Down-Regulation of Annexin A10 in Hepatocellular Carcinoma Is Associated with Vascular Invasion, Early Recurrence, and Poor Prognosis in Synergy with p53 Mutation
2002; Elsevier BV; Volume: 160; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61129-7
ISSN1525-2191
AutoresShu-Hsiang Liu, Chiao-Ying Lin, Shian-Yang Peng, Yung‐Ming Jeng, Hung-Wei Pan, Po-Lin Lai, Chao-Lien Liu, Hey-Chi Hsu,
Tópico(s)Peptidase Inhibition and Analysis
ResumoAnnexins (ANXs) are a large group of calcium-binding proteins participating in diverse important biological processes. ANXA10 is the least expressed new member of unknown function. We showed that ANXA10 mRNA was expressed in adult liver and hepatocellular carcinoma (HCC), but not in multiple adult and fetal tissues, cholangiocarcinoma, and several other common carcinomas. Of 182 unifocal primary HCCs, ANXA10 mRNA was dramatically reduced in 121 (66%), and the down-regulation correlated with p53 mutation (P = 0.024), early intrahepatic tumor recurrence (P = 0.0007), and lower 4-year survival (P = 0.0014). Down-regulation of ANXA10 was twofold more frequent in large than small HCCs (P = 0.0012), in grade II to III than grade I HCC (P < 0.00001), and in stage IIIA to IV than stage I to II HCC (P < 0.00001). Moreover, ANXA10 down-regulation and p53 mutation acted synergistically toward high-grade (P < 0.00001), high-stage HCC (P < 0.00001), and poorer prognosis (P = 0.0025). Our results indicate that the expression of the tissue- and tumor-restricted ANXA10 is a marker of liver cell differentiation and growth arrest, and its down-regulation associated with malignant phenotype of hepatocytes, vascular invasion, and progression of HCC, leading to poor prognosis. Thus, ANXA10 might serve as a new potential target of gene therapy for HCC. Annexins (ANXs) are a large group of calcium-binding proteins participating in diverse important biological processes. ANXA10 is the least expressed new member of unknown function. We showed that ANXA10 mRNA was expressed in adult liver and hepatocellular carcinoma (HCC), but not in multiple adult and fetal tissues, cholangiocarcinoma, and several other common carcinomas. Of 182 unifocal primary HCCs, ANXA10 mRNA was dramatically reduced in 121 (66%), and the down-regulation correlated with p53 mutation (P = 0.024), early intrahepatic tumor recurrence (P = 0.0007), and lower 4-year survival (P = 0.0014). Down-regulation of ANXA10 was twofold more frequent in large than small HCCs (P = 0.0012), in grade II to III than grade I HCC (P < 0.00001), and in stage IIIA to IV than stage I to II HCC (P < 0.00001). Moreover, ANXA10 down-regulation and p53 mutation acted synergistically toward high-grade (P < 0.00001), high-stage HCC (P < 0.00001), and poorer prognosis (P = 0.0025). Our results indicate that the expression of the tissue- and tumor-restricted ANXA10 is a marker of liver cell differentiation and growth arrest, and its down-regulation associated with malignant phenotype of hepatocytes, vascular invasion, and progression of HCC, leading to poor prognosis. Thus, ANXA10 might serve as a new potential target of gene therapy for HCC. The annexin (ANX) family is the largest single category of eukaryotic calcium-binding proteins without EF hands (the helix-loop-helix calcium-binding motif).1Smith PD Moss SE Structural evolution of the annexin supergene family.Trends Genet. 1994; 10: 241-246Abstract Full Text PDF PubMed Scopus (68) Google Scholar The ANXs play an important role in a broad range of physiological processes, including anti-coagulation,2Tait JF Sakata M Mcmullen BA Miao CH Funakoshi T Hendrickson LE Fujikawa K Placental anticoagulant proteins: isolation and comparative characterization of four members of the lipocortin family.Biochemistry. 