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Nie S, Chang L, Huang Y, Zhou H, Yang Q, Kong L, Li Y. β-carboline derivative Z86 attenuates colorectal cancer cell proliferation and migration by directly targeting PI3K. NATURAL PRODUCTS AND BIOPROSPECTING 2024; 14:3. [PMID: 38169019 PMCID: PMC10761647 DOI: 10.1007/s13659-023-00422-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Accepted: 11/22/2023] [Indexed: 01/05/2024]
Abstract
Phosphoinositide 3-kinase (PI3Ks) are lipid kinases widely involved in cell proliferation, metastasis and differentiation. Constitutive activation of the PI3K/Akt/mTOR signaling are well confirmed in colorectal cancers (CRCs). In this study, we identified isopropyl 9-ethyl-1-(naphthalen-1-yl)-9 H-pyrido[3,4-b] indole-3-carboxylate (Z86), as a novel PI3Kα inhibitor with the IC50 value of 4.28 µM. The binding of Z86 to PI3Kα was further confirmed with DARTS and CETSA assay. Immunofluorescence analysis and western blotting data demonstrated that Z86 effectively attenuated PI3K/AKT pathway. Z86 caused dramatic proliferation inhibition of CRCs through G0/G1 cycle arrest rather than apoptosis induction. Besides, the migration of CRCs was also relieved by Z86. The present study not only identified Z86 as a novel PI3Kα inhibitor with potent inhibitory efficiency on PI3K-mediated CRCs growth and migration, but also elucidated a reasonable molecular mechanism of Z86 in the Wnt signaling pathway inhibition.
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Affiliation(s)
- Shiyun Nie
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China
| | - Lizhong Chang
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China
| | - Ying Huang
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China
| | - Heyang Zhou
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China
| | - Qianqing Yang
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China
| | - Lingmei Kong
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China.
| | - Yan Li
- Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan Key Laboratory of Research and Development for Natural Products, School of Pharmacy, Ministry of Education, Yunnan University, Kunming, 650500, People's Republic of China.
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Sugara M, Chowdappa R, Kumar KVV, Gawari R, Swamy SN, Kumar SS. Aberrant Promoter Hypermethylation of p16 and RASSF1a Genes in Colorectal Cancer - Significance in Young Patients. Indian J Surg Oncol 2021; 12:454-459. [PMID: 34658570 PMCID: PMC8490567 DOI: 10.1007/s13193-021-01325-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Accepted: 04/15/2021] [Indexed: 12/31/2022] Open
Abstract
OBJECTIVE The clinical profile of colorectal cancers (CRC) in India is different from that described in western countries. Microsatellite instability and APC mutation explain the molecular biology of up to 50% of colorectal cancers. Global genome hypermethylation may be the cause in at least 20% of cases. Few studies from India have examined the epigenetic profile of colorectal cancers. This study was designed to study aberrant promoter hypermethylation of two select tumour suppressor genes (p16, RASSF1a) in patients with colorectal cancer and their association with clinicopathologic features. METHODS A total of 41 samples including controls were collected from colorectal cancer patients. DNA was isolated from tumour tissue, and methylation-specific PCR was performed for the 2 genes. RESULTS p16 and RASSF1a promoter hypermethylation was found in 26% and 48% of CRC cases, respectively. RASSF1a promoter hypermethylation was more often seen in young CRC patients aged 40 years or less, and this was found to be statistically significant (p value = 0.037). CONCLUSION RASSF1a hypermethylation is peculiar to rectal cancers and left-sided colonic tumours in young patients. Large-scale population-based studies with extensive genetic and epigenetic characterization are required for a better understanding and further validation of our findings. For individuals diagnosed with sporadic CRC, these studies on specimen might help predict prognosis and response to therapy.
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Affiliation(s)
- Medha Sugara
- Department of Surgical oncology, St. John’s Medical College, Bengaluru, Karnataka 560034 India
| | - Ramachandra Chowdappa
- Department of Surgical Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka 560029 India
| | - K. V. Veerendra Kumar
- Department of Surgical Oncology, Jawaharlal Nehru Medical College, Belgaum, Karnataka 590010 India
| | - Ramesh Gawari
- Department of Biochemistry, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka 560029 India
| | - Shalini N. Swamy
- Department of Biochemistry, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka 560029 India
| | - Sandeep S. Kumar
- Department of Biochemistry, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka 560029 India
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Petrillo A, Salati M, Trapani D, Ghidini M. Circulating Tumor DNA as a Biomarker for Outcomes Prediction in Colorectal Cancer Patients. Curr Drug Targets 2021; 22:1010-1020. [PMID: 33155906 DOI: 10.2174/1389450121999201103194248] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2020] [Revised: 08/07/2020] [Accepted: 08/23/2020] [Indexed: 12/24/2022]
Abstract
Circulating tumour DNA (ctDNA) is a novel tool that has been investigated in several types of tumours, including colorectal cancer (CRC). In fact, the techniques based on liquid biopsies are proposed as appealing non-invasive alternatives to tissue biopsy, adding more insights into tumour molecular profile, heterogeneity and for cancer detection and monitoring. Additionally, some analysis showed that in CRC patients, ctDNA seems to act as a biomarker able to predict the outcome (prognostic role) and the response to treatments (predictive role). In particular, in the early stage CRC (stage I-III), it could represent a time marker of adjuvant therapy as well as a marker of minimal residual disease and recurrence risk in addition to the already recognized risk factors. In metastatic CRC, the analysis of molecular tumour profile by ctDNA has shown to have high concordance with the tissue biopsy at diagnosis. Additionally, some studies demonstrated that ctDNA level during the treatment was linked with the early response to treatment and prognosis. Finally, the quantitative analysis of ctDNA and copy number alterations may be useful in order to detect resistance to therapy at the time of progression of disease and to help in finding new therapeutic targets.
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Affiliation(s)
| | - Massimiliano Salati
- Department of Oncology and Hematology, University Hospital of Modena, Modena, Italy
| | - Dario Trapani
- Division of Early Drug Development for Innovative Therapies, European Institute of Oncology IRCCS, Milan, Italy
| | - Michele Ghidini
- Oncology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
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Tabibzadeh A, Tameshkel FS, Moradi Y, Soltani S, Moradi-Lakeh M, Ashrafi GH, Motamed N, Zamani F, Motevalian SA, Panahi M, Esghaei M, Ajdarkosh H, Mousavi-Jarrahi A, Niya MHK. Signal transduction pathway mutations in gastrointestinal (GI) cancers: a systematic review and meta-analysis. Sci Rep 2020; 10:18713. [PMID: 33127962 PMCID: PMC7599243 DOI: 10.1038/s41598-020-73770-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Accepted: 09/02/2020] [Indexed: 02/07/2023] Open
Abstract
The present study was conducted to evaluate the prevalence of the signaling pathways mutation rate in the Gastrointestinal (GI) tract cancers in a systematic review and meta-analysis study. The study was performed based on the PRISMA criteria. Random models by confidence interval (CI: 95%) were used to calculate the pooled estimate of prevalence via Metaprop command. The pooled prevalence indices of signal transduction pathway mutations in gastric cancer, liver cancer, colorectal cancer, and pancreatic cancer were 5% (95% CI: 3-8%), 12% (95% CI: 8-18%), 17% (95% CI: 14-20%), and 20% (95% CI: 5-41%), respectively. Also, the mutation rates for Wnt pathway and MAPK pathway were calculated to be 23% (95% CI, 14-33%) and 20% (95% CI, 17-24%), respectively. Moreover, the most popular genes were APC (in Wnt pathway), KRAS (in MAPK pathway) and PIK3CA (in PI3K pathway) in the colorectal cancer, pancreatic cancer, and gastric cancer while they were beta-catenin and CTNNB1 in liver cancer. The most altered pathway was Wnt pathway followed by the MAPK pathway. In addition, pancreatic cancer was found to be higher under the pressure of mutation compared with others based on pooled prevalence analysis. Finally, APC mutations in colorectal cancer, KRAS in gastric cancer, and pancreatic cancer were mostly associated gene alterations.
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Affiliation(s)
- Alireza Tabibzadeh
- Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Fahimeh Safarnezhad Tameshkel
- Student Research Committee, Iran University of Medical Sciences, Tehran, Iran
- Gastrointestinal and Liver Disease Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Yousef Moradi
- Social Determinants of Health Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
| | - Saber Soltani
- Department of Virology, Tehran University of Medical Sciences, Tehran, Iran
| | - Maziar Moradi-Lakeh
- Gastrointestinal and Liver Disease Research Center, Iran University of Medical Sciences, Tehran, Iran
- Preventive Medicine and Public Health Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - G Hossein Ashrafi
- Cancer Theme SEC Faculty, Kingston University, Penrhyn Road, London, KT1 2EE, UK
| | - Nima Motamed
- Department of Social Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
| | - Farhad Zamani
- Gastrointestinal and Liver Disease Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Seyed Abbas Motevalian
- Department of Epidemiology, School of Public Health, Iran University of Medical Sciences, Tehran, Iran
| | - Mahshid Panahi
- Gastrointestinal and Liver Disease Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Maryam Esghaei
- Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Hossein Ajdarkosh
- Gastrointestinal and Liver Disease Research Center, Iran University of Medical Sciences, Tehran, Iran
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Ta TV, Nguyen QN, Chu HH, Truong VL, Vuong LD. RAS/RAF mutations and their associations with epigenetic alterations for distinct pathways in Vietnamese colorectal cancer. Pathol Res Pract 2020; 216:152898. [PMID: 32089414 DOI: 10.1016/j.prp.2020.152898] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Revised: 01/27/2020] [Accepted: 02/16/2020] [Indexed: 02/07/2023]
Abstract
KRAS, NRAS, and BRAF are potential tumor-driven genes that are involved in the RAS/RAF/MAPK signaling pathway. RAS/RAF mutations importantly contribute to colorectal tumorigenesis since they remain the activated status of downstream pathways without regulation of the upstream EGFR signal. However, it has not been unclear how epigenetic alterations involved in colorectal tumorigenesis mediated by KRAS, NRAS, or BRAF mutations. Therefore, in this study, we investigated the frequency and distribution of KRAS/NRAS/BRAF mutations in Vietnamese colorectal cancer (CRC) and explored the relationship between genetic and epigenetic abnormalities in 156 tumors of CRC. Somatic mutations of KRAS (exon 2, codon 12/13; exon 3, codon 61), NRAS (exon 2, codon 12/13; exon 3, codon 61), and BRAF (exon 15, codon 600) was determined by Cobas® KRAS Mutation Test, Therascreen NRAS Pyro Kit and Cobas® 4800 BRAF V600 Mutation Test, respectively. Methylation status of BRCA1, MLH1, MGMT, p16, RASSF1A, and APC was detected by methylation-specific PCR. Distribution of each abnormality in clinicopathological features was also analyzed. Results showed the mutation rates of KRAS, NRAS, and BRAF were 41.0 %, 9.6 %, 8.3 % respectively, while the methylation rates of BRCA1, MLH1, MGMT, p16, RASSF1A, and APC were 16.7 %, 16.7 %, 32.7 %, 30.1 %, 30.1 %, and 37.2 % respectively. The distribution of KRAS mutation was mutually exclusive against that of NRAS (p < 0.001) and BRAF (p < 0.001) mutations in CRC. RAS/RAF mutations were more common in adenocarcinoma subtype (p = 0.020), whereas RASSF1A methylation was more frequent in mucinous adenocarcinoma subtype (p = 0.007). In addition, the frequency of having KRAS mutations was significantly higher in MGMT (p = 0.035) or RASSF1A (p = 0.043) methylated cases than in those without methylation. BRAF mutations were positively associated with MLH1 hypermethylation (p = 0.028) but were inversely associated with APC hypermethylation (p = 0.032). Overall, our results show specific interactions of genetic and epigenetic alterations and suggest the presence of independent oncogenic pathways in tumorigenesis of CRC.
