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Histone deacetylase 3 promotes liver regeneration and liver cancer cells proliferation through signal transducer and activator of transcription 3 signaling pathway

Histone deacetylase 3 promotes liver regeneration and liver cancer cells proliferation through... Histone deacetylase 3 (HDAC3) plays pivotal roles in cell cycle regulation and is often aberrantly expressed in various cancers including hepatocellular carcinoma (HCC), but little is known about its role in liver regeneration and liver cancer cells proliferation. Using an inducible hepatocyte-selective HDAC3 knockout mouse, we find that lack of HDAC3 dramatically impaired liver regeneration and blocked hepatocyte proliferation in the G1 phase entry. HDAC3 inactivation robustly disrupted the signal transducer and activator of transcription 3 (STAT3) cascade. HDAC3 silencing impaired the ac-STAT3-to-p-STAT3 transition in the cytoplasm, leading to the subsequent breakdown of STAT3 signaling. Furthermore, overexpressed HDAC3 was further associated with increased tumor growth and a poor prognosis in HCC patients. Inhibition of HDAC3 expression reduced liver cancer cells growth and inhibited xenograft tumor growth. Our results suggest that HDAC3 is an important regulator of STAT3-dependent cell proliferation in liver regeneration and cancer. These findings provide novel insights into the HDAC3–STAT3 pathway in liver pathophysiological processes. Introduction chemical injury . In humans, liver regeneration proceeds The mammalian liver has an enormous regenerative within weeks of a major liver resection, whereas in rodents, ability to restore its original mass and function via com- liver recovery can occur within 10 days of PH . Aberrant pensatory hyperplasia in response to diverse injuries . liver regeneration is closely related to the pathogenesis of Quiescent maturity hepatocytes in the G0 phase of the cell liver failure or hepatocellular carcinoma (HCC) . A better cycle can re-enter into the cell cycle rapidly to produce new understanding of the basic mechanisms that regulate liver hepatocytes following partial hepatectomy (PH) or regeneration will have important implications for potential therapies for human liver disease. Histone deacetylase 3 (HDAC3), a member of the highly Correspondence: Yu-Jun Shi (shiyujun@scu.edu.cn) conserved Class I HDAC enzymes, is ubiquitously expressed Laboratory of Pathology, Key Laboratory of Transplant Engineering and 4 in eukaryotic cells . HDAC3 regulates gene transcription by Immunology, NHFPC; West China Hospital, Sichuan University, Chengdu catalyzing the deacetylation of core histones and is involved 610041, China 2 5 Department of Pathology, West China Hospital, Sichuan University, Chengdu in various biological processes in the liver . Inhibition of 610041, China HDAC3 and other HDACs specifically interferes with liver Full list of author information is available at the end of the article These authors contributed equally: Xu-Feng Lu, Xiao-Yue Cao. Edited by Z.-X. Xiao. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Lu et al. Cell Death and Disease (2018) 9:398 Page 2 of 14 development in zebrafish . In mammals, hepatic-specific and notable damage to the DNA are evident at 2 months ablation of HDAC3 dramaticallydisruptsliver function and of age (Supplementary Fig. 1). Therefore, to eliminate causes severe dysregulation of lipid and glucose metabo- post-natal developmental defects upon HDAC3 deletion, 7,8 lism .Conversely, high HDAC3 expression in the liver was we generated an inducible tamoxifen-dependent hepato- ERT2 found to contribute to high-fat-diet-induced metabolic cyte-selective HDAC3 knockout mouse (Alb-Cre ; 9 loxP/loxP syndrome . HDAC3 is also essential for the maintenance of HDAC3 mouse), in which HDAC3 in the liver chromatin structure and genomic stability, and hepatic loss tissues was over 80% ablated after 5 days of tamoxifen of HDAC3 in mice results in an accumulation of DNA treatment (hereafter referred to as the HDAC3 mouse) 10,11 damage and the early onset of spontaneous liver cancer . (Supplementary Fig. 2). Five days after the last injection of Still, the role of HDAC3 in HCC development remains tamoxifen, the inducible HDAC3 mouse did not show unclear because the mRNA and protein levels of HDAC3 obviously impaired liver function or disrupted DNA sta- 11,12 can either increase or decrease in human HCC tissues . bility (Supplementary Fig. 2). Next, we performed two- Furthermore, HDAC3 is a considered biomarker of tumor thirds PH of the livers in male HDAC3 mice and their ERT2 loxP/loxP recurrence following liver transplantation in hepatitis B control littermates (Alb-Cre ;HDAC3 mice virus-associated HCC . HDAC3 also participates in the self- treated with corn oil instead of tamoxifen) to explore the renewal process of liver cancer stem cells through histone specific role of HDAC3 during liver regeneration. Sur- modifications , making HDAC3 suppression a potential prisingly, the post-hepatectomy mutant mice displayed a clinical-therapeutic approach for HCC. severely impaired liver mass reconstitution, which was Multiple in vitro studies have revealed that HDAC3 calculated using the liver weight/body weight ratio plays diverse roles in the regulation of cell cycle pro- (Fig. 1a). Furthermore, HDAC3 silencing interfered with gression. HDAC3 is required to generate a hypoacetylated liver recovery as revealed by the alanine transaminase and H3 tail as the preferred template for the phosphorylation aspartate transaminase serum levels (Fig. 1b). The bro- of H3S10 by Aurora B, which is a crucial step for pro- modeoxyuridine (BrdU) incorporation and Ki67 immu- gression through the G2/M phase and mitosis . HDAC3 nohistochemistry showed that the proliferation peak in is also required for H3K4 deacetylation at the centromere the control littermates appeared between 36 and 48 h 16,17 and sister chromatin cohesion . HDAC3 inhibition post-hepatectomy; strikingly, HDAC3 disruption reduced WAF1/cip1 induces G1 phase arrest by increasing p21 the proliferation rates by approximately 60% during this expression in Hep3B liver cancer cells . HDAC3 deletion time (Fig. 1c, d, f). Phospho-histone H3S10 (p-H3S10), a in mouse embryonic fibroblasts induces S-phase arrest mitotic marker that is specifically expressed during the through DNA damage accumulation, whereas inactivation G2/M phase , showed a corresponding decrease that was of HDAC3 affects replication fork progression in hema- over three-fold in the HDAC3 mice at 48 h post- 10,19 topoietic progenitor cells during the S phase . HDAC3 hepatectomy (Fig. 1e, f). The livers of the control mice also controls G2/M progression in adult neural stem/ were completely reconstituted within 168 h, and mitotic progenitor cells by regulating the CDK1 levels .In hepatocytes were undetectable by 168 h; however, fre- addition, HDAC3 regulates cell cycle progression by quent proliferation was observed in the HDAC3 mice modulating cyclin A acetylation to control cyclin A until 14 days after the PH (Supplementary Fig. 3a). level . The data regarding the role and molecular Similarly, when the mice were challenged with a carbon mechanism of HDAC3 in regulating cell proliferation are tetrachloride (CCl ) injection, the absence of HDAC3 did highly multiple and contradictory. However, the biological not increase sensitivity to the toxin but robustly delayed function of HDAC3 in liver regeneration and liver cancer liver repair (Supplementary Fig. 4). cells proliferation remains unknown. We next examined potential differences in the In this study, we determined that HDAC3 promotes liver expression levels of key cell cycle markers between the regeneration and liver cancer cells proliferation through HDAC3 and control mice during liver regeneration. the signal transducer and activator of transcription 3 Ki67 is expressed from mid-G1 to the end of mitosis , (STAT3) signaling pathway. The role of HDAC3 in the and the low Ki67 staining indicates that the proliferating regulation of cell proliferation through the STAT3 pathway hepatocytes might be arrested in the early G1 phase. provides