Elsevier

Human Pathology

Volume 41, Issue 2, February 2010, Pages 181-189
Human Pathology

Original contribution
H3K4 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

https://doi.org/10.1016/j.humpath.2009.08.007Get rights and content

Summary

Methylation of core histones regulates chromatin structure and gene expression. Recent studies have demonstrated that these methylation patterns have prognostic value for some tumors. Therefore, we investigated dimethylation of histone H3 at lysine 4 (H3K4diMe) and H3K4 methylating (Ash2 complex) and demethylating enzymes (LSD1) in carcinomas of the hepatic and gastrointestinal tract. High levels of H3K4diMe were rarely observed in 15.7% of hepatocellular carcinoma (8/51) unlike other carcinomas including, in ascending order, cholangiocellular carcinoma/adenocarcinoma of the extrahepatic biliary tract, gastric carcinoma, pancreatic ductal adenocarcinoma, and neuroendocrine carcinoma (P < .001). Ash2 was expressed in 84.4% of hepatocellular carcinomas (38/45) and correlated directly with H3K4diMe modification (correlation coefficient r = 0.53) and LSD1 expression (r = 0.35). In contrast to other carcinomas, 65.9% (29/44) of hepatocellular carcinomas analyzed showed no LSD1 expression (P < .001). Interestingly, hepatocellular carcinomas without LSD1 expression appeared to be frequently Ash2 and H3K4diMe weak or negative (P = .004). In summary, high H3K4diMe expression is rare in hepatocellular carcinoma compared with other carcinomas (negative predictive value 92.3%), which may aid in the differential diagnosis. Lack of H3K4diMe is possibly due to complex epigenetic regulation involving Ash2 and 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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