DNA Methylation and Hydroxymethylation in Primary Colon Cancer and Synchronous Hepatic Metastasis.

Silvia Udali,Greta Beschin,Stephanie A Tammen,Simonetta Friso,Alfredo Guglielmi,Andrea Ruzzenente,Tommaso Campagnaro,Oliviero Olivieri,Sang-Woon Choi,Domenica De Santis,Filippo Mazzi,Sara Moruzzi,Patrizia Pattini

doi:10.3389/fgene.2017.00229

Silvia Udali, Greta Beschin + Show 11 more

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https://doi.org/10.3389/fgene.2017.00229

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Abstract

Colon cancer is one of the most frequent solid tumor and simultaneous diagnosis of primary colon cancer and liver metastases occurs in about one fourth of cases. The current knowledge on epigenetic signatures, especially those related to hydroxymethylation in primary cancer tissue, synchronous metastasis, and blood circulating cells is lacking. This study aimed to investigate both methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) status in the DNA of individual patients from colon cancer tissue, synchronous liver metastases, and in cancer-free colon and liver tissues and leukocytes. Patients undergoing curative surgery (n = 16) were enrolled and their laboratory and clinical history data collected. The contents of mCyt and hmCyt were determined by a liquid chromatography/mass spectrometry (LC/MS/MS) method in DNA extracted from primary colon cancer, synchronous hepatic metastatic tissues and homologous cancer-free tissues, i.e., colon and liver tissues as well as leukocytes. The mCyt and hmCyt levels were compared between cancerous and cancer-free tissues, and correlations between leukocytes and colon/liver tissues for both the mCyt and hmCyt levels were evaluated. The mCyt levels were similar in primary colon cancer and liver metastasis tissues (4.69 ± 0.37% vs. 4.77 ± 0.38%, respectively, p = 0.535), and both primary and metastatic tissues were hypomethylated compared to cancer-free colon (4.98 ± 0.26%). The difference in the mCyt content between cancerous and cancer-free colon tissues was significantly lower in primary colon cancer (p = 0.004), but not in liver metastasis (p = 0.148). The hmCyt content was similar in primary colon cancer compared to liver metastasis (0.035%, C.I. 0.024–0.052% versus 0.035%, C.I. 0.021–0.058%, respectively, p = 0.905) and markedly depleted compared to the cancer-free colon (0.081%, C.I. 0.055–0.119%) with a statistically significant difference (p < 0.05) for both comparisons. The mCyt levels showed a borderline correlation between leukocytes and colon cancer tissue (Pearson’s correlation coefficient = 0.51, p = 0.052) while no correlations were detected for the hmCyt levels. In conclusion, primary colon cancer and synchronous liver metastasis tissues showed a similar epigenetic status but were significantly hypomethylated and hypohydroxymethylated as compared to homologous cancer-free colon tissues.

Highlights

Colorectal cancer is the third most frequently diagnosed cancer in males and the second in females (Jemal et al, 2011) and liver tissue is the most common site of colorectal cancer metastasis, observed in up to one fourth of patients at the time of initial diagnosis (Silberhumer et al, 2016).Various studies on the pathogenesis of colorectal cancer and cancer development suggest that, in addition to genetic modifications, there is an important role for epigenetic markers that occur early and manifest frequently in this type of cancer (Okugawa et al, 2015)
The aim of the present study was to quantify mCyt and hmCyt levels by an LC–MS/MS method in patients affected by metastatic colon cancer, by evaluating primary colon cancer tissue, synchronous hepatic metastasis tissues and in homologous cancer-free tissues, i.e., colon, liver and leukocyte DNA, the latter of which to determine whether the status of these epigenetic markers in leukocyte DNA could be a putative biomarker for cancer in specific tissues
Despite the increasing interest in this field, many aspects remain to be clarified since there are little data on the pattern of DNA methylation and DNA hydroxymethylation at a tissue level in primary cancer and metastatic tissues compared to non-affected tissues of the same organ affected by neoplasia together with leukocyte DNA data from the same cancer affected patients

Summary

Introduction

Colorectal cancer is the third most frequently diagnosed cancer in males and the second in females (Jemal et al, 2011) and liver tissue is the most common site of colorectal cancer metastasis, observed in up to one fourth of patients at the time of initial diagnosis (Silberhumer et al, 2016).Various studies on the pathogenesis of colorectal cancer and cancer development suggest that, in addition to genetic modifications, there is an important role for epigenetic markers that occur early and manifest frequently in this type of cancer (Okugawa et al, 2015). Epigenetics refers to the complex of heritable and potentially reversible mechanisms that regulate gene expression without alterations in the DNA sequence, including DNA methylation, histone modifications and microRNAs (Wolffe and Matzke, 1999; Bird, 2007; Carthew and Sontheimer, 2009), all of which are frequently studied phenomena in cancer (Feinberg and Tycko, 2004; Ehrlich, 2006). One of the most extensively evaluated epigenetic phenomenon in cancer is DNA methylation, consisting of the transfer of a methyl group (-CH3) to the 5 position of a cytosine at CpG dinucleotide residues. This process can modulate gene expression by modifying the accessibility of DNA to transcriptional machinery (Jones and Takai, 2001; Heyn and Esteller, 2012). Global DNA hypomethylation is thought to play an important role in carcinogenesis by inducing chromosomal instability and the global loss of imprinting (Gaudet et al, 2003; Holm et al, 2005; Wilson et al, 2007; Ehrlich and Lacey, 2013)

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