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Su L, Bu J, Yu J, Jin M, Meng G, Zhu X. Comprehensive review and updated analysis of DNA methylation in hepatocellular carcinoma: From basic research to clinical application. Clin Transl Med 2024; 14:e70066. [PMID: 39462685 PMCID: PMC11513202 DOI: 10.1002/ctm2.70066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2024] [Revised: 09/30/2024] [Accepted: 10/08/2024] [Indexed: 10/29/2024] Open
Abstract
Hepatocellular carcinoma (HCC) is a primary malignant tumour, ranking second in global mortality rates and posing significant health threats. Epigenetic alterations, particularly DNA methylation, have emerged as pivotal factors associated with HCC diagnosis, therapy, prognosis and malignant progression. However, a comprehensive analysis of the DNA methylation mechanism driving HCC progression and its potential as a therapeutic biomarker remains lacking. This review attempts to comprehensively summarise various aspects of DNA methylation, such as its mechanism, detection methods and biomarkers aiding in HCC diagnosis, treatment and prognostic assessment of HCC. It also explores the role of DNA methylation in regulating HCC's malignant progression and sorafenib resistance, alongside elaborating the therapeutic effects of DNA methyltransferase inhibitors on HCC. A detailed examination of these aspects underscores the significant research on DNA methylation in tumour cells to elucidate malignant progression mechanisms, identify diagnostic markers and develop new tumour-specific inhibitors for HCC. KEY POINTS: A comprehensive summary of various aspects of DNA methylation, such as its mechanism, detection methods and biomarkers aiding in diagnosis and treatment. The role of DNA methylation in regulating hepatocellular carcinoma's (HCC) malignant progression and sorafenib resistance, alongside elaborating therapeutic effects of DNA methyltransferase inhibitors. Deep research on DNA methylation is critical for discovering novel tumour-specific inhibitors for HCC.
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Affiliation(s)
- Lin Su
- Department of Pain ManagementShengjing Hospital of China Medical UniversityShenyangLiaoningChina
| | - Jiawen Bu
- Department of Colorectal SurgeryShengjing Hospital of China Medical UniversityShenyangLiaoningChina
| | - Jiahui Yu
- Department of UltrasoundShengjing Hospital of China Medical UniversityShenyangLiaoningChina
| | - Mila Jin
- Department of Operation RoomThe First Hospital of China Medical UniversityShenyangLiaoningChina
| | - Guanliang Meng
- Department of UrologyShengjing Hospital of China Medical UniversityShenyangLiaoningChina
| | - Xudong Zhu
- Department of OncologyShengjing Hospital of China Medical UniversityShenyangLiaoningChina
- Department of General SurgeryCancer Hospital of China Medical UniversityShenyangLiaoningChina
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Simon C, Stielow B, Nist A, Rohner I, Weber LM, Geller M, Fischer S, Stiewe T, Liefke R. The CpG Island-Binding Protein SAMD1 Contributes to an Unfavorable Gene Signature in HepG2 Hepatocellular Carcinoma Cells. BIOLOGY 2022; 11:557. [PMID: 35453756 PMCID: PMC9032685 DOI: 10.3390/biology11040557] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 03/23/2022] [Accepted: 04/05/2022] [Indexed: 06/14/2023]
Abstract
The unmethylated CpG island-binding protein SAMD1 is upregulated in many human cancer types, but its cancer-related role has not yet been investigated. Here, we used the hepatocellular carcinoma cell line HepG2 as a cancer model and investigated the cellular and transcriptional roles of SAMD1 using ChIP-Seq and RNA-Seq. SAMD1 targets several thousand gene promoters, where it acts predominantly as a transcriptional repressor. HepG2 cells with SAMD1 deletion showed slightly reduced proliferation, but strongly impaired clonogenicity. This phenotype was accompanied by the decreased expression of pro-proliferative genes, including MYC target genes. Consistently, we observed a decrease in the active H3K4me2 histone mark at most promoters, irrespective of SAMD1 binding. Conversely, we noticed an increase in interferon response pathways and a gain of H3K4me2 at a subset of enhancers that were enriched for IFN-stimulated response elements (ISREs). We identified key transcription factor genes, such as IRF1, STAT2, and FOSL2, that were directly repressed by SAMD1. Moreover, SAMD1 deletion also led to the derepression of the PI3K-inhibitor PIK3IP1, contributing to diminished mTOR signaling and ribosome biogenesis pathways. Our work suggests that SAMD1 is involved in establishing a pro-proliferative setting in hepatocellular carcinoma cells. Inhibiting SAMD1's function in liver cancer cells may therefore lead to a more favorable gene signature.
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Affiliation(s)
- Clara Simon
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
| | - Bastian Stielow
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
| | - Andrea Nist
- Genomics Core Facility, Faculty of Medicine, Institute of Molecular Oncology, Member of the German Center for Lung Research (DZL), Philipps University of Marburg, 35043 Marburg, Germany; (A.N.); (T.S.)
| | - Iris Rohner
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
| | - Lisa Marie Weber
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
| | - Merle Geller
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
| | - Sabrina Fischer
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
| | - Thorsten Stiewe
- Genomics Core Facility, Faculty of Medicine, Institute of Molecular Oncology, Member of the German Center for Lung Research (DZL), Philipps University of Marburg, 35043 Marburg, Germany; (A.N.); (T.S.)
| | - Robert Liefke
- Institute of Molecular Biology and Tumor Research (IMT), Faculty of Medicine, Philipps University of Marburg, 35043 Marburg, Germany; (C.S.); (B.S.); (I.R.); (L.M.W.); (M.G.); (S.F.)
- Department of Hematology, Oncology, and Immunology, University Hospital Giessen and Marburg, 35043 Marburg, Germany
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Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines. J Gastrointest Cancer 2021; 52:1320-1335. [PMID: 34463913 DOI: 10.1007/s12029-021-00704-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/23/2021] [Indexed: 10/20/2022]
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines. METHODS Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway. RESULTS We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes. CONCLUSION Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.
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Hayashi T, Shibata T, Nakamura M, Sakurai N, Takano H, Ota M, Nomura-Horita T, Hayashi R, Shimasaki T, Ostuka T, Tahara T, Arisawa T. MAFK Polymorphisms Located in 3'-UTR are Associated with Severity of Atrophy and CDKN2A Methylation Status in the Gastric Mucosa. Genet Test Mol Biomarkers 2021; 25:255-262. [PMID: 33877894 DOI: 10.1089/gtmb.2020.0299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Objective: This study aimed to clarify the association of MAFK polymorphisms (rs4268033, rs3735656, and rs10226620) with the degree of gastric mucosal atrophy and CDKN2A CpG methylation status. Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Results: Either rs3735656 or rs10226620, located in the 3'-UTR of MAFK, was significantly associated with the severity of gastric mucosal atrophy using a dominant genetic model (odds ratio [OR], 2.10; p = 0.0012, and OR, 1.98; p = 0.0027, respectively). However, using a recessive genetic model, no significant association was found between three polymorphisms and gastric mucosal atrophy. The serum pepsinogen I/II ratio was significantly lower in subjects with minor alleles of rs3735656 and rs10226620 than in subjects with the wild homozygous allele (p = 0.018 and 0.013, respectively). In a subgroup including 400 of the 491 subjects, the CpG of p14ARF and p16 INK4a were methylated in 132 and 112 subjects, respectively. Fifty subjects had both CpG methylations and 206 subjects had neither methylation. When comparing the groups with both and neither methylations, there were no significant associations between three polymorphisms and CDKN2A methylation using a dominant genetic model. However, all polymorphisms investigated in this study (rs4268033, rs3735656, and rs10226620) were significantly associated with CDKN2A methylation in a recessive genetic model (OR, 3.58; p = 0.0071, OR, 4.49; p = 0.0004, and OR, 3.45; p = 0.0027, respectively). Conclusions: Our results indicate that carrying the minor allele of the MAFK polymorphisms, particularly when they are located in the 3'-UTR, has a high risk for the severity of gastric mucosal atrophy; furthermore, CDKN2A CpG methylation may develop in subjects with homozygous minor allele of these polymorphisms.
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Affiliation(s)
- Tasuku Hayashi
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Tomoyuki Shibata
- Department of Gastroenterology, Fujita Health University, Kutsukake-cho, Japan
| | - Masakatsu Nakamura
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Naoko Sakurai
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Hikaru Takano
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Masafumi Ota
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Tomoe Nomura-Horita
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Ranji Hayashi
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Takeo Shimasaki
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Toshimi Ostuka
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
| | - Tomomitsu Tahara
- Department of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan
| | - Tomiyasu Arisawa
- Department of Gastroenterology, Kanazawa Medical University, Uchinada-machi, Japan
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Effect of DNMT3A polymorphisms on CpG island hypermethylation in gastric mucosa. BMC MEDICAL GENETICS 2020; 21:205. [PMID: 33066747 PMCID: PMC7562764 DOI: 10.1186/s12881-020-01142-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 10/08/2020] [Indexed: 01/02/2023]
Abstract
Background CpG methylation of tumor suppressor genes occurs in the early stage of carcinogenesis. Detecting risk factors for aberrant CpG methylation is clinically important for predicting cancer development. DNA methyltransferase (DNMT) 3a is considered to play critical roles in the DNA methylation process during pathogenesis. In this study, we evaluated the association between DNMT3A polymorphisms (rs6733868 and rs13428812) and CpG methylation status in non-cancerous gastric mucosa. Methods We determined the DNMT3A genotype and CpG methylation status of 4 genes (p14ARF, p16INK4a, DAPK, and CDH1) in 510 subjects without gastric cancer. Helicobacter pylori (HP) infection status was determined by the rapid urease test, urea breath test, speculum examination, or serum antibody test. We determined the DNMT3A genotype using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). CpG methylation status was determined by methylation-specific polymerase chain reaction (MSP). When the methylated band was stronger than 10 ng/μL according to the DNA marker, we judged CpG island hypermethylation (CIHM) to be present. Associations between genotypes and susceptibilities were assessed by logistic regression analysis. Results The minor allele frequencies of both polymorphisms (rs6733868 and rs13428812) were lower in the CpG methylated groups of each of the 4 genes (p14ARF, p16INK4a, DAPK, and CDH1). Using a dominant genetic model, rs6733868 was significantly associated with the hypermethylation of each gene, whereas rs13428812 was associated with the methylation of 3 genes (all except p14ARF). When low-CIHM was defined as 1 or 2 CpG islands methylated and high-CIHM was defined as 3 or more CpG islands methylated, carrying the minor allele of rs6733868 was associated with both decreased low- and high-CIHM, and that of rs13428812 also was associated with a decrease. Comparing low-CIHM with high-CIHM, carrying the minor alleles of rs6733868 or rs13428812 was related to decreased susceptibility to high-CIHM. In HP-infected subjects, carrying the minor alleles of rs6733868 or rs13428812 had a significantly greater association with decreased susceptibility to high-CIHM. Conclusions Our study indicates that polymorphisms of DNMT3A are associated with the accumulation of gene methylation in gastric mucosa. Carrying the minor alleles of rs6733868 or rs13428812 inhibits aberrant gene methylations, which are typically enhanced by HP infection.
