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Chen SY, Chen X, Zhu S, Xu JJ, Li XF, Yin NN, Xiao YY, Huang C, Li J. miR-324-3p Suppresses Hepatic Stellate Cell Activation and Hepatic Fibrosis Via Regulating SMAD4 Signaling Pathway. Mol Biotechnol 2025; 67:673-688. [PMID: 38407690 PMCID: PMC11711260 DOI: 10.1007/s12033-024-01078-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2023] [Accepted: 01/15/2024] [Indexed: 02/27/2024]
Abstract
In hepatic fibrosis (HF), hepatic stellate cells (HSCs) form the extracellular matrix (ECM), and the pathological accumulation of ECM in the liver leads to inflammation. Our previous research found that miR-324-3p was down-regulated in culture-activated human HSCs. However, the precise effect of miR-324-3p on HF has not been elucidated. In this study, the HF mouse models were induced through directly injecting carbon tetrachloride (CCl4) into mice; the HF cell models were constructed using TGF-β1-treated LX-2 cells. Next, real-time-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and immunohistochemistry (IHC) were applied to assess the expression levels of miR-324-3p, α-smooth muscle actin (α-SMA), Vimentin or SMAD4; hematoxylin and eosin (H&E), Masson' s trichrome and Sirius red staining to evaluate the liver injury; luciferase reporter assay to verify the targeting relationship between miR-324-3p and SMAD4; enzyme-linked immunosorbent assay (ELISA) to determine the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and cell counting kit-8 (CCK-8) and flow cytometry to evaluate the effects of miR-324-3p on cell proliferation and cycle/apoptosis, respectively. The experimental results showed a reduction in miR-324-3p level in CCl4-induced HF mice as well as transforming growth factor (TGF)-β1-activated HSCs. Interestingly, the miR-324-3p level was rescued following the HF recovery process. In HF mice induced by CCl4, miR-324-3p overexpression inhibited liver tissue damage, decreased serum ALT and AST levels, and inhibited fibrosis-related biomarkers (α-SMA, Vimentin) expression, thereby inhibiting HF. Similarly, miR-324-3p overexpression up-regulated α-SMA and Vimentin levels in HF cells, while knockdown of miR-324-3p had the opposite effect. Besides, miR-324-3p played an antifibrotic role through inhibiting the proliferation of hepatocytes. Further experiments confirmed that miR-324-3p targeted and down-regulated SMAD4 expression. SMAD4 was highly expressed in HF cells, and silencing SMAD4 significantly decreased the α-SMA and Vimentin levels in HF cells. Collectively, the miR-324-3p may suppress the activation of HSCs and HF by targeting SMAD4. Therefore, miR-324-3p is identified as a potential and novel therapeutic target for HF.
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Affiliation(s)
- Si-Yu Chen
- Department of Pharmacy, Hefei BOE Hospital, Intersection of Dongfang Avenue and Wenzhong Road, Hefei, China
| | - Xin Chen
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Sai Zhu
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Jin-Jin Xu
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Xiao-Feng Li
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Na-Na Yin
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Yan-Yan Xiao
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Cheng Huang
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China
| | - Jun Li
- School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, 230032, Anhui, China.
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FGF/FGFR Signaling in Hepatocellular Carcinoma: From Carcinogenesis to Recent Therapeutic Intervention. Cancers (Basel) 2021; 13:cancers13061360. [PMID: 33802841 PMCID: PMC8002748 DOI: 10.3390/cancers13061360] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 03/11/2021] [Accepted: 03/13/2021] [Indexed: 12/16/2022] Open
Abstract
Simple Summary As the most common primary liver cancer, HCC is a tricky cancer resistant to systemic therapies. The fibroblast growth factor family and its receptors are gaining more and more attention in various cancers. Noticing an explosion in the number of studies about aberrant FGF/FGFR signaling in HCC being studied, we were encouraged to summarize them. This review discusses how FGF/FGFR signaling influences HCC development and its implications in HCC prediction and target treatment, and combination treatment. Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, ranking third in cancer deaths worldwide. Over the last decade, several studies have emphasized the development of tyrosine kinase inhibitors (TKIs) to target the aberrant pathways in HCC. However, the outcomes are far from satisfactory due to the increasing resistance and adverse effects. The family of fibroblast growth factor (FGF) and its receptors (FGFR) are involved in various biological processes, including embryogenesis, morphogenesis, wound repair, and cell growth. The aberrant FGF/FGFR signaling is also observed in multiple cancers, including HCC. Anti-FGF/FGFR provides delightful benefits for cancer patients, especially those with FGF signaling alteration. More and more multi-kinase inhibitors targeting FGF signaling, pan-FGFR inhibitors, and selective FGFR inhibitors are now under preclinical and clinical investigation. This review summarizes the aberrant FGF/FGFR signaling in HCC initiating, development and treatment status, and provide new insights into the treatment of HCC.
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Wang H, Rao B, Lou J, Li J, Liu Z, Li A, Cui G, Ren Z, Yu Z. The Function of the HGF/c-Met Axis in Hepatocellular Carcinoma. Front Cell Dev Biol 2020; 8:55. [PMID: 32117981 PMCID: PMC7018668 DOI: 10.3389/fcell.2020.00055] [Citation(s) in RCA: 97] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2019] [Accepted: 01/22/2020] [Indexed: 12/17/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, leading to a large global cancer burden. Hepatocyte growth factor (HGF) and its high-affinity receptor, mesenchymal epithelial transition factor (c-Met), are closely related to the onset, progression, and metastasis of multiple tumors. The HGF/c-Met axis is involved in cell proliferation, movement, differentiation, invasion, angiogenesis, and apoptosis by activating multiple downstream signaling pathways. In this review, we focus on the function of the HGF/c-Met axis in HCC. The HGF/c-Met axis promotes the onset, proliferation, invasion, and metastasis of HCC. Moreover, it can serve as a biomarker for diagnosis and prognosis, as well as a therapeutic target for HCC. In addition, it is closely related to drug resistance during HCC treatment.
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Affiliation(s)
- Haiyu Wang
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Benchen Rao
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Jiamin Lou
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Jianhao Li
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Zhenguo Liu
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Ang Li
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Guangying Cui
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Zhigang Ren
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Zujiang Yu
- Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.,Gene Hospital of Henan Province, Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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Li Y, Xu A, Jia S, Huang J. Recent advances in the molecular mechanism of sex disparity in hepatocellular carcinoma. Oncol Lett 2019; 17:4222-4228. [PMID: 30988804 DOI: 10.3892/ol.2019.10127] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2018] [Accepted: 01/25/2019] [Indexed: 12/11/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is more frequently observed and aggressive in men compared with women. Increasing evidence demonstrates that the sex disparity appears to be mediated by the stimulatory effects of androgens and the protective effects of estrogen in the development and progression of HCC. In the past few decades, studies on the sex difference of HCC mainly focused on the effect of sex hormones on the transactivation of hepatitis B virus X protein and the release of inflammatory cytokines, and these studies have further intensified in recent years. Sex hormones are also involved in genetic alterations and DNA damage repair in hepatocytes through binding to their specific cellular receptors and affecting the corresponding signaling pathways. Furthermore, the theory of sex chromosomes participating in HCC has been considered. The present review discussed the recent advances in the molecular mechanisms of sex disparity in HCC, with the aim of improving the understanding of the underlying critical factors and exploring more effective methods for the prevention and treatment of HCC.
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Affiliation(s)
- Yanmeng Li
- Experimental Center, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China.,National Clinical Research Center for Digestive Disease, Beijing 100050, P.R. China
| | - Anjian Xu
- Experimental Center, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China.,National Clinical Research Center for Digestive Disease, Beijing 100050, P.R. China
| | - Siyu Jia
- Experimental Center, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China.,National Clinical Research Center for Digestive Disease, Beijing 100050, P.R. China
| | - Jian Huang
- Experimental Center, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China.,National Clinical Research Center for Digestive Disease, Beijing 100050, P.R. China
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Aristizabal Prada ET, Spöttl G, Maurer J, Lauseker M, Koziolek EJ, Schrader J, Grossman A, Pacak K, Beuschlein F, Auernhammer CJ, Nölting S. The role of GSK3 and its reversal with GSK3 antagonism in everolimus resistance. Endocr Relat Cancer 2018; 25:893-908. [PMID: 29895527 PMCID: PMC7439002 DOI: 10.1530/erc-18-0159] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Accepted: 06/11/2018] [Indexed: 12/11/2022]
Abstract
Pancreatic neuroendocrine tumors (panNETs) are often inoperable at diagnosis. The mTORC1 inhibitor everolimus has been approved for the treatment of advanced NETs. However, the regular development of resistance to everolimus limits its clinical efficacy. We established two independent everolimus-resistant panNET (BON1) cell lines (BON1 RR1, BON1 RR2) to find potential mechanisms of resistance. After 24 weeks of permanent exposure to 10 nM everolimus, BON1 RR1 and BON1 RR2 showed stable resistance with cellular survival rates of 96.70% (IC50 = 5200 nM) and 92.30% (IC50 = 2500 nM), respectively. The control cell line showed sensitivity to 10 nM everolimus with cellular survival declining to 54.70% (IC50 = 34 nM). Both resistant cell lines did not regain sensitivity over time and showed persistent stable resistance after a drug holiday of 13 weeks. The mechanisms of resistance in our cell line model included morphological adaptations, G1 cell cycle arrest associated with reduced CDK1(cdc2) expression and decreased autophagy. Cellular migration potential was increased and indirectly linked to c-Met activation. GSK3 was over-activated in association with reduced baseline IRS-1 protein levels. Specific GSK3 inhibition strongly decreased BON1 RR1/RR2 cell survival. The combination of everolimus with the PI3Kα inhibitor BYL719 re-established everolimus sensitivity through GSK3 inhibition and restoration of autophagy. We suggest that GSK3 over-activation combined with decreased baseline IRS-1 protein levels and decreased autophagy may be a crucial feature of everolimus resistance, and hence, a possible therapeutic target.