1998; 27: 6268-6276Crossref Scopus (172) Google Scholar endocytosis, exocytosis,3Creutz CE The annexins and exocytosis.Science. 1992; 258: 924-931Crossref PubMed Scopus (496) Google Scholar, 4Emans N Gorvel JP Walter C Gerke V Kellner R Griffiths G Gruenberg J Annexin II is a major component of fusogenic endosomal vesicles.J Cell Biol. 1993; 120: 1357-1369Crossref PubMed Scopus (240) Google Scholar immunosuppression,5Sakata T Iwagami S Tsuruta Y Suzuki S Suzuki R Study of natural lipocortin I. 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ANXA1 is overexpressed in breast cancer and hepatocellular carcinoma (HCC),17Masaki T Tokuda M Ohnishi M Watanabe S Fujimura T Miyamoto K Itano T Matsui H Arima K Shirai M Maeba T Sogawa K Konishi R Taniguchi K Hatanaka Y Hatase O Nishioka M Enhanced expression of the protein kinase substrate annexin I in human hepatocellular carcinoma.Hepatology. 1996; 24: 72-81PubMed Google Scholar, 18Ahn SH Sawada H Ro JY Nicolson GL Differential expression of annexin I in human mammary ductal epithelial cells in normal and benign and malignant breast tissues.Clin Exp Metastasis. 1997; 15: 151-156Crossref PubMed Scopus (109) Google Scholar, 19Pencil SD Toh Y Nicolson GL Candidate metastasis-associated genes of the rat 13762NF mammary adenocarcinoma.Breast Cancer Res Treat. 1993; 25: 165-174Crossref PubMed Scopus (94) Google Scholar ANXA2 in brain glial tumors and pancreatic cancer,20Vishwanatha JK Chiang Y Kumble KD Hollingsworth MA Pour PM Enhanced expression of annexin II in human pancreatic carcinoma cells and primary pancreatic cancers.Carcinogenesis. 1993; 14: 2575-2579Crossref PubMed Scopus (125) Google Scholar, 21Roseman BJ Bollen A Hsu J Lamborn K Israel MA Annexin II marks astrocytic brain tumors of high histologic grade.Oncol Res. 1994; 6: 561-567PubMed Google Scholar, 22Nygaard SJ Haugland HK Kristoffersen EK Lund-Johansen M Laerum OD Tysnes OB Expression of annexin II in glioma cell lines and in brain tumor biopsies.J Neurooncol. 1998; 38: 11-18Crossref PubMed Scopus (32) Google Scholar, 23Paciucci R Tora M Diaz VM Real FX The plasminogen activator system in pancreas cancer: role of t-PA in the invasive potential in vitro.Oncogene. 1998; 16: 625-633Crossref PubMed Scopus (66) Google Scholar and ANXA8 in acute promyelocytic leukemia.13Sarkar A Yang P Fan YH Mu ZM Hauptmann R Adolf GR Stass SA Chang KS Regulation of the expression of annexin VIII in acute promyelocytic leukemia.Blood. 1994; 84: 279-286PubMed Google Scholar, 24Chang KS Wang G Freireich EJ Daly M Naylor SL Trujillo JM Stass SA Specific expression of the annexin VIII gene in acute promyelocytic leukemia.Blood. 1992; 79: 1802-1810PubMed Google Scholar Overexpression of ANXA1 correlates positively with metastatic potential of breast cancer,19Pencil SD Toh Y Nicolson GL Candidate metastasis-associated genes of the rat 13762NF mammary adenocarcinoma.Breast Cancer Res Treat. 1993; 25: 165-174Crossref PubMed Scopus (94) Google Scholar and increased growth of tumor cells inoculated in nude mice.25Frey BM Reber BFX Vishwanath B Escher G Frey FJ Annexin I modulates cell functions by controlling intracellular calcium release.FASEB J. 1999; 13: 2235-2245Crossref PubMed Scopus (38) Google Scholar In contrast, ANXA6 has tumor suppressive activity in squamous cell carcinoma,26Theobald J Hanby A Patel K Moss SE Annexin VI has tumor-suppressor activity inhuman A431 squamous epithelial carcinoma cells.Br J Cancer. 