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Journal of Cancer Research and Clinical Oncology

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01-04-2020 | Chronic Pancreatitis | Original Article – Cancer Research

Clinical significance of promoter methylation status of tumor suppressor genes in circulating DNA of pancreatic cancer patients

Authors: Nidhi Singh, Sumaira Rashid, Safoora Rashid, Nihar Ranjan Dash, Surabhi Gupta, Anoop Saraya

Published in: Journal of Cancer Research and Clinical Oncology | Issue 4/2020

Abstract

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive cancer. There are various sub-cellular events (both genetic and epigenetic) that get dysregulated leading to tumorigenesis. Methylation in promoters of tumor suppressor genes is one of these epigenetic phenomena contributing to the pathogenesis of cancer. Genes analyzed for promoter methylation status in this study namely SPARC (Secreted Protein Acidic and Rich in Cysteine, UCHL1 (ubiquitin carboxy-terminal hydrolase L1), NPTX2 (neuronal pentraxin 2), PENK (proenkephalin) had been studied in pancreatic cancer, but there is a need to check methylation in these genes as circulatory non-invasive markers. This study analyzed the absolute quantification of methylation levels of SPARC, UCHL1, PENK, and NPTX2 genes promoters in PDAC patients as well as in chronic pancreatitis (CP) patients and healthy subjects (HC) and evaluated its clinical significance in PDAC.

Materials and methods

The study included 65 PDAC patients, 25 CP patients, and 25 healthy controls. DNA was extracted from their plasma samples and subsequently given bisulfite treatment. Absolute quantization of methylated and unmethylated copies of gene promoters of all the four genes was performed using real-time PCR (SYBR green) by the standard curve method. Methylation levels were expressed as methylation index (MI) for each gene in each patient. MI was calculated from absolute copy numbers as follows: MI-methylated copy number/methylated copy number + unmethylated copy number). These indices were used to compare gene methylation levels within different groups and to correlate with clinicopathological features and survival of pancreatic cancer patients. An appropriate statistical analysis was applied.

Results

Methylation indices for all the four genes in PDAC cases were found to be significantly higher as compared to that in healthy individuals. SPARC MI values were found to differentiate early-stage PDAC patients from CP patients. PDAC patients with the metastasized disease and stage IV disease were found to have high MI for the SPARC gene as well as for the NPTX2 gene, while a higher UCHL1 methylation index was found to correlate with an advanced stage of the disease. Higher MI values for SPARC and NPTX2 genes were found to associate with poor survival in patients with PDAC.

Conclusion

Methylation load in the form of MI for each of the four genes assessed in plasma may emerge as a non-invasive biomarker to differentiate pancreatic cancer from healthy individuals. But only SPARC and NPTX2 hypermethylation were able to distinguish pancreatic cancer from chronic pancreatitis. Association of aberrant methylation in SPARC and NPTX2 gene with metastasis and poor survival of patients suggest the role of methylation in these genes as prognostic markers.

Bheda A, Gullapalli A, Caplow M, Pagano JS, Shackelford J (2010) Ubiquitin editing enzyme UCH L1 and microtubule dynamics: implication in mitosis. Cell Cycle 9(5):980–994PubMed

Feinberg AP, Tycko B (2004) The history of cancer epigenetics. Nat Rev Cancer 4(2):143–153PubMed

Fukushima N, Sato N, Ueki T, Rosty C, Walter KM, Wilentz RE et al (2002) Aberrant methylation of preproenkephalin and p16 genes in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma. Am J Pathol 160(5):1573–1581PubMedPubMedCentral

Goto Y, Zeng L, Yeom CJ, Zhu Y, Morinibu A, Shinomiya K et al (2015) UCHL1 provides diagnostic and antimetastatic strategies due to its deubiquitinating effect on HIF-1α. Nat Commun 23(6):6153

Guo X, Cui Z (2005) Current diagnosis and treatment of pancreatic cancer in China. Pancreas 31(1):13–22PubMed

Henriksen SD, Madsen PH, Larsen AC, Johansen MB, Pedersen IS, Krarup H et al (2017) Cell-free DNA promoter hypermethylation in plasma as a predictive marker for survival of patients with pancreatic adenocarcinoma. Oncotarget 8(55):93942–93956PubMedPubMedCentral

Hingorani SR, Tuveson DA (2003) In search of an early warning system for pancreatic cancer. Cancer Biol Ther 2(1):84–86PubMed

Hong S-M, Kelly D, Griffith M, Omura N, Li A, Li C-P, et al. (2008) Multiple genes are hypermethylated in intraductal papillary mucinous neoplasms of the pancreas. Mod Pathol 21(12):1499–507.PubMedPubMedCentral