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Affiliation(s)
- To Van Ta
- Pathology and Molecular Biology Center, National Cancer Hospital K, 30 Cau Buou Street, Thanh Tri, Hanoi, Viet Nam
| | - Quang Ngoc Nguyen
- Pathology and Molecular Biology Center, National Cancer Hospital K, 30 Cau Buou Street, Thanh Tri, Hanoi, Viet Nam
| | - Ha Hoang Chu
- National Key Laboratory of Gene Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay, Hanoi, Viet Nam
| | - Van-Long Truong
- Department of Smart Food and Drug, College of BNIT, Inje University, Gimhae 50834, South Korea.
| | - Linh Dieu Vuong
- Pathology and Molecular Biology Center, National Cancer Hospital K, 30 Cau Buou Street, Thanh Tri, Hanoi, Viet Nam.
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Germline polymorphisms in the Von Hippel-Lindau and Hypoxia-inducible factor 1-alpha genes, gene-environment and gene-gene interactions and renal cell cancer. Sci Rep 2020; 10:137. [PMID: 31924838 PMCID: PMC6954183 DOI: 10.1038/s41598-019-56980-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Accepted: 11/23/2019] [Indexed: 01/20/2023] Open
Abstract
We investigated the relationship between germline single nucleotide polymorphisms (SNPs) in Von Hippel-Lindau (VHL) and Hypoxia-inducible factor 1-alpha (HIF1A), and their gene-environment and gene-gene interactions, and clear-cell RCC (ccRCC) risk. Furthermore, we assessed the relationship between VHL SNPs and VHL promoter methylation. Three VHL polymorphisms and one HIF1A polymorphism were genotyped in the Netherlands Cohort Study. In 1986, 120,852 participants aged 55–69 completed a self-administered questionnaire on diet and lifestyle and toenail clippings were collected. Toenail DNA was genotyped using the Sequenom MassARRAY platform. After 20.3 years, 3004 subcohort members and 406 RCC cases, of which 263 ccRCC cases, were eligible for multivariate case-cohort analyses. VHL_rs779805 was associated with RCC (Hazard Ratio (HR) 1.53; 95% Confidence Interval (CI) 1.07–2.17) and ccRCC risk (HR 1.88; 95% CI 1.25–2.81). No associations were found for other SNPs. Potential gene-environment interactions were found between alcohol consumption and selected SNPs. However, none remained statistically significant after multiple comparison correction. No gene-gene interactions were observed between VHL and HIF1A. VHL promoter methylation was not associated with VHL SNPs. VHL SNPs may increase (cc)RCC susceptibility. No associations were found between gene-environment and gene-gene interactions and (cc)RCC risk and between VHL promoter methylation and VHL SNPs.
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Parseghian CM, Napolitano S, Loree JM, Kopetz S. Mechanisms of Innate and Acquired Resistance to Anti-EGFR Therapy: A Review of Current Knowledge with a Focus on Rechallenge Therapies. Clin Cancer Res 2019; 25:6899-6908. [PMID: 31263029 PMCID: PMC6891150 DOI: 10.1158/1078-0432.ccr-19-0823] [Citation(s) in RCA: 84] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2019] [Revised: 05/16/2019] [Accepted: 06/26/2019] [Indexed: 02/06/2023]
Abstract
Innate and acquired resistance to anti-EGFR therapy (EGFRi) is a major limitation in the treatment of metastatic colorectal cancer (mCRC). Although RAS genes are the most commonly mutated innate and acquired oncogenes in cancer, there are a number of other mechanisms that limit the effectiveness of EGFRi. Patients with innate resistance have been found to contain BRAFV600E mutations, and possibly MET, MEK, PIK3CA, PTEN, and HER2 alterations. Meanwhile, BRAFV600E mutations may also be involved in acquired resistance to EGFRi, in addition to EGFR ectodomain mutations, MET alterations, and possibly HER2 amplification. In addition, paracrine effects and cell-fate mechanisms of resistance are being increasingly described as contributing to acquired resistance. Utilization of circulating tumor DNA has been paramount in monitoring the dynamic nature of acquired resistance and has helped to guide treatment decisions, particularly in the EGFRi rechallenge setting. Herein, we provide an in-depth review of EGFRi-resistance mechanisms and describe the current therapeutic landscape in the hopes of identifying effective rechallenge strategies.
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Affiliation(s)
- Christine M Parseghian
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
| | - Stefania Napolitano
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | | | - Scott Kopetz
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas
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Oh SJ, Lee MG, Moon JR, Lee CK, Chi SG, Kim HJ. Ras association domain family 1 isoform A suppresses colonic tumor cell growth through p21 WAF1 activation in a p53-dependent manner. J Gastroenterol Hepatol 2019; 34:890-898. [PMID: 30226276 DOI: 10.1111/jgh.14469] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/13/2018] [Revised: 09/02/2018] [Accepted: 09/06/2018] [Indexed: 01/06/2023]
Abstract
BACKGROUND AND AIM Despite the frequent loss of Ras association domain family 1 isoform A (RASSF1A) expression in various cancers, the precise mechanism underlying its tumor-suppressive effect is not fully understood. To elucidate the growth-inhibitory role for RASSF1A in colorectal tumorigenesis, this study investigated the RASSF1A regulation of the p53-p21WAF1 pathway. METHODS Ras association domain family 1 isoform A effect on cellular growth was tested in three human colon cancer cell lines by flow cytometry, cell counting, and [3 H]-thymidine incorporation assay. HCT116 p53+/+ and p53-/- isogenic sublines were utilized to determine the p53 dependence of RASSF1A effect on p21WAF1 . Cycloheximide chase experiment and immunoprecipitation assay were carried out to define RASSF1A effect on p53 stability and mouse double minute 2 (MDM2) homolog ubiquitination. RESULTS Ras association domain family 1 isoform A expression inhibits colonic cell proliferation by preventing the G1 to S phase transition of the cell cycle. The RASSF1A-induced G1 cell cycle arrest is accompanied by the increase in the level of p21WAF1 mRNA expression. The p21WAF -inducing activity of RASSF1A was substantially higher in HCT116 p53+/+ cell compared with isogenic p53-/- cells. The cycloheximide chase assay revealed that RASSF1A expression leads to p53 stabilization and MDM2 homolog degradation. Using p53-/- and p21WAF1-/- subline cells, this study finally validated a crucial role of the p53-p21WAF1 axis in RASSF1A-mediated growth inhibition. CONCLUSIONS RASSF1A suppresses colonic tumor growth through the activation of the p53-p21WAF1 pathway. This finding supports that RASSF1A could be a valuable marker for the assessment of colorectal cancer development and progression.
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Affiliation(s)
- Shin Ju Oh
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea
| | - Min-Goo Lee
- Department of Life Sciences, Korea University, Seoul, Korea
| | - Jung Rock Moon
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea
| | - Chang Kyun Lee
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea
| | - Sung-Gil Chi
- Department of Life Sciences, Korea University, Seoul, Korea
| | - Hyo Jong Kim
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea
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Yu J, Sung JJ. Differential colorectal cancer genomics between east and west. J Gastroenterol Hepatol 2019; 34:811-812. [PMID: 31044476 DOI: 10.1111/jgh.14675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Affiliation(s)
- Jun Yu
- Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong
| | - Joseph Jy Sung
- Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong
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Salama RH, Sayed ZEAA, Ashmawy AM, Elsewify WA, Ezzat GM, Mahmoud MA, Alsanory AA, Alsanory TA. Interrelations of Apoptotic and Cellular Senescence Genes Methylation in Inflammatory Bowel Disease Subtypes and Colorectal Carcinoma in Egyptians Patients. Appl Biochem Biotechnol 2019; 189:330-343. [PMID: 30989570 DOI: 10.1007/s12010-019-03017-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2019] [Accepted: 03/27/2019] [Indexed: 12/24/2022]
Abstract
Ras-related domain family member 1 transcript variant A (RASSF1A) controls apoptosis and cell proliferation while p14/ARF gene has a regulatory role in cellular senescence. Failure of apoptosis and cellular senescence occurs during inflammatory bowel disease (IBD) and colorectal cancer (CRC). To reveal the role of peripheral leukocyte promoter methylation of RASSF1A and p14/ARF in the pathogenesis of IBD subtypes and CRC we investigated the methylation state of the two genes by methylation-specific polymerase chain reaction (MSP-PCR) in 60 CRC patients, 60 patients with IBD; 27 with ulcerative colitis and 33 had Crohn's disease and also in 30 healthy subjects. Methylated RASSF1A and p14/ARF genes were detected in 55% and 60% of CRC, while the frequency of the methylated RASSF1A and p14/ARF genes was 23.3% and 43.3% in IBD patients and 3.3% and 13.3% in the control group (P = 0.000 each). Also, the frequency of methylated RASSF1A gene was significantly higher in ulcerative colitis than in Crohn's disease, while a non-significant frequency of methylated p14/ARF was detected between ulcerative colitis and Crohn's disease. Furthermore, methylated RASSF1A and p14/ARF were associated with the grade of CRC but not associated with the age of patients, family history, or tumor location. Results suggest that methylated RASSF1A and p14/ARF are related to CRC and IBD pathogenesis and may be used as molecular biomarkers for early detection of CRC and IBD.
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Affiliation(s)
- Ragaa H Salama
- Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt
| | | | - Ahmed M Ashmawy
- Department of Internal Medicine, Faculty of Medicine, Assiut University, Assiut, Egypt
| | - Wael A Elsewify
- Department of Internal Medicine, Faculty of Medicine, Aswan University, Aswan, Egypt
| | - Ghada M Ezzat
- Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt.
| | - Mahmoud A Mahmoud
- Department of Internal Medicine, Faculty of Medicine, Assiut University, Assiut, Egypt
| | - Aya A Alsanory
- Students at Faculty of Medicine, Faculty of Pharmacy, Assiut University, Assiut, Egypt
| | - Tasneem A Alsanory
- Students at Faculty of Medicine, Faculty of Pharmacy, Assiut University, Assiut, Egypt
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Sabit H, Abdel-Ghany SE, M Said OA, Mostafa MA, El-Zawahry M. Metformin Reshapes the Methylation Profile in Breast and Colorectal Cancer Cells. Asian Pac J Cancer Prev 2018; 19:2991-2999. [PMID: 30371994 PMCID: PMC6291041 DOI: 10.22034/apjcp.2018.19.10.2991] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
With no sharp cure, breast cancer still be the major and the most serious life-threatening disease worldwide. Colorectal is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women. In the present investigation, colon cancer cells (CaCo-2) and breast cancer cells (MCF-7) were treated with elevated doses of metformin (MET) for 48h. Cell count was assessed using trypan blue test, and the cytotoxicity was evaluated using MTT assay. Methylation-specific PCR was performed on the bisulfite-treated DNA against two tumor suppressor genes; RASSF1A and RB. Results indicated that: in breast cancer, the cell count was decreased significantly (P>0.005) after being treated with 5, 10, 20, 50, and 100 mM of MET. The elevated concentration had increased reduction percentages on the MCF-7 cells, as 5 mM and 100 mM have yielded 35% and 93.3% reduction in cell viability, respectively. Colon cancer cells have responded to the doses of MET differently, as for the 5 mM and the 100 mM, it gave 88% and 60% reduction in cells viability, respectively. Cytotoxicity assay revealed that 5 mM and 100 mM of MET caused breast cancer cells to loss 61.53% and 85.16% of its viability, respectively, whereas colon cancer cells have responded to the 5 mM and 100 mM of MET by reducing the cells viability with 96.91% and 96.24%, respectively. No RB promoter methylation was detected in colon cells, while RASSF1A was partially methylated. In the MCF-7 breast cancer cells, both RASSF1A and RB were partially methylated.
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Affiliation(s)
- Hussein Sabit
- Department of Genetics, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P. O. Box 1982, Dammam, 31441 Saudi Arabia.