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Sakurai N, Shibata T, Nakamura M, Takano H, Hayashi T, Ota M, Nomura-Horita T, Hayashi R, Shimasaki T, Ostuka T, Tahara T, Arisawa T. Influence of MIF polymorphisms on CpG island hyper-methylation of CDKN2A in the patients with ulcerative colitis. BMC MEDICAL GENETICS 2020; 21:201. [PMID: 33046033 PMCID: PMC7552536 DOI: 10.1186/s12881-020-01140-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 08/11/2020] [Accepted: 10/06/2020] [Indexed: 12/31/2022]
Abstract
Background CDKN2A hypermethylation is among the major events associated with carcinogenesis and is also observed in non-neoplastic colonic mucosa in patients with ulcerative colitis (UC). Macrophage migration inhibitory factor (MIF) plays a crucial role in promoting gastrointestinal inflammation characteristic of UC. The aim of this study is to explore associations between CDKN2A methylation status and MIF polymorphisms (rs755622 and rs5844572). Methods One hundred and fifty-nine patients diagnosed with UC were enrolled in this study. The methylation status of p14ARF and p16INK4a was determined by MSP; MIF genotypes were identified by PCR-SSCP. Results We found no differences with respect to mean age, gender, clinical type (chronic continuous or relapse/remitting), or extent of disease among the patients with methylated and unmethylated p14ARF or p16INK4a. Carrying the rs755622 C allele indicated a significantly higher risk for p14ARF methylation (odds ratio (OR), 2.16; 95% confidence interval (CI), 1.08–4.32; p = 0.030); similarly, carrying the rs5844572 7-repeat allele indicated a significantly higher risk for p16INK4a methylation (OR, 2.57; 95% CI, 1.26–5.24; p = 0.0094) after an adjusted regression analysis. The carriers of the rs755662 C allele or the rs5844572 7-repeat allele were both at a significantly higher risk for methylation of both p14ARF and p16INK4a when compared to the cohort in which neither of the genes were methylated (OR, 2.70; 95% CI, 1.22–6.01; p = 0.015 and OR, 2.87; 95% CI, 1.25–6.62; p = 0.013, respectively). Additionally, carrying rs755622 C allele was significantly associated with CIHM in chronic continuous of clinical type and total colitis (OR, 25.9; 95% CI, 2.55–262.6; p = 0.0059 and OR, 4.38; 95% CI, 1.12–17.2; p = 0.034, respectively), and carrying 7-repeat allele of rs5844572 was significantly associated in chronic continuous type (OR, 14.5; 95%CI, 1.46–144.3; p = 0.022). Conclusions Taken together, our findings suggest that MIF genotypes associated with inflammation may also be involved in promoting carcinogenesis via CDKN2A hypermethylation in patients diagnosed with UC.
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Affiliation(s)
- Naoko Sakurai
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Tomoyuki Shibata
- Department of Gastroenterology, Fujita Health University, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake, 470-1192, Japan
| | - Masakatsu Nakamura
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Hikaru Takano
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Tasuku Hayashi
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Masafumi Ota
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Tomoe Nomura-Horita
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Ranji Hayashi
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Takeo Shimasaki
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Toshimi Ostuka
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan
| | - Tomomitsu Tahara
- Department of Gastroenterology and Hepatology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka, 573-1010, Japan
| | - Tomiyasu Arisawa
- Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan.
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Rice A, Del Rio Hernandez A. The Mutational Landscape of Pancreatic and Liver Cancers, as Represented by Circulating Tumor DNA. Front Oncol 2019; 9:952. [PMID: 31608239 PMCID: PMC6769086 DOI: 10.3389/fonc.2019.00952] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2018] [Accepted: 09/09/2019] [Indexed: 02/06/2023] Open
Abstract
The mutational landscapes of pancreatic and liver cancers share many common genetic alterations which drive cancer progression. However, these mutations do not occur in all cases of these diseases, and this tumoral heterogeneity impedes diagnosis, prognosis, and therapeutic development. One minimally invasive method for the evaluation of tumor mutations is the analysis of circulating tumor DNA (ctDNA), released through apoptosis, necrosis, and active secretion by tumor cells into various body fluids. By observing mutations in those genes which promote transformation by controlling the cell cycle and oncogenic signaling pathways, a representation of the mutational profile of the tumor is revealed. The analysis of ctDNA is a promising technique for investigating these two gastrointestinal cancers, as many studies have reported on the accuracy of ctDNA assessment for diagnosis and prognosis using a variety of techniques.
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Affiliation(s)
- Alistair Rice
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Faculty of Engineering, Imperial College London, South Kensington Campus, London, United Kingdom
| | - Armando Del Rio Hernandez
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Faculty of Engineering, Imperial College London, South Kensington Campus, London, United Kingdom
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Serhal R, Saliba N, Hilal G, Moussa M, Hassan GS, El Atat O, Alaaeddine N. Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis. World J Gastroenterol 2019; 25:567-583. [PMID: 30774272 PMCID: PMC6371009 DOI: 10.3748/wjg.v25.i5.567] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/08/2018] [Revised: 12/02/2018] [Accepted: 12/07/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the effect of adipose-derived mesenchymal stem cells (ADMSCs) and their conditioned media (CM) on hepatocellular carcinoma (HCC) cell tumorigenesis.
METHODS The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit 8 (commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.
RESULTS Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.
CONCLUSION These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.
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Affiliation(s)
- Rim Serhal
- Regenerative Medicine Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut 1107 2180, Lebanon
| | - Nagib Saliba
- Surgery Department, Faculty of Medicine, Saint-Joseph University and Hotel-Dieu de France, Beirut 1107 2180, Lebanon
| | - George Hilal
- Cancer and Metabolism Laboratory, Faculty of Medicine, Campus of Medical Sciences, Saint-Joseph University, Beirut 1107 2180, Lebanon
| | - Mayssam Moussa
- Regenerative Medicine Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut 1107 2180, Lebanon
| | - Ghada S Hassan
- Laboratoire d’Immunologie Cellulaire et Moléculaire, Centre Hospitalier de l’Université de Montréal, Montréal, QC H2X 0A9, Canada
| | - Oula El Atat
- Regenerative Medicine Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut 1107 2180, Lebanon
| | - Nada Alaaeddine
- Regenerative Medicine Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut 1107 2180, Lebanon
- Laboratoire d’Immunologie Cellulaire et Moléculaire, Centre Hospitalier de l’Université de Montréal, Montréal, QC H2X 0A9, Canada
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Zhou Y, Wang XB, Qiu XP, Shuai Zhang, Wang C, Zheng F. CDKN2A promoter methylation and hepatocellular carcinoma risk: A meta-analysis. Clin Res Hepatol Gastroenterol 2018; 42:529-541. [PMID: 30143452 DOI: 10.1016/j.clinre.2017.07.003] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2017] [Revised: 07/17/2017] [Accepted: 07/28/2017] [Indexed: 02/04/2023]
Abstract
AIM Lots of studies have explored cyclin-dependent kinase inhibitor 2A (CDKN2A) promoter methylation in hepatocellular carcinoma (HCC), but the established results were controversial. Hence, we conducted the meta-analysis to comprehensively investigate the association between CDKN2A promoter methylation and HCC risk. METHODS A comprehensive search was implemented through searching PubMed, Web of Science and Embase. Associations of CDKN2A promoter methylation with HCC risk, clinicopathological features, and CDKN2A expression were assessed by the pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Subgroup analyses and meta-regression were served for exploring the potential sources of heterogeneity. RESULTS A total of 59 articles including 3067 cases and 2951 controls were incorporated in this meta-analysis. Overall, we observed a high CDKN2A promoter methylation rate (58.18%) in HCC and a significant association between the methylation and HCC risk (OR, 7.07; 95% CI, 5.67-8.80). Furthermore, CDKN2A promoter methylation was robustly associated with decreased mRNA (OR, 13.89; 95% CI, 5.44-35.45) and protein (OR, 48.19; 95% CI, 5.56-417.29). In addition, we found the methylation was related with HBV infection (OR, 3.31; 95% CI, 1.47-7.47), HCV infection (OR, 2.76; 95% CI, 1.80-4.23), cirrhosis status (OR, 1.57; 95% CI, 1.01-2.44) and older age (OR, 1.83; 95% CI, 1.14-2.94). CONCLUSIONS Our results indicated that CDKN2A promoter methylation was associated with an enhancive HCC risk and played a crucial role in the process of HCC with a potential value to being a triage marker for HCC.
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Affiliation(s)
- Ye Zhou
- Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, 430071 Hubei, China
| | - Xue-Bin Wang
- Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, 430071 Hubei, China
| | - Xue-Ping Qiu
- Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, 430071 Hubei, China
| | - Shuai Zhang
- Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, 430071 Hubei, China
| | - Chen Wang
- Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, 430071 Hubei, China
| | - Fang Zheng
- Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, 430071 Hubei, China.