Hruban RH, Takaori K, Klimstra DS, Adsay NV, Albores-Saavedra J, Biankin AV et al (2004) An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms. Am J Surg Pathol 28(8):977–987PubMed

Iacobuzio-Donahue CA (2012) Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project. Gut 61(7):1085–1094PubMed

Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3(6):415–428PubMed

Kato N, Yamamoto H, Adachi Y, Ohashi H, Taniguchi H, Suzuki H et al (2013) Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids. World J Gastroenterol 19(11):1718–1727PubMedPubMedCentral

Kleiman DA, Beninato T, Sultan S, Crowley MJP, Finnerty B, Kumar R et al (2014) Silencing of UCHL1 by CpG promoter hyper-methylation is associated with metastatic gastroenteropancreatic well-differentiated neuroendocrine (carcinoid) tumors. Ann Surg Oncol 21(Suppl 4):S672–679PubMedPubMedCentral

Li L, Tao Q, Jin H, van Hasselt A, Poon FF, Wang X et al (2010) The tumor suppressor UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signalling and is frequently silenced in nasopharyngeal carcinoma. Clin Cancer Res 16(11):2949–2958PubMed

Luo Y, He J, Yang C, Orange M, Ren X, Blair N et al (2018) UCH-L1 promotes invasion of breast cancer cells through activating Akt signalling pathway. J Cell Biochem 119(1):691–700PubMed

Matsubayashi H, Canto M, Sato N, Klein A, Abe T, Yamashita K et al (2006) DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease. Cancer Res 66(2):1208–1217PubMed

Niederhuber JE, Brennan MF, Menck HR (1995) The National Cancer Data Base report on pancreatic cancer. Cancer 76(9):1671–1677PubMed

Ohtsubo K, Watanabe H, Yamaguchi Y, Hu Y-X, Motoo Y, Okai T et al (2003) Abnormalities of tumor suppressor gene p16 in pancreatic carcinoma: immunohistochemical and genetic findings compared with clinicopathological parameters. J Gastroenterol 38(7):663–671PubMed

Pancreatic Cancer-Statistics. Cancer.Net (2012) https://www.cancer.net/cancer-types/pancreatic-cancer/statistics. Accessed Jan 28 2020

Park JK, Ryu JK, Lee KH, Lee JK, Yoon WJ, Lee SH et al (2007) Quantitative analysis of NPTX2 hypermethylation is a promising molecular diagnostic marker for pancreatic cancer. Pancreas 35(3):e9–15PubMed

Park JK, Ryu JK, Yoon WJ, Lee SH, Lee GY, Jeong K-S et al (2012) The role of quantitative NPTX2 hypermethylation as a novel serum diagnostic marker in pancreatic cancer. Pancreas 41(1):95–101PubMed

ppENK Gene Methylation Status in the Development of Pancreatic Carcinoma. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/?term=ppENK+Gene+Methylation+Status+in+the+Development+of+Pancreatic+Carcinoma. Accessed June 27 2019

Sato N, Ueki T, Fukushima N, Iacobuzio-Donahue CA, Yeo CJ, Cameron JL et al (2002) Aberrant methylation of CpG islands in intraductal papillary mucinous neoplasms of the pancreas. Gastroenterology 123(1):365–372PubMed

Sato N, Fukushima N, Maehara N, Matsubayashi H, Koopmann J, Su GH et al (2003) SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor–stromal interactions. Oncogene 22(32):5021–5030PubMed

Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108PubMed

Schneider G, Schmid RM (2003) Genetic alterations in pancreatic carcinoma. Mol Cancer 22(2):15

Sharma S, Kelly TK, Jones PA (2010) Epigenetics in cancer. Carcinogenesis 31(1):27–36PubMed

Singh N, Gupta S, Rashid S, Rashid S, Dash NR, Saraya A (2019) Tu2033—quantitation of methylation load of tumor suppressor gene promoter methylation in pancreatic cancer. Gastroenterology 156(6(Supplement 1)):S1176

Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9(6):465–476PubMed

Tanaka T, Kuramitsu Y, Fujimoto M, Naito S, Oka M, Nakamura K (2008) Downregulation of two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 correlates with high metastatic potentials of human SN12C renal cell carcinoma cell clones. Electrophoresis 29(12):2651–2659PubMed

Thompson MJ, Rubbi L, Dawson DW, Donahue TR, Pellegrini M (2015) Pancreatic cancer patient survival correlates with DNA methylation of pancreas development genes. PLoS ONE 10(6):e0128814PubMedPubMedCentral

Ueki T, Toyota M, Skinner H, Walter KM, Yeo CJ, Issa JP et al (2001) Identification and characterization of differentially methylated CpG islands in pancreatic carcinoma. Cancer Res 61(23):8540–8546PubMed

