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  • br Conclusions br Acknowledgements br

    2020-02-24


    Conclusions
    Acknowledgements
    Introduction Liver cancer was the fifth most common cancer and the second leading cause of cancer-related death worldwide [1]. Most of primary liver cancers (70–90%) occurring worldwide are hepatocellular carcinoma (HCC) [2]. Persistent hepatitis B virus (HBV) infection is one of the major risk factors for HCC development [3]. It has been widely recognized that HBV DNA integration into the genome of hepatocytes is one of the major causes of hepatocarcinogenesis [4], [5]. We previously reported that diacylglycerol kinase (DGK) γ was one of the genes recurrently targeted by HBV integration in HCC [6]. However, DGKγ expression and its role in HCC remain unclear. DGKs are a family of enzymes which phosphorylate diacylglycerol (DAG) and convert it to phosphatidic CC-10004 (PA) [7]. DAG is an established activator of the conventional and novel protein kinase Cs, Ras guanyl nucleotide-releasing protein, Unc-13 and chimaerin [8], [9]. PA has also been reported to control a variety of important signaling proteins, such as phosphatidylinositol-4-phosphate 5-kinase, son of sevenless, Ras GTPase-activating protein, C-Raf and atypical protein kinase C [10]. Therefore, DGKs play a pivotal role in a variety of intracellular signaling by modulating the balance between two bioactive lipids, DAG and PA. To date, a total of ten mammalian DGK isozymes (α–κ) have been identified [11]. These isozymes share two or three characteristic zinc finger-like C1 domains and the catalytic region of the enzyme in common. DGK are divided into five groups according to their structural features. DGKγ belongs to the type I DGKs, containing two sets of Ca2+-binding EF-hand motifs at their N-termini [12]. DGKγ also functions as an upstream suppressor of Rac1 through its catalytic action [13], [14]. It has been reported that DGKγ is epigenetically silenced in colorectal cancer, suggesting that DGKγ may act as a tumor suppressor [15]. However, the function of DGKγ in cancer biology is still not well understood. Cancer cells exhibit profound alterations in their metabolism, mainly characterized by two major changes: Warburg effect, or aerobic glycolysis, and an increased dependence on glutamine [16], [17], [18]. As key metabolic substrates in cancer cells, both glucose and glutamine are critical for cancer development, invasion, and metastases [19], [20], [21]. Previous studies have demonstrated that elevated expression of glucose transporters has been observed in most cancers [20]. Moreover, increased glucose transporter 1 (GLUT1) expression levels in HCC cells functionally promote tumorigenicity [23].
    Materials and methods
    Results
    Discussion Based on our results, we found that DGKγ functioned differently in SNU449 and SK-Hep1 cell lines. DGKγ inhibited cell migration in both SNU449 and SK-Hep1 cell lines, while DGKγ inhibited cell proliferation significantly in SNU449 but marginally in SK-Hep1. The different origin of the two cell lines may attribute to the difference. Because SNU449 in an HCC cell line while SK-Hep1 is derived from liver adenocarcinoma. Furthermore, we found that kinase-dead DGKγ inhibited cell migration but not cell proliferation of HCC cells, indicating that DGKγ inhibited cell migration independent of its kinase activity, and DGKγ inhibited cell migration and proliferation via different pathways. Rac1 is a master regulator of the cytoskeleton and cell motility [33]. It was previously reported that DGKγ acts as an upstream suppressor for Rac1 by activating β2-chimaerin, a Rac-specific GAP, dependent on its kinase activity [13], [14]. In this study, the results showed that DGKγ inhibited cell migration independent of Rac1. DGKγ may suppress cell migration by inhibiting EMT of HCC cells but the mechanism remains unclear. DGKγ silence induced by promoter hypermethylation has been reported in colorectal cancer [15]. To address the underlying mechanism contributed to the aberrant DGKγ down-regulation seen in HCC tumor tissues, we first investigated the methylation status of DGKγ gene promoter in HCC tissues and cell lines. Unexpectedly, no hypermethylation in DGKγ gene promoter and no changes of DGKγ mRNA expression levels after DNA methyltransferase inhibitor treatment were detected in HCC cell lines in the current study. Accordingly, promoter methylation is not associated with DGKγ inactivation in HCC. On the contrary, the down-regulation of DGKγ could be reversed by HDAC inhibitors treatment and meanwhile the acetylation levels of histone H3 and H4 of DGKγ gene promoter were elevated. These results revealed that instead of promoter hypermethylation, histone deacetylation is the main cause of DGKγ aberrantly low expression in HCC. Moreover, it has been reported that HDAC1–5 were upregulated in HCC [34]. Indeed, our results show that respective inhibitors of HDAC1 and 2, HDAC1 and 3 as well as HDAC4 and 5, all of them could upregulate DGKγ mRNA level, indicating that HDAC1–5 may all participated in regulation of DGKγ expression. Furthermore, we found that patients with HBV infection (HBs-Ag positive) exhibited a higher ratio of low DGKγ expression by analyzing the correlation between DGKγ expression and the clinicopathological features in HCC patients. Yoo et al. have reported that Hepatitis B virus X protein (HBx) induces the expression of HDAC1 in HCC [35], suggesting HBx may inhibited DGKγ expression by HDAC1.