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MDM2-p53 Interactions in Human Hepatocellular Carcinoma: What Is the Role of Nutlins and New Therapeutic Options? J Clin Med 2018; 7:jcm7040064. [PMID: 29584707 PMCID: PMC5920438 DOI: 10.3390/jcm7040064] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Revised: 03/22/2018] [Accepted: 03/23/2018] [Indexed: 12/18/2022] Open
Abstract
Human hepatocellular carcinoma (HCC) is the fifth most common cancer and is associated with poor prognosis worldwide. The molecular mechanisms underlying the pathogenesis of HCC have been an area of continuing interest, and recent studies using next generation sequencing (NGS) have revealed much regarding previously unsettled issues. Molecular studies using HCC samples have been mainly targeted with the aim to identify the fundamental mechanisms contributing to HCC and identify more effective treatments. In response to cellular stresses (e.g., DNA damage or oncogenes), activated p53 elicits appropriate responses that aim at DNA repair, genetic stability, cell cycle arrest, and the deletion of DNA-damaged cells. On the other hand, the murine double minute 2 (MDM2) oncogene protein is an important cellular antagonist of p53. MDM2 negatively regulates p53 activity through the induction of p53 protein degradation. However, current research has shown that the mechanisms underlying MDM2-p53 interactions are more complex than previously thought. Microarray data have added new insight into the transcription changes in HCC. Recently, Nutlin-3 has shown potency against p53-MDM2 binding and the enhancement of p53 stabilization as well as an increment of p53 cellular accumulation with potential therapeutic effects. This review outlines the molecular mechanisms involved in the p53-MDM2 pathways, the biological factors influencing these pathways, and their roles in the pathogenesis of HCC. It also discusses the action of Nutlin-3 treatment in inducing growth arrest in HCC and elaborates on future directions in research in this area. More research on the biology of p53-MDM2 interactions may offer a better understanding of these mechanisms and discover new biomarkers, sensitive prognostic indicators as well as new therapeutic interventions in HCC.
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Kopanja D, Huang S, Al Raheed MRH, Guzman G, Raychaudhuri P. p19Arf inhibits aggressive progression of H-ras-driven hepatocellular carcinoma. Carcinogenesis 2018; 39:318-326. [PMID: 29228217 DOI: 10.1093/carcin/bgx140] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Accepted: 11/15/2017] [Indexed: 11/14/2022] Open
Abstract
Arf, a well-established tumor suppressor, is either mutated or downregulated in a wide array of cancers. However, its role in hepatocellular carcinoma (HCC) progression is controversial. Conflicting observations have been published regarding its expression in HCC. In this study, we provide clear genetic evidence demonstrating a protective role of p19Arf in hepatocarcinogenesis. Using Ras-induced mouse model, we show that p19Arf deficiency accelerates progression of aggressive HCC in vivo. To investigate the role of p14ARF in human liver cancers, we analyzed its expression in human HCC using immunohistochemistry (IHC). We observe lack of nucleolar p14ARF in 43.02% of human HCC samples and that low expression of p14ARF strongly correlates with the early onset of HCC. Importantly, cirrhotic livers that did not progress to HCC harbor higher expression of the p14ARF protein in hepatocytes compared with that in cirrhotic livers with HCC. These results are significant because they suggest that nucleolar p14ARF can be used as early prognostic marker in chronic liver disease to reliably identify patients with high risk for developing liver cancer. Currently, there is no effective systemic therapy for advanced liver cancer; hence, more efficient patient screening and early detection of HCC would significantly contribute to the eradication of this devastating disease.
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Affiliation(s)
- Dragana Kopanja
- Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois, College of Medicine, USA
| | - Shuo Huang
- Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois, College of Medicine, USA
| | | | - Grace Guzman
- Department of Pathology, University of Illinois, College of Medicine, USA
| | - Pradip Raychaudhuri
- Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois, College of Medicine, USA.,Department of Research, Jesse Brown VA Medical Center, USA
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12
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Lv X, Ye G, Zhang X, Huang T. p16 Methylation was associated with the development, age, hepatic viruses infection of hepatocellular carcinoma, and p16 expression had a poor survival: A systematic meta-analysis (PRISMA). Medicine (Baltimore) 2017; 96:e8106. [PMID: 28930859 PMCID: PMC5617726 DOI: 10.1097/md.0000000000008106] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Loss of tumor suppressor gene p16 expression via promoter methylation has been reported in hepatocellular carcinoma (HCC). This meta-analysis was conducted to evaluate the correlation between p16 methylation and HCC. Additionally, we also analyzed the potential prognostic role of p16 methylation, expression or alteration-associated HCC. METHODS Online databases based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guideline were performed to analyze the role of p16 gene in HCC. The combined odds ratios (ORs) or hazard ratios (HRs) and their 95% confidence intervals (95% CIs) were summarized. RESULTS Final 3105 HCCs and 808 non-tumor controls (chronic hepatitis and liver cirrhosis) were performed in this meta-analysis. p16 promoter methylation in HCC was significantly higher than in chronic hepatitis and chronic hepatitis in tissue and blood samples. In addition, p16 promoter methylation was notably higher in patients >50 years' old than in patients aged <50 years, and it was higher in hepatitis B virus (HBV) or hepatitis C virus (HCV)-positive HCC than in hepatic viruses-negative HCC. However, p16 promoter methylation was not correlated with sex, cirrhosis, tumor differentiation, clinical stage. No association was found between p16 methylation or alteration and the prognosis of patients with HCC in overall survival (OS) and disease-free survival (DFS). Although p16 expression was significantly correlated with a poor prognosis in OS and DFS (P < .05) CONCLUSIONS:: Our results indicate that p16 methylation was linked to the development, age, HBV, and HCV infection of HCC. p16 methylation or alteration was not associated with the prognosis, but p16 expression was linked to a poor survival.
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Kalatskaya I. Overview of major molecular alterations during progression from Barrett's esophagus to esophageal adenocarcinoma. Ann N Y Acad Sci 2016; 1381:74-91. [PMID: 27415609 DOI: 10.1111/nyas.13134] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Revised: 05/13/2016] [Accepted: 05/19/2016] [Indexed: 12/14/2022]
Abstract
Esophageal adenocarcinoma (EAC) develops in the sequential transformation of normal epithelium into metaplastic epithelium, called Barrett's esophagus (BE), then to dysplasia, and finally cancer. BE is a common condition in which normal stratified squamous epithelium of the esophagus is replaced with an intestine-like columnar epithelium, and it is the most prominent risk factor for EAC. This review aims to impartially systemize the knowledge from a large number of publications that describe the molecular and biochemical alterations occurring over this progression sequence. In order to provide an unbiased extraction of the knowledge from the literature, a text-mining methodology was used to select genes that are involved in the BE progression, with the top candidate genes found to be TP53, CDKN2A, CTNNB1, CDH1, GPX3, and NOX5. In addition, sample frequencies across analyzed patient cohorts at each stage of disease progression are summarized. All six genes are altered in the majority of EAC patients, and accumulation of alterations correlates well with the sequential progression of BE to cancer, indicating that the text-mining method is a valid approach for gene prioritization. This review discusses how, besides being cancer drivers, these genes are functionally interconnected and might collectively be considered a central hub of BE progression.
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Affiliation(s)
- Irina Kalatskaya
- Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada.
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Dauch D, Rudalska R, Cossa G, Nault JC, Kang TW, Wuestefeld T, Hohmeyer A, Imbeaud S, Yevsa T, Hoenicke L, Pantsar T, Bozko P, Malek NP, Longerich T, Laufer S, Poso A, Zucman-Rossi J, Eilers M, Zender L. A MYC-aurora kinase A protein complex represents an actionable drug target in p53-altered liver cancer. Nat Med 2016; 22:744-53. [PMID: 27213815 DOI: 10.1038/nm.4107] [Citation(s) in RCA: 208] [Impact Index Per Article: 23.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Accepted: 04/11/2016] [Indexed: 12/12/2022]
Abstract
MYC oncoproteins are involved in the genesis and maintenance of the majority of human tumors but are considered undruggable. By using a direct in vivo shRNA screen, we show that liver cancer cells that have mutations in the gene encoding the tumor suppressor protein p53 (Trp53 in mice and TP53 in humans) and that are driven by the oncoprotein NRAS become addicted to MYC stabilization via a mechanism mediated by aurora kinase A (AURKA). This MYC stabilization enables the tumor cells to overcome a latent G2/M cell cycle arrest that is mediated by AURKA and the tumor suppressor protein p19(ARF). MYC directly binds to AURKA, and inhibition of this protein-protein interaction by conformation-changing AURKA inhibitors results in subsequent MYC degradation and cell death. These conformation-changing AURKA inhibitors, with one of them currently being tested in early clinical trials, suppressed tumor growth and prolonged survival in mice bearing Trp53-deficient, NRAS-driven MYC-expressing hepatocellular carcinomas (HCCs). TP53-mutated human HCCs revealed increased AURKA expression and a positive correlation between AURKA and MYC expression. In xenograft models, mice bearing TP53-mutated or TP53-deleted human HCCs were hypersensitive to treatment with conformation-changing AURKA inhibitors, thus suggesting a therapeutic strategy for this subgroup of human HCCs.
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Affiliation(s)
- Daniel Dauch
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Ramona Rudalska
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Giacomo Cossa
- Theodor Boveri Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany
| | - Jean-Charles Nault
- Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France
| | - Tae-Won Kang
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany.,Translational Gastrointestinal Oncology Group within the German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Torsten Wuestefeld
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Anja Hohmeyer
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Sandrine Imbeaud
- Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France
| | - Tetyana Yevsa
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Lisa Hoenicke
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Tatu Pantsar
- School of Pharmacy, University of Eastern Finland, Kuopio, Finland
| | - Przemyslaw Bozko
- Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Nisar P Malek
- Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany
| | - Thomas Longerich
- Institute of Pathology, University Hospital RWTH Aachen, Aachen, Germany
| | - Stefan Laufer
- Department of Pharmaceutical Chemistry, University of Tuebingen, Tuebingen, Germany
| | - Antti Poso
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany.,School of Pharmacy, University of Eastern Finland, Kuopio, Finland
| | - Jessica Zucman-Rossi
- Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France
| | - Martin Eilers
- Theodor Boveri Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany.,Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Wuerzburg, Germany
| | - Lars Zender
- Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany.,Translational Gastrointestinal Oncology Group within the German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany
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15
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Ju HL, Han KH, Lee JD, Ro SW. Transgenic mouse models generated by hydrodynamic transfection for genetic studies of liver cancer and preclinical testing of anti-cancer therapy. Int J Cancer 2016; 138:1601-1608. [PMID: 26220477 DOI: 10.1002/ijc.29703] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Accepted: 07/15/2015] [Indexed: 01/04/2025]
Abstract
Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide; however, the genetic mechanisms underlying its pathogenesis are incompletely understood. Genetically engineered mouse (GEM) models of HCC have been developed to elucidate the role of individual cancer-related genes in hepatocarcinogenesis. However, the expensive and time-consuming processes related to generating a GEM model discourage the development of diverse genotype models. Recently, a simple and inexpensive liver-specific transgenic approach was developed, in which a hydrodynamics-based transfection (HT) method was coupled with the Sleeping Beauty transposase system. Various HT models in which different oncogenic pathways are activated and/or tumor-suppressing pathways inactivated have been developed in recent years. The applicability of HT models in liver cancer research is expected to broaden and ultimately elucidate the cooperation between oncogenic signaling pathways and aid in designing molecular therapy to target altered pathways.