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Nunes SP, Moreira-Barbosa C, Salta S, Palma de Sousa S, Pousa I, Oliveira J, Soares M, Rego L, Dias T, Rodrigues J, Antunes L, Henrique R, Jerónimo C. Cell-Free DNA Methylation of Selected Genes Allows for Early Detection of the Major Cancers in Women. Cancers (Basel) 2018; 10:cancers10100357. [PMID: 30261643 PMCID: PMC6210550 DOI: 10.3390/cancers10100357] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2018] [Revised: 09/21/2018] [Accepted: 09/25/2018] [Indexed: 02/06/2023] Open
Abstract
Background: Breast (BrC), colorectal (CRC) and lung (LC) cancers are the three most common and deadly cancers in women. Cancer screening entails an increase in early stage disease detection but is hampered by high false-positive rates and overdiagnosis/overtreatment. Aberrant DNA methylation occurs early in cancer and may be detected in circulating cell-free DNA (ccfDNA), constituting a valuable biomarker and enabling non-invasive testing for cancer detection. We aimed to develop a ccfDNA methylation-based test for simultaneous detection of BrC, CRC and LC. Methods: CcfDNA from BrC, CRC and LC patients and asymptomatic controls were extracted from plasma, sodium-bisulfite modified and whole-genome amplified. APC, FOXA1, MGMT, RARβ2, RASSF1A, SCGB3A1, SEPT9, SHOX2 and SOX17 promoter methylation levels were determined by multiplex quantitative methylation-specific PCR. Associations between methylation and standard clinicopathological parameters were assessed. Biomarkers’ diagnostic performance was also evaluated. Results: A “PanCancer” panel (APC, FOXA1, RASSF1A) detected the three major cancers with 72% sensitivity and 74% specificity, whereas a “CancerType” panel (SCGB3A1, SEPT9 and SOX17) indicated the most likely cancer topography, with over 80% specificity, although with limited sensitivity. Conclusions: CcfDNA’s methylation assessment allows for simultaneous screening of BrC, CRC and LC, complementing current modalities, perfecting cancer suspects’ triage, increasing compliance and cost-effectiveness.
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Affiliation(s)
- Sandra P Nunes
- Cancer Biology & Epigenetics Group-Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal.
- Master in Oncology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), 4050-313 Porto, Portugal.
| | - Catarina Moreira-Barbosa
- Cancer Biology & Epigenetics Group-Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal.
| | - Sofia Salta
- Cancer Biology & Epigenetics Group-Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal.
| | - Susana Palma de Sousa
- Breast Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Inês Pousa
- Lung Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Júlio Oliveira
- Lung Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Marta Soares
- Lung Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Licínio Rego
- Digestive Tract Pathology Clinic and Surgical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Teresa Dias
- Digestive Tract Pathology Clinic and Surgical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Jéssica Rodrigues
- Department of Epidemiology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Luís Antunes
- Department of Epidemiology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
| | - Rui Henrique
- Cancer Biology & Epigenetics Group-Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal.
- Department of Pathology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal.
- Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), 4050-313 Porto, Portugal.
| | - Carmen Jerónimo
- Cancer Biology & Epigenetics Group-Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal.
- Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), 4050-313 Porto, Portugal.
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13
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Abstract
The peritoneum protects the intraabdominal organs. This function is exploited by aggressive cancers originating from organs within the abdomen, resulting in peritoneal metastasis. We discuss genomic variants that may lead to peritoneal metastasis from multiple cancers. Peritoneal malignancies are attributed to epithelial-mesenchymal transition. These metastatic lesions harbor similar genetic mutations to the primary tumor yet may manifest clone-specific aberrations that promote propagation. Peritoneal metastasis are increasingly being treated with surgical resection as an adjunct to radiation, chemotherapy, and other biologic therapies. We describe genetic and genomic variances that are predictive markers for metastasis and burgeoning indicators for peritoneal malignancies.
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Affiliation(s)
- Enusha Karunasena
- Department of Oncology, GI Clinical Cancer Research and Cancer Immunology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Medical Institute, 600 North Wolfe Street, Baltimore, MD 21287, USA
| | - Jonathan Sham
- Department of Surgery, Johns Hopkins Medical Institute, 600 North Wolfe Street, Baltimore, MD 21287, USA
| | - Kevin Wyatt McMahon
- Department of Surgery, Johns Hopkins Medical Institute, 600 North Wolfe Street, Baltimore, MD 21287, USA
| | - Nita Ahuja
- Department of Surgery, Johns Hopkins Medical Institute, 600 North Wolfe Street, Baltimore, MD 21287, USA; Cancer Biology, Department of Oncology, Sidney Kimmel Comprehensive Cancer, Johns Hopkins Medical Institute, 600 North Wolfe Street, Baltimore, MD 21287, USA; Department of Surgery, Yale School of Medicine, PO Box 208062, New Haven, CT 06520-8062, USA.
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14
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Schmidt ML, Hobbing KR, Donninger H, Clark GJ. RASSF1A Deficiency Enhances RAS-Driven Lung Tumorigenesis. Cancer Res 2018; 78:2614-2623. [PMID: 29735543 DOI: 10.1158/0008-5472.can-17-2466] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2017] [Revised: 01/26/2018] [Accepted: 03/01/2018] [Indexed: 12/30/2022]
Abstract
Mutant K-RAS has been shown to have both tumor-promoting and -suppressing functions, and growing evidence suggests that the RASSF family of tumor suppressors can act as RAS apoptosis and senescence effectors. It has been hypothesized that inactivation of the RASSF1A tumor suppressor facilitates K-RAS-mediated transformation by uncoupling it from apoptotic pathways such as the Hippo pathway. In human lung tumors, combined activation of K-RAS and inactivation of RASSF1A is closely associated with the development of the most aggressive and worst prognosis tumors. Here, we describe the first transgenic mouse model for activation of K-RAS in the lung in a RASSF1A-defective background. RASSF1A deficiency profoundly enhanced the development of K-RAS-driven lung tumors in vivo Analysis of these tumors showed loss of RASSF1A-uncoupled RAS from the proapoptotic Hippo pathway as expected. We also observed an upregulation of AKT and RALGEF signaling in the RASSF1A- tumors. Heterozygosity of RASSF1A alone mimicked many of the effects of RAS activation on mitogenic signaling in lung tissue, yet no tumors developed, indicating that nonstandard Ras signaling pathways may be playing a key role in tumor formation in vivo In addition, we observed a marked increase in inflammation and IL6 production in RASSF1A-deficient tumors. Thus, RASSF1A loss profoundly affects RAS-driven lung tumorigenesis and mitogenic signaling in vivo Deregulation of inflammatory pathways due to loss of RASSF1A may be essential for RAS-mediated tumorigenesis. These results may have considerable ramifications for future targeted therapy against RAS+/RASSF1A- tumors.Significance: A transgenic mouse model shows that suppression of RASSF1A dramatically enhances Ras-driven tumorigenesis and alters Ras signaling pathway activity.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/10/2614/F1.large.jpg Cancer Res; 78(10); 2614-23. ©2018 AACR.
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Affiliation(s)
- M Lee Schmidt
- Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky
| | - Katharine R Hobbing
- Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky
| | - Howard Donninger
- Department of Medicine, University of Louisville, Louisville, Kentucky
| | - Geoffrey J Clark
- Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky.
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15
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Deckers IA, van Engeland M, van den Brandt PA, Van Neste L, Soetekouw PM, Aarts MJ, Baldewijns MM, Keszei AP, Schouten LJ. Promoter CpG island methylation in ion transport mechanisms and associated dietary intakes jointly influence the risk of clear-cell renal cell cancer. Int J Epidemiol 2018; 46:622-631. [PMID: 27789672 DOI: 10.1093/ije/dyw266] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/25/2016] [Indexed: 12/12/2022] Open
Abstract
Background Sodium intake, but not potassium or fluid intake, has been associated with higher renal cell cancer (RCC) risk. However, risk factors may differ by molecular subtypes of the tumour. In renal physiology, electrolyte and water homeostasis is facilitated by ion transport mechanisms (ITM). Aberrant regulation of ITM genes, for example by promoter CpG island methylation, may modify associations between sodium, potassium and fluid intake and RCC risk. Methods We identified ARHGDIG , ATP1A1 , SCNN1B and SLC8A3 as ITM genes exhibiting RCC-specific promoter methylation and down-regulation. Methylation-specific polymerase chain reaction (PCR) was used to analyse promoter CpG island methylation in tumour DNA of 453 RCC cases from the Netherlands Cohort Study ( n = 120 852) after 20.3 years of follow-up. Diet was measured at baseline using food-frequency questionnaires. Cox regression analyses were restricted to clear-cell (cc)RCC ( n = 306) and stratified by tumours with no, low (1 gene) and high (≥ 2 genes) methylation. Results Sodium intake (high vs low) increased ccRCC risk particularly in tumours with a high methylation index: hazard ratio (HR) [95% confidence interval (CI)]: 2.04 (1.16-3.58), whereas heterogeneity across the methylation index was not significant ( P -heterogeneity = 0.26). Potassium intake was differentially associated with ccRCC risk ( P -heterogeneity = 0.008); the risk for high (vs low) potassium intake was low for unmethylated tumours [HR (95% CI): 0.60 (0.36-1.01)], but high for tumours with a high methylation index [HR (95% CI): 1.60 (0.96-2.65)]. Risks similarly differed for fluid intake, though not significantly ( P -heterogeneity = 0.54). Conclusions Our findings suggest for the first time that dietary intakes are differentially associated with ccRCC risk according to molecular subtypes defined by ITM gene-specific promoter methylation.
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Affiliation(s)
| | | | | | | | - Patricia Mmb Soetekouw
- Department of Medical Oncology, Maastricht University Medical Centre (MUMC), Maastricht, The Netherlands
| | - Maureen Jb Aarts
- Department of Medical Oncology, Maastricht University Medical Centre (MUMC), Maastricht, The Netherlands
| | - Marcella Mll Baldewijns
- Department of Pathology.,Department of Pathology, Antwerp University Hospital, Antwerp, Belgium
| | - András P Keszei
- Department of Epidemiology.,Department of Medical Informatics, Uniklinik RWTH Aachen University, Aachen, Germany
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16
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Rattanapan Y, Korkiatsakul V, Kongruang A, Chareonsirisuthigul T, Rerkamnuaychoke B, Wongkularb A, Wilailak S. EGFL7 and RASSF1 promoter hypermethylation in epithelial ovarian cancer. Cancer Genet 2018; 224-225:37-40. [PMID: 29778234 DOI: 10.1016/j.cancergen.2018.04.117] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Revised: 03/06/2018] [Accepted: 04/13/2018] [Indexed: 01/22/2023]
Abstract
DNA methylation is one of the epigenetic mechanisms associated with gene expression and plays a key role as in activation and deactivation of oncogenes and tumor suppressor genes, respectively. This study employed DNA methylation array to identify methylated genes which are highly correlated with various phenotypes of epithelial ovarian cancer (EOC) in Thai patients and to quantify promoter CpG-island methylation of candidate genes. Tissues from patients with serous and non-serous EOC showed significantly higher promoter methylation of EGFL7 and RASSF1 compared to benign cases. These results indicate the potential of investigating promoter CpG-island methylation of cancer-associated genes as biomarkers of disease progression and even possibly of early detection.
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Affiliation(s)
- Yanisa Rattanapan
- Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand
| | - Veerawat Korkiatsakul
- Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand
| | - Adcharee Kongruang
- Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand
| | - Takol Chareonsirisuthigul
- Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand.
| | - Budsaba Rerkamnuaychoke
- Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand
| | - Anna Wongkularb
- Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand
| | - Sarikapan Wilailak
- Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 10400, Thailand
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17
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Schirosi L, Mazzotta A, Opinto G, Pinto R, Graziano G, Tommasi S, Fucci L, Simone G, Mangia A. β-catenin interaction with NHERF1 and RASSF1A methylation in metastatic colorectal cancer patients. Oncotarget 2018; 7:67841-67850. [PMID: 27765918 PMCID: PMC5356523 DOI: 10.18632/oncotarget.12280] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 09/21/2016] [Indexed: 01/10/2023] Open
Abstract
There is an increasing need to identify new biomarkers in colorectal cancer (CRC) to further characterize this malignancy. β-catenin plays a central role in the Wnt signaling pathway. It also binds Na+/H+ exchanger regulating factor 1 (NHERF1) and interacts with the RAS-association domain family 1, isoform A (RASSF1A), but the mechanisms of this possible crosstalk are still not fully understood. In this study, we analyzed for the first time the different subcellular expression of β-catenin, NHERF1, and RASSF1A and their relationships with RASSF1A methylation in the progression of CRC. We assessed immunohistochemical expression and RASSF1A methylation in 51 patients with stage IV colorectal cancer. Biomarker expression analysis was carried out considering the tumor-adjacent normal tissue, the primary tumor, and the paired liver metastases. Regarding the tumor compartment, it was found that cytoplasmic β-catenin expression was positively correlated to membranous (r = 0.3002, p = 0.0323) and nuclear NHERF1 (r = 0.293, p = 0.0368). In the liver metastases, instead, we found a positive correlation of cytoplasmic and nuclear β-catenin expression with RASSF1A methylation (r = 0.4019, p = 0.0068 and r = 0.3194, p = 0.0345, respectively). In conclusion, our results showed that β-catenin was the crucial protagonist in metastatic CRC through different effector proteins involved in this developing process. In tumor tissues, β-catenin was predominantly associated with NHERF1 in a dynamic context, while interestingly in liver metastases, we noted an increase of its oncogenic function through RASSF1A inactivation.