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Affiliation(s)
- Elke Tatjana Aristizabal Prada
- Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany
| | - Gerald Spöttl
- Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany
| | - Julian Maurer
- Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany
| | - Michael Lauseker
- Institute for Medical Information SciencesBiometry, and Epidemiology, Campus Grosshadern, Ludwig-Maximilians-University of Munich, Munich, Germany
| | - Eva Jolanthe Koziolek
- Department of Nuclear MedicineUniversity Medical Center Charité, Berlin, Germany
- German Cancer Consortium (DKTK)Heidelberg, Germany
- German Cancer Research Center (DKFZ)Heidelberg, Germany
| | - Jörg Schrader
- I. Medizinische Klinik und PoliklinikUniversitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
| | - Ashley Grossman
- Oxford Centre for DiabetesEndocrinology and Metabolism, University of Oxford, Oxford, UK
- Royal Free Hospital ENETS Centre of ExcellenceLondon, UK
| | - Karel Pacak
- Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNational Institutes of Health, Bethesda, Maryland, USA
| | - Felix Beuschlein
- Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany
- Klinik für EndokrinologieDiabetologie und Klinische Ernährung, Universitätsspital Zürich, Zurich, Switzerland
| | - Christoph Joseph Auernhammer
- Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany
| | - Svenja Nölting
- Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany
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6
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Activated HGF-c-Met Axis in Head and Neck Cancer. Cancers (Basel) 2017; 9:cancers9120169. [PMID: 29231907 PMCID: PMC5742817 DOI: 10.3390/cancers9120169] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Revised: 12/06/2017] [Accepted: 12/07/2017] [Indexed: 12/14/2022] Open
Abstract
Head and neck squamous cell carcinoma (HNSCC) is a highly morbid disease. Recent developments including Food and Drug Administration (FDA) approved molecular targeted agent’s pembrolizumab and cetuximab show promise but did not improve the five-year survival which is currently less than 40%. The hepatocyte growth factor receptor; also known as mesenchymal–epithelial transition factor (c-Met) and its ligand hepatocyte growth factor (HGF) are overexpressed in head and neck squamous cell carcinoma (HNSCC); and regulates tumor progression and response to therapy. The c-Met pathway has been shown to regulate many cellular processes such as cell proliferation, invasion, and angiogenesis. The c-Met pathway is involved in cross-talk, activation, and perpetuation of other signaling pathways, curbing the cogency of a blockade molecule on a single pathway. The receptor and its ligand act on several downstream effectors including phospholipase C gamma (PLCγ), cellular Src kinase (c-Src), phosphotidylinsitol-3-OH kinase (PI3K) alpha serine/threonine-protein kinase (Akt), mitogen activate protein kinase (MAPK), and wingless-related integration site (Wnt) pathways. They are also known to cross-talk with other receptors; namely epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) and specifically contribute to treatment resistance. Clinical trials targeting the c-Met axis in HNSCC have been undertaken because of significant preclinical work demonstrating a relationship between HGF/c-Met signaling and cancer cell survival. Here we focus on HGF/c-Met impact on cellular signaling in HNSCC to potentiate tumor growth and disrupt therapeutic efficacy. Herein we summarize the current understanding of HGF/c-Met signaling and its effects on HNSCC. The intertwining of c-Met signaling with other signaling pathways provides opportunities for more robust and specific therapies, leading to better clinical outcomes.
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7
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Berretta M, Cavaliere C, Alessandrini L, Stanzione B, Facchini G, Balestreri L, Perin T, Canzonieri V. Serum and tissue markers in hepatocellular carcinoma and cholangiocarcinoma: clinical and prognostic implications. Oncotarget 2017; 8:14192-14220. [PMID: 28077782 PMCID: PMC5355172 DOI: 10.18632/oncotarget.13929] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Accepted: 10/28/2016] [Indexed: 12/12/2022] Open
Abstract
HCC represents the sixth most common cancer worldwide and the second leading cause of cancer-related death. Despite the high incidence, treatment options for advanced HCC remain limited and unsuccessful, resulting in a poor prognosis. Despite the major advances achieved in the diagnostic management of HCC, only one third of the newly diagnosed patients are presently eligible for curative treatments. Advances in technology and an increased understanding of HCC biology have led to the discovery of novel biomarkers. Improving our knowledge about serum and tissutal markers could ultimately lead to an early diagnosis and better and early treatment strategies for this deadly disease. Serum biomarkers are striking potential tools for surveillance and early diagnosis of HCC thanks to the non-invasive, objective, and reproducible assessments they potentially enable. To date, many biomarkers have been proposed in the diagnosis of HCC. Cholangiocarcinoma (CCA) is an aggressive malignancy, characterized by early lymph node involvement and distant metastasis, with 5-year survival rates of 5%-10%. The identification of new biomarkers with diagnostic, prognostic or predictive value is especially important as resection (by surgery or combined with a liver transplant) has shown promising results and novel therapies are emerging. However, the relatively low incidence of CCA, high frequency of co-existing cholestasis or cholangitis (primary sclerosing cholangitis –PSC- above all), and difficulties with obtaining adequate samples, despite advances in sampling techniques and in endoscopic visualization of the bile ducts, have complicated the search for accurate biomarkers. In this review, we attempt to analyze the existing literature on this argument.
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Affiliation(s)
| | - Carla Cavaliere
- Department of Onco-Ematology Medical Oncology, S.G. Moscati Hospital of Taranto Taranto, Italy
| | - Lara Alessandrini
- Division of Pathology, National Cancer Institute, Aviano (PN), Italy
| | - Brigida Stanzione
- Department of Medical Oncology, National Cancer Institute, Aviano (PN), Italy
| | - Gaetano Facchini
- Department of Medical Oncology, National Cancer Institute, "G. Pascale" Foundation, Naples, Italy
| | - Luca Balestreri
- Department of Radiology, National Cancer Institute, Aviano (PN), Italy
| | - Tiziana Perin
- Division of Pathology, National Cancer Institute, Aviano (PN), Italy
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8
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Szparecki G, Ilczuk T, Gabzdyl N, Stocka-Łabno E, Górnicka B. Expression of c-MET Protein in Various Subtypes of Hepatocellular Adenoma Compared to Hepatocellular Carcinoma and Non-Neoplastic Liver in Human Tissue. Folia Biol (Praha) 2017; 63:146-154. [PMID: 29256857 DOI: 10.14712/fb2017063040146] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Hepatocellular adenoma (HA) is a benign neoplasm of the liver, whose aetiopathogenesis is little known. Newest research allowed dividing all cases into three types based on molecular characteristics: inflammatory HA, HA with HNF1A mutation, β-catenin-mutated HA. The clinical significance of HA is chiefly due to the possibility of malignant transformation into hepatocellular carcinoma (HCC). The aim of the present study was to immunohistochemically assess the expression pattern and level of c-MET protein in hepatocellular adenoma (taking into account its status of Wnt/β-catenin pathway functioning) and intertwining the results into a wider pattern of expression in non-neoplastic liver and hepatocellular carcinoma of various histological grades. It was found that expression of c-MET in poorly-differentiated HCC was significantly higher than in non-neoplastic liver and well- to moderately-differentiated HCC. The expression in HA was variable and differed between molecular subtypes of this neoplasm: inflammatory and HNF1A mutation-associated type are characterized by overexpression of c-MET to an extent comparable with poorly-differentiated HCC, whereas Wnt/β-catenin dysfunction-associated type lacks overexpression, and the amount of c-MET protein accumulated in its cells is similar to the levels in non-neoplastic tissue and well- to moderately-differentiated HCC. These findings suggest that c-MET overexpression in HA is not an early event in hepatocarcinogenesis, but constitutes a divergent molecular pathway leading to neoplastic change compared to overexpression observed in the late stages of tumour progression.
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Affiliation(s)
- G Szparecki
- Department of Pathology, Medical University of Warsaw, Poland
| | - T Ilczuk
- Department of Pathology, Medical University of Warsaw, Poland
| | - N Gabzdyl
- Department of Pathology, Medical University of Warsaw, Poland
| | - E Stocka-Łabno
- Department of Pathology, Medical University of Warsaw, Poland
| | - B Górnicka
- Department of Pathology, Medical University of Warsaw, Poland
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9
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Aptamers Binding to c-Met Inhibiting Tumor Cell Migration. PLoS One 2015; 10:e0142412. [PMID: 26658271 PMCID: PMC4676636 DOI: 10.1371/journal.pone.0142412] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2015] [Accepted: 10/21/2015] [Indexed: 01/04/2023] Open
Abstract
The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.