1995; 71: 786-788Crossref PubMed Scopus (56) Google Scholar and a decrease in ANXA6 and ANXA7 proteins is associated with malignant phenotype and the metastatic potential of melanoma.27Francia G Mitchell SD Moss SE Hanby AM Marshall JF Hart IR Identification by differential display of annexin-VI, a gene differentially expressed during melanoma progression.Cancer Res. 1996; 56: 3855-3858PubMed Google Scholar, 28Kataoka TR Ito A Asada H Watabe K Nishiyama K Nakamoto K Itami S Yoshikawa K Ito M Nojima H Kitamura Y Annexin VII as a novel marker for invasive phenotype of malignant melanoma.Jpn J Cancer Res. 2000; 91: 75-83Crossref PubMed Scopus (35) Google Scholar Moreover, ANXA4 plays a role in chemoresistance.29Han EKH Tahir SK Cherian SP Collins N Ng SC Modulation of pacitaxel resistance by annexin IV in human cancer cell lines.Br J Cancer. 2000; 83: 83-88Crossref PubMed Scopus (98) Google Scholar Hence, there are at least two categories of ANXs implicated in tumor progression and tumor suppression, and different ANXs play important and complex roles in cancer. ANXA10, a novel member of the ANX family, has several distinct features, including rare expression, codon deletion in conserved repeat 3, and an unusual ablation of the type II calcium-binding sites in tetrad core repeats.15Morgan RO Jenkins NA Gilbert DJ Copeland NG Balsara BR Testa JR Fernandez MP Novel human and mouse annexin A10 are linked to the genome duplications during early chordate evolution.Genomics. 1999; 60: 40-49Crossref PubMed Scopus (59) Google Scholar The loss of type II calcium-binding sites is a fundamental deviation from ANX structure conservation and undoubtedly has functional consequences for membrane association and aggregation properties.30Bitto E Cho W Roles of individual domains of annexin I in its vesicle binding and vesicle aggregation: a comprehensive study.Biochemistry. 1998; 37: 10231-10237Crossref PubMed Scopus (24) Google Scholar ANXA10 is mapped to chromosome 4q33.31Deloukas P Schuler GD Gyapay G Beasley EM Soderlund C Rodriguez-Tomé P Hui L Matise TC McKusick KB Beckman JS et al.A physical map of 30,000 human genes.Science. 1998; 282: 744-746Crossref PubMed Scopus (592) Google Scholar Chromosome 4q is one of the most common regions with a high frequency of allelic loss in HCC.32Yeh SH Chen PJ Lai MY Chen DS Allelic loss on chromosomes 4q and 16q in hepatocellular carcinoma: association with elevated alpha-fetoprotein production.Gastroenterology. 1996; 110: 184-192Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 33Nagai H Pineau P Tiollais P Buendia MA Dejean A Comprehensive allelotyping of human hepatocellular carcinoma.Oncogene. 1997; 14: 2927-2933Crossref PubMed Scopus (268) Google Scholar, 34Bando K Nagai H Matsumoto S Koyama M Kawamura N Onda M Emi M Identification of a 1-cM region of common deletion on 4q35 associated with progression of hepatocellular carcinoma.Genes Chromosom Cancer. 1999; 25: 284-289Crossref PubMed Scopus (46) Google Scholar, 35Wong N Lai P Lee SW Fan S Pang E Liew CT Sheng Z Lau JW Johnson PJ Assessment of genetic changes in hepatocellular carcinoma detected by comparative genomic hybridization: relationship to disease stage, tumor size, and cirrhosis.Am J Pathol. 1999; 154: 37-43Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar Introduction of normal human chromosome 4 leads to suppressed tumorigenicity of teratocarcinoma PA-1 cells in nude mice, suggestive of a putative tumor suppressor gene on chromosome 4.35Wong N Lai P Lee SW Fan S Pang E Liew CT Sheng Z Lau JW Johnson PJ Assessment of genetic changes in hepatocellular carcinoma detected by comparative genomic hybridization: relationship to disease stage, tumor size, and cirrhosis.Am J Pathol. 