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Affiliation(s)
- Hye-Lim Ju
- Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, Seoul, Korea
| | - Kwang-Hyub Han
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea
| | - Jong Doo Lee
- Department of Nuclear Medicine, Catholic Kwandong University, Seoul, Korea
| | - Simon Weonsang Ro
- Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, Seoul, Korea
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea
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16
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Chung SI, Moon H, Kim DY, Cho KJ, Ju HL, Kim DY, Ahn SH, Han KH, Ro SW. Development of a transgenic mouse model of hepatocellular carcinoma with a liver fibrosis background. BMC Gastroenterol 2016; 16:13. [PMID: 26821924 PMCID: PMC4731926 DOI: 10.1186/s12876-016-0423-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Accepted: 01/25/2016] [Indexed: 01/29/2023] Open
Abstract
Background Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for hepatocellular carcinoma (HCC) and present in 80 to 90 % of patients with HCC. Current genetically engineered mouse models for HCC, however, generally do not feature liver fibrosis, which is a critical discrepancy between human HCC and murine models thereof. In this study, we developed a simple transgenic mouse model of HCC within the context of a fibrotic liver. Methods Employing hydrodynamic transfection (HT), coupled with the Sleeping Beauty (SB) transposon system, liver was stably transfected with transposons expressing cMyc and a short hairpin RNA down-regulating p53 (shp53). A chronic liver injury model, induced by hepatotoxic carbon tetrachloride (CCl4), was applied to the transgenic mice, allowing cells expressing cMyc plus shp53 to become malignant in the background of liver fibrosis. Results Livers harvested about 3 months after HT had excessive collagen deposition and activated hepatic stellate cells surrounding the tumors. Hepatocarcinogenesis was significantly accelerated in the fibrotic livers compared to those of the control, significantly decreasing the life span of the mice. The tumor incidence and average number of tumors per mouse were significantly higher in the group treated with CCl4 compared to the vehicle-treated control mice, following HT (p < 0.01). Conclusions Considering the simplicity and efficiency in generating HCC for fibrotic livers, the transgenic HCC model has the potential to be effectively used in preclinical testing of HCC anticancer therapy and in studies of hepatocarcinogenesis in fibrotic livers. Electronic supplementary material The online version of this article (doi:10.1186/s12876-016-0423-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Sook In Chung
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 120-752, South Korea. .,Brain Korea 21 Project for Medical Science College of Medicine, Yonsei University, Seoul, 120-752, South Korea.
| | - Hyuk Moon
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 120-752, South Korea. .,Brain Korea 21 Project for Medical Science College of Medicine, Yonsei University, Seoul, 120-752, South Korea.
| | - Dae Yeong Kim
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 120-752, South Korea.
| | - Kyung Joo Cho
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 120-752, South Korea.
| | - Hye-Lim Ju
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 120-752, South Korea.
| | - Do Young Kim
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, 120-752, South Korea.
| | - Sang Hoon Ahn
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, 120-752, South Korea.
| | - Kwang-Hyub Han
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, 120-752, South Korea.
| | - Simon Weonsang Ro
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 120-752, South Korea. .,Room 407, ABMRC, Severance Hospital, Yonsei University College of Medicine, Yonsei-ro 50-1, Seoul, 120-752, South Korea.
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17
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Cantore A, Ranzani M, Bartholomae CC, Volpin M, Valle PD, Sanvito F, Sergi LS, Gallina P, Benedicenti F, Bellinger D, Raymer R, Merricks E, Bellintani F, Martin S, Doglioni C, D'Angelo A, VandenDriessche T, Chuah MK, Schmidt M, Nichols T, Montini E, Naldini L. Liver-directed lentiviral gene therapy in a dog model of hemophilia B. Sci Transl Med 2016; 7:277ra28. [PMID: 25739762 DOI: 10.1126/scitranslmed.aaa1405] [Citation(s) in RCA: 105] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.
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Affiliation(s)
- Alessio Cantore
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy. Vita-Salute San Raffaele University, 20132 Milan, Italy
| | - Marco Ranzani
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy. Vita-Salute San Raffaele University, 20132 Milan, Italy
| | - Cynthia C Bartholomae
- Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany
| | - Monica Volpin
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy. Vita-Salute San Raffaele University, 20132 Milan, Italy
| | - Patrizia Della Valle
- Coagulation Service and Thrombosis Research Unit, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Francesca Sanvito
- Pathology Unit, Department of Oncology, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Lucia Sergi Sergi
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Pierangela Gallina
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Fabrizio Benedicenti
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Dwight Bellinger
- Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Robin Raymer
- Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Elizabeth Merricks
- Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | | | | | - Claudio Doglioni
- Pathology Unit, Department of Oncology, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Armando D'Angelo
- Coagulation Service and Thrombosis Research Unit, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Thierry VandenDriessche
- Department of Gene Therapy and Regenerative Medicine, Free University of Brussels, 1050 Brussels, Belgium. Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium
| | - Marinee K Chuah
- Department of Gene Therapy and Regenerative Medicine, Free University of Brussels, 1050 Brussels, Belgium. Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium
| | - Manfred Schmidt
- Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany
| | - Timothy Nichols
- Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Eugenio Montini
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Luigi Naldini
- San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy. Vita-Salute San Raffaele University, 20132 Milan, Italy.
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Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2014; 1849:247-56. [PMID: 25497382 DOI: 10.1016/j.bbagrm.2014.12.004] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Revised: 12/01/2014] [Accepted: 12/03/2014] [Indexed: 12/12/2022]
Abstract
Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2'-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5' CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.
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Li CC, Yu Z, Cui LH, Piao JM, Liu M. Role of P14 and MGMT Gene Methylation in Hepatocellular Carcinomas: a Meta-analysis. Asian Pac J Cancer Prev 2014; 15:6591-6. [DOI: 10.7314/apjcp.2014.15.16.6591] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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Evangelou K, Havaki S, Kotsinas A. E2F transcription factors and digestive system malignancies: How much do we know? World J Gastroenterol 2014; 20:10212-10216. [PMID: 25110451 PMCID: PMC4123353 DOI: 10.3748/wjg.v20.i29.10212] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/21/2013] [Revised: 02/22/2014] [Accepted: 04/16/2014] [Indexed: 02/06/2023] Open
Abstract
The E2F proteins comprise a family of 8 members that function as transcription factors. They are key targets of the retinoblastoma protein (RB) and were initially divided into groups of activators and repressors. Accumulating data suggest that there is no specific role for each individual E2F member. Instead, each E2F can exert a variety of cellular effects, some of which represent opposing ones. For instance, specific E2Fs can activate transcription and repression, promote or hamper cell proliferation, augment or inhibit apoptosis, all being dependent on the cellular context. This complexity reflects the importance that these transcription factors have on a cell’s fate. Thus, delineating the specific role for each E2F member in specific malignancies, although not easy, is a challenging and continuously pursued task, especially in view of potential E2F targeted therapies. Therefore, several reviews are continuously trying to evaluate available data on E2F status in various malignancies. Such reviews have attempted to reach a consensus, often in the simplistic form of oncogenes or tumor suppressor genes for the E2Fs. However they frequently miss spatial and temporal alterations of these factors during tumor development, which should also be considered in conjunction with the status of the regulatory networks that these factors participate in. In the current ‘‘Letter to the Editor’’, we comment on the flaws, misinterpretations and omissions in one such review article published recently in the World Journal of Gastroenterology regarding the role of E2Fs in digestive system malignancies.
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Nakayama M, Ogasawara S, Akiba J, Ueda K, Koura K, Todoroki K, Kinoshita H, Yano H. Side population cell fractions from hepatocellular carcinoma cell lines increased with tumor dedifferentiation, but lack characteristic features of cancer stem cells. J Gastroenterol Hepatol 2014; 29:1092-101. [PMID: 24325739 DOI: 10.1111/jgh.12484] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 11/15/2013] [Indexed: 12/13/2022]
Abstract
BACKGROUND AND AIM Cancer stem cells (CSCs), a minority population with stem cell-like characteristics, play important roles in cancer development and progression. Putative CSC markers, such as CD13, CD90, CD133, and epithelial cell adhesion molecule (EpCAM), and side population (SP) technique are generally used in an attempt to isolate CSCs. We aimed to clarify the relationship between CSCs and clonal dedifferentiation in hepatocellular carcinoma (HCC). METHODS We used a well-differentiated HCC cell line (HAK-1A) and a poorly differentiated HCC cell line (HAK-1B) established from a single nodule with histological heterogeneity. HAK-1B arose because of clonal dedifferentiation of HAK-1A. The SP cells and non-SP (NSP) cells were isolated from the two cell lines with a FACSAria II and used for the analyses. RESULTS The SP cell fractions in HAK-1A and HAK-1B were 0.2% and 0.9%, respectively. CD90 or EpCAM was not expressed in either HAK-1A or HAK-1B, while CD13 and CD133 were expressed in HAK-1B alone. Although sphere forming ability, tumorigenicity, growth rate, and CD13 expression were higher in HAK-1B SP cells than HAK-1B NSP cells, there were no differences in drug resistance, colony forming ability, or cell cycle rates between HAK-1B SP and NSP cells, suggesting HAK-1B SP cells do not fulfill CSC criteria. CONCLUSIONS Our findings suggested a possible relationship between the expression of CSC markers and clonal dedifferentiation. However, the complete features of CSC could not be identified in SP cells, and the concept of SP cells as a universal marker for CSC may not apply to HAK-1A and HAK-1B.