a potential drug target for the treatment of HCC. Consistently, the levels of cyclin D1 and CDK4, which arethe keymarkers of theearly G1 phase , peaked at 24 Results h after PH in the control livers; however, the cyclin D1 HDAC3 deletion severely impairs liver regeneration by and CDK4 levels were dramatically suppressed in the delaying the G1 phase entry HDAC3 livers (Fig. 1g; Supplementary Fig. 3b). The 7,10 As described previously , in the Alb-Cre; levels of CDK2 and cyclin E1, which function at the G1/S loxP/loxP 22 HDAC3 mouse, where HDAC3 is constitutively transition , peaked at 36 h post-hepatectomy, but the inactivated, dramatic dysregulation of hepatic metabolism levels were correspondingly lower in the mutant livers Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 3 of 14 Fig. 1 Impaired liver regeneration in HDAC3 mice following 70% PH. a The liver weight/body weight ratio was calculated at different time points after PH, and liver reconstitution was notably delayed in the HDAC3 livers. b The serum AST and ALT levels after PH. c–f Immunohistochemistry for BrdU, Ki67, and p-Histone H3 (Ser10) shows that the proliferation rates are reduced in the HDAC3 mice after PH (scale bar: 50 μm). g Western blot analysis of cell cycle proteins at different times after PH shows that mitotic hepatocytes are delayed at the G1 phase entrance in the HDAC3 mice. The levels of the indicated proteins are expressed relative to GAPDH. All data represent the mean ± SD; n = 3–8; *p < 0.05; **p < 0.01; ***p < 0.001 (Fig. 1g; Supplementary Fig. 3b). Notably, the protein deficient hepatocytes were robustly arrested in the G1 levels of cyclin A2, an essential marker for S-phase phase entry. progression ,weresignificantly reduced in the HDAC3 livers at 48 h after PH (Fig. 1g; Supplementary HDAC3 inactivation impairs STAT3 signaling Fig. 3b). Additionally, the levels of CDK1, cyclin B1, and Because the rapid transitions of the cell cycle activate p-H3S10, which appear in the G2/M phase ,werealso multiple signaling cascades and numerous genes during △ 1 markedly decreased in the HDAC3 livers during the liver regeneration , we performed a microarray analysis to peak period of mitosis (Fig. 1g; Supplementary Fig. 3b). screen the molecular defects in the HDAC3 liver. The Taken together, our findings suggested that HDAC3- inducible short-term absence of HDAC3 did not Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 4 of 14 Fig. 2 (See legend on next page.) Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 5 of 14 (see figure on previous page) Fig. 2 The gene expression profile demonstrates the broad and multiple effects of HDAC3 on gene expression after PH. a A heat map of 1557 differentially expressed genes (fold changes ≥1.5 and p < 0.05) in the control and mutant livers at 36 h after PH compared with their corresponding baseline values (0 h). b mRNA microarray analysis of cell cycle-related genes. Differentially expressed genes (fold changes ≥1.25 and p < 0.05) in the control and HDAC3 livers at 36 h after PH. c GO term analysis of the list of differentially expressed genes (fold changes ≥1.5 and p < 0.05) indicated that the IL6/STAT3 pathway is dramatically changed during liver regeneration in the HDAC3 mice after PH. d Heat map of the selected genes from the microarray analysis of the control and HDAC3 livers. e Quantitative RT-PCR confirmation of the downregulated early- response genes in the HDAC3 livers vs. the control liver at 36 h after PH. f qRT-PCR confirms downregulation of cyclin D1 and c-myc in the HDAC3 livers vs. the control liver at 36 h after PH. All data represent the mean ± SD; n = 3; **p < 0.01; ***p < 0.001; n.s. not significant obviously alter the gene expression profiles of the quies- phase post-hepatectomy (Fig. 3b; Supplementary Fig. 6a). cent hepatocytes (Supplementary Fig. 5a–c); however, the Intriguingly, the level of p-STAT3(S727), which is phos- gene profiles of the mutant liver were dramatically phorylated by the c-src non-receptor tyrosine kinase , changed 36 h after the PH (Fig. 2a). Among these genes, was not obviously impaired in the HDAC3-deficient livers 131 cell cycle-related genes were identified, and cyclin D1, (Fig. 3b), indicating that HDAC3 ablation might selec- cyclin A2, cyclin B1, and CDK1 were significantly down- tively target p-STAT3(Y705). To further determine whe- regulated in the HDAC3 liver (Fig. 2b; Supplementary ther the low level of p-STAT3(Y705) was caused by the Fig. 5d), which was consistent with the proliferation HDAC3 deletion, we examined p-STAT3(Y705) expres- defects observed in the mutant liver. sion in HDAC3-deficient mice by simultaneously chal- A Gene Ontology (GO) term analysis of the differen- lenging the mice with PH and intraperitoneal injections of tially regulated genes showed that the interleukin 6 (IL6)/ lipopolysaccharide (LPS) (Supplementary Fig. 6b, c). IL6 is STAT3 signaling pathway, which regulates approximately extraordinarily increased by LPS stimulation , whereas 36% of the immediate-early genes during the acute the levels of p-STAT3(Y705) and c-myc remained notably 1 △ hepatic response , was extremely disturbed (Fig. 2c, d). As depressed in the HDAC3 livers, regardless of the expected, the immediate early-response genes, including upregulation of p-JAK2 (Fig. 3c; Supplementary Fig. 6c). c-fos, c-jun, Egr2/3, Gadd45a, junb, and Mdm2 , were Upon ligand stimulation, acetylation of STAT3 (ac- significantly downregulated in the HDAC3 livers STAT3) is critical for enhancing the transcription of cell 27,28 (Fig. 2e, Supplementary Fig. 5d). Additionally, c-myc and growth-related genes . Co-activator p300, which is a cyclin D1, which are components of the STAT3 path- histone acetyltransferase that primarily functions in the 24 29,30 way , were among the most reduced genes, and this nucleus, is required for STAT3 acetylation .Our observation was further confirmed by qRT-PCR (Fig. 2f). immunoprecipitation assay showed a strong interaction Collectively, our microarray data supported the idea that between p300 and STAT3 in both the control and △ △ breakdown of STAT3 signaling in the HDAC3 mice HDAC3 livers (Fig. 3d). As STAT3 activity is modified by might account for the G1 phase arrest during liver phosphorylation and acetylation at post-translational regeneration. level , we next examined whether HDAC3 deficiency results in impaired STAT3 activation. In the mutant liver, HDAC3 silencing impairs the STAT3 transition from the protein that was co-immunoprecipitated by the STAT3 acetylation to phosphorylation antibody overwhelmingly consisted of ac-STAT3 with STAT3 mediates the expression of various genes in extremely low p-STAT3(Y705), while the control liver response to diverse stimuli and thus plays a key role in showed the opposite dynamic (Fig. 3e). The absence of p- multiple cellular processes such as cell growth, pro- STAT3(Y705) in the nucleus prompted us to investigate liferation, and apoptosis . When IL6 combines with its whether HDAC3 deprivation impaired the STAT3 transi- membrane receptor glycoprotein 130 (gp130), STAT3 tion from acetylation to phosphorylation during liver becomes phosphorylated at tyrosine (Y705) by regeneration. The cytoplasmic and nuclear fractions were receptor-associated Janus kinases (JAKs) . Compared prepared from the livers after PH at indicated time points, with the control littermates, in which a large number of p- and the immunoblotting analysis showed that during the STAT3(Y705) nuclear-positive hepatocytes were observed early phase of liver regeneration, p-STAT3(Y705) was at 3 h after PH, nuclear p-STAT3(Y705) was abolished in notably increased both in the cytoplasm and nucleus of the HDAC3-deficient mice (Fig. 3a–c; Supplementary Fig. 6a). control hepatocytes whereas ac-STAT3 was maintained at Additionally, the level of c-myc, which is a major down- a low level in the cytoplasm (Fig. 3f). Conversely, HDAC3- stream gene of STAT3 signaling and an important tran- deficient hepatocytes showed substantial hyperacetylation scription factor for successful initiation of cell mitosis , of STAT3 but notably reduced p-STAT3(Y705) in both the was also reduced in HDAC3-deficient mice at the early cytoplasm and the nucleus (Fig. 3f, g). Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 6 of 14 Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 7 of 14 (see figure on previous page) Fig. 3 The HDAC3 deficiency inhibited STAT3(Y705) phosphorylation and enhanced STAT3 acetylation in hepatocytes. a Immunofluorescence demonstrates that p-STAT3(Y705) is not activated in the HDAC3 hepatocytes (scale bar: 50 μm). b Western blot analysis shows that p-STAT3(Y705) and c-myc are reduced in the HDAC3 mice during the early phase of liver regeneration. GAPDH was used as the loading control. c Western blot analysis shows that p-STAT3 remains inhibited in the mutant hepatocytes after the LPS/PH treatment. d Co- immunoprecipitation shows that STAT3 is associated with p300 in both the control and HDAC3 liver. e Immunoprecipitation confirms that the protein complex overwhelmingly consists of ac-STAT3 with extremely low p-STAT3(Y705) in the HDAC3 liver. f Western blot analysis shows that the HDAC3 deficiency leads to prolonged acetylation of STAT3 in the cytoplasm during liver regeneration. β-Actin and histone H3 were used as the loading controls. g Immunofluorescence demonstrates that ac-STAT3 significantly accumulates in the cytoplasm of the HDAC3 hepatocytes (scale bar: 50 μm). n = 3–5; ***p < 0.001 HDAC3 deletion impairs cell growth by inhibiting nuclear of ac-STAT3 in vivo remain undefined. We previously translocation of STAT3 reported that loss of HDAC1 and/or HDAC2 did not To determine whether the HDAC3 deficiency inhibited disturb cell cycle progression before the M phase , which the nuclear translocation of STAT3, we cultured fluor- highlighted the negative effects of HDAC1 and HDAC2 escently labeled primary diploid hepatocytes that were on the IL6/STAT3 signaling cascade. Consistently, dele- isolated from the (Gt(ROSA)26Sortm4(ACTB-tdTomato- tions of HDAC1, HDAC2, or both did not increase the ,EGFP) mice (hereafter referred to as mT/mG mice) and level of ac-STAT3 compared to that of the wild-type HDAC3 ;mT/mG mice, respectively (Supplementary (WT) liver during the early phase of liver regeneration Fig. 7). Cell viability assays revealed that compared to the (Fig. 5a, b). Moreover, loss of HDAC8, another member of diploid hepatocytes that were isolated from the mT/mG the Class I HDACs, did not have a noticeable effect on the mice, cell number of the HDAC3 mutant hepatocytes was progression of liver regeneration (Supplementary Fig. 9). significantly decreased (Fig. 4a, b). Consistently, HDAC3 Therefore, we did not further analyze the role of HDAC8 knockdown in human liver cancer cells HepG2 using in STAT3 signaling during liver regeneration. siRNA significantly increased ac-STAT3 expression level It has been suggested that ac-STAT3 is likely to be 28,32 and remarkably inhibited cell growth (Fig. 4c; Supple- deacetylated by Class I HDACs in the nucleus . To test mentary Fig. 8a–c). this, we performed immunoprecipitations to examine the Notably, the mutant hepatocytes displayed robustly associations between STAT3 and HDAC1, HDAC2, and decreased p-STAT3(Y705) with significantly increased HDAC3 in the nucleus. All three Class I HDAC members cytoplasmic ac-STAT3; additionally, IL6 stimulation interacted with ac-STAT3 (Fig. 5c); however, compared to failed to upregulate p-STAT3(Y705) in the HDAC3- the WT liver, single disruptions to each HDAC or a deficient cells (Fig. 4d). Because STAT3 localizes to the combined disruption of HDAC1 and HDAC2 did not nucleus after it is phosphorylated , we tested whether ac- noticeably increase the nuclear level of ac-STAT3 fol- STAT3 prevented the nuclear translocation of STAT3. lowing PH (Fig. 5d). This observation strongly suggested Indeed, the immunofluorescence analysis showed that the that HDACs do not catalyze the deacetylation of ac- nuclear localization of total STAT3 was significantly STAT3 in the nucleus. This observation was also vali- suppressed in the HDAC3-deficient hepatocytes after IL6 dated by the finding that high levels of ac-STAT3 were treatment (Fig. 4e). Moreover, p-STAT3(Y705) was evident in the cytoplasm of hepatocytes from each gen- noticeably localized to the nuclei of the control hepato- otypic mouse, though the ac-STAT3 levels were much cytes, whereas p-STAT3(Y705) remained absent from the higher in the HDAC3-deficient livers than in the HDAC1- HDAC3-deficient hepatocyte nuclei, even with and/or HDAC2-deficient livers (Figs. 3g and 5d). IL6 stimulation (Fig. 4f). Meanwhile, the phosphorylation The cytoplasmic but not nuclear accumulation of ac- of STAT3(Y705) after IL6 stimulation was also prevented STAT3 in the HDAC3-deficient liver strongly indicated in response to HDAC3 knockdown (Supplementary that ac-STAT3 is primarily deacetylated in the cyto- Fig. 8b–d), which indicated that loss of HDAC3 remark- plasm rather than in the nucleus, and HDAC3 might ably disrupted the transduction of STAT3 signaling from function a powerful role in the deacetylation of ac- the cytoplasm to the nucleus. STAT3. Although HDAC3 is a nuclear protein, HDAC3 contains a nuclear export signal in its C terminus HDAC3 is essential for deacetylation of ac-STAT3 in the (residues 180–313) which facilitates the nucleoplasm 33,34 cytoplasm transport of HDAC3 .Weconfirmed the cyto- Class I HDACs have been suggested to contribute to plasmic distribution of HDAC3 by immunoblotting, STAT3 deacetylation in 293T cells , but the individual andincontrasttoHDAC3,HDAC1 andHDAC2 were roles of the Class I HDAC members in the deacetylation nearly absent from the hepatocyte cytoplasm (Fig. 5e). Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 8 of 14 Fig. 4 The HDAC3 deficiency suppresses hepatocyte proliferation and STAT3 signaling in vitro.a Living hepatocyte numbers show that cell number of the mutant diploid hepatocyte is continuously decreased. The values are normalized to the average cell numbers on 0 h. b The cell cycle of in vitro diploid hepatocytes following mitogen stimulation for 48 h was determined by flow cytometry analysis. c Cell growth in HepG2 cells was tested after HDAC3 knockdown using a CCK-8 kit. d Western blot analysis demonstrates that STAT3(Y705) fails to be phosphorylated due to the high level of ac-STAT3 in the primary mutant hepatocytes after the IL6 treatment. β-Actin and histone H3 were used as the loading controls. e Immunofluorescence shows that STAT3 is unable to accumulate in the nuclei of the mutant hepatocytes following IL6 stimulation for 30 min (scale bar: 5 μm). f Immunofluorescence demonstrates that p-STAT3(Y705) remains absent in the mutant hepatocytes after the IL6 treatment (scale bar: 5 μm). All data represent the mean ± SD; n = 3–5; *p < 0.05; **p < 0.01; ***p < 0.001 Moreover, HDAC3, but not HDAC1 or HDAC2, was HDAC3 enhances p-STAT3(Y705) and Ki67 index in HDAC3- associated with ac-STAT3 in the cytoplasm (Fig. 5f; positive HCC Supplementary Fig. 8e), suggesting that HDAC3 played Active proliferation of tumor cells is a major feature of a unique role in the deacetylation of ac-STAT3 in the HCC that often indicates a poor prognosis . The IL6/ cytoplasm. STAT3 signaling pathway plays crucial roles in the Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 9 of 14 Fig. 5 HDAC3 specifically interacts with STAT3 in the cytoplasm. a, b Western blot analysis demonstrates that the level of ac-STAT3 is high in the HDAC3 liver at 6 h after PH. Histone H3 was used as the loading control. c Immunoprecipitation shows that HDAC1, HDAC2, and HDAC3 can each combine with ac-STAT3 in the nucleus following PH. d Immunoblotting to determination of the cytoplasmic and nuclear distribution shows that ac- STAT3 is primarily localized to the cytoplasm at 6 h after PH. β-Actin and histone H3 were used as the loading controls. e Immunoblotting to determination of the cytoplasmic and nuclear distributions of HDAC1, HDAC2, and HDAC3 in the control and HDAC3 mice demonstrates that HDAC3 localizes to both the cytoplasm and nucleus, whereas HDAC1 and HDAC2 strictly localize to the nucleus. f Co-immunoprecipitation assays demonstrate that HDAC3, but not HDAC1 or HDAC2, combines with STAT3 in