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Affiliation(s)
- Laura Schirosi
- Functional Biomorphology Laboratory, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Annalisa Mazzotta
- Functional Biomorphology Laboratory, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Giuseppina Opinto
- Functional Biomorphology Laboratory, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Rosamaria Pinto
- Molecular Genetics Laboratory, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Giusi Graziano
- Scientific Direction, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Stefania Tommasi
- Molecular Genetics Laboratory, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Livia Fucci
- Pathology Department, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Giovanni Simone
- Pathology Department, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
| | - Anita Mangia
- Functional Biomorphology Laboratory, IRCCS Istituto Tumori "Giovanni Paolo II", Bari, Italy
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18
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Battaglia P, Baritono E, Remo A, Vendraminelli R, Conti A. KRAS Mutations and M2PK Upregulation in Stool Samples from Individuals with Positive Fecal Occult Blood Tests Screened for Colorectal Cancer. TUMORI JOURNAL 2018; 100:122-7. [DOI: 10.1177/030089161410000202] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Background Screening for colorectal cancer (CRC) requires non-invasive methods of high diagnostic sensitivity and specificity. We evaluated the measurement of genetic and protein biomarkers of CRC in stool samples with the aim of testing their clinical utility in a CRC screening program. Patients and Methods Individuals aged 53–75 years who were at risk of CRC and immunochemical fecal occult blood test (iFOBT) positive were invited to submit stool samples for molecular testing prior to colonoscopy. KRAS codon 12 Gly→Asp, Gly, Val, and codon 13 Gly→Cys gene mutations were tested using an in-house real-time ARMS PCR method. M2PK levels in stool samples were measured utilizing a commercial ELISA kit. Results At colonoscopy, 7.6% of patients were found to have CRC, 50% had adenomas, 10.6% had hyperplastic polyps, 20.2% had diverticulosis and hemorrhoids, and 11.6% had normal mucosa. The best sensitivity for CRC (50%) was found in those cases where M2PK and KRAS abnormalities coexisted. M2PK showed a detection rate of 40.3% for adenomas but the combination of M2PK and KRAS abnormalities was found in only 5.7% of adenomas (P <0.01). iFOBT was false positive in 31.8% of cases in which colonoscopy excluded neoplastic lesions, while the coexistence of molecular and enzymatic abnormalities was more specific with false positive rates between 8.3% and 9.0% (P <0.05). Conclusion Our molecular screening approach demonstrates that detection of cancer-associated biomarkers measured in iFOBT-positive stool samples could help separate true from false positives in a FOBT-based screening process. M2PK showed particular promise for the detection of CRC and adenomas.
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Affiliation(s)
- Paolo Battaglia
- Department of Laboratory Medicine, Mater Salutis Hospital, Legnago (Verona), Italy
| | - Elisabetta Baritono
- Department of Laboratory Medicine, Mater Salutis Hospital, Legnago (Verona), Italy
| | - Andrea Remo
- Department of Clinical Pathology, Mater Salutis Hospital, Legnago (Verona), Italy
| | | | - Antonio Conti
- Department of Laboratory Medicine, Mater Salutis Hospital, Legnago (Verona), Italy
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19
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Bai J, Zhang X, Hu K, Liu B, Wang H, Li A, Lin F, Zhang L, Sun X, Du Z, Song J. Silencing DNA methyltransferase 1 (DNMT1) inhibits proliferation, metastasis and invasion in ESCC by suppressing methylation of RASSF1A and DAPK. Oncotarget 2018; 7:44129-44141. [PMID: 27286455 PMCID: PMC5190084 DOI: 10.18632/oncotarget.9866] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2015] [Accepted: 03/31/2016] [Indexed: 11/25/2022] Open
Abstract
Our previous study showed DNMT1 is up-regulated in esophageal squamous cell carcinoma (ESCC), which is associated with methylation of tumor suppressors. In the current study, we investigate the role of DNMT1 in ESCC. We found silencing DNMT1 inhibited proliferation, metastasis and invasion of three different ESCC cells, K150, K410 and K450. We also found silencing DNMT1 induced G1 arrest and cell apoptosis in K150, K410 and K450 cells. In vivo study showed silencing DNMT1 suppressed tumor growth in nude mice. In addition, silencing DNMT1 increased expression of tumor suppressor genes, RASSF1A and DAPK, in ESCC cells and ESCC xenograft in nude mice. Moreover, silencing DNMT1 decreased methylation in promoter of RASSF1A and DAPK. In conclusion, our data demonstrated that silencing DNMT1 inhibits proliferation, metastasis and invasion in ESCC by suppressing methylation of RASSF1A and DAPK.
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Affiliation(s)
- Jian Bai
- Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Xue Zhang
- Department of ICU, The Second Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Kai Hu
- Department of Thoracic & Cardiovascular Surgery, The Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Bangqing Liu
- Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Haiyong Wang
- Department of Thoracic & Cardiovascular Surgery, The Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Angui Li
- Department of Thoracic & Cardiovascular Surgery, The Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Feng Lin
- Department of Thoracic & Cardiovascular Surgery, The Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Lifei Zhang
- Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Xiaolin Sun
- Department of Thoracic & Cardiovascular Surgery, The Affiliated Hospital of Guilin Medical University, Guilin, China
| | - Zhenzong Du
- Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Guilin, China.,Current address: Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Lingui District, Guilin, China
| | - Jianfei Song
- Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Guilin, China.,Current address: Department of Thoracic & Cardiovascular Surgery, The Second Affiliated Hospital of Guilin Medical University, Lingui District, Guilin, China
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20
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Sebio A, Stintzing S, Heinemann V, Sunakawa Y, Zhang W, Ichikawa W, Tsuji A, Takahashi T, Parek A, Yang D, Cao S, Ning Y, Stremitzer S, Matsusaka S, Okazaki S, Barzi A, Berger MD, Lenz HJ. A genetic variant in Rassf1a predicts outcome in mCRC patients treated with cetuximab plus chemotherapy: results from FIRE-3 and JACCRO 05 and 06 trials. THE PHARMACOGENOMICS JOURNAL 2018; 18:43-48. [PMID: 27698403 PMCID: PMC5378677 DOI: 10.1038/tpj.2016.69] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Revised: 05/17/2016] [Accepted: 08/25/2016] [Indexed: 12/22/2022]
Abstract
The Hippo pathway is involved in colorectal cancer (CRC) development and progression. The Hippo regulator Rassf1a is also involved in the Ras signaling cascade. In this work, we tested single nucleotide polymorphisms within Hippo components and their association with outcome in CRC patients treated with cetuximab. Two cohorts treated with cetuximab plus chemotherapy were evaluated (198 RAS wild-type (WT) patients treated with first-line FOLFIRI plus Cetuximab within the FIRE-3 trial and 67 Ras WT patients treated either with first-line mFOLFOX6 or SOX plus Cetuximab). In these two populations, Rassf1a rs2236947 was associated with overall survival (OS), as patients with a CC genotype had significantly longer OS compared with those with CA or AA genotypes. This association was stronger in patients with left-side CRC (hazard ratio (HR): 1.79 (1.01-3.14); P=0.044 and HR: 2.83 (1.14-7.03); P=0.025, for Fire 3 and JACCRO cohorts, respectively). Rassf1a rs2236947 is a promising biomarker for patients treated with cetuximab plus chemotherapy.
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Affiliation(s)
- A Sebio
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
- Medical Oncology Department, Santa Creu i Sant Pau Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - S Stintzing
- Department of Hematology and Oncology, Klinikum der Universitat, University of Munich, Munich, Germany
| | - V Heinemann
- Department of Hematology and Oncology, Klinikum der Universitat, University of Munich, Munich, Germany
| | - Y Sunakawa
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - W Zhang
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - W Ichikawa
- Department of Medical Oncology, Showa University, Yokohama, Japan
| | - A Tsuji
- Department of Clinical Oncology, Kagawa University, Kagawa, Japan
| | - T Takahashi
- Department of Medical Oncology, Showa University, Yokohama, Japan
| | - A Parek
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - D Yang
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - S Cao
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Y Ning
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - S Stremitzer
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - S Matsusaka
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - S Okazaki
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - A Barzi
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - M D Berger
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - H-J Lenz
- Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
- Department of Preventive Medicine, Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
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21
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Tse JWT, Jenkins LJ, Chionh F, Mariadason JM. Aberrant DNA Methylation in Colorectal Cancer: What Should We Target? Trends Cancer 2017; 3:698-712. [PMID: 28958388 DOI: 10.1016/j.trecan.2017.08.003] [Citation(s) in RCA: 82] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2017] [Revised: 08/10/2017] [Accepted: 08/11/2017] [Indexed: 12/16/2022]
Abstract
Colorectal cancers (CRCs) are characterized by global hypomethylation and promoter-specific DNA methylation. A subset of CRCs with extensive and co-ordinate patterns of promoter methylation has also been identified, termed the CpG-island methylator phenotype. Some genes methylated in CRC are established tumor suppressors; however, for the majority, direct roles in disease initiation or progression have not been established. Herein, we examine functional evidence of specific methylated genes contributing to CRC pathogenesis, focusing on components of commonly deregulated signaling pathways. We also review current knowledge of the mechanisms underpinning promoter methylation in CRC, including genetic events, altered transcription factor binding, and DNA damage. Finally, we summarize clinical trials of DNA methyltransferase inhibitors in CRC, and propose strategies for enhancing their efficacy.
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Affiliation(s)
- Janson W T Tse
- Olivia Newton-John Cancer Research Institute, Melbourne, Australia; These authors contributed equally
| | - Laura J Jenkins
- Olivia Newton-John Cancer Research Institute, Melbourne, Australia; School of Cancer Medicine, La Trobe University, Melbourne, Australia; These authors contributed equally
| | - Fiona Chionh
- Olivia Newton-John Cancer Research Institute, Melbourne, Australia
| | - John M Mariadason
- Olivia Newton-John Cancer Research Institute, Melbourne, Australia; School of Cancer Medicine, La Trobe University, Melbourne, Australia.
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22
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Fumagalli C, Bianchi F, Raviele PR, Vacirca D, Bertalot G, Rampinelli C, Lazzeroni M, Bonanni B, Veronesi G, Fusco N, Barberis M, Guerini-Rocco E. Circulating and tissue biomarkers in early-stage non-small cell lung cancer. Ecancermedicalscience 2017; 11:717. [PMID: 28194229 PMCID: PMC5295844 DOI: 10.3332/ecancer.2017.717] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Indexed: 12/31/2022] Open
Abstract
OBJECTIVE We sought to characterise circulating and tissue tumour biomarkers of patients who developed early-stage non-small cell lung cancer (NSCLC) during long-term follow-up of a chemoprevention trial (NCT00321893). MATERIALS AND METHODS Blood and sputum samples were collected from 202 high-risk asymptomatic individuals with CT-detected stable lung nodules. Real-time PCR was performed on plasma to quantify free circulating DNA. Baseline serum was investigated with a previously validated test based on 13 circulating miRNAs (miR-Test). Promoter methylation status of p16, RASSF1a and RARβ2 and telomerase activity were assessed in sputum samples. DNA was extracted from each tumour developed during follow-up and subjected to a mutation survey using the LungCarta panel on the Sequenom MassARRAY platform. RESULTS During follow-up (9 years) six individuals underwent surgery for stage I NSCLC with a median time of disease onset of 20.5 months. MiR-Test scores were positive (range: 0.14-7.24) in four out of six baseline pre-disease onset sera. No association was identified between free circulating DNA or sputum biomarkers and disease onset. All tumours harboured at least one somatic mutation in well-known cancer genes, including KRAS (n = 4), BRAF (n = 1), and TP53 (n = 3). CONCLUSION Circulating miRNA tests may represent valuable tools to detect clinically-silent tumours. Early-stage lung adenocarcinomas harbour recurrent genetic events similar to those described in advanced-stage NSCLCs.