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Affiliation(s)
- Masamichi Nakayama
- Department of Pathology, Kurume University School of Medicine, Kurume, Japan; Department of Surgery, Kurume University School of Medicine, Kurume, Japan
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Lee SC, Tan HT, Chung MCM. Prognostic biomarkers for prediction of recurrence of hepatocellular carcinoma: Current status and future prospects. World J Gastroenterol 2014; 20:3112-3124. [PMID: 24696598 PMCID: PMC3964383 DOI: 10.3748/wjg.v20.i12.3112] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2013] [Revised: 12/03/2013] [Accepted: 01/02/2014] [Indexed: 02/07/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with region specific etiologies. Despite improvements made in the diagnosis of HCC, the prognosis of HCC patients remains poor due to the high recurrence rate of HCC. There is an urgent need for development of prognostic biomarkers to predict the risk of recurrence in HCC patients after “curative” treatment. Such stratification may aid in patient management and development of personalized medicine for HCC treatment. Omics based studies facilitate the study of global changes in biomolecules in a disease in a high throughput manner, and hence are well poised to understand the complex changes which led to HCC recurrence. The quantitative nature of data obtained from omics based studies allow for development of prognostic biomarkers based on changes in gene, protein and metabolite expression. In this review, we surveyed the application of transcriptomics, proteomics and metabolomics in the study of HCC recurrence. We summarised the data in the literature from these three fields of studies that claimed to be prognostic for HCC recurrence. We critiqued on the limitations of each area of research and the challenges faced in translating the research results for clinical application in predicting HCC recurrence.
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Zhang L, Yang Z, Ma A, Qu Y, Xia S, Xu D, Ge C, Qiu B, Xia Q, Li J, Liu Y. Growth arrest and DNA damage 45G down-regulation contributes to Janus kinase/signal transducer and activator of transcription 3 activation and cellular senescence evasion in hepatocellular carcinoma. Hepatology 2014; 59:178-89. [PMID: 23897841 DOI: 10.1002/hep.26628] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Accepted: 07/07/2013] [Indexed: 12/19/2022]
Abstract
UNLABELLED Growth arrest and DNA damage 45G (GADD45G), a stress sensor with multiple implications in various biological processes, is down-regulated in a broad spectrum of cancers. However, little is known about the biological effects of GADD45G on hepatocellular carcinoma (HCC) cells and the related mechanisms. In the present study, we found that GADD45G was commonly down-regulated in oncogene-transformed mouse liver cells and in human and mouse HCC. Ectopic expression of GADD45G robustly elicited senescence in HCC cells and suppressed tumor growth in vivo. Furthermore, GADD45G-induced senescence occurred in HCC cells independently of p53, p16(INK4a) (p16), and retinoblastoma (Rb). Instead, the prompt inhibition of Janus kinase 2 (Jak2), tyrosine kinase 2 (Tyk2), and signal transducer and activator of transcription 3 (Stat3) activation was observed in cells undergoing senescence. Impairment of Jak-Stat3 activation caused by GADD45G expression was associated with activation of SH2 domain-containing protein tyrosine phosphatase-2 (Shp2). Expression of constitutively activated Stat3 or human telomerase reverse transcriptase (hTERT), as well as knockdown of Shp2f, efficiently counteracted GADD45G-induced senescence. More important, in clinical HCC specimens, we found that GADD45G expression was inversely correlated with phosphorylated Stat3 expression in tumor cells and disease progression. CONCLUSION GADD45G functions as a negative regulator of the Jak-Stat3 pathway and inhibits HCC by inducing cellular senescence. The decrease or absence of GADD45G expression may be a key event for tumor cells or premalignant liver cells to bypass cellular senescence.
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Affiliation(s)
- Li Zhang
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, Shanghai, China
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Chaar I, Amara S, Elamine OE, Khiari M, Ounissi D, Khalfallah T, Ben Hmida A, Mzabi S, Bouraoui S. Biological significance of promoter hypermethylation of p14/ARF gene: relationships to p53 mutational status in Tunisian population with colorectal carcinoma. Tumour Biol 2013; 35:1439-49. [PMID: 24065196 PMCID: PMC3932170 DOI: 10.1007/s13277-013-1198-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2013] [Accepted: 09/11/2013] [Indexed: 11/26/2022] Open
Abstract
One of the most important pathways which are frequently affected in colorectal cancer is p53/ (MDM2)/p14ARF pathway. We aim to determine the methylation pattern of p14/ARF in relation to mutation of p53. This correlation was studied to investigate whether their alterations could be considered as a predictor factor of prognosis in colorectal cancer and whether it can be useful in early-stage diagnosis. Statistical analyses show that p14/ARF hypermethylation was correlated with rectum location (p = 0.004), primary TNM stage (p = 0.016), and advanced Astler–Coller stage (p = 0.024). The RT-PCR that revel 31 % of patients did not express p14/ARF mRNA or at very low level. A high concordance between CpG hypermethylation and the low levels (p < 0.005) was shown. In addition, our analyses demonstrate that patients with mutation in the p53 gene have a lack of the protein expression (p < 0.005). This category with negative expression of p53 had a shorter survival rate (p < 0.005). On the one hand, MSP pattern of p14/ARF were correlated with a lack of p53 expression (p = 0.007). We found that p53/p14ARF pathway was frequently deregulated among our patients. In our study, we demonstrate that hypermethylation of p14/ARF occurs early during CRC tumorogenesis. However, we did not find correlation between p14/ARF and survival. These results suggest that p14/ARF methylation pattern may constitute a predictor factor of CRC in early stage but it could not be considered as a prognostic factor. On the other hand and because of the reversibility of the methylation mechanism, it may be appropriate to target the demethylation of p14/ARF to develop new drogues for CRC.
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Affiliation(s)
- Ines Chaar
- Laboratory of Colorectal Cancer Research UR03ES04, Science University Tunis, Tunis, Tunisia,
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25
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Hepatocellular carcinoma and other malignancies in autoimmune hepatitis. Dig Dis Sci 2013; 58:1459-76. [PMID: 23306849 DOI: 10.1007/s10620-012-2525-5] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2012] [Accepted: 12/03/2012] [Indexed: 12/13/2022]
Abstract
Hepatocellular carcinoma and extrahepatic malignancies can complicate the course of autoimmune hepatitis, and these occurrences may increase in frequency as the survival of patients with cirrhosis is extended and the prospect of new nonstandard immune-modifying intervention is realized. The frequency of hepatocellular carcinoma in patients with autoimmune hepatitis and cirrhosis is 1-9 %, and annual occurrence in patients with cirrhosis is 1.1-1.9 %. The standardized incidence ratio for hepatocellular carcinoma in autoimmune hepatitis is 23.3 (95 % confidence interval (CI) 7.5-54.3) in Sweden, and the standardized mortality ratio for hepatobiliary cancer is 42.3 (95 % CI 20.3-77.9) in New Zealand. The principal risk factor is long-standing cirrhosis, and patients at risk are characterized mainly by cirrhosis for ≥ 10 years, manifestations of portal hypertension, persistent liver inflammation, and immunosuppressive therapy for ≥ 3 years. Multiple molecular disturbances, including the accumulation of senescent hepatocytes because of telomere shortening, step-wise accumulation of chromosomal injuries, and aberrations in transcription factors and genes, may contribute to the risk. Extraheptic malignancies of diverse cell types occur in 5 % in an unpredictable fashion. The standardized incidence ratio is 2.7 (95 % CI 1.8-3.9) in New Zealand, and non-melanoma skin cancers are most common. Outcomes are related to the nature and stage of the tumor at diagnosis. Surveillance recommendations have not been promulgated, but hepatic ultrasonography every six months in patients with cirrhosis is a consideration. Routine health screening measures for other malignancies should be applied diligently.
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Low expression of the putative tumour suppressor spinophilin is associated with higher proliferative activity and poor prognosis in patients with hepatocellular carcinoma. Br J Cancer 2013; 108:1830-7. [PMID: 23591196 PMCID: PMC3658515 DOI: 10.1038/bjc.2013.165] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
BACKGROUND Spinophilin, a multifunctional intracellular scaffold protein, is reduced in certain types of cancer and is regarded as a novel putative tumour suppressor protein. However, the role of spinophilin in hepatocellular carcinoma (HCC) has never been explored before. METHODS In this study, we determined for the first time the expression pattern of spinophilin in human HCC by immunohistochemistry and quantitative reverse transcriptase-PCR analysis. In addition, we performed immunohistochemical analysis of p53, p14(ARF) and the proliferation marker Ki-67. Kaplan-Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence spinophilin and to explore the effects of reduced spinophilin expression on cellular growth. RESULTS In our study, complete loss of spinophilin immunoreactivity was found in 44 of 104 HCCs (42.3%) and reduced levels were found in an additional 37 (35.6%) cases. After adjusting for other prognostic factors, multivariate Cox regression analysis identified low expression of spinophilin as an independent prognostic factor with respect to disease-free (hazard ratio (HR)=1.8; 95% confidence interval (CI)=1.04-3.40; P=0.043) and cancer-specific survival (HR=2.0; CI=1.1-3.8; P=0.025). Reduced spinophilin expression significantly correlated with higher Ki-67 index in HCC (P=0.014). Reducing spinophilin levels by siRNA induced a higher cellular growth rate and increased cyclin D2 expression in tumour cells (P<0.05). CONCLUSION This is the first study of the expression pattern and distribution of spinophilin in HCC. According to our data, the loss of spinophilin is associated with higher proliferation and might be useful as a prognostic marker in patients with HCC.
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Shiraha H, Yamamoto K, Namba M. Human hepatocyte carcinogenesis (review). Int J Oncol 2013; 42:1133-8. [PMID: 23426905 PMCID: PMC3622653 DOI: 10.3892/ijo.2013.1829] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2012] [Accepted: 10/22/2012] [Indexed: 02/07/2023] Open
Abstract
Hepatocellular carcinoma is the third most frequent cause of cancer-related death worldwide; and its incidence rate is increasing. Clinical and molecular medical analyses have revealed substantial information on hepatocarcinogenesis. Hepatocarcinogenesis is a stepwise process during which multiple genes are altered. Genetic changes and their biological consequences in human HCC can be divided into at least 4 groups: i) tumor suppressor genes (p53, retinoblastoma, phosphatase tensin homolog and runt-related transcription factor 3), ii) oncogenes (myc, K-ras, BRAF), iii) reactivation of developmental pathways (Wnt, hedgehog), and iv) growth factors and their receptors (transforming growth factor-α, insulin-like growth factor-2 receptor). An experimental model of human hepatocarcinogenesis such as in vitro neoplastic transformation of human hepatocytes has not been successfully achieved yet, but several immortalized human hepatocyte cell lines have been established. These immortalized human hepatocytes will become useful tools for the elucidation of hepatocarcinogenesis, especially for the initial step of multistep hepatocarcinogenesis.