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10
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Granito A, Guidetti E, Gramantieri L. c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma. J Hepatocell Carcinoma 2015; 2:29-38. [PMID: 27508192 PMCID: PMC4918282 DOI: 10.2147/jhc.s77038] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
c-MET is the membrane receptor for hepatocyte growth factor (HGF), also known as scatter factor or tumor cytotoxic factor, a mitogenic growth factor for hepatocytes. HGF is mainly produced by cells of mesenchymal origin and it mainly acts on neighboring epidermal and endothelial cells, regulating epithelial growth and morphogenesis. HGF/MET signaling has been identified among the drivers of tumorigenesis in human cancers. As such, c-MET is a recognized druggable target, and against it, targeted agents are currently under clinical investigation. c-MET overexpression is a common event in a wide range of human malignancies, including gastric, lung, breast, ovary, colon, kidney, thyroid, and liver carcinomas. Despite c-MET overexpression being reported by a large majority of studies, no evidence for a c-MET oncogenic addiction exists in hepatocellular carcinoma (HCC). In particular, c-MET amplification is a rare event, accounting for 4%–5% of cases while no mutation has been identified in c-MET oncogene in HCC. Thus, the selection of patient subgroups more likely to benefit from c-MET inhibition is challenging. Notwithstanding, c-MET overexpression was reported to be associated with increased metastatic potential and poor prognosis in patients with HCC, providing a rationale for its therapeutic inhibition. Here we summarize the role of activated HGF/MET signaling in HCC, its prognostic relevance, and the implications for therapeutic approaches in HCC.
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Affiliation(s)
- Alessandro Granito
- Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy
| | - Elena Guidetti
- Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy
| | - Laura Gramantieri
- Dipartimento dell'Apparato Digerente, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; Centro di Ricerca Biomedica Applicata (CRBA), Azienda Ospedaliero-Universitaria Policlinico S Orsola-Malpighi e Università di Bologna, Bologna, Italy
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Abstract
Liver cancer is the third leading cause of cancer-related death worldwide. Advances in sequencing technologies have enabled the examination of liver cancer genomes at high resolution; somatic mutations, structural alterations, HBV integration, RNA editing and retrotransposon changes have been comprehensively identified. Furthermore, integrated analyses of trans-omics data (genome, transcriptome and methylome data) have identified multiple critical genes and pathways implicated in hepatocarcinogenesis. These analyses have uncovered potential therapeutic targets, including growth factor signalling, WNT signalling, the NFE2L2-mediated oxidative pathway and chromatin modifying factors, and paved the way for new molecular classifications for clinical application. The aetiological factors associated with liver cancer are well understood; however, their effects on the accumulation of somatic changes and the influence of ethnic variation in risk factors still remain unknown. The international collaborations of cancer genome sequencing projects are expected to contribute to an improved understanding of risk evaluation, diagnosis and therapy for this cancer.
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Affiliation(s)
- Tatsuhiro Shibata
- Division of Cancer Genomics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan
| | - Hiroyuki Aburatani
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8904, Japan
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12
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Giordano S, Columbano A. Met as a therapeutic target in HCC: facts and hopes. J Hepatol 2014; 60:442-52. [PMID: 24045150 DOI: 10.1016/j.jhep.2013.09.009] [Citation(s) in RCA: 139] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2013] [Revised: 08/13/2013] [Accepted: 09/03/2013] [Indexed: 12/15/2022]
Abstract
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and its burden is expected to increase further in the next years. In spite of the advances of classical therapies, such as surgery, transplantation, use of radiofrequency and transarterial embolization, the prognosis of this neoplasm has not considerably improved over the past few years. The advent of targeted therapies and the approval of the systemic treatment of advanced HCC with the kinase inhibitor sorafenib have provided some hope for the future. Even if the molecular mechanisms responsible for the onset and progression of HCC are still largely unknown, new therapeutic targets have recently come to the spotlight. One of these targets is the tyrosine kinase receptor for the Hepatocyte Growth Factor, encoded by the MET gene, known to promote tumor growth and metastasis in many human organs. In this review we will summarize the contrasting results obtained in vitro (in HCC cell lines) and in animal experimental models and we will also try to analyze the reasons for the opposite findings, suggesting that the HGF/MET axis can have either a promoting or a suppressive role in the development of HCC. We will also reconsider the evidence of activation of this pathway in human HCCs and discuss the results of the clinical trials performed with MET inhibitors. The final purpose is to better clarify which can be the role of MET as a therapeutic target in HCC.
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Affiliation(s)
- Silvia Giordano
- Department of Oncology, University of Torino, Institute for Cancer Research and Treatment (IRCC), 10060 Candiolo (Torino), Italy.
| | - Amedeo Columbano
- Department of Biomedical Sciences, Unit of Oncology and Molecular Pathology, University of Cagliari, Cagliari, Italy.
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Arao T, Ueshima K, Matsumoto K, Nagai T, Kimura H, Hagiwara S, Sakurai T, Haji S, Kanazawa A, Hidaka H, Iso Y, Kubota K, Shimada M, Utsunomiya T, Hirooka M, Hiasa Y, Toyoki Y, Hakamada K, Yasui K, Kumada T, Toyoda H, Sato S, Hisai H, Kuzuya T, Tsuchiya K, Izumi N, Arii S, Nishio K, Kudo M. FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma. Hepatology 2013; 57:1407-15. [PMID: 22890726 DOI: 10.1002/hep.25956] [Citation(s) in RCA: 122] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/07/2011] [Accepted: 06/25/2012] [Indexed: 12/13/2022]
Abstract
UNLABELLED The response rate to sorafenib in hepatocellular carcinoma (HCC) is relatively low (0.7%-3%), however, rapid and drastic tumor regression is occasionally observed. The molecular backgrounds and clinico-pathological features of these responders remain largely unclear. We analyzed the clinical and molecular backgrounds of 13 responders to sorafenib with significant tumor shrinkage in a retrospective study. A comparative genomic hybridization analysis using one frozen HCC sample from a responder demonstrated that the 11q13 region, a rare amplicon in HCC including the loci for FGF3 and FGF4, was highly amplified. A real-time polymerase chain reaction-based copy number assay revealed that FGF3/FGF4 amplification was observed in three of the 10 HCC samples from responders in which DNA was evaluable, whereas amplification was not observed in 38 patients with stable or progressive disease (P = 0.006). Fluorescence in situ hybridization analysis confirmed FGF3 amplification. In addition, the clinico-pathological features showed that multiple lung metastases (5/13, P = 0.006) and a poorly differentiated histological type (5/13, P = 0.13) were frequently observed in responders. A growth inhibitory assay showed that only one FGF3/FGF4-amplified and three FGFR2-amplified cancer cell lines exhibited hypersensitivity to sorafenib in vitro. Finally, an in vivo study revealed that treatment with a low dose of sorafenib was partially effective for stably and exogenously expressed FGF4 tumors, while being less effective in tumors expressing EGFP or FGF3. CONCLUSION FGF3/FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib. Our findings may provide a novel insight into the molecular background of HCC and sorafenib responders, warranting further prospective biomarker studies.
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Affiliation(s)
- Tokuzo Arao
- Department of Genome Biology, Kinki University Faculty of Medicine, Osaka, Japan
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14
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Locatelli A, Lofgren KA, Daniel AR, Castro NE, Lange CA. Mechanisms of HGF/Met signaling to Brk and Sam68 in breast cancer progression. Discov Oncol 2012; 3:14-25. [PMID: 22124844 DOI: 10.1007/s12672-011-0097-z] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Signal transduction pathways downstream of receptor tyrosine kinases (RTKs) are often deregulated during oncogenesis, tumor progression, and metastasis. In particular, the peptide growth factor hormone, hepatocyte growth factor (HGF), and its specific receptor, Met tyrosine kinase, regulate cancer cell migration, thereby conferring an aggressive phenotype (Nakamura et al., J Clin Invest 106(12):1511-1519, 2000; Huh et al., Proc Natl Acad Sci U S A 101:4477-4482, 2004). Additionally, overexpression of Met is associated with enhanced invasiveness of breast cancer cells (Edakuni et al., Pathol Int 51(3):172-178, 2001; Jin et al., Cancer 79(4):749-760, 1997; Tuck et al., Am J Pathol 148(1):225-232, 1996). Here, we review the regulation of recently identified novel downstream mediators of HGF/Met signaling, Breast tumor kinase (Brk/PTK6), and Src-associated substrate during mitosis of 68 kDa (Sam68), and discuss their relevance to mechanisms of breast cancer progression.
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Affiliation(s)
- Alessia Locatelli
- Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, 55455, USA
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15
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Goodman ZD, Terracciano LM, Wee A. Tumours and tumour-like lesions of the liver. MACSWEEN'S PATHOLOGY OF THE LIVER 2012:761-851. [DOI: 10.1016/b978-0-7020-3398-8.00014-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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16
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Jung S, Lee S, Lee J, Li C, Ohk JY, Jeong HK, Lee S, Kim S, Choi Y, Kim S, Lee H, Lee MS. Protein expression pattern in response to ionizing radiation in MCF-7 human breast cancer cells. Oncol Lett 2011; 3:147-154. [PMID: 22740871 DOI: 10.3892/ol.2011.444] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2011] [Accepted: 09/26/2011] [Indexed: 01/06/2023] Open
Abstract
Breast cancer is one of the most common types of cancer in women and is highly treatable by radiotherapy. However, repeated exposure to radiation results in tumor cell resistance. Understanding the molecular mechanisms involved in the response of tumors to γ-irradiation is important for improving radiotherapy. For this reason, we aimed to identify radiation-responsive genes at the protein level. In the present study, we observed differentially expressed proteins using 2D-PAGE and MALDI-TOF-MS for the global analysis of protein expression patterns in response to ionizing radiation (IR). When the expression patterns of proteins were compared to a control gel, numerous spots were found that differed greatly. Among them, 11 spots were found to be significantly different. One set of proteins (GH2, RGS17, BAK1, CCNH, TSG6, RAD51B, IGFBP1 and CASP14) was upregulated and another set of proteins (C1QRF, PLSCR2 and p34(SE1-1)) was downregulated after exposure to γ-rays. These proteins are known to be related to cell cycle control, apoptosis, DNA repair, cell proliferation and other signaling pathways.