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Affiliation(s)
- Caterina Fumagalli
- Division of Pathology, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Fabrizio Bianchi
- Institute for Stem-cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), IRCCS Casa Sollievo della Sofferenza, Viale Cappuccini 1, 71013, San Giovanni Rotondo, Foggia, Italy
| | - Paola Rafaniello Raviele
- Division of Pathology, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Davide Vacirca
- Division of Pathology, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Giovanni Bertalot
- Molecular Medicine Programme IEO, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Cristiano Rampinelli
- Department of Radiology, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Matteo Lazzeroni
- Division of Cancer Prevention and Genetics, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Bernardo Bonanni
- Division of Cancer Prevention and Genetics, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Giulia Veronesi
- Division of Thoracic Surgery, Humanitas Research Hospital, Via Manzoni 56, 20089, Rozzano Milan, Italy
| | - Nicola Fusco
- Division of Pathology, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, University of Milan, Via Francesco Sforza 35, 20122, Milan, Italy
| | - Massimo Barberis
- Division of Pathology, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
| | - Elena Guerini-Rocco
- Division of Pathology, European Institute of Oncology, Via Giuseppe Ripamonti 435, 20141, Milan, Italy
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23
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Abstract
Stems cells of the colon crypt are the origin of colon mature cells. Colorectal cancer cells are also suggested to originate from crypt stem cells undergoing a series of epigenetic and genetic alterations. Aberrant methylation plays important roles in early carcinogenesis and lead to altered gene expression and regulation, resulting in accumulation of damages to cell function and ultimately, malignant transformation. Aberrances in hypermethylation and hypomethylation act in different mechanism through the regulation of various genes during CSC carcinogenesis, and both of them play crucial roles in stem cell differentiation towards cancer cells. A large majority of epigenetic and genetic abnormalities that work coordinately in colorectal carcinogenesis are related to cell growth and division, indicating that the intrinsic abnormalities of CRC lie in dysregulation of basic cellular processes. Detection of abnormal methylation can be used in cancer screening and early detection, while reversal of aberrant methylation using drugs may have potential in cancer therapy. This review will provide an overview on the roles of aberrant methylation and a summary of genes that are affected during CRC carcinogenesis.
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Affiliation(s)
- Lele Song
- Department of Radiotherapy, The Chinese PLA 309th Hospital, No. 17, Heishanhu Road, Haidian District, Beijing, 100091, People's Republic of China.
- BioChain (Beijing) Science and Technology, Inc, Beijing, 100176, People's Republic of China.
| | - Yuemin Li
- Department of Radiotherapy, The Chinese PLA 309th Hospital, No. 17, Heishanhu Road, Haidian District, Beijing, 100091, People's Republic of China.
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24
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Defining, distinguishing and detecting the contribution of heterogeneous methylation to cancer heterogeneity. Semin Cell Dev Biol 2016; 64:5-17. [PMID: 27582426 DOI: 10.1016/j.semcdb.2016.08.030] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2016] [Accepted: 08/24/2016] [Indexed: 01/07/2023]
Abstract
DNA methylation is a fundamental means of epigenetic gene regulation that occurs in virtually all cell types. In many higher organisms, including humans, it plays vital roles in cell differentiation and homeostatic maintenance of cell phenotype. The control of DNA methylation has traditionally been attributed to a highly coordinated, linear process, whose dysregulation has been associated with numerous pathologies including cancer, where it occurs early in, and even prior to, the development of neoplastic tissues. Recent experimental evidence has demonstrated that, contrary to prevailing paradigms, methylation patterns are actually maintained through inexact, dynamic processes. These processes normally result in minor stochastic differences between cells that accumulate with age. However, various factors, including cancer itself, can lead to substantial differences in intercellular methylation patterns, viz. methylation heterogeneity. Advancements in molecular biology techniques are just now beginning to allow insight into how this heterogeneity contributes to clonal evolution and overall cancer heterogeneity. In the current review, we begin by presenting a didactic overview of how the basal bimodal methylome is established and maintained. We then provide a synopsis of some of the factors that lead to the accrual of heterogeneous methylation and how this heterogeneity may lead to gene silencing and impact the development of cancerous phenotypes. Lastly, we highlight currently available methylation assessment techniques and discuss their suitability to the study of heterogeneous methylation.
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25
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Expression Pattern and Clinicopathological Relevance of the Indoleamine 2,3-Dioxygenase 1/Tryptophan 2,3-Dioxygenase Protein in Colorectal Cancer. DISEASE MARKERS 2016; 2016:8169724. [PMID: 27578919 PMCID: PMC4992785 DOI: 10.1155/2016/8169724] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/27/2016] [Accepted: 07/03/2016] [Indexed: 02/08/2023]
Abstract
Aims. Cancer cells use the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to suppress the host's immune response in order to facilitate survival, growth, invasion, and metastasis of malignant cells. Higher IDO1 expression was shown to be involved in colorectal cancer (CRC) progression and to be correlated with impaired clinical outcome. However, the potential correlation between the expression of IDO1 in a CRC population with a low mutation rate of the APC gene remains unknown. Material and Methods. Tissues and blood samples were collected from 192 CRC patients. The expressions of IDO1, tryptophan 2,3-dioxygenase (TDO2), and beta-catenin proteins were analyzed by immunohistochemistry. Microsatellite instability (MSI) was determined by PCR amplification of microsatellite loci. Results. The results showed that high IDO1 or TDO2 protein expression was associated with characteristics of more aggressive phenotypes of CRC. For the first time, they also revealed a positive correlation between the abnormal expression of beta-catenin and IDO1 or TDO2 proteins in a CRC population with a low mutation rate of APC. Conclusion. We concluded that an IDO1-regulated molecular pathway led to abnormal expression of beta-catenin in the nucleus/cytoplasm of CRC patients with low mutation rate of APC, making IDO1 an interesting target for immunotherapy in CRC.
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26
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Sebio A, Matsusaka S, Zhang W, Yang D, Ning Y, Stremitzer S, Stintzing S, Sunakawa Y, Yamauchi S, Fujimoto Y, Ueno M, Lenz HJ. Germline polymorphisms in genes involved in the Hippo pathway as recurrence biomarkers in stages II/III colon cancer. THE PHARMACOGENOMICS JOURNAL 2016; 16:312-319. [PMID: 26370619 PMCID: PMC4792794 DOI: 10.1038/tpj.2015.64] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/15/2015] [Revised: 06/04/2015] [Accepted: 08/04/2015] [Indexed: 01/16/2023]
Abstract
The Hippo pathway regulates tissue growth and cell fate. In colon cancer, Hippo pathway deregulation promotes cellular quiescence and resistance to 5-Fluorouracil (5-Fu). In this study, 14 polymorphisms in 8 genes involved in the Hippo pathway (MST1, MST2, LATS1, LATS2, YAP, TAZ, FAT4 and RASSF1A) were evaluated as recurrence predictors in 194 patients with stages II/III colon cancer treated with 5-Fu-based adjuvant chemotherapy. Patients with a RASSF1A rs2236947 AA genotype had higher 3-year recurrence rate than patients with CA/CC genotypes (56 vs 33%, hazard ratio (HR): 1.87; P=0.017). Patients with TAZ rs3811715 CT or TT genotypes had lower 3-year recurrence rate than patients with a CC genotype (28 vs 40%; HR: 0.66; P=0.07). In left-sided tumors, this association was stronger (HR: 0.29; P=0.011) and a similar trend was found in an independent Japanese cohort. These promising results reveal polymorphisms in the Hippo pathway as biomarkers for stages II and III colon cancer.The Pharmacogenomics Journal advance online publication, 15 September 2015; doi:10.1038/tpj.2015.64.
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Affiliation(s)
- Ana Sebio
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
- Medical Oncology Department; Santa Creu i Sant Pau Hospital; Universitat Autònoma de Barcelona; Barcelona; Spain
| | - Satoshi Matsusaka
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Wu Zhang
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Dongyun Yang
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Yan Ning
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Stefan Stremitzer
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Sebastian Stintzing
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
- Department of Hematology and Oncology, Klinikum der Universitat, University of Munich, Munich, Germany
| | - Yu Sunakawa
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Shinichi Yamauchi
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
| | - Yoshiya Fujimoto
- Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Masashi Ueno
- Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Heinz-Josef Lenz
- Division of Medical Oncology; Sharon A. Carpenter Laboratory; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
- Department of Preventive Medicine; Norris Comprehensive Cancer Center; Keck School of Medicine, University of Southern California, Los Angeles, USA
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27
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Schouten LJ, Deckers IAG, van den Brandt PA, Baldewijns MMLL, van Engeland M. Alcohol and Dietary Folate Intake and Promoter CpG Island Methylation in Clear-Cell Renal Cell Cancer. Nutr Cancer 2016; 68:1097-107. [PMID: 27340879 DOI: 10.1080/01635581.2016.1187283] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
We investigated whether alcohol and dietary folate intakes were associated with promoter methylation in clear-cell renal cell carcinoma (ccRCC). The Netherlands Cohort Study with a case-cohort design included 120,852 subjects aged 55-69 yr in 1986. Diet was measured with a food-frequency questionnaire. After 20.3 yr of follow-up, paraffin-embedded tumor blocks were collected. Methylation-specific polymerase chain reaction (MSP) was used to analyze promoter methylation of 11 genes. ccRCC cases were classified into low (0-19% of the genes), intermediate (20-39%), and high (40%+) methylation. Multivariable Cox regression analyses were conducted, stratified according to methylation, including 3980 subcohort members and 297 ccRCC cases. Increasing alcohol intake was associated with decreased ccRCC risk, but was not statistically significant; multivariable adjusted hazard ratio (HR) for ≥30 g alcohol/day versus 0 g/day was 0.78 [95% confidence interval (CI): 0.48-1.24], and P-value for trend was 0.46. In strata according to methylation index, no significant heterogeneity was observed. Dietary folate intake was not associated with ccRCC risk. There was no significant heterogeneity between strata according to methylation index. There was no effect modification of alcohol and dietary folate intake on ccRCC risk, nor in strata according to methylation index. Our findings do not support the hypothesis that alcohol and dietary folate intakes are involved in ccRCC.
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Affiliation(s)
- Leo J Schouten
- a Department of Epidemiology , School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre , Maastricht , The Netherlands
| | - Ivette A G Deckers
- a Department of Epidemiology , School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre , Maastricht , The Netherlands
| | - Piet A van den Brandt
- a Department of Epidemiology , School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre , Maastricht , The Netherlands.,b Department of Epidemiology , School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Centre , Maastricht , The Netherlands
| | | | - Manon van Engeland
- d Department of Pathology , School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre , Maastricht , The Netherlands
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28
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Matthaios D, Balgkouranidou I, Karayiannakis A, Bolanaki H, Xenidis N, Amarantidis K, Chelis L, Romanidis K, Chatzaki A, Lianidou E, Trypsianis G, Kakolyris S. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer. Oncol Lett 2016; 12:748-756. [PMID: 27347211 DOI: 10.3892/ol.2016.4649] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Accepted: 04/29/2016] [Indexed: 01/26/2023] Open
Abstract
DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated RASSF1A. The observed significant correlations between APC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.