1999; 154: 37-43Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar The biological function of ANXA10 and its role in HCC are not known. Using differential display, we found a cDNA clone identical to ANXA1015 that was expressed in liver and often down-regulated in HCC. In this study, we correlate the mRNA expression of ANXA10 in HCC with clinicopathological features. We show that ANXA10 might be a potential tumor suppressor gene because its down-regulation is associated with malignant phenotype of liver cells, and correlates with vascular invasion and progression of HCC. Between 1987 and 1997, 909 surgically resected primary and 127 recurrent HCCs were pathologically assessed at the National Taiwan University Hospital. The tissue samples were immediately cut into small pieces, snap-frozen in liquid nitrogen, and stored in deep freezers. Of these, 187 patients who already had RNA samples taken from resected HCCs were analyzed for ANXA10 mRNA levels, including 182 unifocal and 5 multifocal primary HCCs. To elucidate the clinical implication of ANXA10 expression, the 182 cases of unifocal primary HCC, as previously defined,36Hsu HC Chiou TJ Chen ZY Lee CS Lee PH Peng SY Clonality and clonal evolution of hepatocellular carcinoma with multiple nodules.Hepatology. 1991; 13: 923-928Crossref PubMed Scopus (113) Google Scholar, 37Hsu HC Tseng HJ Lai PL Lee PH Chen DS Peng SY Expression of p53 gene in 184 unifocal hepatocellular carcinomas (HCC): association with tumor growth and invasiveness.Cancer Res. 1993; 53: 4691-4694PubMed Google Scholar, 38Peng SY Lai PL Chu JS Lee PH Tsung PT Chen DS Hsu HC Expression and hypomethylation of alpha-fetoprotein gene in unicentric and multicentric human hepatocellular carcinomas.Hepatology. 1993; 17: 35-41Crossref PubMed Scopus (70) Google Scholar, 39Hsu HC Peng SY Lai PL Chu JS Lee PH Mutations of p53 gene in hepatocellular carcinoma (HCC) correlate with tumor progression and patient prognosis: a study of 138 patients with unifocal HCC.Int J Oncol. 1994; 4: 1341-1347PubMed Google Scholar were selected for clinicopathological analysis. Multifocal HCC was excluded from this correlation because of incomplete sampling of the tumor nodules for analysis and their variation in pathological features. To study the tissue and tumor distribution, we also examined the mRNA expression of ANXA10 in multiple adult tissues and several common tumor tissues obtained from surgical pathology specimens, and multiple fetal tissues obtained from autopsy. The intrahepatic tumor recurrence (IHTR) was based on imaging diagnosis with ultrasonography and/or computed tomography, supplemented with elevated serum α-fetoprotein (AFP). Until the end of October 2001, tumor recurrence was detected in 113 patients. Among the 182 patients studied, 156 were eligible for the evaluation of early IHTR (≤1 year). Twenty-six patients who died within 1 year after resection and had no information about or were negative for IHTR were excluded from the evaluation of early recurrence. The tumor grade was simply divided into three groups: well (grade I), moderately (grade II), and poorly differentiated HCC (grades III and IV). Resected unifocal HCC was staged as stage I, II, IIIA, IIIB, and IV as previously described.40Hsu HC Jeng YM Mao TL Chu JS Lai PL Peng SY β-Catenin mutations are associated with a subset of low stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis.Am J Pathol. 