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Affiliation(s)
- Samil Jung
- Research Center for Women's Diseases, Sookmyung Women's University, Seoul
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17
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Napieralski R, Brünner N, Mengele K, Schmitt M. Emerging biomarkers in breast cancer care. Biomark Med 2010; 4:505-22. [DOI: 10.2217/bmm.10.73] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Currently, decision-making for breast cancer treatment in the clinical setting is mainly based on clinical data, histomorphological features of the tumor tissue and a few cancer biomarkers such as steroid hormone receptor status (estrogen and progesterone receptors) and oncoprotein HER2 status. Although various therapeutic options were introduced into the clinic in recent decades, with the objective of improving surgery, radiotherapy, biochemotherapy and chemotherapy, varying response of individual patients to certain types of therapy and therapy resistance is still a challenge in breast cancer care. Therefore, since breast cancer treatment should be based on individual features of the patient and her tumor, tailored therapy should be an option by integrating cancer biomarkers to define patients at risk and to reliably predict their course of the disease and/or response to cancer therapy. Recently, candidate-marker approaches and genome-wide transcriptomic and epigenetic screening of different breast cancer tissues and bodily fluids resulted in new promising biomarker panels, allowing breast cancer prognosis, prediction of therapy response and monitoring of therapy efficacy. These biomarkers are now subject of validation in prospective clinical trials.
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Affiliation(s)
- Rudolf Napieralski
- Clinical Research Unit, Department of Obstetrics & Gynecology, Klinikum rechts der Isar, Technische Universitaet Muenchen, Germany
| | - Nils Brünner
- University of Copenhagen, Faculty of Life Sciences, Department of Veterinary Disease Biology, Ridebanevej 9, DK-1870 Frederiksberg C, Denmark
| | - Karin Mengele
- Clinical Research Unit, Department of Obstetrics & Gynecology, Klinikum rechts der Isar, Technische Universitaet Muenchen, Germany
| | - Manfred Schmitt
- Clinical Research Unit, Department of Obstetrics & Gynecology, Ismaninger Strasse 22, Klinikum rechts der Isar, Technische Universitaet Muenchen, D-81675 Munich, Germany
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18
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He AR, Soe K, El Zouhairi M. Current problems with systemic treatment of advanced hepatocellular cancer. Curr Probl Cancer 2010; 34:131-49. [PMID: 20417353 DOI: 10.1016/j.currproblcancer.2010.03.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Aiwu Ruth He
- Department of Medicine, Lombardi Cancer Center, Georgetown University, Washington, DC, USA
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19
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Chochi Y, Kawauchi S, Nakao M, Furuya T, Hashimoto K, Oga A, Oka M, Sasaki K. A copy number gain of the 6p arm is linked with advanced hepatocellular carcinoma: an array-based comparative genomic hybridization study. J Pathol 2009; 217:677-84. [PMID: 19097070 DOI: 10.1002/path.2491] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
In accordance with cancer progression, genomic aberrations accumulate in cancer cells in a stepwise fashion. However, whether there are genomic changes linked with tumour progression remains unclarified. The purpose of this study is to elucidate the relationship between genomic alterations and clinical stages in hepatocellular carcinoma (HCC). A technology of array-based CGH using DNA chips spotted with 1440 BAC clones was applied to 42 surgically removed HCCs to examine the DNA copy number aberrations. A frequent copy number gain was detected on chromosomal regions 1q, 8q and Xq. In particular, gains of 1q42.12, 1q43 and 8q24.3 were detected in more than 65% of tumours. A frequent copy number loss was detected on chromosomal regions 1p, 4q, 6q, 8p and 17p. Losses of 8p21 and 17p13 were detected in more than 55% of HCCs. However, the DNA copy number gains of clones on 6p and 8q24.12 were more frequent in stage III/IV tumours than in stage I/II tumours (p < 0.001). In particular, the gain of the whole 6p was virtually limited to advanced-staged HCCs. The gain of the whole 6p is suggested to be a genomic marker for the late stages in HCCs. These observations therefore support the concept of genomic staging in HCC.
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Affiliation(s)
- Yasuyo Chochi
- Department of Pathology, Yamaguchi University Graduate School of Medicine, Ube 755-8505, Japan
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20
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21
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Midorikawa Y, Sugiyama Y, Aburatani H. Screening of liver-targeted drugs. Expert Opin Drug Discov 2008; 3:643-54. [DOI: 10.1517/17460441.3.6.643] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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22
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Shah S. Statistical framework for quantitative analysis of array CGH. CONFERENCE PROCEEDINGS : ... ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. ANNUAL CONFERENCE 2007; 2006:5806-9. [PMID: 17946722 DOI: 10.1109/iembs.2006.259843] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Over the last several years there has been an explosion of microarray technology in the biosciences, medical sciences, biotechnology, and pharmaceutical industry. The technology has centered on providing a platform for determining the gene expression profiles of hundreds to tens of thousands of genes (or transcript levels of RNA species) in tissue, tumors, cells, or biological fluids in a single experiment. In recent years, this technology has been extended to include the use of microarrays to study genomic DNA for gains and losses of chromosomal regions. This has become possible through the attachment of large genomic fragments such as BACs (bacterial artificial chromosomes). In this paper, we present a methodology to model a CGH (comparative genomic hybridization) profile as a statistical process and solve for distribution parameters to determine genomic changes across the genome, including whole chromosome gains and losses, and focal point variations that are commonly seen in solid tumors and genetic disorders.
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Affiliation(s)
- Shishir Shah
- Dept. of Comput. Sci., Houston Univ., TX 77204, USA.
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23
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Lei KF, Wang YF, Zhu XQ, Lu PC, Sun BS, Jia HL, Ren N, Ye QH, Sun HC, Wang L, Tang ZY, Qin LX. Identification of MSRA gene on chromosome 8p as a candidate metastasis suppressor for human hepatitis B virus-positive hepatocellular carcinoma. BMC Cancer 2007; 7:172. [PMID: 17784942 PMCID: PMC2000900 DOI: 10.1186/1471-2407-7-172] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2007] [Accepted: 09/04/2007] [Indexed: 01/19/2023] Open
Abstract
Background The prognosis of patients with hepatocellular carcinoma (HCC) still remains very dismal, which is mainly due to metastasis. In our previous studies, we found that chromosome 8p deletions might contribute to metastasis of HCC. In this study, we aimed to identify the candidate metastatic suppressor gene on chromosome 8p. Methods Oligo-nucleotide microarrays which included 322 genes on human chromosome 8p were constructed to analyze the difference in gene expression profiles between HCC tissues with and without metastasis. The leading differentially expressed genes were identified and selected for further analysis by real-time PCR and Western blotting. Recombinant expression plasmid vectors for each target gene were constructed and transfected into HCC cells and its in vitro effects on proliferation and invasion of HCC cells were also investigated. Results Sixteen leading differentially expressed genes were identified from the HCC tissues with metastasis compared with those without metastasis (p < 0.01, q < 16 %). Among of the 10 significantly down-regulated genes in HCC with metastasis, methionine sulfoxide reductase A (MSRA) had the lowest p value and false discovery rate (FDR), and was considered as a potential candidate for metastasis suppressor gene. Real-time PCR and Western blotting confirmed that the mRNA and protein expression levels of MSRA were significantly decreased in HCC with metastasis compared with those without metastasis (p < 0.001), and MSRA mRNA level in HCCLM6 cells (with high metastatic potential) was also much lower than that of other HCC cell lines. Transfection of a recombinant expression plasmid vector and overexpression of MSRA gene could obviously inhibit cell colony formation (4.33 ± 2.92 vs. 9.17 ± 3.38, p = 0.008) and invasion (7.40 ± 1.67 vs. 17.20 ± 2.59, p= 0.0001) of HCCLM6 cell line. Conclusion MSRA gene on chromosome 8p might possess metastasis suppressor activity in HCC.
