Original contributionH3K4 dimethylation in hepatocellular carcinoma is rare compared with other hepatobiliary and gastrointestinal carcinomas and correlates with expression of the methylase Ash2 and the demethylase LSD1
Introduction
Epigenetic changes have been recognized to play an important role in the development and progression of cancer [1], [2], [3]. In addition to DNA methylation, posttranslational modifications of core histones, including methylation and acetylation, have critical roles in the organization of chromatin structure and the regulation of transcription [4]. Enzymes that modify histone H3K4 show altered activity in cancer [5], [6]. The consequence of altered activity of histone modifying enzymes can be increased or decreased gene expression [7], [8]. It has been shown that dimethylation of histone H3 at lysine 4 (H3K4diMe) in combination with trimethylation of histone H3 at lysine 4 (H3K4triMe) is associated with an activated transcriptional state [9]. An increase of H3K4diMe in neoplastic tissues is predictive of clinical outcome [10], [11]. In particular, tumor recurrence occurs earlier in low-grade prostate carcinoma patients with low H3K4diMe, independently of other clinical and pathologic parameters [11]. Similarly, in large cell and squamous cell carcinomas, low H3K4diMe expression correlates with short survival [10]. In other carcinomas, however, H3K4diMe has not been investigated comprehensively. Here, we compared H3K4diMe expression in gastrointestinal and hepatobiliary carcinomas. Interestingly, H3K4diMe was frequently and highly expressed in pancreatic ductal adenocarcinomas (PDACs) and stomach and neuroendocrine carcinomas, whereas it was less abundant in hepatocellular carcinomas (HCCs).
One explanation of the differential expression in carcinomas of different origin could be a difference in specific histone modification determined by local levels and activities of opposing enzyme pairs such as histone methylases and demethylases. Therefore, we assessed whether H3K4diMe correlated with demethylating and methylating enzymatic complexes in HCC and/or clinicopathologic parameters.
Ash2 (absent, small or homeotic discs2) is a trithorax protein that as Ash2 complex trimethylates H3K4 and has been implicated as an oncoprotein [12]. The resulting H3K4triMe is a histone mark that is closely associated with transcribed genes [13], [14], [15]. However, histone methylation can be reversed by histone demethylases. The lysine-specific histone demethylase 1, a nuclear amine oxidase homolog, is of special interest, as it demethylates H3K4diMe [16] and, thus, could contribute to the overall methylation state of H3K4 in carcinomas. Demethylation of this mark is associated with repression of CoREST target genes [9]. LSD1 is a coactivator of the androgen receptor and can, depending on the nature of the interacting transcriptional regulator, either activate or repress gene transcription [17]. In prostate carcinomas, LSD1 represents a negative prognostic marker [18]. Therefore, we compared levels of H3K4diMe in hepatobiliary and gastrointestinal tract carcinomas and, subsequently, with expression levels of the methylase Ash2 and demethylase LSD1.
Section snippets
Selection of tumor specimens and tissue microarrays
Tissue microarrays (TMAs) were prepared from formalin-fixed, paraffin-embedded primary resection specimens (n = 193), including HCC (n = 51), intrahepatic cholangiocarcinomas, and extrahepatic adenocarcinomas of the biliary tract (CCCs) (n = 29), pancreatic adenocarcinomas (n = 69), gastric adenocarcinomas (n = 28), and primary intestinal neuroendocrine carcinomas (n = 16). The cases were retrieved from the archives of the Institute of Pathology, University of Bonn, from 1997 to 2007. Clinical
Results
The staining patterns of H3K4diMe, Ash2, and LSD1 in carcinomas were assessed using tissue microarrays and confirmed using representative standard paraffin sections.
H3K4diMe, LSD1, and Ash2 were expressed in a spectrum of carcinomas of the hepatobiliary and gastrointestinal tract as summarized in Table 2. Frequency of expression ranged from 0% to 100% of tumor nuclei analyzed. Interestingly, high SI correlated with high frequency of positively staining tumor cells (P < .001).
Examples of
Discussion
Recent studies demonstrate that chromatin regulation by posttranslational means, for example, the methylation and acetylation of core histones, is an essential control mechanism in tumorigenesis and tumor progression [27]. Clinical importance comes from the observation that patients with carcinomas that show dimethylation of histone mark H3K4 have a lower risk of recurrent disease [11]. In the present study, we examined the H3K4 methylation status in a panel of carcinomas of the
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