2000; 157: 763-770Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar Briefly, stage I HCC is encapsulated and without parenchymal invasion. Stage II HCC has liver invasion, but no vascular invasion. Stage IIIA HCC has invasion of small vessels in the tumor capsule. Stage IIIB and IV HCCs have invasion of portal vein branches. The pathological stage of HCC correlates closely with survival.40Hsu HC Jeng YM Mao TL Chu JS Lai PL Peng SY β-Catenin mutations are associated with a subset of low stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis.Am J Pathol. 2000; 157: 763-770Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar Differential display was performed according to the recommendation of the manufacturer using the RNAimage kit (GenHunter Corp., Nashville, TN). Total RNA (0.2 μg) was reverse-transcribed in a mixture containing 100 U MMLV reverse transcriptase, 20 μmol/L dNTP, and 0.2 μmol/L anchor primer H-T11C (5′-AAGCT11C-3′). The reverse transcription products (2 μl) were subjected to polymerase chain reaction (PCR) amplification in a PCR mixture containing 2 μmol/L dNTP, 0.2 μmol/L H-T11C (5′-AAGCT11C-3′), 25 mCi/mmol [α-33P]dATP (Amersham, Buckinghamshire, England), 1 U Vent DNA polymerase (New England Biolabs, Inc. Beverly, MA), 0.2 μmol/L arbitrary primer HAP35 (5′-AAGCTTCAGGGCA-3′). PCR was done in Perkin-Elmer's 9600 thermocycler (94°C for 30 seconds, 40°C for 2 minutes, 72°C for 1 minute for 40 cycles, and a final elongation at 72°C for 5 minutes). The amplification products were separated by 6% denaturing polyacrylamide gel electrophoresis. The dried gel was then exposed by film autoradiography. The differentially expressed band was cut, eluted, and sequenced. RT-PCR assays were used for large-scale quantitative measurements for ANXA10 mRNA levels, as previously described,41Peng SY Chou SP Hsu HC Association of downregulation of cyclin D1 and of overexpression of cyclin E with p53 mutation, high tumor grade and poor prognosis in hepatocellular carcinoma.J Hepatol. 1998; 29: 281-289Abstract Full Text PDF PubMed Scopus (64) Google Scholar using S26 ribosomal protein mRNA as internal control.42Vincent S Marty L Fort P S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eucaryotic cells and tissues.Nucleic Acids Res. 1993; 21: 1498Crossref PubMed Scopus (202) Google Scholar Briefly, 2 μl of RT product, 1.25 U Pro Taq DNA polymerase (Protech Technology Enterprise Co, Taipei, Taiwan), Pro Taq buffer, and 200 μmol/L each dATP, dTTP, dGTP, and dCTP were mixed with primer pairs for ANXA10 and S26 in a total volume of 30 μl. The PCR was performed in an automated DNA thermal cycler 480 (Perkin-Elmer/Cetus). The PCR was stopped at the exponential phases, 29 cycles for ANXA10 and 22 cycles for S26. These experiments were repeated when the S26 loading showed inappropriate template amount for PCR. The primers used included the sense primer ANXA10-A: 5′-TTGTTCTCTGTGTTCGAGAGAAACC-3′, and anti-sense primer ANXA10-F: 5′-GTAGGCAAATTCAGGATAGTAGGC-3′. The products were electrophoresed on 2% agarose gel. The ANXA10 mRNA expression level was determined by the ratio of signal intensity of ANXA10 to that of S26 measured by 1D Image Analysis Software (Kodak Digital Science, Rochester, NY), and scored as high (ratio ≥ 1.0), intermediate (0.5 ≤ ratio < 1.0), trace (ratio < 0.5), and negative (or immeasurable). An ANXA10/S26 ratio 4 years or until death. The analyses were performed using the Statistica for Windows software (Statsoft, Inc., Chicago, IL). Two-tailed chi-square and Fisher exact tests were used for univariate analysis. The cumulative survival after tumor removal was calculated with the log-rank test. P