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Affiliation(s)
- Ke-Feng Lei
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Yan-Fang Wang
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Xiao-Qun Zhu
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Peng-Cheng Lu
- Statistics Graduate Program, Iowa State University, Ames, Iowa, USA
| | - Bing-Sheng Sun
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Hu-Liang Jia
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Ning Ren
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Qing-Hai Ye
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Hui-Chuan Sun
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Lu Wang
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Zhao-You Tang
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Lun-Xiu Qin
- Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China
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24
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Chen Y, Takita J, Mizuguchi M, Tanaka K, Ida K, Koh K, Igarashi T, Hanada R, Tanaka Y, Park MJ, Hayashi Y. Mutation and expression analyses of the MET and CDKN2A genes in rhabdomyosarcoma with emphasis on MET overexpression. Genes Chromosomes Cancer 2007; 46:348-58. [PMID: 17243166 DOI: 10.1002/gcc.20416] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. The simultaneous loss of Ink4a/Arf function and disruption of Met signaling in Ink4a/Arf-/- mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF) induces RMS with extremely high penetrance and short latency. To address the roles of MET and CDKN2A (p16INK4A/p14ARF) in human RMS, we performed mutational analyses in 39 samples of RMS by PCR-SSCP. No mutations were detected in exons 14-21 of MET whereas a nonsense mutation at codon 80 of p16(INK4A) was identified in an alveolar RMS cell line. We also quantified the relative expression levels and DNA copy numbers of these genes in seven cell lines and 17 fresh tumors by real-time quantitative PCR. Expression of MET was detected in all samples; however, more than 10-fold difference was found in the samples with higher or lower expression level, despite a normal DNA copy number. The protein expression level was consistent with that of mRNA, and in cell lines with a higher expression level, MET was constitutively activated. Notably, the expression level of MET was significantly higher in patients who died (P = 0.02), in patients with stage IV (P = 0.04), as well as in patients with PAX3-FKHR chimeric transcript (P = 0.04). On the other hand, reduced or absent expression of p16INK4A and/or p14(ARF) showed no significant correlation with the clinicopathological parameters, except for the age at diagnosis. Our data suggest that MET plays a role in the progression of RMS.
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Affiliation(s)
- Yuyan Chen
- Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
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25
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Midorikawa Y, Makuuchi M, Tang W, Aburatani H. Microarray-based analysis for hepatocellular carcinoma: From gene expression profiling to new challenges. World J Gastroenterol 2007; 13:1487-92. [PMID: 17461438 PMCID: PMC4146888 DOI: 10.3748/wjg.v13.i10.1487] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Accumulation of mutations and alterations in the expression of various genes result in carcinogenesis, and the development of microarray technology has enabled us to identify the comprehensive gene expression alterations in oncogenesis. Many studies have applied this technology for hepatocellular carcinoma (HCC), and identified a number of candidate genes useful as biomarkers in cancer staging, prediction of recurrence and prognosis, and treatment selection. Some of these target molecules have been used to develop new serum diagnostic markers and therapeutic targets against HCC to benefit patients. Previously, we compared gene expression profiling data with classification based on clinicopathological features, such as hepatitis viral infection or liver cancer progression. The next era of gene expression analysis will require systematic integration of expression profiles with other types of biological information, such as genomic locus, gene function, and sequence information. We have reported integration between expression profiles and locus information, which is effective in detecting structural genomic abnormalities, such as chromosomal gains and losses, in which we showed that gene expression profiles are subject to chromosomal bias. Furthermore, array-based comparative genomic hybridization analysis and allelic dosage analysis using genotyping arrays for HCC were also reviewed, with comparison of conventional methods.
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Affiliation(s)
- Yutaka Midorikawa
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, The University of Tokyo, Tokyo 113-8655, Japan.
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26
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Abstract
Various cytokines and soluble growth factors upon interaction with their membrane receptors are responsible for inducing cellular proliferation, differentiation, movement, and protection from anoikis (a planned suicide activated by normal cells in absence of attachment to neighboring cells or extracellular matrix (EMC)). Among those soluble factors a major position is exerted by hepatocyte growth factor (HGF) together with its receptor MET and macrophage-stimulating protein (MSP) in cooperation with its receptor RON.
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Affiliation(s)
- Silvia Benvenuti
- Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Candiolo (Torino), Italy
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Abstract
Hepatocellular carcinoma is among the most lethal and prevalent cancers in the human population. Despite its significance, there is only an elemental understanding of the molecular, cellular and environmental mechanisms that drive disease pathogenesis, and there are only limited therapeutic options, many with negligible clinical benefit. This Review summarizes the current state of knowledge of this, the most common and dreaded liver neoplasm, and highlights the principal challenges and scientific opportunities that are relevant to controlling this accelerating global health crisis.
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Affiliation(s)
- Paraskevi A Farazi
- Department of Genetics, Division of Medical Sciences, Harvard University, Boston, Massachusetts 02115, USA
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Park SJ, Jeong SY, Kim HJ. Y chromosome loss and other genomic alterations in hepatocellular carcinoma cell lines analyzed by CGH and CGH array. ACTA ACUST UNITED AC 2006; 166:56-64. [PMID: 16616112 DOI: 10.1016/j.cancergencyto.2005.08.022] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2005] [Revised: 08/16/2005] [Accepted: 08/23/2005] [Indexed: 11/26/2022]
Abstract
Hepatocellular carcinoma (HCC) is one of the most frequently occurring malignant tumors worldwide. The incidence of HCC is much higher in males than in females. In order to clarify the molecular basis of the male predominance in HCC, we have characterized the detailed genomic alterations in 5 hepatitis B virus integrated Korean HCC cell lines using G-banding, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), PCR, and CGH array. The commonest alterations were observed in chromosome 7 and Y, as well as chromosomal regions 1q, 8q, 4q, and 16q. The most frequent aberration of genomic material was gain of 1q and loss of chromosome Y. Significant loss of DNA copy number of the cancer related genes that are located on chromosome Y was detected by CGH array. By investigating the karyotypes of the previously reported 21 male HCC cell lines, we found 18 HCC cell lines with Y chromosome loss, indicating that this loss is a significant feature of HCC cell lines. We propose that Y chromosome loss in HCC cell lines may be responsible for the preponderance of males in HCC and its significance may lead to further studies for better understanding of carcinogenesis in HCC.
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Affiliation(s)
- Sang-Jin Park
- Department of Medical Genetics, School of Medicine, Ajou University, Suwon 442-721, Korea
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Wu X, Jia HL, Wang YF, Ren N, Ye QH, Sun HC, Wang L, Liu YK, Tang ZY, Qin LX. HTPAP gene on chromosome 8p is a candidate metastasis suppressor for human hepatocellular carcinoma. Oncogene 2006; 25:1832-40. [PMID: 16261160 DOI: 10.1038/sj.onc.1209191] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Our previous studies suggested that chromosome 8p deletion is associated with metastasis of hepatocellular carcinoma (HCC), in which some novel metastasis suppressor genes might be harbored. The present study aimed to identify the metastatic suppressor gene(s). A cDNA chip was constructed with the expressed sequence tags (ESTs) from chromosome 8p and used to compare the difference of expression profiling between the MHCC97-H and MHCC97-L cell lines with different metastatic potentials and similar genetic backgrounds. In all, 10 ESTs were significantly downregulated in MHCC97-H cell line with higher metastatic potential. One full-length gene, HTPAP (phosphatidic acid phosphatase type 2 domain containing 1B), was identified at chromosome 8p12. Sequencing and bioinformatic analyses revealed that HTPAP has 826 bp and encodes a putative protein of 175 amino acids with a transmembrane segment at the NH2 terminus, two protein kinase C phosphorylation site and one tyrosine kinase phosphorylation site. Its expression level in metastatic tumor tissues was much lower than that of primary HCC tissues. Both in vitro and in vivo assays suggested that HTPAP could suppress the invasion and metastasis of HCC. These suggested that HTPAP is a novel metastatic suppressor gene for HCC. The mechanism of the effect of HTPAP on HCC metastasis is not clear yet and deserves further investigation.
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Affiliation(s)
- X Wu
- Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai 200032, China
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Oh BK, Kim YJ, Park YN, Choi J, Kim KS, Park C. Quantitative assessment of hTERT mRNA expression in dysplastic nodules of HBV-related hepatocarcinogenesis. Am J Gastroenterol 2006; 101:831-8. [PMID: 16494581 DOI: 10.1111/j.1572-0241.2006.00532.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND Telomerase reverse transcriptase (hTERT) is the rate-limiting determinant of telomerase, which is critical for carcinogenesis. Dysplastic nodules (DNs) appear to be preneoplastic lesions of hepatocellular carcinomas (HCCs). In this study, in order to characterize DNs, hTERT mRNA, hTERT gene dosage, and mRNA for c-myc, a transcriptional activator of hTERT were studied in human multi-step hepatocarcinogenesis. METHODS Fifty four hepatic nodules including 5 large regenerative nodules, 14 low-grade DNs, 7 high-grade DNs, 11 DNs with HCC foci and 17 HCCs, 23 livers with chronic hepatitis/cirrhosis, and 6 normal livers were examined. Transcript levels were measured by real-time quantitative RT-PCR and gene dosages by real-time PCR and Southern blotting. RESULTS The hTERT mRNA levels increased with the progression of hepatocarcinogenesis, and a significant induction in the transition between low- and high-grade DNs was seen. Most high-grade DNs strongly expressed hTERT mRNA at levels similar to those of HCCs. Twenty-one percent of low-grade DNs had high levels of hTERT mRNA, up to those of high-grade DNs and there was no difference in the pathological features between low-grade DNs with and without increased hTERT mRNA levels. No correlation was found between hTERT mRNA levels, hTERT gene dosage, and c-myc mRNA levels. CONCLUSIONS These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.