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Affiliation(s)
- Dimitrios Matthaios
- Department of Medical Oncology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Ioanna Balgkouranidou
- Department of Medical Oncology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Anastasios Karayiannakis
- Second Department of Surgery, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Helen Bolanaki
- Second Department of Surgery, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Nikolaos Xenidis
- Department of Medical Oncology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Kyriakos Amarantidis
- Department of Medical Oncology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Leonidas Chelis
- Department of Medical Oncology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Konstantinos Romanidis
- Second Department of Surgery, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Aikaterini Chatzaki
- Laboratory of Pharmacology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Evi Lianidou
- Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens 15771, Greece
| | - Grigorios Trypsianis
- Laboratory of Statistics, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
| | - Stylianos Kakolyris
- Department of Medical Oncology, Medical School, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis 68100, Greece
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Hashimoto Y, Zumwalt TJ, Goel A. DNA methylation patterns as noninvasive biomarkers and targets of epigenetic therapies in colorectal cancer. Epigenomics 2016; 8:685-703. [PMID: 27102979 DOI: 10.2217/epi-2015-0013] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Aberrant DNA methylation is frequently detected in gastrointestinal tumors, and can therefore potentially be used to screen, diagnose, prognosticate, and predict colorectal cancers (CRCs). Although colonoscopic screening remains the gold standard for CRC screening, this procedure is invasive, expensive, and suffers from poor patient compliance. Methylated DNA is an attractive choice for a biomarker substrate because CRCs harbor hundreds of aberrantly methylated genes. Furthermore, abundance in extracellular environments and resistance to degradation and enrichment in serum, stool, and other noninvasive bodily fluids, allows quantitative measurements of methylated DNA biomarkers. This article describes the most important studies that investigated the efficacy of serum- or stool-derived methylated DNA as population-based screening biomarkers in CRC, details several mechanisms and factors that control DNA methylation, describes a better use of prevailing technologies that discover novel DNA methylation biomarkers, and illustrates the diversity of demethylating agents and their applicability toward clinical impact.
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Affiliation(s)
- Yutaka Hashimoto
- Center for Translational Genomics & Oncology, Baylor Scott & White Research Institute & Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA
| | - Timothy J Zumwalt
- Center for Translational Genomics & Oncology, Baylor Scott & White Research Institute & Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA
| | - Ajay Goel
- Center for Translational Genomics & Oncology, Baylor Scott & White Research Institute & Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA
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30
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Deckers IAG, Schouten LJ, Van Neste L, van Vlodrop IJH, Soetekouw PMMB, Baldewijns MMLL, Jeschke J, Ahuja N, Herman JG, van den Brandt PA, van Engeland M. Promoter Methylation of CDO1 Identifies Clear-Cell Renal Cell Cancer Patients with Poor Survival Outcome. Clin Cancer Res 2015; 21:3492-500. [PMID: 25904753 PMCID: PMC4612631 DOI: 10.1158/1078-0432.ccr-14-2049] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2014] [Accepted: 04/10/2015] [Indexed: 12/31/2022]
Abstract
PURPOSE In this era of molecular diagnostics, prediction of clear-cell renal cell cancer (ccRCC) survival requires optimization, as current prognostic markers fail to determine individual patient outcome. Epigenetic events are promising molecular markers. Promoter CpG island methylation of cysteine dioxygenase type 1 (CDO1), which was identified as prognostic marker for breast cancer, is studied as a potential marker for ccRCC survival. EXPERIMENTAL DESIGN We collected primary tissues of 365 ccRCC cases identified within the prospective Netherlands Cohort Study (NLCS). In this population-based series, CDO1 promoter methylation was observed in 124 of 324 (38.3%) patients with successful methylation-specific PCR analysis. Kaplan-Meier curves and Wilcoxon tests were used to evaluate 10-year ccRCC-specific survival. Cox regression analysis was used to obtain crude and multivariate HRs and 95% confidence intervals (CI). The relative prognostic value of multivariate models with and without CDO1 promoter methylation was compared using likelihood-ratio tests. RESULTS Patients with CDO1 promoter methylation have a significantly poorer survival than those without (Wilcoxon P = 0.006). Differences in survival were independent of other prognostic factors, including age and sex (HR, 1.66; 95% CI, 1.12-2.45) and TNM stage, tumor size, and Fuhrman grade (HR, 1.89; 95% CI, 1.25-2.85). Multivariate models performed better with than without CDO1 promoter methylation status (likelihood-ratio P = 0.003). Survival curves were validated in an independent series of 280 ccRCC cases from The Cancer Genome Atlas (TCGA; Wilcoxon P < 0.001). CONCLUSIONS CDO1 promoter methylation may not substitute common prognostic makers to predict ccRCC survival, but offers additional, relevant prognostic information, indicating that it might be a novel molecular marker to determine ccRCC prognosis.
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Affiliation(s)
- Ivette A G Deckers
- Department of Epidemiology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Leo J Schouten
- Department of Epidemiology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Leander Van Neste
- Department of Pathology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Iris J H van Vlodrop
- Department of Pathology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Patricia M M B Soetekouw
- Division of Medical Oncology, Department of Internal Medicine, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Marcella M L L Baldewijns
- Department of Pathology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Jana Jeschke
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Laboratory of Cancer Epigenetics, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium
| | - Nita Ahuja
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - James G Herman
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Piet A van den Brandt
- Department of Epidemiology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands
| | - Manon van Engeland
- Department of Pathology, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands.
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31
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Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays. J Transl Med 2015; 95:833-42. [PMID: 25867767 DOI: 10.1038/labinvest.2015.53] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2015] [Revised: 03/06/2015] [Accepted: 03/07/2015] [Indexed: 02/07/2023] Open
Abstract
Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffin-embedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n=9) and breast cancer (n=11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ=0.9927 (s.d. ±0.0015)). Matched FF/FFPEr correlation was also high (ρ=0.9590 (s.d. ±0.0184)) compared with matched FF/FFPE (ρ=0.8051 (s.d. ±0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ±0.66) was comparable to FF samples (99.98%, s.d. ±0.019) and substantially lower in FFPE samples (82.31%, s.d. ±18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for molecular pathological epidemiology research on archived samples with limited tissue amount.
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Breugom AJ, Swets M, Bosset JF, Collette L, Sainato A, Cionini L, Glynne-Jones R, Counsell N, Bastiaannet E, van den Broek CBM, Liefers GJ, Putter H, van de Velde CJH. Adjuvant chemotherapy after preoperative (chemo)radiotherapy and surgery for patients with rectal cancer: a systematic review and meta-analysis of individual patient data. Lancet Oncol 2015; 16:200-7. [PMID: 25589192 DOI: 10.1016/s1470-2045(14)71199-4] [Citation(s) in RCA: 416] [Impact Index Per Article: 41.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
BACKGROUND The role of adjuvant chemotherapy for patients with rectal cancer after preoperative (chemo)radiotherapy and surgery is uncertain. We did a meta-analysis of individual patient data to compare adjuvant chemotherapy with observation for patients with rectal cancer. METHODS We searched PubMed, Medline, Embase, Web of Science, the Cochrane Library, CENTRAL, and conference abstracts to identify European randomised, controlled, phase 3 trials comparing observation with adjuvant chemotherapy after preoperative (chemo)radiotherapy and surgery for patients with non-metastatic rectal cancer. The primary endpoint of interest was overall survival. FINDINGS We analysed data from four eligible trials, including data from 1196 patients with (y)pTNM stage II or III disease, who had an R0 resection, had a low anterior resection or an abdominoperineal resection, and had a tumour located within 15 cm of the anal verge. We found no significant differences in overall survival between patients who received adjuvant chemotherapy and those who underwent observation (hazard ratio [HR] 0.97, 95% CI 0.81-1.17; p=0.775); there were no significant differences in overall survival in subgroup analyses. Overall, adjuvant chemotherapy did not significantly improve disease-free survival (HR 0.91, 95% CI 0.77-1.07; p=0.230) or distant recurrences (0.94, 0.78-1.14; p=0.523) compared with observation. However, in subgroup analyses, patients with a tumour 10-15 cm from the anal verge had improved disease-free survival (0.59, 0.40-0.85; p=0.005, p(interaction)=0.107) and fewer distant recurrences (0.61, 0.40-0.94; p=0.025, p(interaction)=0.126) when treated with adjuvant chemotherapy compared with patients undergoing observation. INTERPRETATION Overall, adjuvant fluorouracil-based chemotherapy did not improve overall survival, disease-free survival, or distant recurrences. However, adjuvant chemotherapy might benefit patients with a tumour 10-15 cm from the anal verge in terms of disease-free survival and distant recurrence. Further studies of preoperative and postoperative treatment for this subgroup of patients are warranted. FUNDING None.
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Affiliation(s)
- Anne J Breugom
- Department of Surgery, Leiden University Medical Centre, Leiden, Netherlands
| | - Marloes Swets
- Department of Surgery, Leiden University Medical Centre, Leiden, Netherlands
| | - Jean-François Bosset
- Department of Radiation Oncology, Besançon University Hospital J Minjoz, Besançon, France
| | - Laurence Collette
- Department of Statistics, European Organisation for Research and Treatment of Cancer, Brussels, Belgium
| | - Aldo Sainato
- Department of Radiotherapy, Azienda Ospedaliera Universitaria Pisana, Pisa, Italy
| | - Luca Cionini
- Department of Radiotherapy, Azienda Ospedaliera Universitaria Pisana, Pisa, Italy
| | - Rob Glynne-Jones
- Department of Medical Oncology, Mount Vernon Centre for Cancer Treatment, London, UK
| | | | - Esther Bastiaannet
- Department of Surgery, Leiden University Medical Centre, Leiden, Netherlands; Department of Gerontology and Geriatrics, Leiden University Medical Centre, Leiden, Netherlands
| | | | - Gerrit-Jan Liefers
- Department of Surgery, Leiden University Medical Centre, Leiden, Netherlands
| | - Hein Putter
- Department of Medical Statistics and Bio-informatics, Leiden University Medical Centre, Leiden, Netherlands
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Wang HL, Liu P, Zhou PY, Zhang Y. Promoter methylation of the RASSF1A gene may contribute to colorectal cancer susceptibility: a meta-analysis of cohort studies. Ann Hum Genet 2014; 78:208-16. [PMID: 24621386 DOI: 10.1111/ahg.12059] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2014] [Accepted: 01/27/2014] [Indexed: 01/11/2023]
Abstract
This meta-analysis of published cohort studies was conducted to evaluate whether promoter methylation of the RASSF1A gene contributes to colorectal cancer (CRC) susceptibility. A range of electronic databases were searched without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude risk differences (RD) with their 95% confidence intervals (95%CI) were calculated. In this meta-analysis, 11 clinical cohort studies with a total of 630 CRC patients were included. The pooled results revealed that the frequency of RASSF1A gene methylation in cancer tissues was significantly higher than that in benign, adjacent, and normal tissues (cancer tissues vs. benign tissues: RD = 0.25, 95%CI = 0.13-0.38, P < 0.001; cancer tissues vs. adjacent tissues: RD = 0.32, 95%CI: 0.20-0.45, P < 0.001; cancer tissues vs. normal tissues: RD = 0.38, 95%CI: 0.26-0.50, P < 0.001; respectively). Subgroup analysis by ethnicity demonstrated that RASSF1A promoter methylation also exhibited a higher frequency in cancer tissues among both Asians and Caucasians (all P < 0.05). Our meta-analysis has shown positive correlations between RASSF1A promoter methylation and CRC susceptibility. Thus, detection of RASSF1A promoter methylation may be utilized as a valuable diagnostic marker for CRC.
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Affiliation(s)
- He-Ling Wang
- Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, 110022, P.R. China
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Furlan D, Sahnane N, Bernasconi B, Frattini M, Tibiletti MG, Molinari F, Marando A, Zhang L, Vanoli A, Casnedi S, Adsay V, Notohara K, Albarello L, Asioli S, Sessa F, Capella C, La Rosa S. APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation. Virchows Arch 2014; 464:553-64. [PMID: 24590585 DOI: 10.1007/s00428-014-1562-1] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2013] [Revised: 01/24/2014] [Accepted: 02/16/2014] [Indexed: 12/15/2022]
Abstract
Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels.
