (3.576.3-fold). Moreover, recurrence after resection was common in cases that displayed zyxin overexpressions in the initial resected tumor (P ¼ 0.05). Functional examination of zyxin by small interfering RNA-mediated knockdown in Hep3B cell line indicated a significant inhibition on cell migration through porous membrane (P ¼ 0.002) and invasion through matrigel-coated membrane (P ¼ 0.005). In summary, mapping of chromosome 7q34–q36 has led to the identification of frequent zyxin overexpressions in hepatocellular carcinoma, and a potential role for zyxin in conferring a motile phenotype. Modern Pathology (2006) 19, 1108–1116. doi:10.1038/modpathol.3800626; published online 5 May 2006 Keywords: hepatocellular carcinoma; complementary DNA-based comparative genomic hynridization; transcrip- tional mapping; zyxin; cell migration; cell invasion Extensive genetic characterizations on hepatocellu- derived from early and advanced stages hepato- lar carcinoma have indicated common copy number cellular carcinoma tumors with an aim to define the gains on chromosomes 1q, 8q, 17q and 20q, and genetic aberrations involved in disease progression.4 losses on 1p, 4q 8p, 13q, 16q and 17p.1–3 While these A number of regional copy gains were indicated, changes have been considered early genetic events among which chromosome 7q34–q36 gains were in liver carcinogenesis, the molecular events by common in the advanced metastatic hepatocellular which specific genomic alterations contribute to carcinoma.4 the progression of hepatocellular carcinoma remain Distal chromosome 7q gains have been suggested elusive. Our group has previously conducted large- in association with biliary tract carcinoma progres- scale correlative analysis on the genomic data sion,5 and higher-stage non-small cell lung cancer with nodal involvement.6 Previous studies have also demonstrated a positive association between pros- Correspondence: Dr N Wong, D Phil, Department of Anatomical tate cancer progression and copy gains of distal and Cellular Pathology, Chinese University of Hong Kong, Prince chromosome 7q,7 and the amplification of chromo- of Wales Hospital, Shatin, N.T., SAR Hong Kong, China. some 7q31–q36 region in the progression of gliomas E-mail: [email protected] 8 Received 24 January 2006; revised 10 April 2006; accepted 11 to grade III or IV. Taken together, it is likely that April 2006; published online 5 May 2006 gene(s) residing on chromosome 7q34–q36 may have Zyxin in hepatocellular carcinoma progression SM-H Sy et al 1109 a role in cancer progression. In the current study, (lymphocytic DNA from three healthy subjects) were a combined analysis using of high-resolution geno- differentially labeled with Cy5-dCTP and Cy3-dCTP mic and transcriptional mapping was performed using the RadPrime DNA labeling system (Gibco to identify potential tumor-related gene(s) residing Invitrogen Corporation). Labeled DNA was com- on chromosome 7q34–q36. Using a complementary bined with 30 mg human Cot-1 DNA, 20 mg poly DNA array platform with an average resolution of (dA-dT) and 100 mg yeast tRNA in DIGhyb buffer B91 kb on chromosome 7, mapping studies where prior to overnight hybridization in a dark chamber at performed on a hepatocellular carcinoma tumor that 371C. Following posthybridization washes in 1 Â displayed regional chromosome 7q34–q36 high- saline sodium citrate/0.1% sodium dodecyl sulfate level gains from metaphase comparative genomic at 451C and gentle rinsing in 1 Â saline sodium hybridization analysis. This integrated analysis has citrate at room temperature, hybridized signals were permitted recognition of candidate genes and mini- captured by the ScanArray 5000 scanner (GSI mized the number of genes required for subsequent Lumonics, Billerica, MA) and analyzed by the quantitative reverse transcription-polymerase chain GenePix Pro 3.0. Data manipulation was facilitated reaction validations. The functional effects of an using the Normalization Suite v1.63 (http://www. identified candidate gene on hepatocellular carci- utoronto.ca/cancyto/). Results from duplicate spots noma cell proliferation and motility were further and dye swap experiments were calculated and investigated. incorporated with the physical map locations of complementary DNA in sequential order of mega- base distances. ‘Test to reference’ ratios greater than Materials and methods twofold were considered as copy number gain. Patients Expression Microarray Tumorous hepatocellular carcinoma and the corre- sponding adjacent nonmalignant liver tissues were Using complementary DNA microarray chip as the obtained from 52 patients who underwent curative array-comparative genomic hybridization study surgery for hepatocellular carcinoma at the Prince of (Ontario Cancer Institute, Toronto, Canada), expres- Wales Hospital, Hong Kong between 1997 and 2002. sion array analysis was performed according to Histological examination confirmed the diagnosis of procedure previously described from our group.10 hepatocellular carcinoma, and the presence of Ten micrograms of total RNA from HKCI-6 and underlying cirrhosis in 80.8%. Serological analysis reference pool (3 normal livers acquired from indicated chronic viral hepatitis B carriage in 94.0% CloneTech, Stratagene and Ambion) were reverse of patients. The American Joint Cancer Center tumor transcribed by AncT messenger RNA primer using staging system classified one case as T1, 30 cases as Supescript II reverse transcriptase separately (Gibco T2, 12 cases as T3 and 9 cases as T4. Statistical Invitrogen Corporation). Complementary DNA dif- analysis did not suggest differences in the clinico- ferentially labeled with Cy3-dCTP and Cy5-dCTP pathological characteristics between the early T1/T2 were combined with 20 mg calf thymus DNA and and advanced T3/T4 stage patients (P40.05), except 100 mg yeast tRNA prior to hybridization onto for the presence of multifocal feature (Po0.0001) array slides. Following posthybridization washes, (Table 2). Five year follow-up information on 32 image acquisition and data analysis were as des- cases showed tumor recurrence in 15 patients. cribed for complementary DNA microarray-based comparative genomic hybridization. ‘Test to refer- ence’ ratios greater than twofold were considered as Complementary DNA Microarray-Based Comparative upregulations. Genomic Hybridization The complementary DNA microarray chip utilized Quantitative Reverse Transcription-Polymerase contained 1738 complementary DNA clones that Chain Reaction spanned an average resolution of B91 kb along the entire human chromosome 7 (Ontario Cancer In- Total RNA extracted from HKCI-6 and primary stitute, Toronto, Canada). Complementary DNA hepatocellular carcinoma tumors using TRIzol microarray-based comparative genomic hybridiza- reagent (Gibco Invitrogen Corporation) were treated tion was performed according to method described with 1.0 U Rq1 RNase-free DNase (Promega Corpora- in Beheshti et al.9 A hepatocellular carcinoma cell tion) prior to reverse transcription. To ensure no line, HKCI-6, that displayed high-level gains of genomic DNA carryover, no-reverse transcription regional chromosome 7q34–q36 was employed in polymerase chain reaction was first performed using the mapping analyses. HKCI-6 was established from primer sets for genomic beta-globin gene (sense: 50- the tumorous liver tissue of a chronic hepatitis B GAA GAG CCA AGG ACA GGT AC-30 and antisense: virus infected male patient, who presented a stage 50-CAA CTT CAT CCA CGT TCA CC-30). First strand T3 hepatocellular carcinoma tumor with multifocal complementary DNA was prepared from total RNA lesions. DNA from HKCI-6, and
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