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Affiliation(s)
- Bong-Kyeong Oh
- Department of Pathology, Center for Chronic Metabolic Disease Research and Yonsei Biomedical Science and Technology Initiative, Yonsei University College of Medicine, Seoul, Korea
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Katoh H, Shibata T, Kokubu A, Ojima H, Loukopoulos P, Kanai Y, Kosuge T, Fukayama M, Kondo T, Sakamoto M, Hosoda F, Ohki M, Imoto I, Inazawa J, Hirohashi S. Genetic profile of hepatocellular carcinoma revealed by array-based comparative genomic hybridization: identification of genetic indicators to predict patient outcome. J Hepatol 2005; 43:863-74. [PMID: 16139920 DOI: 10.1016/j.jhep.2005.05.033] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/04/2005] [Revised: 05/17/2005] [Accepted: 05/18/2005] [Indexed: 01/22/2023]
Abstract
BACKGROUND/AIMS We conducted an analysis of chromosomal numerical aberrations and their clinical significance in hepatocellular carcinoma. METHODS We analyzed 87 hepatocellular carcinomas by array-based comparative genomic hybridization with an array containing 800 bacterial artificial chromosome clones. RESULTS Frequent (>30%) chromosomal losses on 1p36.1, 4q21-25, 4q34-35.1, 8p23.3b-11.1, 13q14.1-14.3, 16p13.3, 16q22.1-24.3b, 17p13.3-13.1 and 17p13.3-11, and gains on 1q21-44f, 2q21.2, 2q34, 3q11.2, 5p14.2, 5q13.2-14, 7p22, 7p14.2, 7q21.1, 7q22.3, 7q34, 8q12-24.3 and 17q23, were observed. Recurrent (>5%) amplifications were detected on 1q25, 8q11 and 11q11, and we discovered a novel homozygous deletion at 14q32.11. The extent of chromosomal aberrations correlated significantly with various clinicopathological characteristics of the tumors, and increased in a stepwise manner with the progression of hepatocellular carcinoma. We also identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Multivariate analysis revealed that both chromosomal loss on 17p13.3 and gain on 8q11 are independent prognostic indicators. CONCLUSIONS Our results contribute to a complete description of genomic structural aberrations in relation to hepatocarcinogenesis and provide a valuable basis from which we can begin to understand the characteristics of tumors, predict patient outcomes and discover novel therapeutic targets for hepatocellular carcinoma.
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Affiliation(s)
- Hiroto Katoh
- Pathology Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, and Department of Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
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Fang H, Tong W, Perkins R, Shi L, Hong H, Cao X, Xie Q, Yim SH, Ward JM, Pitot HC, Dragan YP. Bioinformatics approaches for cross-species liver cancer analysis based on microarray gene expression profiling. BMC Bioinformatics 2005; 6 Suppl 2:S6. [PMID: 16026603 PMCID: PMC1637037 DOI: 10.1186/1471-2105-6-s2-s6] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Background The completion of the sequencing of human, mouse and rat genomes and knowledge of cross-species gene homologies enables studies of differential gene expression in animal models. These types of studies have the potential to greatly enhance our understanding of diseases such as liver cancer in humans. Genes co-expressed across multiple species are most likely to have conserved functions. We have used various bioinformatics approaches to examine microarray expression profiles from liver neoplasms that arise in albumin-SV40 transgenic rats to elucidate genes, chromosome aberrations and pathways that might be associated with human liver cancer. Results In this study, we first identified 2223 differentially expressed genes by comparing gene expression profiles for two control, two adenoma and two carcinoma samples using an F-test. These genes were subsequently mapped to the rat chromosomes using a novel visualization tool, the Chromosome Plot. Using the same plot, we further mapped the significant genes to orthologous chromosomal locations in human and mouse. Many genes expressed in rat 1q that are amplified in rat liver cancer map to the human chromosomes 10, 11 and 19 and to the mouse chromosomes 7, 17 and 19, which have been implicated in studies of human and mouse liver cancer. Using Comparative Genomics Microarray Analysis (CGMA), we identified regions of potential aberrations in human. Lastly, a pathway analysis was conducted to predict altered human pathways based on statistical analysis and extrapolation from the rat data. All of the identified pathways have been known to be important in the etiology of human liver cancer, including cell cycle control, cell growth and differentiation, apoptosis, transcriptional regulation, and protein metabolism. Conclusion The study demonstrates that the hepatic gene expression profiles from the albumin-SV40 transgenic rat model revealed genes, pathways and chromosome alterations consistent with experimental and clinical research in human liver cancer. The bioinformatics tools presented in this paper are essential for cross species extrapolation and mapping of microarray data, its analysis and interpretation.
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Affiliation(s)
- H Fang
- Division of Bioinformatics, Z-Tech Corporation, 3900 NCTR Road, Jefferson, AR 72079
| | - W Tong
- Division of Systems Toxicology, National Center for Toxicological Research (NCTR), FDA, 3900 NCTR Road, Jefferson, AR 72079
| | - R Perkins
- Division of Bioinformatics, Z-Tech Corporation, 3900 NCTR Road, Jefferson, AR 72079
| | - L Shi
- Division of Systems Toxicology, National Center for Toxicological Research (NCTR), FDA, 3900 NCTR Road, Jefferson, AR 72079
| | - H Hong
- Division of Bioinformatics, Z-Tech Corporation, 3900 NCTR Road, Jefferson, AR 72079
| | - X Cao
- Division of Bioinformatics, Z-Tech Corporation, 3900 NCTR Road, Jefferson, AR 72079
| | - Q Xie
- Division of Bioinformatics, Z-Tech Corporation, 3900 NCTR Road, Jefferson, AR 72079
| | - SH Yim
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892
| | - JM Ward
- Verterinary and Tumor Pathology Section, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702
| | - HC Pitot
- McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706
| | - YP Dragan
- Division of Systems Toxicology, National Center for Toxicological Research (NCTR), FDA, 3900 NCTR Road, Jefferson, AR 72079
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Tsubosa Y, Sugihara H, Mukaisho KI, Kamitani S, Peng DF, Ling ZQ, Tani T, Hattori T. Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization. ACTA ACUST UNITED AC 2005; 158:156-66. [PMID: 15796963 DOI: 10.1016/j.cancergencyto.2004.08.033] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2004] [Revised: 07/28/2004] [Accepted: 08/20/2004] [Indexed: 10/25/2022]
Abstract
Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.
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Affiliation(s)
- Yasuhiro Tsubosa
- Department of Pathology, Shiga University of Medical Science, Otsu 520-2192, Japan
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Kawaguchi K, Honda M, Yamashita T, Shirota Y, Kaneko S. Differential gene alteration among hepatoma cell lines demonstrated by cDNA microarray-based comparative genomic hybridization. Biochem Biophys Res Commun 2005; 329:370-80. [PMID: 15721316 DOI: 10.1016/j.bbrc.2005.01.128] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2005] [Indexed: 11/22/2022]
Abstract
We assayed chromosomal abnormalities in hepatoma cell lines using the microarray-based comparative genomic hybridization (array-CGH) method and investigated the relationship between genomic copy number alterations and expression profiles in these hepatoma cell lines. We modified a cDNA array-CGH assay to compare genomic DNAs from seven hepatoma cell lines, as well as DNA from two non-hepatoma cell lines and from normal cells. The mRNA expression of each sample was assayed in parallel by cDNA microarray. We identified small amplified or deleted chromosomal regions, as well as alterations in DNA copy number not previously described. We predominantly found alterations of apoptosis-related genes in Hep3B and HepG2, cell adhesion and receptor molecules in HLE, and cytokine-related genes in PLC/PRF/5. About 40% of the genes showing amplification or loss showed altered levels of mRNA (p < 0.05). Hierarchical clustering analysis showed that the expression of these genes allows differentiation between alpha-fetoprotein (AFP)-producing and AFP-negative cell lines. cDNA array-CGH is a sensitive method that can be used to detect alterations in genomic copy number in tumor cells. Differences in DNA copy alterations between AFP-producing and AFP-negative cells may lead to differential gene expression and may be related to the phenotype of these cells.
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Affiliation(s)
- Kazunori Kawaguchi
- Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
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Rae JM, Ramus SJ, Waltham M, Armes JE, Campbell IG, Clarke R, Barndt RJ, Johnson MD, Thompson EW. Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties. Clin Exp Metastasis 2005; 21:543-52. [PMID: 15679052 DOI: 10.1007/s10585-004-3759-1] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analysis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.
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Affiliation(s)
- James M Rae
- Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA
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Tsuda H, Birrer MJ, Ito YM, Ohashi Y, Lin M, Lee C, Wong WH, Rao PH, Lau CC, Berkowitz RS, Wong KK, Mok SC. Identification of DNA copy number changes in microdissected serous ovarian cancer tissue using a cDNA microarray platform. ACTA ACUST UNITED AC 2005; 155:97-107. [PMID: 15571795 DOI: 10.1016/j.cancergencyto.2004.03.002] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2003] [Revised: 02/26/2004] [Accepted: 03/03/2004] [Indexed: 11/30/2022]
Abstract
We have established a method for using a cDNA array platform in combination with degenerate oligonucleotide primer polymerase chain reaction (DOP-PCR) and taramide signal amplification (TSA) to identify DNA copy number abnormalities (CNA) in cancer cell lines and cancer cells procured with laser-based microdissection. To determine the sensitivity and specificity for detecting single-copy gain and loss, receiver-operator curve analysis was performed on hybridization signal ratios generated from non-DOP and DOP amplified female and male DNA using a 10,816-element cDNA microarray. A cutoff value of 1.12 and 1.07 average signal ratio for X-chromosomal genes versus autosomal genes provided a sensitivity and specificity of 50 and 79%, respectively, for non-DOP amplified DNA and a sensitivity and specificity of 50 and 72%, respectively, for DOP amplified DNA. We used this approach to identify DNA copy number abnormalities in the ovarian cancer cell line OVCA633, which has previously been shown to have 12p amplification. Transcription profiling of OVCA633 was also performed. Two amplified and overexpressed genes located on 12p11, KRAS2 and LRMP, were identified; these were validated with quantitative real-time PCR. Subsequently, the same approach was used to identify CNAs and gene expression alterations in 11 microdissected serous ovarian adenocarcinoma cases. Validated data revealed amplification and overexpression of ERBB3 and FOS and deletion and underexpression of KRT6 and APXL in more than 50% of the tissue samples. These results show the feasibility of using the cDNA array platform to identify changes in DNA and mRNA copy number simultaneously in microdissected tumor tissues.
