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LINE-1 is preferentially hypomethylated within adenomatous polyps in the presence of synchronous colorectal cancer.

ABSTRACT: Conventional tubular adenomas are frequently detected in patients undergoing average risk screening colonoscopy and are over-represented in patients who will develop colorectal cancer (CRC). Whether features of adenomas could serve as predictors of synchronous CRC is not known. Here, we investigate whether global methylation markers, including LINE-1, differ within adenomas in patients with and without synchronous CRC.Colorectal tubular/tubulovillous adenomatous polyps in the absence (P group, n?=?45) and in the presence of synchronous CRC (PC group, n?=?32) were identified. Global methylation and demethylation by ELISA for 5-methylcytosine (5-mC) and 5-hydroxymethyl cytosine (5-hmC), respectively, were assessed in polyps and adjacent normal non-neoplastic tissue. LINE-1 hypomethylation was assessed by pyrosequencing of bisulfite-converted DNA as well.Global methylation (5-mC) showed no differences in overall methylation status in the adenomatous polyps in the two groups (5-mC relative to control %, PC group 0.117; P group 0.161, p?=?0.148). Global hydroxymethylation 5-hmC was also not significantly different in adenomatous polyps of the PC group than in those of the P group (0.0059 vs 0.0097, p?=?0.681). Similarly, global 5-hmC was not different between normal tissues from patients without neoplasia in comparison to those from CRC patients (0.0461?±?0.080 vs 0.039?±?0.159, p?=?0.215). In contrast, adenomatous polyps of the PC group had lower levels of LINE-1 methylation compared to the adenomas in the P group (53.07?±?4.5 vs 59.95?±?5.4, p?

SUBMITTER: Jiang AC

PROVIDER: S-EPMC5345219 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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LINE-1 is preferentially hypomethylated within adenomatous polyps in the presence of synchronous colorectal cancer.

Jiang Alice Chu AC Buckingham Lela L Barbanera William W Korang Amoah Yeboah AY Bishesari Faraz F Melson Joshua J

Clinical epigenetics 20170309

<h4>Background</h4>Conventional tubular adenomas are frequently detected in patients undergoing average risk screening colonoscopy and are over-represented in patients who will develop colorectal cancer (CRC). Whether features of adenomas could serve as predictors of synchronous CRC is not known. Here, we investigate whether global methylation markers, including LINE-1, differ within adenomas in patients with and without synchronous CRC.<h4>Methods</h4>Colorectal tubular/tubulovillous adenomatou ...[more]

PMID: 28293326

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Project description:Colorectal tumors mostly arise from sporadic adenomatous polyps. Polyps are defined as a mass of cells that protrudes into the lumen of the colon. Adenomatous polyps are benign neoplasms that, by definition display some characteristics of dysplasia. It has been shown that polyps were benign tumors which may undergo malignant transformation. Adenomatous polyps have been classified into three histologic types; tubular, tubulovillous, and villous with increasing malignant potential. The ability to differentially diagnose these colorectal adenomatous polyps is important for therapeutic intervention. To date, little efforts have been directed to identifying genetic changes involved in adenomatous polyps. This study was designed to examine the relevance of mitochondrial genome alterations in the three adenomatous polyps. Using high resolution restriction endonucleases and PCR-based sequencing, fifty-seven primary fresh frozen tissues of adenomatous polyps (37 tumors and 20 matched surrounding normal tissues) obtained from the southern regional Cooperative Human Tissue Network (CHTN) and Grady Memorial Hospital at Atlanta were screened with three mtDNA regional primer pairs that spanned 5.9 kbp. Results from our data analyses revealed the presence of forty-four variants in some of these mitochondrial genes that the primers spanned; COX I, II, III, ATP 6, 8, CYT b, ND 5, 6 and tRNAs. Based on the MITODAT database as a sequence reference, 25 of the 44 (57%) variants observed were unreported. Notably, a heteroplasmic variant C8515G/T in the MT-ATP 8 gene and a germline variant 8327delA in the tRNAlys was observed in all the tissue samples of the three adenomatous polyps in comparison to the referenced database sequence. A germline variant G9055A in the MT-ATP 6 gene had a frequency of 100% (17/17) in tubular and 57% (13/23) in villous adenomas; no corresponding variant was in tubulovillous adenomas. Furthermore, A9006G variant at MT-ATP 6 gene was observed at frequency of 57% (13/23) in villous adenomas only. Interestingly, variants A9006G and G9055A were absent in the villous tissue samples that were clinicopathological designated as "polyvillous adenomas". Our current data provide a basis for continued investigation of certain mtDNA variants as predictors of the three adenomatous polyps in a larger number of clinicopathological specimens.

Project description:BackgroundEnergy metabolism in carcinogenesis is poorly understood. It is widely accepted the majority of colorectal cancers (CRCs) arise from adenomatous polyps (APs). We aimed to characterize the bioenergetic alterations in APs and CRCs.MethodsFifty-six APs, 93 CRCs and adjacent normal mucosae were tested. Oxygen consumption rate (OCR) was measured representing mitochondrial oxidative phosphorylation (OxPhos), and extracellular acidification rate (ECAR)was measured representing glycolysis. Mitochondrial DNA (mtDNA) variants and mutations were studied. Over-expressed metabolic genes in APs were identified by microarray and validated by qRT-PCR, Western blots and immunohistochemistry. Identified genes were knocked down in WiDr and colo205 CRC cell lines, and their expression was analyzed in APs/CRCs with enhanced glycolysis.FindingsECAR, not OCR, was significantly increased in APs. While no difference of ECAR was found between CRCs and normal mucosae, OCR was significantly reduced in CRCs. OCR/ECAR ratio was decreased in APs over 1 cm, APs with a villous component and CRCs, indicating their glycolytic tendencies. The number of mtDNA mutations was increased in APs and CRCs, but not correlated with metabolic profiles. Two metabolic genes ALDOB and SLC16A4 were up-regulated in APs. Both ALDOB-knockdown and SLC16A4-knockdown CRC cell lines showed increased basal motichondrial OxPhos and decreased basal glycolysis. Moreover, the increase of mitochondrial ATP-linked respiration and the decrease of glycolytic capacity were showed in SLC16A4-knockdown cells. Finally, APs/CRCs with enhanced glycolysis had increased SLC16A4 expression.InterpretationATP production shifts from OxPhos to glycolysis in the process of AP enlargement and villous transformation. OxPhos defects are present in CRCs but not in APs. APs and CRCs tend to accumulate mtDNA mutations, but these are not correlated with bioenergetic profiles. Finally, the ALDOB and SLC16A4 may contribute to the glycolytic shift in APs/CRCs.

Project description:BackgroundLong interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed.MethodsFormalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples.ResultsLINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC.ConclusionsLINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.

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LINE-1 is preferentially hypomethylated within adenomatous polyps in the presence of synchronous colorectal cancer.

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LINE-1 is preferentially hypomethylated within adenomatous polyps in the presence of synchronous colorectal cancer.

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