Ueki T, Walter KM, Skinner H, Jaffee E, Hruban RH, Goggins M (2002) Aberrant CpG island methylation in cancer cell lines arises in the primary cancers from which they were derived. Oncogene 21(13):2114–2117PubMed

Wang L, Yang G-H, Lu X-H, Huang Z-J, Li H (2003) Pancreatic cancer mortality in China (1991–2000). World J Gastroenterol 9(8):1819–1823PubMedPubMedCentral

Wang W-J, Li Q-Q, Xu J-D, Cao X-X, Li H-X, Tang F et al (2008) Over-expression of ubiquitin carboxy terminal hydrolase-L1 induces apoptosis in breast cancer cells. Int J Oncol 33(5):1037–1045PubMed

Xiang T, Li L, Yin X, Yuan C, Tan C, Su X et al (2012) The ubiquitin peptidase UCHL1 induces G0/G1 cell cycle arrest and apoptosis through stabilizing p53 and is frequently silenced in breast cancer. PLoS ONE 7(1):e29783PubMedPubMedCentral

Yadav D, Lowenfels AB (2013) The epidemiology of pancreatitis and pancreatic cancer. Gastroenterology 144(6):1252–1261PubMed

Yan W-J, Guo M-Z, Yang Y-S (2012) The role of hypermethylation in promoter region of ubiquitin carboxyl-terminal hydrolase L1 in human esophageal cancer. Zhonghua Nei Ke Za Zhi 51(5):390–393PubMed

Yao F, Sun M, Dong M, Jing F, Chen B, Xu H, Wang S (2013) NPTX2 hypermethylation in pure pancreatic juice predicts pancreatic neoplasms. Am J Med Sci 346:175–180. https://doi.org/10.1097/MAJ.0b013e31827b94b6 CrossRefPubMed

Yi JM, Guzzetta AA, Bailey VJ, Downing SR, Van Neste L, Chiappinelli KB et al (2013) Novel methylation biomarker panel for the early detection of pancreatic cancer. Clin Cancer Res 19(23):6544–6555PubMedPubMedCentral

Yu J, Tao Q, Cheung KF, Jin H, Poon FF, Wang X et al (2008) Epigenetic identification of ubiquitin carboxyl-terminal hydrolase L1 as a functional tumor suppressor and biomarker for hepatocellular carcinoma and other digestive tumors. Hepatology 48(2):508–518PubMed

Zhang L, Gao J, Li L, Li Z, Du Y, Gong Y (2011) The neuronal pentraxin II gene (NPTX2) inhibit proliferation and invasion of pancreatic cancer cells in vitro. Mol Biol Rep 38(8):4903–4911PubMed

Zhang L, Gao J, Li Z, Gong Y (2012) Neuronal pentraxin II (NPTX2) is frequently down-regulated by promoter hypermethylation in pancreatic cancers. Dig Dis Sci 57(10):2608–2614PubMed

Zhang Y, Yang B, Du Z, Bai T, Gao Y-T, Wang Y-J et al (2012) Aberrant methylation of SPARC in human hepatocellular carcinoma and its clinical implication. World J Gastroenterol 18(17):2043–2052PubMedPubMedCentral

Zhou Y-F, Xu W, Wang X, Sun J-S, Xiang J-J, Li Z-S et al (2014) Negative methylation status of vimentin predicts improved prognosis in pancreatic carcinoma. World J Gastroenterol 20(36):13172–13177PubMedPubMedCentral

Zhou C, Qin Y, Xie Z, Zhang J, Yang M, Li S et al (2015) NPTX1 is a novel epigenetic regulation gene and associated with prognosis in lung cancer. Biochem Biophys Res Commun 458(2):381–386PubMed

Title: Clinical significance of promoter methylation status of tumor suppressor genes in circulating DNA of pancreatic cancer patients
Authors: Nidhi Singh
Sumaira Rashid
Safoora Rashid
Nihar Ranjan Dash
Surabhi Gupta
Anoop Saraya
Publication date: 01-04-2020
Publisher: Springer Berlin Heidelberg
Keywords: Chronic Pancreatitis
Pancreatic Cancer
Pancreatic Cancer
Published in: Journal of Cancer Research and Clinical Oncology / Issue 4/2020
Print ISSN: 0171-5216
Electronic ISSN: 1432-1335
DOI: https://doi.org/10.1007/s00432-020-03169-y

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Clinical significance of promoter methylation status of tumor suppressor genes in circulating DNA of pancreatic cancer patients

Abstract

Introduction

Materials and methods

Results

Conclusion

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