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Affiliation(s)
- Hiroshi Tsuda
- Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Laboratory of Gynecologic Oncology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, BLI-447, 221 Longwood Avenue, Boston, MA 02115, USA
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Scaruffi P, Parodi S, Mazzocco K, Defferrari R, Fontana V, Bonassi S, Tonini GP. Detection of MYCN amplification and chromosome 1p36 loss in neuroblastoma by cDNA microarray comparative genomic hybridization. ACTA ACUST UNITED AC 2004; 8:93-100. [PMID: 15527323 DOI: 10.1007/bf03260051] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
BACKGROUND In the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identify MYCN gene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma. AIM The aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma, MYCN amplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer. METHODS DNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region and MYCN gene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control and Arabidopsis thaliana DNA was spotted to control unspecific hybridization. Fluorescence in situ hybridization analysis was also performed to validate results from the microarray CGH. RESULTS Both MYCN amplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detect MYCN amplification. DISCUSSION Our results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.
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Affiliation(s)
- Paola Scaruffi
- Laboratory of Neuroblastoma, National Cancer Research Institute (IST), Genoa, Italy
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Nakahara Y, Shiraishi T, Okamoto H, Mineta T, Oishi T, Sasaki K, Tabuchi K. Detrended fluctuation analysis of genome-wide copy number profiles of glioblastomas using array-based comparative genomic hybridization. Neuro Oncol 2004; 6:281-9. [PMID: 15494095 PMCID: PMC1872007 DOI: 10.1215/s1152851703000632)] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
We examined whole genomic aberrations of biopsied samples from 19 independent glioblastomas by array-based comparative genomic hybridization analysis. The highest frequencies of copy number gains were observed on RFC2 (73.3%), EGFR (63.2%), and FGR, ELN, CDKN1C , FES, TOP2A, and ARSA (57.9% each). The highest frequencies of copy number losses were detected on TBR1 (52.6%), BMI1 (52.6%), EGR2 (47.4%), DMBT1 (47.4%), MTAP (42.1%), and FGFR2 (42.1%). The copy number gains of CDKN1C and INS and the copy number losses of TBR1 were significantly correlated with longer survival of patients. High-level amplifications were identified on EGFR, SAS/CDK4, PDGFRA, MDM2, and ARSA. These genes are assumed to be involved in tumorigenesis or progression of glioblastomas. The first attempts to apply detrended fluctuation analysis to copy number profiles by considering the reading direction as the time axis demonstrated that higher long-term fractal scaling exponents (alpha2) correlated well with longer survival of glioblastoma patients. The present study indicates that array-based comparative genomic hybridization analysis has great potential for assessment of copy number changes and altered chromosomal regions of brain tumors. Furthermore, we show that nonlinear analysis methods of whole genome copy number profiles may provide prognostic information about glioblastoma patients.
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Affiliation(s)
- Yukiko Nakahara
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-850, Japan.
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Nakahara Y, Shiraishi T, Okamoto H, Mineta T, Oishi T, Sasaki K, Tabuchi K. Detrended fluctuation analysis of genome-wide copy number profiles of glioblastomas using array-based comparative genomic hybridization. Neuro Oncol 2004. [PMID: 15494095 DOI: 10.1215/s1152851703000632] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
We examined whole genomic aberrations of biopsied samples from 19 independent glioblastomas by array-based comparative genomic hybridization analysis. The highest frequencies of copy number gains were observed on RFC2 (73.3%), EGFR (63.2%), and FGR, ELN, CDKN1C , FES, TOP2A, and ARSA (57.9% each). The highest frequencies of copy number losses were detected on TBR1 (52.6%), BMI1 (52.6%), EGR2 (47.4%), DMBT1 (47.4%), MTAP (42.1%), and FGFR2 (42.1%). The copy number gains of CDKN1C and INS and the copy number losses of TBR1 were significantly correlated with longer survival of patients. High-level amplifications were identified on EGFR, SAS/CDK4, PDGFRA, MDM2, and ARSA. These genes are assumed to be involved in tumorigenesis or progression of glioblastomas. The first attempts to apply detrended fluctuation analysis to copy number profiles by considering the reading direction as the time axis demonstrated that higher long-term fractal scaling exponents (alpha2) correlated well with longer survival of glioblastoma patients. The present study indicates that array-based comparative genomic hybridization analysis has great potential for assessment of copy number changes and altered chromosomal regions of brain tumors. Furthermore, we show that nonlinear analysis methods of whole genome copy number profiles may provide prognostic information about glioblastoma patients.
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Affiliation(s)
- Yukiko Nakahara
- Department of Neurosurgery, Faculty of Medicine, Saga University, Saga 849-850, Japan.
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Dong G, Lee TL, Yeh NT, Geoghegan J, Van Waes C, Chen Z. Metastatic squamous cell carcinoma cells that overexpress c-Met exhibit enhanced angiogenesis factor expression, scattering and metastasis in response to hepatocyte growth factor. Oncogene 2004; 23:6199-208. [PMID: 15221009 DOI: 10.1038/sj.onc.1207851] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2003] [Revised: 04/06/2004] [Accepted: 04/28/2004] [Indexed: 12/23/2022]
Abstract
We previously performed gene expression profiling in a multistep squamous cell carcinoma (SCC) progression model, and identified growth-regulated oncogene-1 (Gro-1/KC) as a factor that contributes to enhanced angiogenesis, tumorigenesis and metastasis. In the present study, we explored molecular pathways coactivated with Gro-1/KC, and identified a transcript that encodes c-Met, the receptor for hepatocyte growth factor/scatter factor (HGF). Northern, Western blot, and immunohistochemical analyses confirm that expression of c-Met mRNA and protein is increased with SCC progression. In vitro, HGF preferentially promoted scattering in the metastatic LY-1 and LY-2 lines, and enhanced angiogenesis factors Gro-1/KC and vascular endothelial growth factor (VEGF) production by all tumor cell lines. In vivo, tumor growth and lung metastasis were promoted by transfection and overexpression of HGF cDNA in metastatic LY-1 cells. Our data indicate that metastatic SCC cells that overexpress c-Met exhibit angiogenesis factor expression and enhanced scattering in response to HGF in vitro, and tumorigenesis and metastasis in response to HGF in the tumor microenvironment in vivo.
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Affiliation(s)
- Gang Dong
- Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 10/5D55, MSC-1419, Bethesda, MD 20892-1419, USA
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Hashimoto K, Mori N, Tamesa T, Okada T, Kawauchi S, Oga A, Furuya T, Tangoku A, Oka M, Sasaki K. Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH. Mod Pathol 2004; 17:617-22. [PMID: 15133472 DOI: 10.1038/modpathol.3800107] [Citation(s) in RCA: 87] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
To clarify the genetic aberrations involved in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCV-HCC), we investigated DNA copy number aberrations (DCNAs) in 19 surgically resected HCCs by conventional CGH and array CGH. Conventional CGH revealed that increases of DNA copy number were frequent at 1q (79% of the cases), 8q (37%), 6p (32%), and 10p (32%) and that decreases were frequent at 17p (79%), 16q (58%), 4q (53%), 13q (42%), 10q (37%), 1p (32%), and 8p (32%). In general, genes that showed DCNAs by array CGH were usually located in chromosomal regions with DCNAs detected by conventional CGH analysis. Increases in copy numbers of the LAMC2, TGFB2, and AKT3 genes (located on 1q) and decreases in copy numbers of FGR/SRC2 and CYLD (located on 1p and 16q, respectively) were observed in more than 30% of tumors, including small, well-differentiated carcinomas. These findings suggest that these genes are associated with the development of HCV-HCC. Increases of MOS, MYC, EXT1, and PTK2 (located on 8q) were detected exclusively in moderately and poorly differentiated tumors, suggesting that these alterations contribute to tumor progression. In conclusion, chromosomal and array CGH technologies allow identification of genes involved in the development and progression of HCV-HCC.