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Affiliation(s)
- Hidenori Shiraha
- Department of Gastroenterology and Hepatology, Okayama University Faculty of Medicine, Okayama 700-8558, Japan.
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28
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Malik S, Bhatnagar S, Chaudhary N, Katare DP, Jain SK. DEN+2-AAF-induced multistep hepatotumorigenesis in Wistar rats: supportive evidence and insights. PROTOPLASMA 2013; 250:175-183. [PMID: 22456951 DOI: 10.1007/s00709-012-0392-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/02/2012] [Accepted: 02/21/2012] [Indexed: 05/31/2023]
Abstract
Diethylnitrosamine (DEN), found in many commonly consumed foods, has been reported to induce cancers in animals and humans. Several models have been developed to study multistage carcinogenesis in rat liver; these include the Solt-Farber-resistant hepatocyte model. In the Solt-Farber model, the initiation consists of either a necrogenic dose of a hepatocarcinogen or a non-necrogenic dose in conjunction with partial hepatectomy (PH). We report a novel protocol for tumor induction in liver which eliminates the need for PH. Male Wistar rats were injected with single i.p. dose of DEN (200 mg/kg body weight), controls received saline only. After 1 week of recovery, the DEN-treated animals were administered with the repeated doses of 2-acetyamino fluorine (150 mg/kg body weight) orally in 1 % carboxymethyl cellulose that served as promoting agent. Thirty days after the DEN administration, hepatocellular damage was observed as evident by histopathological analysis. The marker enzyme analysis showed elevated levels of serum AST, ALT, and alkaline phosphatase and a decrease in the levels of liver superoxide dismutase and catalase. The oxidative stress in liver was confirmed by elevated levels of lipid peroxidation and a decrease in antioxidant parameters.
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Affiliation(s)
- Shabnam Malik
- Department of Biotechnology, Hamdard University, Hamdard Nagar, New Delhi 110062, India
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29
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Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat Methods 2013; 10:155-61. [PMID: 23314173 PMCID: PMC3589714 DOI: 10.1038/nmeth.2331] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2012] [Accepted: 10/22/2012] [Indexed: 01/05/2023]
Abstract
Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.
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Tentner AR, Lee MJ, Ostheimer GJ, Samson LD, Lauffenburger DA, Yaffe MB. Combined experimental and computational analysis of DNA damage signaling reveals context-dependent roles for Erk in apoptosis and G1/S arrest after genotoxic stress. Mol Syst Biol 2012; 8:568. [PMID: 22294094 PMCID: PMC3296916 DOI: 10.1038/msb.2012.1] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2011] [Accepted: 12/23/2011] [Indexed: 11/24/2022] Open
Abstract
Following DNA damage, cells display complex multi-pathway signaling dynamics that connect cell-cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell-cycle re-entry and proliferation, permanent cell-cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time-resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin-induced DNA damage in the presence or absence of TNFα co-treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell-cycle regulatory responses. Two relational modeling approaches were then used to identify network-level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term 'time-interval stepwise regression.' Taken together, the results from these analysis methods revealed complex, cytokine-modulated inter-relationships among multiple signaling pathways following DNA damage, and identified an unexpected context-dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug.
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Affiliation(s)
- Andrea R Tentner
- Departments of Biology and Biological Engineering, David H Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Michael J Lee
- Departments of Biology and Biological Engineering, David H Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Gerry J Ostheimer
- Departments of Biology and Biological Engineering, David H Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Leona D Samson
- Departments of Biology and Biological Engineering, David H Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Douglas A Lauffenburger
- Departments of Biology and Biological Engineering, David H Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Michael B Yaffe
- Departments of Biology and Biological Engineering, David H Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
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El-Serag HB, Lechel A, Rudolph KL. Epidemiology and Molecular Mechanisms of Hepatocarcinogenesis. ZAKIM AND BOYER'S HEPATOLOGY 2012:142-156. [DOI: 10.1016/b978-1-4377-0881-3.00010-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Prasad ML, Patel SG, Shah JP, Hoshaw-Woodard S, Busam KJ. Prognostic significance of regulators of cell cycle and apoptosis, p16(INK4a), p53, and bcl-2 in primary mucosal melanomas of the head and neck. Head Neck Pathol 2011; 6:184-90. [PMID: 22160615 PMCID: PMC3370030 DOI: 10.1007/s12105-011-0319-2] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2011] [Accepted: 12/03/2011] [Indexed: 11/30/2022]
Abstract
Abnormalities in cell cycle regulation, tumor suppressor gene functions and apoptosis are frequent events in tumorigenesis. Their role in the pathogenesis and prognosis of primary mucosal melanomas (MM) of the upper aerodigestive tract remains unknown. Sixty-four patients (40 men, 24 women, median age 64 years) with MM were included in this study; 32 had tumors in the nasal/paranasal cavities, 28 in the oral cavity and 4 in the pharynx. Archival tissues from 47 initial mucosal tumors, 17 mucosal recurrences, and 13 nodal/distant metastases were subjected to immunohistochemistry using antibodies against p16, p53, and bcl-2. The results were correlated with histological features and survival data. Expressions of p16, p53, and bcl-2 proteins were seen in 25% (N=19/76), 21% (N=16/76), and 74% (N=56/76) of all tumors, respectively. bcl-2 expression in the initial tumors was associated with significantly longer overall and disease specific survival (3.3 vs. 1.5 years, P ≤ 0.05). Expression of p16 was increasingly lost, from 32% in initial tumors to 12% in recurrent and 15% in metastatic tumors (P=0.06). Tumors comprised of undifferentiated cells were significantly more p53 positive than epithelioid or spindle cells (80% vs. 33%, P=0.02). Expression of these markers did not correlate with necrosis, or vascular and/or deep tissue invasion. Expression of bcl-2 is associated with better survival in MM. Loss of p16 was seen with tumor progression whereas aberrant p53 expression was frequent in undifferentiated tumor cells.
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Affiliation(s)
- Manju L. Prasad
- Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY USA ,Department of Pathology, Yale University School of Medicine, 310 Cedar St, PO Box 208070, New Haven, CT 06520 USA
| | - Snehal G. Patel
- Division of Head and Neck Surgery, Memorial Sloan Kettering Cancer Center, New York, NY USA
| | - Jatin P. Shah
- Division of Head and Neck Surgery, Memorial Sloan Kettering Cancer Center, New York, NY USA
| | - Stacy Hoshaw-Woodard
- Department of Biostatistics, Ohio State University Medical Center, Columbus, OH USA
| | - Klaus J. Busam
- Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY USA
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Nishida N, Goel A. Genetic and epigenetic signatures in human hepatocellular carcinoma: a systematic review. Curr Genomics 2011; 12:130-7. [PMID: 21966251 PMCID: PMC3129047 DOI: 10.2174/138920211795564359] [Citation(s) in RCA: 147] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2010] [Revised: 01/04/2011] [Accepted: 01/18/2011] [Indexed: 12/17/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is the third most common cause of cancer deaths worldwide, and the incidence of this fatal disease is still on rise. The majority of HCCs emerge in the background of a chronic liver disease, such as chronic hepatitis and liver cirrhosis. The current understanding is that majority of HCCs evolve as a consequence of chronic inflammation and due to the presence of infection with hepatitis viruses. These underlying pathogenic stimuli subsequently induce a spectrum of genetic and epigenetic alterations in several cancer-related genes, which are involved in cell-cycle regulation, cell growth and adhesion. Such widespread genomic alterations cause disruption of normal cellular signaling and finally lead to the acquisition of a malignant phenotype in HCC. In general, the type of gene alterations, such as point mutations, deletion of chromosomal regions and abnormal methylation of gene promoters differ according to the individual targeted gene. In HCC, incidence of genetic alterations is relatively rare and is limited to a subset of few cancer-specific genes, such as the tumor suppressor p53, RB genes and oncogenes such as the CTNNB1. In contrast, epigenetic changes that involve aberrant methylation of genes and other post-transcriptional histone modifications occur far more frequently, and some of these epigenetic alterations are now being exploited for the development of molecular diagnostic signatures for HCC. In addition, recent findings of unique microRNA expression profiles also provide an evidence for the existence of novel mechanisms for gene expression regulation in HCC. In this review article, we will review the current state of knowledge on the activation of various oncogenic pathways and the inactivation of tumor suppressor pathways in HCC that result in the disruption of cancer-related gene function. In addition, we will specifically emphasize the clinical implication of some of these genetic and epigenetic alterations in the management of hepatocarcinogenesis.
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Affiliation(s)
- Naoshi Nishida
- Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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Calvisi DF. Dr. Jekyll and Mr. Hyde: a paradoxical oncogenic and tumor suppressive role of signal transducer and activator of transcription 3 in liver cancer. Hepatology 2011; 54:9-12. [PMID: 21608001 DOI: 10.1002/hep.24435] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
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35
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Schneller D, Machat G, Sousek A, Proell V, van Zijl F, Zulehner G, Huber H, Mair M, Muellner MK, Nijman SMB, Eferl R, Moriggl R, Mikulits W. p19(ARF) /p14(ARF) controls oncogenic functions of signal transducer and activator of transcription 3 in hepatocellular carcinoma. Hepatology 2011; 54:164-72. [PMID: 21452288 DOI: 10.1002/hep.24329] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
UNLABELLED Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.