the cytoplasm of hepatocytes from control livers. All data represent the mean ± SD; n = 3–5; ***p < 0.001 Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 10 of 14 Fig. 6 HDAC3 is highly expressed in HCC subtypes and is correlated with a poor prognosis in HCC patients. a Representative microphotograph and statistical analysis of HDAC3 and p-STAT3(Y705) expression in HCC tissues (n = 90) by immunohistochemistry (scale bar: 50 μm). b Co-immunoprecipitation shows the combination between STAT3 and HDAC3 or p300 in HDAC3-positive (+) or HDAC3-negative (−) HCC tissues. c Immunoblotting demonstrates that p-STAT3(Y705) increases in HDAC3-positive HCC tissues. NL normal liver, T tumor tissue, NT adjacent non-tumor tissues. GAPDH was used as the loading control. d Kaplan–Meier analysis shows that single upregulation of HDAC3 (n = 90) or combined upregulation of HDAC3 and p-STAT3(Y705) (n = 42) significantly reduces the overall survival rate of HCC patients. e Analysis of TCGA data revealed that HCC patients with high HDAC3 expression (Z score >1) were significantly associated with a poorer overall survival. FPKM fragments per kilobase of exon per million fragments mapped. All data represent the mean ± SD; ***p < 0.001 tumorigenesis and development of HCC . Because corresponding adjacent non-tumor tissues (Fig. 6a; Sup- HDAC3 is a critical regulator of STAT3 signaling during plementary Table 4). As observed by immunoprecipita- liver regeneration, we investigated whether elevated tion, HDAC3 was also detected in combination with HDAC3 in HCC was correlated with a higher tumor cell STAT3 in HDAC3-positive HCC (Fig. 6b), highlighting proliferation rate. We performed immunohistochemistry their interaction. Remarkably, HDAC3 was positively to assess HDAC3 expression in tissues from 90 HCC correlated with p-STAT3 and the Ki67 index in the cases, and observed that 27 cases exhibited strong nuclear HDAC3-positive HCCs (Pearson correction = 0.622, and cytoplasmic HDAC3 positivity, and that HDAC3 P < 0.001, and Pearson correction = 0.760, P < 0.001, was either negatively or weakly expressed in the respectively) (Fig. 6c; Supplementary Fig. 10). The Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 11 of 14 Kaplan–Meier analysis showed that the high expression of a critical signaling cascade that promotes the transcrip- HDAC3 alone or in combination with high p-STAT3 tion of numerous immediate-early genes in response to (Y705) remarkably reduced recurrence-free survival acute liver injury. (Fig. 6d; Supplementary Tables 5 and 6). Consistently, the As a nucleocytoplasmic shuttling transcription factor, Cancer Genome Atlas (TCGA) data also revealed that STAT3 is acetylated by p300 in the nucleus ; however, HCC patients with high HDAC3 expression have lower the details regarding deacetylation of STAT3 remain overall survival rates (Fig. 6e). unclear. Our data show that HDAC3 deficiency remark- ably increases the p300-dependent STAT3 acetylation in Inhibition of HDAC3 expression decreases HCC xenografts the cytoplasm of hepatocytes. It has been suggested that growth ac-STAT3 is likely to be deacetylated by class I HDAC To elucidate the effect of elevated HDAC3 on HCC members in the nucleus . Unlike HDAC1 or HDAC2, growth, SMMC-7721 cells were injected subcutaneously which predominantly function in the nucleus, HDAC3 is a into nude mice for xenograft assay. We treated xenograft unique Class I HDAC subfamily member that regulates with panobinostat, a pan-HDAC-inhibitor for clinical acetylation status of non-histone proteins in the cyto- 12 33,34 treatment of lymphoma and multiple myeloma ,to plasm . Our results revealed that HDAC1 and/or reduce HDAC3 expression in SMMC-7721 cells. HDAC3 HDAC2 deficiency does not change the acetylation status inhibition significantly reduced tumor growth and of STAT3 in the liver, while loss of HDAC3 results in a decreased the expression of Ki67 in HCC xenograft substantial cytoplasmic accumulation of ac-STAT3, tumors (Fig. 7a–d). Consistently, the expression of p- indicating that the deacetylation of ac-STAT3 might be STAT3(Y705) in panobinostat-treated xenograft tumors selectively dependent on HDAC3 in the cytoplasm rather was significantly reduced (Fig. 7d, e). In addition, HDAC3 than in the nucleus. knockdown remarkably suppressed cell cycle progression After IL6 stimulation, STAT3 that resides in the cyto- and inhibited the phosphorylation of STAT3(Y705) plasm rapidly becomes phosphorylated and translocates (Supplementary Fig. 8). Therefore, increased HDAC3 into the nucleus to induce target genes expression . Our enhanced tumor growth that may occur through data show that HDAC3 deficiency significantly increases STAT3 signaling in liver cancer cells. ac-STAT3 level and decreases p-STAT3(Y705) level together with a decrease in STAT3 nucleus entry, sug- Discussion gesting that HDAC3-mediated deacetylation of ac-STAT3 HDAC3 is a crucial deacetylase component of the is essential for STAT3(Y705) phosphorylation and STAT3 NCOR1/SMRT complexes, which is involved in histone reactivation cycle. It has been shown that the conserved modifications, chromatin remodeling, and transcriptional lysine residues for acetylation in STAT3 are all near the 5,37 29 regulation . In addition to the important role of Y705 residual of STAT3 , supporting the possibility that HDAC3 in DNA repair and in the maintenance of the STAT3 acetylation induced by HDAC3 deficiency could circadian rhythm of metabolism homeostasis, the essen- prevent STAT3(Y705) phosphorylation and block tial non-transcriptional function of HDAC3 during STAT3 signaling activation. However, the detail mitosis is still largely unknown. Here, we have established mechanisms of HDAC3-mediated modifications of for the first time the importance of HDAC3 for liver STAT3 deacetylation and phosphorylation in hepatocytes regeneration together with liver cancer cells proliferation. remain further studies. Regardless of other mechanisms Two types of transmembrane enzyme-linked receptor- that might be regulated by HDAC3 during the whole mediated signaling cascades, namely, the cytokine- process of hepatocyte proliferation, HDAC3 is undoubt- dependent pathway and the growth factor-dependent edly critical to the initiation of the cell cycle progression pathway, which transduce extracellular growth stimuli through the proper transition of ac-STAT3 to p-STAT3. into the nucleus, play crucial roles in priming liver Therefore, HDAC3 may act as a molecular switch in the 1,23 regeneration . Our previous study showed that loss of STAT3 signaling cascade that transmits growth stimuli the stimulatory G protein α subunit (G α), which activates from the cytoplasm to the nucleus. the cAMP-dependent pathway, leads to a breakdown in Multiple studies have shown that HDAC3 is aberrantly growth factor signaling and dramatically arrests the expressed in various human cancers and that the aberrant 38 37 regenerating hepatocytes in the G1 phase extension . expression is often associated with a poor prognosis , Lack of HDAC3 results in a similar proliferative defect at making HDAC3 inhibition a potential strategy for cancer the cell cycle entrance, which indicates a breakdown in therapy. The HDAC3 mRNA level is lacked in one-sixth the cytokine-dependent or growth factor-dependent sig- of human HCC tissues . The inactivation of HDAC3 may naling cascade. Using a microarray assay analysis and contribute to HCC development by increasing global LPS/PH model, we showed that HDAC3 deficiency dra- histone acetylation, defective DNA damage repair, and matically impairs the STAT3 signaling pathway, which is mutation accumulation . HDAC3 is also upregulated in a Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 12 of 14 Fig. 7 (See legend on next page.) Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 13 of 14 (see figure on previous page) Fig. 7 Inhibition of HDAC3 expression decreases HCC xenografts growth. a–c HCC growth in mouse xenograft model. SMMC-7721 cells (2 × 10 ) were injected subcutaneously into nude mice for xenograft assay. Tumor volume and average weight of HCC xenografts in each group were shown (n = 4) (scale bar: 1 cm). d Immunohistochemical analysis of control and panobinostat-treated tumors with p-STAT3(Y705), Ki-67, and γ-H2A.X antibodies (scale bar: 50 μm). Quantitative analysis of p-STAT3(Y705), Ki-67, and γ-H2A.X-positive rates in each group of tumors were shown. e Western blot analysis demonstrates that p-STAT3(Y705) is inhibited in panobinostat-treated tumors. Histone H3 were used as the loading controls. f Diagram of the HDAC3–STAT3 axis in the IL6/STAT3 signaling pathway and the signaling cascades that regulate hepatocyte proliferation and tumorigenesis. All data represent the mean ± SD; *p < 0.05; **p < 0.01; n.s. not significant 12,13 specific subset of HCCs ; however, the role of HDAC3 For inducible deletion of HDAC3, tamoxifen was dis- in the pathogenesis of HCC remains unknown. The solved in corn oil containing 10% (vol/vol) ethanol to 20 upregulated expression of HDAC3 in human HCC mg/ml and shaken overnight at 37 °C . Six-week-old ERT2 loxP/loxP prompts us to determine whether the HDAC3–STAT3 male Alb-Cre ;HDAC3 mice were injected pathway enhances HCC tumor growth. Notably, IL6, a intraperitoneally with tamoxifen (2 mg per mouse) for five cytokine that is essential for activating STAT3 signaling in consecutive days. Experiments on mice were performed liver inflammation, enhances HCC development .We 5 days after the last injection. Then, these mice were found that overexpression of HDAC3 is significantly treated with either classical 2/3 PH surgery or intraper- associated with an increased p-STAT3(Y705) level and itoneal injection of CCl (10 ml/kg body weight) . BrdU Ki67 index in HCC. Moreover, our xenograft assay shows (1 mg/kg body weight) was intraperitoneally injected 60 that a low dose of panobinostat could significantly min before sacrifice. decrease HCC xenografts growth along with a significant reduction in p-STAT3(Y705) level. Our data suggest that Statistical analysis the drug panobinostat approved by FDA might also pre- Data were expressed as the mean ± standard deviation vent liver cancer cells proliferation through (SD). Statistical comparisons were assessed with an HDAC3–STAT3 signaling pathway in HDAC3- unpaired, one-tailed Student’s t-test with Welch correc- overexpressed HCC, while the detail effects and tion. A p-value < 0.05 was considered significant. mechanisms of panobinostat for the clinical treatment of Acknowledgements HCC remain further studies. We thank Dr. Qing Richard Lu for kindly providing transgenic mice. We also In summary, we have demonstrated that HDAC3 acts thank Li Li, Fei Chen and Lei Zhang for pathology technique assistance. This work was supported by grants from the Natural Science Foundation of China an essential role in the enhancement of the (No. 81472303), the National Key Clinical Project, and the Major Science and STAT3 signaling pathway by catalyzing the deacetylation Technology Project of Science & Technology Department of Sichuan Province of ac-STAT3 in the cytoplasm, thereby, promoting liver (2017SZ0003). regeneration and tumor growth, as illustrated in Fig. 7f. Author details Our findings highlight the importance of the Laboratory of Pathology, Key Laboratory of Transplant Engineering and HDAC3–STAT3 signaling cascade in promoting cell cycle Immunology, NHFPC; West China Hospital, Sichuan University, Chengdu progression and support HDAC3 as a potential ther- 610041, China. Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China. Department of Pediatrics, Division of apeutic target for the treatment of HCC as well as other Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati solid cancers whose HDAC3 is overexpressed. 4 Children’s Hospital Medical Center, Cincinnati, OH 25229, USA. Department of Liver and Vascular Surgery, West China Hospital, Sichuan University, Chengdu 610041, China Materials and methods Mice Conflict of interest All mouse experimental procedures were approved by The authors declare that they have no conflict of interest. the Animal Care and Use Committee of Sichuan Uni- loxP/loxP versity. HDAC3 frozen embryos were purchased Publisher's note from the European Mouse Mutant Archive (EMMA), Springer Nature remains neutral with regard to jurisdictional claims in University of Veterinary Medicine, Vienna. Alb-Cre and published maps and institutional affiliations. ERT2 Alb-Cre transgenic mice were purchased from Supplementary Information accompanies this paper at https://doi.org/ Shanghai Biomodel Organism Science & Technology 10.1038/s41419-018-0428-x. Development Co., Ltd, China. The genotypes of Alb-Cre; loxP/loxP ERT2 loxP/loxP HDAC3 and Alb-Cre ;HDAC3 mice Received: 30 October 2017 Revised: 4 January 2018 Accepted: 12 January were determined using PCR amplification of tail DNA. The sequences of genotyping primers are presented in Supplementary Table 1. Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 14 of 14 References 21. Vidal-Laliena, M. et al. Histone deacetylase 3 regulates cyclin A stability. J. Biol. 1. Taub, R. Liver regeneration: from myth to mechanism. Nat. Rev. Mol. Cell Biol. 5, Chem. 288,21096–21104 (2013). 836–847 (2004). 22. Berridge, M. J. Module 2: cell signalling pathways. Cell Signal. Biol. 6,1–138 2. Michalopoulos, G. K. Liver regeneration. J. 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D. & Hsieh, J. HDAC3 controls gap 2/mitosis progression in adult 1924–1931 (2013). neural stem/progenitor cells by regulating CDK1 levels. Proc. Natl. Acad. Sci. USA 111, 13541–13546 (2014). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Disease Springer Journals

Histone deacetylase 3 promotes liver regeneration and liver cancer cells proliferation through signal transducer and activator of transcription 3 signaling pathway

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Copyright © 2018 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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2041-4889
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10.1038/s41419-018-0428-x
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Abstract

Histone deacetylase 3 (HDAC3) plays pivotal roles in cell cycle regulation and is often aberrantly expressed in various cancers including hepatocellular carcinoma (HCC), but little is known about its role in liver regeneration and liver cancer cells proliferation. Using an inducible hepatocyte-selective HDAC3 knockout mouse, we find that lack of HDAC3 dramatically impaired liver regeneration and blocked hepatocyte proliferation in the G1 phase entry. HDAC3 inactivation robustly disrupted the signal transducer and activator of transcription 3 (STAT3) cascade. HDAC3 silencing impaired the ac-STAT3-to-p-STAT3 transition in the cytoplasm, leading to the subsequent breakdown of STAT3 signaling. Furthermore, overexpressed HDAC3 was further associated with increased tumor growth and a poor prognosis in HCC patients. Inhibition of HDAC3 expression reduced liver cancer cells growth and inhibited xenograft tumor growth. Our results suggest that HDAC3 is an important regulator of STAT3-dependent cell proliferation in liver regeneration and cancer. These findings provide novel insights into the HDAC3–STAT3 pathway in liver pathophysiological processes. Introduction chemical injury . In humans, liver regeneration proceeds The mammalian liver has an enormous regenerative within weeks of a major liver resection, whereas in rodents, ability to restore its original mass and function via com- liver recovery can occur within 10 days of PH . Aberrant pensatory hyperplasia in response to diverse injuries . liver regeneration is closely related to the pathogenesis of Quiescent maturity hepatocytes in the G0 phase of the cell liver failure or hepatocellular carcinoma (HCC) . A better cycle can re-enter into the cell cycle rapidly to produce new understanding of the basic mechanisms that regulate liver hepatocytes following partial hepatectomy (PH) or regeneration will have important implications for potential therapies for human liver disease. Histone deacetylase 3 (HDAC3), a member of the highly Correspondence: Yu-Jun Shi (shiyujun@scu.edu.cn) conserved Class I HDAC enzymes, is ubiquitously expressed Laboratory of Pathology, Key Laboratory of Transplant Engineering and 4 in eukaryotic cells . HDAC3 regulates gene transcription by Immunology, NHFPC; West China Hospital, Sichuan University, Chengdu catalyzing the deacetylation of core histones and is involved 610041, China 2 5 Department of Pathology, West China Hospital, Sichuan University, Chengdu in various biological processes in the liver . Inhibition of 610041, China HDAC3 and other HDACs specifically interferes with liver Full list of author information is available at the end of the article These authors contributed equally: Xu-Feng Lu, Xiao-Yue Cao. Edited by Z.