values <0.05 were considered statistically significant. We used differential display for the analysis of genes aberrantly expressed in HCC, and cloned a 192-bp cDNA fragment that was consistently expressed in liver and often down-regulated in HCC. This cDNA clone was identical to ANXA1015 (GenBank GI no. 9625245). A nearly full-length cDNA of 1268-bp long was subsequently cloned from Hep3B cell line by RT-PCR amplification; the sequence was identical to ANXA10.15Morgan RO Jenkins NA Gilbert DJ Copeland NG Balsara BR Testa JR Fernandez MP Novel human and mouse annexin A10 are linked to the genome duplications during early chordate evolution.Genomics. 1999; 60: 40-49Crossref PubMed Scopus (59) Google Scholar The preferential down-regulation of ANXA10 in HCC was verified by Northern blotting (Figure 1). Using RT-PCR for large-scale analysis, ANXA10 mRNA was expressed abundantly in liver, and in low level in only 12 (6.4%) of 176 livers harboring benign (focal nodular hyperplasia) and malignant tumors (HCC, cholangiocarcinoma, metastatic carcinoma). In contrast, ANXA10 mRNA was dramatically decreased in 121 of 182 unifocal primary HCCs (66%) as compared to paired nontumor livers (Table 1; Figure 2).Table 1ANXA10 mRNA Expression in Benign and Malignant Human TumorsANXA10 mRNA expressionTumor typeExpressedNot expressed or dramatically decreasedHepatocellular carcinoma (n = 182)61121 (66%)Hepatoblastoma (n = 5)05 (100%)Cholangiocarcinoma (n = 2)02Metastatic colorectal carcinoma (n = 10)*Includes one ovarian and one breast, and eight metastatic colorectal carcinomas.010 (100%)Focal nodular hyperplasia (n = 9)81 (11%)Ovarian carcinoma (n = 16)016 (100%)Lung carcinoma (n = 3)03Breast carcinoma (n = 10)010 (100%)* Includes one ovarian and one breast, and eight metastatic colorectal carcinomas. Open table in a new tab Figure 2RT-PCR of ANXA10 mRNA in paired HCC and liver. ANXA10 mRNA (A10) was dramatically decreased in HCC (T) in 16 of 22 representative cases. Stage, tumor stage (I to IV); size, tumor size (cm).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To elucidate the tissue-specific expression of the ANXA10, RNA samples taken from multiple fetal and adult tissues were analyzed. Using polyA+ RNA blot obtained from multiple adult tissues (RPN4800; Amersham Pharmacia Biotech, Buckinghamshire, UK), a single ANXA10 transcript of ∼1.3 kb was identified in adult liver alone (Figure 3). By RT-PCR analysis, ANXA10 mRNA was expressed only in a very limited number of adult tissues, abundant in liver and stomach, low in pancreas, and not expressed in other adult tissues, such as lung, heart, breast, kidney, and spleen (Figure 4). ANXA10 mRNA was also undetectable in most fetal tissues, such as brain, thyroid, lung, heart, liver, kidney, adrenal gland, and testes. ANXA10 mRNA was expressed abundantly in fetal stomach (Figure 4). Among three fetal livers taken from fetuses less than gestation age of 22 weeks and without congenital anomalies, ANXA10 mRNA was expressed in low level in one and not detected in two (Figure 4).Figure 4Expression of ANXA10 in multiple adult and fetal tissues. ANXA10 mRNA (A10) was expressed in abundance in adult and fetal stomach, in low level in adult pancreas, but undetectable in most adult tissues and fetal tissues.