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Raidl M, Pirker C, Schulte-Hermann R, Aubele M, Kandioler-Eckersberger D, Wrba F, Micksche M, Berger W, Grasl-Kraupp B. Multiple chromosomal abnormalities in human liver (pre)neoplasia. J Hepatol 2004; 40:660-8. [PMID: 15030983 DOI: 10.1016/j.jhep.2003.12.020] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/14/2003] [Revised: 12/15/2003] [Accepted: 12/29/2003] [Indexed: 12/20/2022]
Abstract
BACKGROUND/AIMS In human hepatocarcinogenesis the tumor precursor lesions and the sequence of genetic aberrations are not known. We therefore compared genetic alterations of different types of benign liver lesions to those of hepatocellular carcinoma. METHODS By comparative genomic hybridisation (CGH) 40 cases, including cirrhotic liver (CL), focal nodular hyperplasia (FNHs), hepatocellular adenoma (HCAs), dysplastic nodules (DNs), primary hepatocellular carcinoma (HCCs), and hepatocellular metastases to the lung were studied. RESULTS FNHs and HCAs exhibited few chromosomal abnormalities. Frequency and pattern of genetic alterations in DNs highly resembled those in HCCs: gains of DNA clustered in chromosome arms 1p/q, 7q, 15q, 16p, 17q, and 20q and losses were often found at 3p, 4q, 9p, and 11q. Aberrations on 1p, 6q, 8p/q, and 13q occurred almost exclusively in HCCs; the gain at 8q encompassed amplification of c-myc, as verified by fluorescence in situ hybridisation. CONCLUSIONS The pattern of genetic alterations in HCCs resembled more the alterations found in DNs than in FNHs and HCAs, suggesting that DNs may be the actual tumor precursors. Furthermore, alterations at 4q, 9p, 11q, 16p, and 17q appear as early genetic events being crucial for hepatocarcinogenesis.
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Affiliation(s)
- Maria Raidl
- Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria
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Pontisso P, Calabrese F, Benvegnù L, Lise M, Belluco C, Ruvoletto MG, Marino M, Valente M, Nitti D, Gatta A, Fassina G. Overexpression of squamous cell carcinoma antigen variants in hepatocellular carcinoma. Br J Cancer 2004; 90:833-7. [PMID: 14970861 PMCID: PMC2410161 DOI: 10.1038/sj.bjc.6601543] [Citation(s) in RCA: 97] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (score⩽1) was associated to lower MIB-1 labelling index, compared to cases with SCCA score ⩾2 (mean±s.d.: 2%±2.4 vs 7.5%±10.3, P<0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G351 to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.
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Affiliation(s)
- P Pontisso
- Department of Clinical and Experimental Medicine, Via Giustiniani, 2 35123, Padova, Italy.
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van Dekken H, Wink J, Alers JC, de Man RA, IJzermans JN, Zondervan PE. Genetic evaluation of the dysplasia-carcinoma sequence in chronic viral liver disease: a detailed analysis of two cases and a review of the literature. Acta Histochem 2003; 105:29-41. [PMID: 12666986 DOI: 10.1078/0065-1281-00694] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies, especially in Asia and Africa, but also in the Western world its incidence is increasing. HCC is a complication of chronic liver disease with cirrhosis as the most important risk factor. Viral co-pathogenesis due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection seems to be an important factor in the development of HCC. Curative therapy is often not possible due to the late detection of HCC. Thus, it is attractive to find parameters which predict malignant transformation in HBV- and HCV-infected livers. In the past decade, preneoplastic lesions, i.e. dysplastic foci or nodules, have gained interest as possible markers for imminent malignancy. Noteworthy, dysplastic liver lesions are increasingly detected by imaging techniques. We describe here two cases of chronic viral liver disease, one HBV-and one HCV-related, in which dysplastic lesions were present adjacent to HCC. In the HBV case, a (smaller) satellite of HCC was present as well. The neoplastic specimens were investigated by comparative genomic hybridization (CGH) and in situ hybridization (ISH). Both methods revealed multiple genetic alterations in the HCCs. The genetic patterns of the HBV-related HCC and the satellite tumor showed many shared alterations suggesting a clonal relationship. A subset of genetic changes were already present in dysplasias illustrating their preneoplastic nature. Surrounding liver cirrhosis samples did not display chromosomal aberrations. A literature survey illustrates the relative paucity of information concerning genetic alterations in preneoplastic liver lesions. However, all the data strongly suggests a role for liver cell dysplasia as a precursor condition of liver cell cancer.
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Affiliation(s)
- Herman van Dekken
- Department of Pathology, Erasmus MC, Rotterdam University Medical Center, Rotterdam, The Netherlands.
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Abstract
Molecular cytogenetic methods including fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) can be used for surgically removed solid tumors to obtain information valuable for both biomedical research and clinical oncology. FISH allows cytogenetic analysis even of cells in interphase. In addition, because CGH analysis permits comprehensive analysis of alterations in DNA copy number in a single experiment, it is possible to estimate not only the genetic pathways of carcinogenesis but also the biological characteristics, such as metastatic potential and patient prognosis at the time of diagnosing the solid tumor. The number of DNA copy number aberrations increases with tumor progression, leading to the concept of genetic staging of malignant tumors. Molecular cytogenetic analysis aids in realizing individualized, tailored medicine in cancer patients; therapeutic strategies are constructed for individual patients based on specific genetic alterations.
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Affiliation(s)
- Kohsuke Sasaki
- Department of Pathology, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube 755-8505, Japan
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Sasaki T, Arai H, Beppu T, Ogasawara K. Detection of gene amplification and deletion in high-grade gliomas using a genome DNA microarray (GenoSensor Array 300). Brain Tumor Pathol 2003; 20:59-63. [PMID: 14756442 DOI: 10.1007/bf02483448] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Glioblastoma is a rapidly growing tumor that accounts for more than 50% of all primary gliomas. Amplification of oncogenes and deletion of tumor suppressor genes frequently affects tumor progression. Thus, the goal of this study was to conduct a comprehensive analysis of gene aberrations of individual glioblastomas. A genome DNA microarray (GenoSensor Array 300), spotted with 287 target genes, was used to analyze resected tissue from 11 different high-grade gliomas. The average number of gene aberrations was 9.0 per case (WHO grade III) and 13.3 per case (WHO grade IV). EGFR was the most frequent amplified gene in this series (4 of 11 cases), and high-level amplification was also detected for EGFR, SAS/CDK4, and AKT1. A high frequency of deleted genes was observed in 6 of 11 cases (54.5%), including FGFR2, MTAP, and DMBT1. The detected gene aberrations were matched to the classical primary glioblastoma pathway in five of nine cases. We conclude that the GenoSensor Array 300 genomic DNA microarray is a useful method for the comprehensive identification of amplified and deleted genes in glioblastoma.
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Affiliation(s)
- Teruo Sasaki
- Department of Neurosurgery, Iwate Medical University, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan.
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Wu Y, Han B, Sheng H, Lin M, Moore PA, Zhang J, Wu J. Clinical significance of detecting elevated serum DcR3/TR6/M68 in malignant tumor patients. Int J Cancer 2003; 105:724-32. [PMID: 12740925 DOI: 10.1002/ijc.11138] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
TR6/DcR3/M68 is a soluble receptor that belongs to the TNF receptor family. It is expressed in malignant cells of several tumor types and has been postulated to help tumor cells to gain survival advantage by inhibiting apoptosis and by interfering with immune surveillance. In our study, we assessed for the first time serum TR6 in tumor patients to explore its diagnostic and prognostic value. We examined serum TR6 levels with ELISA in 146 tumor patients, 19 patients with acute infection, 5 patients with liver cirrhosis and 29 healthy individuals. TR6 expression in tumor mass was studied with immunohistochemistry. TR6 gene copy number in tumor tissues was evaluated by real time PCR. Ninety-seven point nine percent (47 of 48 cases) of healthy individuals and patients with acute infection were serum TR6-negative. In contrast, 56.2% (82 of 146 cases) of the tumor patients were serum TR6-positive. Almost all serum TR6-positive individuals (98.8%, 82 out of 83 cases) had malignancy, excluding the cases of liver cirrhosis. In gastric carcinomas, serum TR6 levels were closely correlated with tumor differentiation status and TNM classification. Tumor mass was the source of serum TR6 because its levels decreased drastically after curative tumor resection. TR6 gene amplification occurred in about half of liver carcinomas, but not in gastric or pancreatic carcinomas, indicating plural mechanisms of TR6 upregulation. Our study demonstrated that serum TR6 should be considered as a novel parameter for the diagnosis, treatment and prognosis of malignancies.
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Affiliation(s)
- Yulian Wu
- Laboratory of Transplantation Immunology, Research Center, Centre Hospitalier de l'Universite de Montreal (CHUM)-Notre Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, Canada
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Abstract
The genome of an organism is a dynamic physical entity, comprising genomic DNA bound to many different proteins and organized into chromosomes. A thorough characterization of the physical genome is relevant to our understanding of processes such as the regulation of gene expression, DNA replication and repair, recombination, chromosome segregation, epigenetic inheritance and genomic instability. Methods based on microarrays are beginning to provide a detailed picture of this physical genome, and they complement the genome-wide studies of mRNA expression profiling that have previously been so successful.
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Affiliation(s)
- Jonathan R Pollack
- Department of Pathology, Stanford University School of Medicine, CCSR Building, Room 3245A, 269 Campus Drive, Stanford, California 94305-5176, USA.
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Armour JAL, Barton DE, Cockburn DJ, Taylor GR. The detection of large deletions or duplications in genomic DNA. Hum Mutat 2002; 20:325-37. [PMID: 12402329 DOI: 10.1002/humu.10133] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications.
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Affiliation(s)
- J A L Armour
- Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, UK
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