-X. Xiao. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Lu et al. Cell Death and Disease (2018) 9:398 Page 2 of 14 development in zebrafish . In mammals, hepatic-specific and notable damage to the DNA are evident at 2 months ablation of HDAC3 dramaticallydisruptsliver function and of age (Supplementary Fig. 1). Therefore, to eliminate causes severe dysregulation of lipid and glucose metabo- post-natal developmental defects upon HDAC3 deletion, 7,8 lism .Conversely, high HDAC3 expression in the liver was we generated an inducible tamoxifen-dependent hepato- ERT2 found to contribute to high-fat-diet-induced metabolic cyte-selective HDAC3 knockout mouse (Alb-Cre ; 9 loxP/loxP syndrome . HDAC3 is also essential for the maintenance of HDAC3 mouse), in which HDAC3 in the liver chromatin structure and genomic stability, and hepatic loss tissues was over 80% ablated after 5 days of tamoxifen of HDAC3 in mice results in an accumulation of DNA treatment (hereafter referred to as the HDAC3 mouse) 10,11 damage and the early onset of spontaneous liver cancer . (Supplementary Fig. 2). Five days after the last injection of Still, the role of HDAC3 in HCC development remains tamoxifen, the inducible HDAC3 mouse did not show unclear because the mRNA and protein levels of HDAC3 obviously impaired liver function or disrupted DNA sta- 11,12 can either increase or decrease in human HCC tissues . bility (Supplementary Fig. 2). Next, we performed two- Furthermore, HDAC3 is a considered biomarker of tumor thirds PH of the livers in male HDAC3 mice and their ERT2 loxP/loxP recurrence following liver transplantation in hepatitis B control littermates (Alb-Cre ;HDAC3 mice virus-associated HCC . HDAC3 also participates in the self- treated with corn oil instead of tamoxifen) to explore the renewal process of liver cancer stem cells through histone specific role of HDAC3 during liver regeneration. Sur- modifications , making HDAC3 suppression a potential prisingly, the post-hepatectomy mutant mice displayed a clinical-therapeutic approach for HCC. severely impaired liver mass reconstitution, which was Multiple in vitro studies have revealed that HDAC3 calculated using the liver weight/body weight ratio plays diverse roles in the regulation of cell cycle pro- (Fig. 1a). Furthermore, HDAC3 silencing interfered with gression. HDAC3 is required to generate a hypoacetylated liver recovery as revealed by the alanine transaminase and H3 tail as the preferred template for the phosphorylation aspartate transaminase serum levels (Fig. 1b). The bro- of H3S10 by Aurora B, which is a crucial step for pro- modeoxyuridine (BrdU) incorporation and Ki67 immu- gression through the G2/M phase and mitosis . HDAC3 nohistochemistry showed that the proliferation peak in is also required for H3K4 deacetylation at the centromere the control littermates appeared between 36 and 48 h 16,17 and sister chromatin cohesion . HDAC3 inhibition post-hepatectomy; strikingly, HDAC3 disruption reduced WAF1/cip1 induces G1 phase arrest by increasing p21 the proliferation rates by approximately 60% during this expression in Hep3B liver cancer cells . HDAC3 deletion time (Fig. 1c, d, f). Phospho-histone H3S10 (p-H3S10), a in mouse embryonic fibroblasts induces S-phase arrest mitotic marker that is specifically expressed during the through DNA damage accumulation, whereas inactivation G2/M phase , showed a corresponding decrease that was of HDAC3 affects replication fork progression in hema- over three-fold in the HDAC3 mice at 48 h post- 10,19 topoietic progenitor cells during the S phase . HDAC3 hepatectomy (Fig. 1e, f). The livers of the control mice also controls G2/M progression in adult neural stem/ were completely reconstituted within 168 h, and mitotic progenitor cells by regulating the CDK1 levels .In hepatocytes were undetectable by 168 h; however, fre- addition, HDAC3 regulates cell cycle progression by quent proliferation was observed in the HDAC3 mice modulating cyclin A acetylation to control cyclin A until 14 days after the PH (Supplementary Fig. 3a). level . The data regarding the role and molecular Similarly, when the mice were challenged with a carbon mechanism of HDAC3 in regulating cell proliferation are tetrachloride (CCl ) injection, the absence of HDAC3 did highly multiple and contradictory. However, the biological not increase sensitivity to the toxin but robustly delayed function of HDAC3 in liver regeneration and liver cancer liver repair (Supplementary Fig. 4). cells proliferation remains unknown. We next examined potential differences in the In this study, we determined that HDAC3 promotes liver expression levels of key cell cycle markers between the regeneration and liver cancer cells proliferation through HDAC3 and control mice during liver regeneration. the signal transducer and activator of transcription 3 Ki67 is expressed from mid-G1 to the end of mitosis , (STAT3) signaling pathway. The role of HDAC3 in the and the low Ki67 staining indicates that the proliferating regulation of cell proliferation through the STAT3 pathway hepatocytes might be arrested in the early G1 phase. provides a potential drug target for the treatment of HCC. Consistently, the levels of cyclin D1 and CDK4, which arethe keymarkers of theearly G1 phase , peaked at 24 Results h after PH in the control livers; however, the cyclin D1 HDAC3 deletion severely impairs liver regeneration by and CDK4 levels were dramatically suppressed in the delaying the G1 phase entry HDAC3 livers (Fig. 1g; Supplementary Fig. 3b). The 7,10 As described previously , in the Alb-Cre; levels of CDK2 and cyclin E1, which function at the G1/S loxP/loxP 22 HDAC3 mouse, where HDAC3 is constitutively transition , peaked at 36 h post-hepatectomy, but the inactivated, dramatic dysregulation of hepatic metabolism levels were correspondingly lower in the mutant livers Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 3 of 14 Fig. 1 Impaired liver regeneration in HDAC3 mice following 70% PH. a The liver weight/body weight ratio was calculated at different time points after PH, and liver reconstitution was notably delayed in the HDAC3 livers. b The serum AST and ALT levels after PH. c–f Immunohistochemistry for BrdU, Ki67, and p-Histone H3 (Ser10) shows that the proliferation rates are reduced in the HDAC3 mice after PH (scale bar: 50 μm). g Western blot analysis of cell cycle proteins at different times after PH shows that mitotic hepatocytes are delayed at the G1 phase entrance in the HDAC3 mice. The levels of the indicated proteins are expressed relative to GAPDH. All data represent the mean ± SD; n = 3–8; *p < 0.05; **p < 0.01; ***p < 0.001 (Fig. 1g; Supplementary Fig. 3b). Notably, the protein deficient hepatocytes were robustly arrested in the G1 levels of cyclin A2, an essential marker for S-phase phase entry. progression ,weresignificantly reduced in the HDAC3 livers at 48 h after PH (Fig. 1g; Supplementary HDAC3 inactivation impairs STAT3 signaling Fig. 3b). Additionally, the levels of CDK1, cyclin B1, and Because the rapid transitions of the cell cycle activate p-H3S10, which appear in the G2/M phase ,werealso multiple signaling cascades and numerous genes during △ 1 markedly decreased in the HDAC3 livers during the liver regeneration , we performed a microarray analysis to peak period of mitosis (Fig. 1g; Supplementary Fig. 3b). screen the molecular defects in the HDAC3 liver. The Taken together, our findings suggested that HDAC3- inducible short-term absence of HDAC3 did not Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 4 of 14 Fig. 2 (See legend on next page.) Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 5 of 14 (see figure on previous page) Fig. 2 The gene expression profile demonstrates the broad and multiple effects of HDAC3 on gene expression after PH. a A heat map of 1557 differentially expressed genes (fold changes ≥1.5 and p < 0.05) in the control and mutant livers at 36 h after PH compared with their corresponding baseline values (0 h). b mRNA microarray analysis of cell cycle-related genes. Differentially expressed genes (fold changes ≥1.25 and p < 0.05) in the control and HDAC3 livers at 36 h after PH. c GO term analysis of the list of differentially expressed genes (fold changes ≥1.5 and p < 0.05) indicated that the IL6/STAT3 pathway is dramatically changed during liver regeneration in the HDAC3 mice after PH. d Heat map of the selected genes from the microarray analysis of the control and HDAC3 livers. e Quantitative RT-PCR confirmation of the downregulated early- response genes in the HDAC3 livers vs. the control liver at 36 h after PH. f qRT-PCR confirms downregulation of cyclin D1 and c-myc in the HDAC3 livers vs. the control liver at 36 h after PH. All data represent the mean ± SD; n = 3; **p < 0.01; ***p < 0.001; n.s. not significant obviously alter the gene expression profiles of the quies- phase post-hepatectomy (Fig. 3b; Supplementary Fig. 6a). cent hepatocytes (Supplementary Fig. 5a–c); however, the Intriguingly, the level of p-STAT3(S727), which is phos- gene profiles of the mutant liver were dramatically phorylated by the c-src non-receptor tyrosine kinase , changed 36 h after the PH (Fig. 2a). Among these genes, was not obviously impaired in the HDAC3-deficient livers 131 cell cycle-related genes were identified, and cyclin D1, (Fig. 3b), indicating that HDAC3 ablation might selec- cyclin A2, cyclin B1, and CDK1 were significantly down- tively target p-STAT3(Y705). To further determine whe- regulated in the HDAC3 liver (Fig. 2b; Supplementary ther the low level of p-STAT3(Y705) was caused by the Fig. 5d), which was consistent with the proliferation HDAC3 deletion, we examined p-STAT3(Y705) expres- defects observed in the mutant liver. sion in HDAC3-deficient mice by simultaneously chal- A Gene Ontology (GO) term analysis of the differen- lenging the mice with PH and intraperitoneal injections of tially regulated genes showed that the interleukin 6 (IL6)/ lipopolysaccharide (LPS) (Supplementary Fig. 6b, c). IL6 is STAT3 signaling pathway, which regulates approximately extraordinarily increased by LPS stimulation , whereas 36% of the immediate-early genes during the acute the levels of p-STAT3(Y705) and c-myc remained notably 1 △ hepatic response , was extremely disturbed (Fig. 2c, d). As depressed in the HDAC3 livers, regardless of the expected, the immediate early-response genes, including upregulation of p-JAK2 (Fig. 3c; Supplementary Fig. 6c). c-fos, c-jun, Egr2/3, Gadd45a, junb, and Mdm2 , were Upon ligand stimulation, acetylation of STAT3 (ac- significantly downregulated in the HDAC3 livers STAT3) is critical for enhancing the transcription of cell 27,28 (Fig. 2e, Supplementary Fig. 5d). Additionally, c-myc and growth-related genes . Co-activator p300, which is a cyclin D1, which are components of the STAT3 path- histone acetyltransferase that primarily functions in the 24 29,30 way , were among the most reduced genes, and this nucleus, is required for STAT3 acetylation .Our observation was further confirmed by qRT-PCR (Fig. 2f). immunoprecipitation assay showed a strong interaction Collectively, our microarray data supported the idea that between p300 and STAT3 in both the control and △ △ breakdown of STAT3 signaling in the HDAC3 mice HDAC3 livers (Fig. 3d). As STAT3 activity is modified by might account for the G1 phase arrest during liver phosphorylation and acetylation at post-translational regeneration. level , we next examined whether HDAC3 deficiency results in impaired STAT3 activation. In the mutant liver, HDAC3 silencing impairs the STAT3 transition from the protein that was co-immunoprecipitated by the STAT3 acetylation to phosphorylation antibody overwhelmingly consisted of ac-STAT3 with STAT3 mediates the expression of various genes in extremely low p-STAT3(Y705), while the control liver response to diverse stimuli and thus plays a key role in showed the opposite dynamic (Fig. 3e). The absence of p- multiple cellular processes such as cell growth, pro- STAT3(Y705) in the nucleus prompted us to investigate liferation, and apoptosis . When IL6 combines with its whether HDAC3 deprivation impaired the STAT3 transi- membrane receptor glycoprotein 130 (gp130), STAT3 tion from acetylation to phosphorylation during liver becomes phosphorylated at tyrosine (Y705) by regeneration. The cytoplasmic and nuclear fractions were receptor-associated Janus kinases (JAKs) . Compared prepared from the livers after PH at indicated time points, with the control littermates, in which a large number of p- and the immunoblotting analysis showed that during the STAT3(Y705) nuclear-positive hepatocytes were observed early phase of liver regeneration, p-STAT3(Y705) was at 3 h after PH, nuclear p-STAT3(Y705) was abolished in notably increased both in the cytoplasm and nucleus of the HDAC3-deficient mice (Fig. 3a–c; Supplementary Fig. 6a). control hepatocytes whereas ac-STAT3 was maintained at Additionally, the level of c-myc, which is a major down- a low level in the cytoplasm (Fig. 3f). Conversely, HDAC3- stream gene of STAT3 signaling and an important tran- deficient hepatocytes showed substantial hyperacetylation scription factor for successful initiation of cell mitosis , of STAT3 but notably reduced p-STAT3(Y705) in both the was also reduced in HDAC3-deficient mice at the early cytoplasm and the nucleus (Fig. 3f, g). Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 6 of 14 Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 7 of 14 (see figure on previous page) Fig. 3 The HDAC3 deficiency inhibited STAT3(Y705) phosphorylation and enhanced STAT3 acetylation in hepatocytes. a Immunofluorescence demonstrates that p-STAT3(Y705) is not activated in the HDAC3 hepatocytes (scale bar: 50 μm). b Western blot analysis shows that p-STAT3(Y705) and c-myc are reduced in the HDAC3 mice during the early phase of liver regeneration. GAPDH was used as the loading control. c Western blot analysis shows that p-STAT3 remains inhibited in the mutant hepatocytes after the LPS/PH treatment. d Co- immunoprecipitation shows that STAT3 is associated with p300 in both the control and HDAC3 liver. e Immunoprecipitation confirms that the protein complex overwhelmingly consists of ac-STAT3 with extremely low p-STAT3(Y705) in the HDAC3 liver. f Western blot analysis shows that the HDAC3 deficiency leads to prolonged acetylation of STAT3 in the cytoplasm during liver regeneration. β-Actin and histone H3 were used as the loading controls. g Immunofluorescence demonstrates that ac-STAT3 significantly accumulates in the cytoplasm of the HDAC3 hepatocytes (scale bar: 50 μm). n = 3–5; ***p < 0.001 HDAC3 deletion impairs cell growth by inhibiting nuclear of ac-STAT3 in vivo remain undefined. We previously translocation of STAT3 reported that loss of HDAC1 and/or HDAC2 did not To determine whether the HDAC3 deficiency inhibited disturb cell cycle progression before the M phase , which the nuclear translocation of STAT3, we cultured fluor- highlighted the negative effects of HDAC1 and HDAC2 escently labeled primary diploid hepatocytes that were on the IL6/STAT3 signaling cascade. Consistently, dele- isolated from the (Gt(ROSA)26Sortm4(ACTB-tdTomato- tions of HDAC1, HDAC2, or both did not increase the ,EGFP) mice (hereafter referred to as mT/mG mice) and level of ac-STAT3 compared to that of the wild-type HDAC3 ;mT/mG mice, respectively (Supplementary (WT) liver during the early phase of liver regeneration Fig. 7). Cell viability assays revealed that compared to the (Fig. 5a, b). Moreover, loss of HDAC8, another member of diploid hepatocytes that were isolated from the mT/mG the Class I HDACs, did not have a noticeable effect on the mice, cell number of the HDAC3 mutant hepatocytes was progression of