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ANXA10 mRNA was expressed in benign liver lesions such as focal nodular hyperplasia, and decreased dramatically in hepatoblastoma (Table 1). To clarify the tumor-specific distribution, we analyzed the ANXA10 mRNA expression in several common carcinomas of other anatomical sites. As shown in Table 1, ANXA10 mRNA was undetectable or weakly expressed in carcinomas of the ovary, breast, colon, and lung, and metastatic carcinomas of the liver (Figure 5). To elucidate the biological significance of its expression, we correlated the ANXA10 expression with clinicopathological features. As shown in Table 2, the decreased ANXA10 expression was closely associated with serum AFP elevation (P < 0.00001). The ANXA10 expression did not correlate with age, gender, hepatitis B virus (HBV) infection, and liver cirrhosis (data not shown). Histopathologically, down-regulation of ANXA10 was found more often in large HCCs than in HCCs <3 cm (P = 0.0012), in grade II and III than in grade I HCCs (P < 0.00001), and in HCCs with vascular invasion (stage IIIA to IV) than in stage I to II HCCs that has no vascular invasion (P < 0.00001).Table 2Correlation of ANXA10 mRNA Expression with Clinicopathological Features in 182 Patients with Unifocal Primary Hepatocellular CarcinomaANXA10 mRNA expressionNormal expression (n = 61)Down-regulation (n = 121)P valueAFP <320 ng/ml (n = 97)5146 (47%) 3 cm (n = 137)37100 (73%)Tumor grade I (n = 39)2514 (36%)<0.00001 II–III (n = 142)36106 (75%)Tumor stage I–II (n = 83)4241 (49%)<0.00001 IIIA–IV (n = 99)1980 (81%)Early recurrence (≤1-yr)*Tumor recurrence in the liver was detected ≤12 months after resection of the primary HCC.14/52 (27%)58/104 (56%)0.0007* Tumor recurrence in the liver was detected ≤12 months after resection of the primary HCC. Open table in a new tab Down-regulation of ANXA10 was associated with higher frequency of early intrahepatic tumor recurrence (P = 0.0007). Furthermore, HCC with down-regulation of ANXA10 was associated with a lower 4-year patient survival rate than HCC with normal ANXA10 expression, 54% versus 33%, P = 0.0014 (Figure 6). Because p53 mutation correlates closely with tumor aggressiveness and unfavorable prognosis of HCC,37Hsu HC Tseng HJ Lai PL Lee PH Chen DS Peng SY Expression of p53 gene in 184 unifocal hepatocellular carcinomas (HCC): association with tumor growth and invasiveness.Cancer Res. 1993; 53: 4691-4694PubMed Google Scholar, 39Hsu HC Peng SY Lai PL Chu JS Lee PH Mutations of p53 gene in hepatocellular carcinoma (HCC) correlate with tumor progression and patient prognosis: a study of 138 patients with unifocal HCC.Int J Oncol. 1994; 4: 1341-1347PubMed Google Scholar we analyzed the potential interplay between these two important unfavorable prognostic factors. In this study, p53 mutation was found in 77 (49%) of 156 HCCs examined. Down-regulation of ANXA10 correlated with p53 mutation, P = 0.024 (Table 2). HCC with both ANXA10 down-regulation and p53 mutation had the highest frequencies of grade II to III and stage IIIA to IV tumors than other groups, P < 0.00001 and P < 0.00001, respectively (Table 3). Moreover, HCCs with both ANXA10 down-regulation and p53 mutation also had the most frequent early intrahepatic tumor recurrence, P = 0.0001 (Table 3), and the worst 4-year survival, P = 0.0025 (Figure 7).Table 3Synergistic Effect between ANXA10 Down-Regulation and p53 Mutation in Unifocal Hepatocellular CarcinomaDown-Regulation of ANXA10/p53 mutationFeatureNo/No n = 33Yes/No n = 46No/Yes n = 19Yes/Yes n = 58P valueTumor grade I16 (48%)9 (20%)4 (21%)4 (7%)<0.00001 II–III17371554Tumor stage I–II26 (79%)23 (50%)10 (53%)13 (22%)<0.00001 III–IV823945Early intrahepatic tumor recurrence*Tumor recurs in the liver ≤12 months after resection of the primary HCC.7/30 (23%)15/38 (39%)6/15 (40%)36/53 (68%)0.0001* Tumor recurs
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