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Affiliation(s)
- Doris Schneller
- Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, Vienna, Austria
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Recent advances in the research of hepatitis B virus-related hepatocellular carcinoma: epidemiologic and molecular biological aspects. Adv Cancer Res 2011; 108:21-72. [PMID: 21034965 DOI: 10.1016/b978-0-12-380888-2.00002-9] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Hepatocellular carcinoma (HCC) is one of the commonest cancers worldwide, and more than half of HCC patients are attributable to persistent hepatitis B virus (HBV) infections. The best and cheapest way to prevent HBV-related HCC is the implementation of universal hepatitis B vaccination program, by which the incidence rates of childhood HCC have been reduced in several countries, including Taiwan. However, there are still hundreds of millions of HBV carriers in the world that remain a global health challenge. In the past decade, several hepatitis B viral factors such as serum HBV DNA level, genotype, and naturally occurring mutants have already been identified to influence liver disease progression and HCC development in HBV carriers. Several easy-to-use scoring systems based on clinical and viral characteristics are developed to predict HCC risk in HBV carriers and may facilitate the communication between practicing physicians and patients in clinical practice. In addition, the role of nonviral factors in HBV-related HCC has also been increasingly recognized. On the basis of these emerging data, it is recommended that HBV carriers should be screened and monitored to identify those who have a higher risk of liver disease progression and require antiviral treatments. Regarding the molecular carcinogenesis of HCC development, despite some progress in the research of cell biology of HCC in the past decade, aberrant pathways involved in maintaining HCC phenotypes have not been completely elucidated yet. In the future, through comprehensive and integrated approaches to analyze the genomes of human HCC, novel target genes or pathways critically involved in hepatocarcinogenesis may hopefully be identified.
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Nakanishi Y, Shiraha H, Nishina SI, Tanaka S, Matsubara M, Horiguchi S, Iwamuro M, Takaoka N, Uemura M, Kuwaki K, Hagihara H, Toshimori J, Ohnishi H, Takaki A, Nakamura S, Kobayashi Y, Nouso K, Yagi T, Yamamoto K. Loss of runt-related transcription factor 3 expression leads hepatocellular carcinoma cells to escape apoptosis. BMC Cancer 2011; 11:3. [PMID: 21205319 PMCID: PMC3022884 DOI: 10.1186/1471-2407-11-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2010] [Accepted: 01/04/2011] [Indexed: 01/27/2023] Open
Abstract
Background Runt-related transcription factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC). Methods RUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis. Results RUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 ± 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 ± 4% and 4 ± 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation. Conclusion RUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis.
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Affiliation(s)
- Yutaka Nakanishi
- Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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Association between cyclin D1 polymorphism with CpG island promoter methylation status of tumor suppressor genes in gastric cancer. Dig Dis Sci 2010; 55:3449-57. [PMID: 20397048 DOI: 10.1007/s10620-010-1206-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/14/2009] [Accepted: 03/18/2010] [Indexed: 12/09/2022]
Abstract
BACKGROUND CpG island hypermethylation of tumor suppressor genes is highly involved in gastric carcinogenesis, and enhanced cell proliferation could accelerate this process. Cyclin D1 regulates cell cycle function and may play a role in methylation-related carcinogenesis. AIMS We investigated the association between Cyclin D1 gene G870A polymorphism and the methylation status of tumor suppressor genes in gastric cancer. METHODS Polymorphisms at G870A in the Cyclin D1 gene were genotyped, and methylation status of the p14, p16, DAP-kinase, and CDH1 genes were determined by methylation-specific-polymerase chain reaction in 139 gastric cancer tissues. CIHM high was defined as three or more methylated CpG islands. RESULTS Although no association was found between methylation status and different stages and Lauren's subtypes, patients with CIHM of DAP-kinase showed significantly worse survival than those without (p = 0.017). In addition, the number of methylated sites was also associated with survival curves (p = 0.0397). The 870G carrier a significantly lower prevalence of CIHM high compared to the AA genotype in advanced-stage gastric cancer (adjusted OR = 0.32, p = 0.047). A weak correlation between the same genotypes and CIHM of p14 were found in the same subtype (adjusted OR = 0.32, p = 0.052). The mean methylation number was significantly lower in G carriers than in AA genotypes in advanced-stage gastric cancer (p = 0.017). CONCLUSIONS Genetic polymorphism of CCND1 is associated with CIHM status in gastric cancer, especially in the advanced stage, but is independent of clinico-pathological features.
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Chiba T, Seki A, Aoki R, Ichikawa H, Negishi M, Miyagi S, Oguro H, Saraya A, Kamiya A, Nakauchi H, Yokosuka O, Iwama A. Bmi1 promotes hepatic stem cell expansion and tumorigenicity in both Ink4a/Arf-dependent and -independent manners in mice. Hepatology 2010; 52:1111-23. [PMID: 20648475 DOI: 10.1002/hep.23793] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
UNLABELLED We previously reported that forced expression of Bmi1 (B lymphoma Moloney murine leukemia virus insertion region 1 homolog) in murine hepatic stem/progenitor cells purified from fetal liver enhances their self-renewal and drives cancer initiation. In the present study, we examined the contribution of the Ink4a/Arf tumor suppressor gene locus, one of the major targets of Bmi1, to stem cell expansion and cancer initiation. Bmi1(-/-) Delta-like protein (Dlk)(+) hepatic stem/progenitor cells showed de-repression of the Ink4a/Arf locus and displayed impaired growth activity. In contrast, Ink4a/Arf(-/-) Dlk(+) cells gave rise to considerably larger colonies containing a greater number of bipotent cells than wild-type Dlk(+) cells. Although Ink4a/Arf(-/-) Dlk(+) cells did not initiate tumors in recipient nonobese diabetic/severe combined immunodeficiency mice, enforced expression of Bmi1 in Ink4a/Arf(-/-) Dlk(+) cells further augmented their self-renewal capacity and resulted in tumor formation in vivo. Microarray analyses successfully identified five down-regulated genes as candidate downstream targets for Bmi1 in hepatic stem/progenitor cells. Of these genes, enforced expression of sex determining region Y-box 17 (Sox17) in Dlk(+) cells strongly suppressed colony propagation and tumor growth. CONCLUSION These results indicate that repression of targets of Bmi1 other than the Ink4a/Arf locus plays a crucial role in the oncogenic transformation of hepatic stem/progenitor cells. Functional analyses of Bmi1 target genes would be of importance to elucidate the molecular machinery underlying hepatic stem cell system and explore therapeutic approaches for the eradication of liver cancer stem cells.
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Affiliation(s)
- Tetsuhiro Chiba
- Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan
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Lee SA, Ladu S, Evert M, Dombrowski F, De Murtas V, Chen X, Calvisi DF. Synergistic role of Sprouty2 inactivation and c-Met up-regulation in mouse and human hepatocarcinogenesis. Hepatology 2010; 52:506-17. [PMID: 20683950 PMCID: PMC2920762 DOI: 10.1002/hep.23681] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
UNLABELLED Sprouty2 (Spry2), a negative feedback regulator of the Ras/mitogen-activated protein kinase (MAPK) pathway, is frequently down-regulated in human hepatocellular carcinoma (HCC). We tested the hypothesis that loss of Spry2 cooperates with unconstrained activation of the c-Met protooncogene to induce hepatocarcinogenesis via in vitro and in vivo approaches. We found coordinated down-regulation of Spry2 protein expression and activation of c-Met as well as its downstream effectors extracellular signal-regulated kinase (ERK) and v-akt murine thymoma viral oncogene homolog (AKT) in a subset of human HCC samples with poor outcome. Mechanistic studies revealed that Spry2 function is disrupted in human HCC via multiple mechanisms at both transcriptional and post-transcriptional level, including promoter hypermethylation, loss of heterozygosity, and proteosomal degradation by neural precursor cell expressed, developmentally down-regulated 4 (NEDD4). In HCC cell lines, Spry2 overexpression inhibits c-Met-induced cell proliferation as well as ERK and AKT activation, whereas loss of Spry2 potentiates c-Met signaling. Most importantly, we show that blocking Spry2 activity via a dominant negative form of Spry2 cooperates with c-Met to promote hepatocarcinogenesis in the mouse liver by sustaining proliferation and angiogenesis. The tumors exhibited high levels of activated ERK and AKT, recapitulating the subgroup of human HCC with a clinically aggressive phenotype. CONCLUSION The occurrence of frequent genetic, epigenetic, and biochemical events leading to Spry2 inactivation provides solid evidence that Spry2 functions as a tumor suppressor gene in liver cancer. Coordinated deregulation of Spry2 and c-Met signaling may be a pivotal oncogenic mechanism responsible for unrestrained activation of ERK and AKT pathways in human hepatocarcinogenesis.
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Affiliation(s)
- Susie A. Lee
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA
| | - Sara Ladu
- Department of Medicine and Aging, University of Chieti, Chieti, Italy
| | - Matthias Evert
- Institut fur Pathologie, Ernst-Moritz-Arndt-Universitat, Greifswald, Germany
| | - Frank Dombrowski
- Institut fur Pathologie, Ernst-Moritz-Arndt-Universitat, Greifswald, Germany
| | - Valentina De Murtas
- Institut fur Pathologie, Ernst-Moritz-Arndt-Universitat, Greifswald, Germany
| | - Xin Chen
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, Liver Center, University of California, San Francisco, CA
| | - Diego F. Calvisi
- Institut fur Pathologie, Ernst-Moritz-Arndt-Universitat, Greifswald, Germany
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van Zijl F, Zulehner G, Petz M, Schneller D, Kornauth C, Hau M, Machat G, Grubinger M, Huber H, Mikulits W. Epithelial-mesenchymal transition in hepatocellular carcinoma. Future Oncol 2010; 5:1169-79. [PMID: 19852728 DOI: 10.2217/fon.09.91] [Citation(s) in RCA: 263] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The transition of epithelial cells to a mesenchymal phenotype is of paramount relevance for embryonic development and adult wound healing. During the past decade, the epithelial-mesenchymal transition (EMT) has been increasingly recognized to occur during the progression of various carcinomas such as hepatocellular carcinoma (HCC). Here, we focus on EMT in both experimental liver models and human HCC, emphasizing the underlying molecular mechanisms which show partial recurrence of embryonic programs such as TGF-beta and Wnt/ beta-catenin signaling, including collaboration with hepatitis viruses. We further discuss the differentiation repertoire of malignant hepatocytes with respect to the potential acquisition of stemness, and the involvement of the mesenchymal to epithelial transition, the reversal of EMT, in cancer dissemination and metastatic colonization. The strong evidence for EMT in HCC patients demands novel strategies in pathological assessments and therapeutic concepts to efficiently combat HCC progression.
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Affiliation(s)
- Franziska van Zijl
- Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria
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Zulehner G, Mikula M, Schneller D, van Zijl F, Huber H, Sieghart W, Grasl-Kraupp B, Waldhör T, Peck-Radosavljevic M, Beug H, Mikulits W. Nuclear beta-catenin induces an early liver progenitor phenotype in hepatocellular carcinoma and promotes tumor recurrence. THE AMERICAN JOURNAL OF PATHOLOGY 2009; 176:472-81. [PMID: 20008139 DOI: 10.2353/ajpath.2010.090300] [Citation(s) in RCA: 73] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.