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Affiliation(s)
- Daniela Furlan
- Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy
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RETRACTED ARTICLE: Aberrant promoter methylation of RASSF1A gene may be correlated with colorectal carcinogenesis: a meta-analysis. Mol Biol Rep 2014; 41:3991-9. [DOI: 10.1007/s11033-014-3267-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2014] [Accepted: 02/12/2014] [Indexed: 12/21/2022]
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Sumita K, Yoshino H, Sasaki M, Majd N, Kahoud ER, Takahashi H, Takeuchi K, Kuroda T, Lee S, Charest PG, Takeda K, Asara JM, Firtel RA, Anastasiou D, Sasaki AT. Degradation of activated K-Ras orthologue via K-Ras-specific lysine residues is required for cytokinesis. J Biol Chem 2013; 289:3950-9. [PMID: 24338482 DOI: 10.1074/jbc.m113.531178] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.
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Affiliation(s)
- Kazutaka Sumita
- From the Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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Korah R, Healy JM, Kunstman JW, Fonseca AL, Ameri AH, Prasad ML, Carling T. Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma. Mol Cancer 2013; 12:87. [PMID: 23915220 PMCID: PMC3750604 DOI: 10.1186/1476-4598-12-87] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2013] [Accepted: 08/03/2013] [Indexed: 12/17/2022] Open
Abstract
Background Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with high mutational heterogeneity and a generally poor clinical outcome. Despite implicated roles of deregulated TP53, IGF-2 and Wnt signaling pathways, a clear genetic association or unique mutational link to the disease is still missing. Recent studies suggest a crucial role for epigenetic modifications in the genesis and/or progression of ACC. This study specifically evaluates the potential role of epigenetic silencing of RASSF1A, the most commonly silenced tumor suppressor gene, in adrenocortical malignancy. Results Using adrenocortical tumor and normal tissue specimens, we show a significant reduction in expression of RASSF1A mRNA and protein in ACC. Methylation-sensitive and -dependent restriction enzyme based PCR assays revealed significant DNA hypermethylation of the RASSF1A promoter, suggesting an epigenetic mechanism for RASSF1A silencing in ACC. Conversely, the RASSF1A promoter methylation profile in benign adrenocortical adenomas (ACAs) was found to be very similar to that found in normal adrenal cortex. Enforced expression of ectopic RASSF1A in the SW-13 ACC cell line reduced the overall malignant behavior of the cells, which included impairment of invasion through the basement membrane, cell motility, and solitary cell survival and growth. On the other hand, expression of RASSF1A/A133S, a loss-of-function mutant form of RASSF1A, failed to elicit similar malignancy-suppressing responses in ACC cells. Moreover, association of RASSF1A with the cytoskeleton in RASSF1A-expressing ACC cells and normal adrenal cortex suggests a role for RASSF1A in modulating microtubule dynamics in the adrenal cortex, and thereby potentially blocking malignant progression. Conclusions Downregulation of RASSF1A via promoter hypermethylation may play a role in the malignant progression of adrenocortical carcinoma possibly by abrogating differentiation-promoting RASSF1A- microtubule interactions.
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Affiliation(s)
- Reju Korah
- Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT 06520, USA
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Ashktorab H, Rahi H, Wansley D, Varma S, Shokrani B, Lee E, Daremipouran M, Laiyemo A, Goel A, Carethers JM, Brim H. Toward a comprehensive and systematic methylome signature in colorectal cancers. Epigenetics 2013; 8:807-15. [PMID: 23975090 DOI: 10.4161/epi.25497] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
CpG Island Methylator Phenotype (CIMP) is one of the underlying mechanisms in colorectal cancer (CRC). This study aimed to define a methylome signature in CRC through a methylation microarray analysis and a compilation of promising CIMP markers from the literature. Illumina HumanMethylation27 (IHM27) array data was generated and analyzed based on statistical differences in methylation data (1st approach) or based on overall differences in methylation percentages using lower 95% CI (2nd approach). Pyrosequencing was performed for the validation of nine genes. A meta-analysis was used to identify CIMP and non-CIMP markers that were hypermethylated in CRC but did not yet make it to the CIMP genes' list. Our 1st approach for array data analysis demonstrated the limitations in selecting genes for further validation, highlighting the need for the 2nd bioinformatics approach to adequately select genes with differential aberrant methylation. A more comprehensive list, which included non-CIMP genes, such as APC, EVL, CD109, PTEN, TWIST1, DCC, PTPRD, SFRP1, ICAM5, RASSF1A, EYA4, 30ST2, LAMA1, KCNQ5, ADHEF1, and TFPI2, was established. Array data are useful to categorize and cluster colonic lesions based on their global methylation profiles; however, its usefulness in identifying robust methylation markers is limited and rely on the data analysis method. We have identified 16 non-CIMP-panel genes for which we provide rationale for inclusion in a more comprehensive characterization of CIMP+ CRCs. The identification of a definitive list for methylome specific genes in CRC will contribute to better clinical management of CRC patients.
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Affiliation(s)
- Hassan Ashktorab
- Department of Medicine and Cancer Center; Department of Pathology; Howard University College of Medicine; Washington, D.C. USA
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Gou HF, Li X, Qiu M, Cheng K, Li LH, Dong H, Chen Y, Tang Y, Gao F, Zhao F, Men HT, Ge J, Su JM, Xu F, Bi F, Gao JJ, Liu JY. Epidermal growth factor receptor (EGFR)-RAS signaling pathway in penile squamous cell carcinoma. PLoS One 2013; 8:e62175. [PMID: 23637996 PMCID: PMC3634795 DOI: 10.1371/journal.pone.0062175] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2012] [Accepted: 03/17/2013] [Indexed: 02/05/2023] Open
Abstract
Penile Squamous Cell Carcinoma (SCC) is a rare cancer with poor prognosis and limited response to conventional chemotherapy. The genetic and epigenetic alterations of Epidermal Growth Factor Receptor (EGFR)-RAS-RAF signaling in penile SCC are unclear. This study aims to investigate four key members of this pathway in penile SCC. We examined the expression of EGFR and RAS-association domain family 1 A (RASSF1A) as well as the mutation status of K-RAS and BRAF in 150 cases of penile SCC. EGFR and RASSF1A expression was evaluated by immunohistochemistry. KRAS mutations at codons 12 and 13, and the BRAF mutation at codon 600 were analyzed on DNA isolated from formalin fixed paraffin embedded tissues by direct genomic sequencing. EGFR expression was positive in all specimens, and its over-expression rate was 92%. RASSF1A expression rate was only 3.42%. Significant correlation was not found between the expression of EGFR or RASSF1A and tumor grade, pT stage or lymph node metastases. The detection of KRAS and BRAF mutations analysis was performed in 94 and 83 tumor tissues, respectively. We found KRAS mutation in only one sample and found no BRAF V600E point mutation. In summary, we found over-expression of EGFR in the majority cases of penile SCC, but only rare expression of RASSF1A, rare KRAS mutation, and no BRAF mutation in penile SCC. These data suggest that anti-EGFR agents may be potentially considered as therapeutic options in penile SCC.
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Affiliation(s)
- Hong-Feng Gou
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Xiang Li
- Department of Urology, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Meng Qiu
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Ke Cheng
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Long-Hao Li
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Hang Dong
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Ye Chen
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Yuan Tang
- Department of Pathology, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Feng Gao
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Feng Zhao
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Hai-Tao Men
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Jun Ge
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Jing-Mei Su
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Feng Xu
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Feng Bi
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
| | - Jian-Jun Gao
- Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
| | - Ji-Yan Liu
- Department of Medical Oncology, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
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Sinha R, Hussain S, Mehrotra R, Kumar RS, Kumar K, Pande P, Doval DC, Basir SF, Bharadwaj M. Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation: indicators of tumor staging and metastasis in adenocarcinomatous sporadic colorectal cancer in Indian population. PLoS One 2013; 8:e60142. [PMID: 23573237 PMCID: PMC3616004 DOI: 10.1371/journal.pone.0060142] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2012] [Accepted: 02/21/2013] [Indexed: 12/24/2022] Open
Abstract
OBJECTIVE Colorectal cancer (CRC) development involves underlying modifications at genetic/epigenetic level. This study evaluated the role of Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation together/independently in sporadic CRC in Indian population and correlation with clinicopathological variables of the disease. METHODS One hundred and twenty four consecutive surgically resected tissues (62 tumor and equal number of normal adjacent controls) of primary sporadic CRC were included and patient details including demographic characteristics, lifestyle/food or drinking habits, clinical and histopathological profiles were recorded. Polymerase chain reaction - Restriction fragment length polymorphism and direct sequencing for Kras gene mutation and Methylation Specific-PCR for RASSF1A, FHIT and MGMT genes was performed. RESULTS Kras gene mutation at codon 12 & 13 and methylated RASSF1A, FHIT and MGMT gene was observed in 47%, 19%, 47%, 37% and 47% cases, respectively. Alcohol intake and smoking were significantly associated with presence of Kras mutation (codon 12) and MGMT methylation (p-value <0.049). Tumor stage and metastasis correlated with presence of mutant Kras codon 12 (p-values 0.018, 0.044) and methylated RASSF1A (p-values 0.034, 0.044), FHIT (p-values 0.001, 0.047) and MGMT (p-values 0.018, 0.044) genes. Combinatorial effect of gene mutation/methylation was also observed (p-value <0.025). Overall, tumor stage 3, moderately differentiated tumors, presence of lymphatic invasion and absence of metastasis was more frequently observed in tumors with mutated Kras and/or methylated RASSF1A, FHIT and MGMT genes. CONCLUSION Synergistic interrelationship between these genes in sporadic CRC may be used as diagnostic/prognostic markers in assessing the overall pathological status of CRC.
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Affiliation(s)
- Rupal Sinha
- Department of Research, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
- Division of Molecular Genetics and Biochemistry, Institute of Cytology and Preventive Oncology, Noida, India
- Department of Surgical Oncology, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
| | - Showket Hussain
- Division of Molecular Genetics and Biochemistry, Institute of Cytology and Preventive Oncology, Noida, India
| | - Ravi Mehrotra
- Division of Cytopathology, Institute of Cytology and Preventive Oncology, Noida, India
| | - R. Suresh Kumar
- Division of Molecular Genetics and Biochemistry, Institute of Cytology and Preventive Oncology, Noida, India
| | - Kapil Kumar
- Department of Surgical Oncology, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
| | - Pankaj Pande
- Department of Surgical Oncology, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
| | - Dinesh Chandra Doval
- Department of Research, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
- Department of Medical Oncology, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
| | | | - Mausumi Bharadwaj
- Division of Molecular Genetics and Biochemistry, Institute of Cytology and Preventive Oncology, Noida, India
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Malhotra P, Anwar M, Nanda N, Kochhar R, Wig JD, Vaiphei K, Mahmood S. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians. Tumour Biol 2013; 34:1901-11. [PMID: 23526092 DOI: 10.1007/s13277-013-0734-y] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2013] [Accepted: 03/04/2013] [Indexed: 02/06/2023] Open
Abstract
The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort.
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Affiliation(s)
- Pooja Malhotra
- Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India
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Brinkhuizen T, van den Hurk K, Winnepenninckx VJL, de Hoon JP, van Marion AM, Veeck J, van Engeland M, van Steensel MAM. Epigenetic changes in Basal Cell Carcinoma affect SHH and WNT signaling components. PLoS One 2012; 7:e51710. [PMID: 23284750 PMCID: PMC3524166 DOI: 10.1371/journal.pone.0051710] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2012] [Accepted: 11/05/2012] [Indexed: 01/08/2023] Open
Abstract
Background The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies. Objective We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR. Results Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples). Conclusions We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.
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Affiliation(s)
- Tjinta Brinkhuizen
- Department of Dermatology, Maastricht University Medical Center, Maastricht, The Netherlands.