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Affiliation(s)
- Gudrun Zulehner
- Department of Internal Medicine I, Centre of Public Health, Medical University of Vienna, Vienna, Austria
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Tillmann HL, Plentz RR, Begus‐Nahrmann Y, Lechel A, Rudolph LK. Telomeres and Aging, Cancer, and Hepatic Fibrosis. THE LIVER 2009:1105-1119. [DOI: 10.1002/9780470747919.ch68] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Aurora kinase inhibitor PHA-739358 suppresses growth of hepatocellular carcinoma in vitro and in a xenograft mouse model. Neoplasia 2009; 11:934-44. [PMID: 19724687 DOI: 10.1593/neo.09664] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2009] [Revised: 06/05/2009] [Accepted: 06/06/2009] [Indexed: 01/16/2023] Open
Abstract
Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50 nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G(2)/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.
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Tahara T, Shibata T, Nakamura M, Yamashita H, Yoshioka D, Okubo M, Maruyama N, Kamano T, Kamiya Y, Fujita H, Nakagawa Y, Nagasaka M, Iwata M, Takahama K, Watanabe M, Hirata I, Arisawa T. MTHFR 677T carrier influences the methylation status of H. pylori-infected gastric mucosa in older subjects. Dig Dis Sci 2009; 54:2391-8. [PMID: 19082889 DOI: 10.1007/s10620-008-0624-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/05/2008] [Accepted: 11/03/2008] [Indexed: 12/29/2022]
Abstract
DNA methylation is one of the major events in the early process of gastric carcinogenesis and it also occurs in non-neoplastic gastric mucosa. MTHFR plays a central role in biotransformation of folate to form S-adenosylmethionine, the universal methyl donor in cells and affects DNA methylation status. We investigated the association between common functional polymorphism of MTHFR C677T and DNA methylation status in H. pylori-infected non-neoplastic gastric mucosa. For 99 gastric mucosa samples from H. pylori positive non-cancer subjects, we assessed the association between MTHFR C677T genetic polymorphism and promoter methylation status of the four candidate promoters (p14, p16, DAP-kinase, and CDH1). In most all of the subjects, weak correlation was found between the p16 promoter methylation and MTHFR 677T carriers (age, sex-adjusted OR = 2.57, P = 0.053). When subjects were divided into two groups according to age, the MTHFR T carrier held a significantly higher risk of p16 promoter methylation, especially in 66 years or older generation (sex-adjusted OR = 14.28, P = 0.02). In addition, mean number of methylated CpG cites were significantly higher in T carrier than CC genotype in the same generation (P = 0.0418). Our data suggest that MTHFR 677T carrier influences the risk of DNA methylation in gastric mucosa in the long-term outcome of the H. pylori infection.
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Affiliation(s)
- Tomomitsu Tahara
- Department of Gastroenterology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
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Lahsnig C, Mikula M, Petz M, Zulehner G, Schneller D, van Zijl F, Huber H, Csiszar A, Beug H, Mikulits W. ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression. Oncogene 2008; 28:638-50. [PMID: 19015638 DOI: 10.1038/onc.2008.418] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In human hepatocellular carcinoma (HCC), epithelial to mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. We employed a model of EMT based on immortalized p19(ARF) null hepatocytes (MIM), which display tumor growth upon expression of oncogenic Ras and undergo EMT through the synergism of Ras and transforming growth factor (TGF)-beta. Here, we show that the interleukin-related protein interleukin-like EMT inducer (ILEI), a novel EMT-, tumor- and metastasis-inducing protein, cooperates with oncogenic Ras to cause TGF-beta-independent EMT. Ras-transformed MIM hepatocytes overexpressing ILEI showed cytoplasmic E-cadherin, loss of ZO-1 and induction of alpha-smooth muscle actin as well as platelet-derived growth factor (PDGF)/PDGF-R isoforms. As shown by dominant-negative PDGF-R expression in these cells, ILEI-induced PDGF signaling was required for enhanced cell migration, nuclear accumulation of beta-catenin, nuclear pY-Stat3 and accelerated growth of lung metastases. In MIM hepatocytes expressing the Ras mutant V12-C40, ILEI collaborated with PI3K signaling resulting in tumor formation without EMT. Clinically, human HCC samples showed granular or cytoplasmic localization of ILEI correlating with well and poorly differentiated tumors, respectively. In conclusion, these data indicate that ILEI requires cooperation with oncogenic Ras to govern hepatocellular EMT through mechanisms involving PDGF-R/beta-catenin and PDGF-R/Stat3 signaling.
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Affiliation(s)
- C Lahsnig
- Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Borschke-Gasse 8a, Vienna, Austria
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Zhang C, Guo X, Zhang L, Lu Z, Ma N, Cheng Y, Shen F, Zhang B, Wu M, Wei L. Methylation-Related silencing of p14ARF gene correlates with telomerase activity and mRNA expression of human telomerase reverse transcriptase in hepatocellular carcinoma. J Surg Oncol 2008; 98:462-8. [DOI: 10.1002/jso.21131] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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Kondo I, Iida S, Takagi Y, Sugihara K. MDM2 mRNA expression in the p53 pathway may predict the potential of invasion and liver metastasis in colorectal cancer. Dis Colon Rectum 2008; 51:1395-402. [PMID: 18607552 DOI: 10.1007/s10350-008-9382-5] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2007] [Revised: 12/23/2007] [Accepted: 01/05/2008] [Indexed: 02/08/2023]
Abstract
PURPOSE The p53/MDM2/p14ARF pathway is one of the major signaling cascades involved in the regulation of apoptosis. Although many tumors have been reported to show disruption of the p53/MDM2/p14ARF pathway, few studies have examined p53, MDM2, and p14ARF simultaneously in colorectal carcinoma. The present study was undertaken to clarify whether correlations exist among MDM2, p53, and p14ARF in colorectal cancer. METHODS We determined the presence of mutations in the p53 gene, MDM2 expression, and methylation status of the p14ARF in 97 primary colorectal carcinoma specimens. Associations with survival and clinicopathologic factors were investigated. RESULTS At least one abnormality of these three molecules was found in 82 (84 percent) tumors. We observed a significant inverse association between MDM2 expression and tumor invasion (P = 0.01). Furthermore, the presence of liver metastasis was also significantly associated with low MDM2 expression (P = 0.02). CONCLUSIONS The results suggest that disruption of the p53/MDM2/p14ARF pathway may frequently participate in colonic carcinogenesis and that MDM2 expression status may be a factor in the prediction of potential invasion and liver metastasis of colorectal carcinomas.
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Affiliation(s)
- Ito Kondo
- Department of Surgical Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo, Japan.
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Qin XG, Luo DZ, Lv ZL, Lin J, Su CL. Expression of P14ARF, ARF-BP1 and c-myc mRNAs in hepatocellular carcinoma and their significances. Shijie Huaren Xiaohua Zazhi 2008; 16:2656-2660. [DOI: 10.11569/wcjd.v16.i23.2656] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the expression of P14ARF, ARF-BP1 and c-myc mRNAs in hepatocellular carcinoma (HCC) and their clinical significances.
METHODS: Abundance of P14ARF, ARF-BP1 and c-myc mRNAs was detected by semi-quantification reverse transcription polymerase chain reaction (RT-PCR) technique in the samples from 52 HCCs and their 45 non-tumorous liver tissues, and their clinical significances were assayed with the clinical parameters.
RESULTS: The expression levels of P14ARF, ARF-BP1 and c-myc mRNAs (77.0%, 77.0%, 75.0%) were higher in HCC tissues than those in the non-tumorous liver tissues (11.1%, 20.0%, 53.3%), respectively. The expression of P14ARF mRNA and ARF-BP1 mRNAs were related to tumor size (t = 2.169, 2.087; both P < 0.05), but the expression of c-myc mRNA was not. The expression of P14ARF, ARF-BP1 and c-myc mRNAs were not related to the gender, age, AFP level, clinical stageing, HBV infection, envelopes, infiltration and metastasis. There was a positive correlation between P14ARF and ARF-BP1 expression, P14ARF and c-myc expression as well as ARF-BP1 and c-myc expression (r = 0.565, 0.436, 0.584; all P < 0.01).
CONCLUSION: Over-expression of P14ARF, ARF-BP1 and c-myc mRNAs are markers in the early stage of HCC, and ARF-BP1 over-expression play an important role in the genesis and development of HCC. ARF-BP1 may become a novel therapeutic target against HCC.
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Guo Y, Pajovic S, Gallie BL. Expression of p14ARF, MDM2, and MDM4 in human retinoblastoma. Biochem Biophys Res Commun 2008; 375:1-5. [PMID: 18644346 DOI: 10.1016/j.bbrc.2008.07.055] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2008] [Accepted: 07/09/2008] [Indexed: 12/19/2022]
Abstract
It is still not clear whether the p53 pathway is altered in retinoblastoma development. We assessed the expression of the p53 pathway genes p14(ARF), mouse double minute 2 (MDM2), and mouse double minute 4 (MDM4) in human retinoblastoma compared to normal retina. Primary human retinoblastomas, retinoblastoma cell lines and normal retinas were assessed for p14(ARF) and MDM4 mRNA by quantitative RT-PCR. p14(ARF), MDM2, and MDM4 protein were measured by immunoblot and immunohistochemistry. Compared to retina, p14(ARF) mRNA expression was notably increased in retinoblastoma but p14(ARF) protein was undetectable. MDM2 and MDM4 proteins were expressed in 22/22 retinoblastomas. MDM2 was expressed in 3/10 retinas tested, and MDM4 in 10/10 retinas. The expression level of MDM2 protein in retinoblastomas and retina was comparable, while MDM4 protein was overexpressed in one retinoblastoma cell line Y79 and two primary retinoblastomas. We observe that overexpression of MDM2 and MDM4 is not a necessary step in retinoblastoma development. However, loss of detectable p14(ARF) protein and resultant lack of functional inactivation of these p53 inhibitors may contribute to retinoblastoma development by constitutive inhibition of p53.
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Affiliation(s)
- Ying Guo
- Division of Applied Molecular Oncology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Room 8-415, 610 University Avenue, Toronto, ON, Canada
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