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Nilsson TK, Löf-Öhlin ZM, Sun XF. DNA methylation of the p14ARF, RASSF1A and APC1A genes as an independent prognostic factor in colorectal cancer patients. Int J Oncol 2012; 42:127-33. [PMID: 23128528 PMCID: PMC3583697 DOI: 10.3892/ijo.2012.1682] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2012] [Accepted: 09/28/2012] [Indexed: 01/05/2023] Open
Abstract
We quantitated the methylated fraction of CpG sites in the promoter regions of O6-MGMT, p14ARF, p16INK4a, RASSF1A and APC1A in tumor tissue from patients with colorectal cancer (CRC) in order to determine if promoter hypermethylation of any of these genes predicts survival. DNA was isolated from 111 primary CRC and 46 matched normal colorectal mucosa samples from the same patients, obtained at primary surgery and DNA methylation was examined by Pyrosequencing®. Follow-up time was up to 20 years. Patients showed partial promoter methylation in the following frequencies: O6-MGMT, 34%; p14ARF, 29%; p16INK4a, 28%; RASSF1A, 14%; and APC1A, 27%. Normal mucosa was always unmethylated. CRC patients with methylated p14ARF gene promoter had significantly worse prognosis (p=0.036), whereas those with methylated O6-MGMT had significantly better prognosis through the first 60 months post-treatment (RR 0.36; p=0.023). Methylation of one or more of the genes from the set p14ARF, RASSF1A and APC1A, was significantly (p= 0.021) associated with worse prognosis even adjusting for tumor stage and differentiation (RR 2.2, p=0.037). Thus, DNA methylation of the p14ARF, RASSF1A and APC1A genes, diagnosed by Pyrosequencing, defines a poor prognosis subset of CRC patients independently of both tumor stage and differentiation. O6-MGMT methylation may play a protective role.
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Affiliation(s)
- Torbjörn K Nilsson
- Department of Laboratory Medicine, Örebro University Hospital, Örebro, Sweden.
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Chen SP, Wu CC, Huang SY, Kang JC, Chiu SC, Yang KL, Pang CY. β-catenin and K-ras mutations and RASSF1A promoter methylation in Taiwanese colorectal cancer patients. Genet Test Mol Biomarkers 2012; 16:1277-81. [PMID: 23009572 DOI: 10.1089/gtmb.2012.0126] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
AIMS The purpose of this study was to investigate the associations of β-catenin mutations, K-ras mutations, methylations of the RASSF1A promoter, and the survival of Taiwanese colorectal cancer (CRC) subjects who received 5-fluorouracil (5-FU) adjuvant chemotherapy. RESULTS The complete coding region of the K-ras gene and exon 3 and exon 4 of the β-catenin gene isolated from tumor tissues and adjacent normal colon tissues from 117 CRC subjects were sequenced, respectively. Methylations in the RASSF1A promoter region were also investigated. Various characteristics of the 117 subjects were recorded and used in the Cox proportional-hazard model analyses. Three missense mutations, one nonsense mutation, and one deletion were identified in the β-catenin gene. A 2 bp deletion was identified in the K-ras gene. We found that the frequencies of mutations in the β-catenin and K-ras genes were less pronounced in Taiwanese CRC subjects as compared with other populations. Methylations in the RASSF1A promoter region were detected in 73.5% (n=86/117) of the subjects, which was higher than in other studies. Methylations in the RASSF1A promoter have no significant effect on hazards for all CRC deaths caused in Taiwanese CRC patients. No interaction between 5-FU adjuvant chemotherapy and methylations of the RASSF1A promoter was observed. CONCLUSIONS The mutation frequencies of β-catenin and K-ras genes in Taiwanese CRC patients are very low, which may suggest that they are not the dominant factors for CRC occurrence and prognosis in Taiwanese CRC patients. Methylation of RASSF1A promoter is independent of the prognosis for Taiwanese CRC patients. Taiwanese subjects differ from subjects of other populations with regard to β-catenin, K-ras, and RASSF1A presentations for CRC.
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Affiliation(s)
- Shee-Ping Chen
- Tzu Chi Stem Cells Center, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, Republic of China
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Fernandes MS, Carneiro F, Oliveira C, Seruca R. Colorectal cancer and RASSF family--a special emphasis on RASSF1A. Int J Cancer 2012; 132:251-8. [PMID: 22733432 DOI: 10.1002/ijc.27696] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2012] [Revised: 05/23/2012] [Accepted: 06/11/2012] [Indexed: 12/14/2022]
Abstract
The RAS-association domain family, commonly referred to as RASSF, is a family of 10 members (RASSF1-10) implicated in a variety of key biological processes, including cell cycle regulation, apoptosis and microtubule stability. Furthermore, RASSFs have been implicated in tumorigenesis and several family members are now thought to be tumor suppressors. As opposed to the KRAS oncogene, for which mutational activation is frequent in colorectal cancer (CRC), RASSFs are found to be silenced mainly by aberrant promoter methylation. In particular, RASSF1A, RASSF2 and RASSF5 methylation has been associated with CRC development, though the mechanisms of action remain poorly understood. This review focus on the current knowledge of RASSF inactivation in CRC, particularly RASSF1A, and on the implications RASSFs may have as potential biomarkers and for the development of new targeted therapies for CRC.
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Affiliation(s)
- Maria Sofia Fernandes
- Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal
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Xue ZT, Sjögren HO, Salford LG, Widegren B. An epigenetic mechanism for high, synergistic expression of indoleamine 2,3-dioxygenase 1 (IDO1) by combined treatment with zebularine and IFN-γ: potential therapeutic use in autoimmune diseases. Mol Immunol 2012; 51:101-11. [PMID: 22424783 DOI: 10.1016/j.molimm.2012.01.006] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2011] [Accepted: 01/14/2012] [Indexed: 12/21/2022]
Abstract
IDO1 can be induced by interferon gamma (IFN-γ) in multiple cell types. We have earlier described that the DNA methyltransferase inhibitor zebularine also induces IDO1 in several rat cell clones. We now describe a synergistic induction of IDO1 expression by IFN-γ and zebularine. To elucidate the mechanism of the IDO1 induction we have studied the methylation status in the promoter region of the IDO1 gene from both human monocytic THP-1 cells and H1D2 rat colon cancer cells. Interestingly, the IDO1 promoter is hypermethylated and IFN-γ is shown to induce a significant demethylation. The synergism in effect of zebularine and IFN-γ on IDO1 expression is paralleled by a similar synergistic effect on expression of two other IFN-γ-responsive genes, the transcription factors STAT1 and IRF1 with binding sites in the IDO1 promoter region. The demonstrated synergistic activation of IDO1 expression has implications in relation to therapeutic induction of immunosuppression in autoimmunity and chronic inflammation.
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Affiliation(s)
- Zhong-Tian Xue
- The Rausing Laboratory, Division of Neurosurgery, Department of Clinical Sciences, Lund University, Lund, Sweden
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Development of a novel approach, the epigenome-based outlier approach, to identify tumor-suppressor genes silenced by aberrant DNA methylation. Cancer Lett 2012; 322:204-12. [PMID: 22433712 DOI: 10.1016/j.canlet.2012.03.016] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2012] [Revised: 03/10/2012] [Accepted: 03/12/2012] [Indexed: 12/22/2022]
Abstract
Identification of tumor-suppressor genes (TSGs) silenced by aberrant methylation of promoter CpG islands (CGIs) is important, but hampered by a large number of genes methylated as passengers of carcinogenesis. To overcome this issue, we here took advantage of the fact that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. First, we demonstrated that three of six known TSGs in breast cancer and two of three in colon cancer had Pol II and lacked H3K27me3 in normal cells, being outliers to the general rule. BRCA1, HOXA5, MLH1, and RASSF1A had high Pol II, but were expressed only at low levels in normal cells, and were unlikely to be identified as outliers by their expression statuses in normal cells. Then, using epigenome statuses (Pol II binding and H3K27me3) in normal cells, we made a genome-wide search for outliers in breast cancers, and identified 14 outlier promoter CGIs. Among these, DZIP1, FBN2, HOXA5, and HOXC9 were confirmed to be methylated in primary breast cancer samples. Knockdown of DZIP1 in breast cancer cell lines led to increases of their growth, suggesting it to be a novel TSG. The outliers based on their epigenome statuses contained unique TSGs, including DZIP1, compared with those identified by the expression microarray data. These results showed that the epigenome-based outlier approach is capable of identifying a different set of TSGs, compared to the expression-based outlier approach.
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Epigenetic changes induced by oxidative stress in colorectal cancer cells: methylation of tumor suppressor RUNX3. Tumour Biol 2012; 33:403-12. [DOI: 10.1007/s13277-012-0322-6] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2011] [Accepted: 01/04/2012] [Indexed: 01/18/2023] Open
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Kontic M, Stojsic J, Jovanovic D, Bunjevacki V, Ognjanovic S, Kuriger J, Puumala S, Nelson HH. Aberrant promoter methylation of CDH13 and MGMT genes is associated with clinicopathologic characteristics of primary non-small-cell lung carcinoma. Clin Lung Cancer 2011; 13:297-303. [PMID: 22169480 DOI: 10.1016/j.cllc.2011.11.003] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2011] [Revised: 10/31/2011] [Accepted: 11/09/2011] [Indexed: 01/02/2023]
Abstract
UNLABELLED Non–small-cell lung carcinoma (NSCLC) (n = 65) were analyzed for promoter methylation of RASSF1A, CDH13, MGMT, ESR1, and DAPK genes in matching lung tumors, normal lung tissue, and blood samples. Aberrant methylation in CDH13 and MGMT was associated with clinicopathologic features of NSCLC. Hypermethylation detected in primary tumors was not observed in corresponding blood samples, which rendered this an unsuitable blood-based test for NSCLC detection. INTRODUCTION Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue and whether DNA methylation changes can be detected in paired blood samples. MATERIAL AND METHODS Sixty-five patients were enrolled in a surgical case series of non-small-cell lung carcinoma at a single institution. By using bisulfite pyrosequencing, CpG methylation was quantified at 5 genes (RASSF1A, CDH13, MGMT, ESR1, and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases. RESULTS The analyses of methylation in tumors compared with normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, whereas ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the 3 aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, which indicated a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly, we probed whether tumor methylation was associated with clinical and demographic characteristics. Histology and sex were associated with methylation at the CDH13 gene, whereas, stage was associated with methylation at MGMT. CONCLUSION Our results showed higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared with normal lung. The lack of reflection of these methylation changes in blood samples from patients with non-small-cell lung carcinoma indicates their poor suitability for a screening test.
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Affiliation(s)
- Milica Kontic
- Pulmonology Clinic, Clinical Centre of Serbia, Medical Faculty, University in Belgrade, Belgrade, Serbia
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Tommasi S, Pinto R, Petriella D, Pilato B, Lacalamita R, Santini D, Zito F, Colucci G, Paradiso A, Silvestris N. Oncosuppressor methylation: a possible key role in colon metastatic progression. J Cell Physiol 2011; 226:1934-9. [PMID: 21506124 DOI: 10.1002/jcp.22524] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
K-RAS and BRAF gene mutations are mandatory to set anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients. Due to the relationship of these mutations with tumor epigenotype, we hypothesized the potential role of oncosuppressor methylation of genes involved in K-RAS/BRAF pathway (CDKN2A, RASSF1A, and RARbeta suppressor genes) in inhibiting EGFR signaling cascade. Primary tumor and synchronous liver metastatic tissues of 75 mCRC patients were characterized for promoter methylation by QMSP and for K-RAS and BRAF mutations. RARbeta, RASSF1A, and CDKN2A genes were methylated in 82%, 35%, and 26% of primary tumors, respectively. RASSF1A resulted significantly more frequently methylated in liver metastasis than in primary site (P=0.015), while RARbeta was significantly lower methylated in distant metastasis (P=1.2 × 10(-6)). As regards methylation content, RASSF1A methylation status was significantly higher in liver metastasis with respect to primary tumor (P=0.000) underlying the role of this gene in liver metastatic progression. In our series K-RAS and BRAF were mutated in 39% and 4% of cases, respectively. Methylation frequencies seemed to be unrelated to gene mutations; on the other hand, RASSF1A mean content methylation resulted significantly higher in liver than in primary tumor (288.78 vs. 56.23, respectively, P=0.05) only in K-RAS wild-type cases sustaining a specific role of this gene in metastatic site thus supporting its function in strengthening the apoptotic role of K-RAS. These evidences held the role of oncosuppressor methylation in both colon tumorigenesis and progression and suggested that epigenetic events should be taken into account when biological therapies in mCRC patients have to be set.
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Affiliation(s)
- Stefania Tommasi
- Clinical Experimental Oncology Laboratory, National Cancer Centre Giovanni Paolo II, Bari, Italy.
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