liver regeneration (Supplementary Fig. 9). significantly decreased (Fig. 4a, b). Consistently, HDAC3 Therefore, we did not further analyze the role of HDAC8 knockdown in human liver cancer cells HepG2 using in STAT3 signaling during liver regeneration. siRNA significantly increased ac-STAT3 expression level It has been suggested that ac-STAT3 is likely to be 28,32 and remarkably inhibited cell growth (Fig. 4c; Supple- deacetylated by Class I HDACs in the nucleus . To test mentary Fig. 8a–c). this, we performed immunoprecipitations to examine the Notably, the mutant hepatocytes displayed robustly associations between STAT3 and HDAC1, HDAC2, and decreased p-STAT3(Y705) with significantly increased HDAC3 in the nucleus. All three Class I HDAC members cytoplasmic ac-STAT3; additionally, IL6 stimulation interacted with ac-STAT3 (Fig. 5c); however, compared to failed to upregulate p-STAT3(Y705) in the HDAC3- the WT liver, single disruptions to each HDAC or a deficient cells (Fig. 4d). Because STAT3 localizes to the combined disruption of HDAC1 and HDAC2 did not nucleus after it is phosphorylated , we tested whether ac- noticeably increase the nuclear level of ac-STAT3 fol- STAT3 prevented the nuclear translocation of STAT3. lowing PH (Fig. 5d). This observation strongly suggested Indeed, the immunofluorescence analysis showed that the that HDACs do not catalyze the deacetylation of ac- nuclear localization of total STAT3 was significantly STAT3 in the nucleus. This observation was also vali- suppressed in the HDAC3-deficient hepatocytes after IL6 dated by the finding that high levels of ac-STAT3 were treatment (Fig. 4e). Moreover, p-STAT3(Y705) was evident in the cytoplasm of hepatocytes from each gen- noticeably localized to the nuclei of the control hepato- otypic mouse, though the ac-STAT3 levels were much cytes, whereas p-STAT3(Y705) remained absent from the higher in the HDAC3-deficient livers than in the HDAC1- HDAC3-deficient hepatocyte nuclei, even with and/or HDAC2-deficient livers (Figs. 3g and 5d). IL6 stimulation (Fig. 4f). Meanwhile, the phosphorylation The cytoplasmic but not nuclear accumulation of ac- of STAT3(Y705) after IL6 stimulation was also prevented STAT3 in the HDAC3-deficient liver strongly indicated in response to HDAC3 knockdown (Supplementary that ac-STAT3 is primarily deacetylated in the cyto- Fig. 8b–d), which indicated that loss of HDAC3 remark- plasm rather than in the nucleus, and HDAC3 might ably disrupted the transduction of STAT3 signaling from function a powerful role in the deacetylation of ac- the cytoplasm to the nucleus. STAT3. Although HDAC3 is a nuclear protein, HDAC3 contains a nuclear export signal in its C terminus HDAC3 is essential for deacetylation of ac-STAT3 in the (residues 180–313) which facilitates the nucleoplasm 33,34 cytoplasm transport of HDAC3 .Weconfirmed the cyto- Class I HDACs have been suggested to contribute to plasmic distribution of HDAC3 by immunoblotting, STAT3 deacetylation in 293T cells , but the individual andincontrasttoHDAC3,HDAC1 andHDAC2 were roles of the Class I HDAC members in the deacetylation nearly absent from the hepatocyte cytoplasm (Fig. 5e). Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 8 of 14 Fig. 4 The HDAC3 deficiency suppresses hepatocyte proliferation and STAT3 signaling in vitro.a Living hepatocyte numbers show that cell number of the mutant diploid hepatocyte is continuously decreased. The values are normalized to the average cell numbers on 0 h. b The cell cycle of in vitro diploid hepatocytes following mitogen stimulation for 48 h was determined by flow cytometry analysis. c Cell growth in HepG2 cells was tested after HDAC3 knockdown using a CCK-8 kit. d Western blot analysis demonstrates that STAT3(Y705) fails to be phosphorylated due to the high level of ac-STAT3 in the primary mutant hepatocytes after the IL6 treatment. β-Actin and histone H3 were used as the loading controls. e Immunofluorescence shows that STAT3 is unable to accumulate in the nuclei of the mutant hepatocytes following IL6 stimulation for 30 min (scale bar: 5 μm). f Immunofluorescence demonstrates that p-STAT3(Y705) remains absent in the mutant hepatocytes after the IL6 treatment (scale bar: 5 μm). All data represent the mean ± SD; n = 3–5; *p < 0.05; **p < 0.01; ***p < 0.001 Moreover, HDAC3, but not HDAC1 or HDAC2, was HDAC3 enhances p-STAT3(Y705) and Ki67 index in HDAC3- associated with ac-STAT3 in the cytoplasm (Fig. 5f; positive HCC Supplementary Fig. 8e), suggesting that HDAC3 played Active proliferation of tumor cells is a major feature of a unique role in the deacetylation of ac-STAT3 in the HCC that often indicates a poor prognosis . The IL6/ cytoplasm. STAT3 signaling pathway plays crucial roles in the Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 9 of 14 Fig. 5 HDAC3 specifically interacts with STAT3 in the cytoplasm. a, b Western blot analysis demonstrates that the level of ac-STAT3 is high in the HDAC3 liver at 6 h after PH. Histone H3 was used as the loading control. c Immunoprecipitation shows that HDAC1, HDAC2, and HDAC3 can each combine with ac-STAT3 in the nucleus following PH. d Immunoblotting to determination of the cytoplasmic and nuclear distribution shows that ac- STAT3 is primarily localized to the cytoplasm at 6 h after PH. β-Actin and histone H3 were used as the loading controls. e Immunoblotting to determination of the cytoplasmic and nuclear distributions of HDAC1, HDAC2, and HDAC3 in the control and HDAC3 mice demonstrates that HDAC3 localizes to both the cytoplasm and nucleus, whereas HDAC1 and HDAC2 strictly localize to the nucleus. f Co-immunoprecipitation assays demonstrate that HDAC3, but not HDAC1 or HDAC2, combines with STAT3 in the cytoplasm of hepatocytes from control livers. All data represent the mean ± SD; n = 3–5; ***p < 0.001 Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 10 of 14 Fig. 6 HDAC3 is highly expressed in HCC subtypes and is correlated with a poor prognosis in HCC patients. a Representative microphotograph and statistical analysis of HDAC3 and p-STAT3(Y705) expression in HCC tissues (n = 90) by immunohistochemistry (scale bar: 50 μm). b Co-immunoprecipitation shows the combination between STAT3 and HDAC3 or p300 in HDAC3-positive (+) or HDAC3-negative (−) HCC tissues. c Immunoblotting demonstrates that p-STAT3(Y705) increases in HDAC3-positive HCC tissues. NL normal liver, T tumor tissue, NT adjacent non-tumor tissues. GAPDH was used as the loading control. d Kaplan–Meier analysis shows that single upregulation of HDAC3 (n = 90) or combined upregulation of HDAC3 and p-STAT3(Y705) (n = 42) significantly reduces the overall survival rate of HCC patients. e Analysis of TCGA data revealed that HCC patients with high HDAC3 expression (Z score >1) were significantly associated with a poorer overall survival. FPKM fragments per kilobase of exon per million fragments mapped. All data represent the mean ± SD; ***p < 0.001 tumorigenesis and development of HCC . Because corresponding adjacent non-tumor tissues (Fig. 6a; Sup- HDAC3 is a critical regulator of STAT3 signaling during plementary Table 4). As observed by immunoprecipita- liver regeneration, we investigated whether elevated tion, HDAC3 was also detected in combination with HDAC3 in HCC was correlated with a higher tumor cell STAT3 in HDAC3-positive HCC (Fig. 6b), highlighting proliferation rate. We performed immunohistochemistry their interaction. Remarkably, HDAC3 was positively to assess HDAC3 expression in tissues from 90 HCC correlated with p-STAT3 and the Ki67 index in the cases, and observed that 27 cases exhibited strong nuclear HDAC3-positive HCCs (Pearson correction = 0.622, and cytoplasmic HDAC3 positivity, and that HDAC3 P < 0.001, and Pearson correction = 0.760, P < 0.001, was either negatively or weakly expressed in the respectively) (Fig. 6c; Supplementary Fig. 10). The Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 11 of 14 Kaplan–Meier analysis showed that the high expression of a critical signaling cascade that promotes the transcrip- HDAC3 alone or in combination with high p-STAT3 tion of numerous immediate-early genes in response to (Y705) remarkably reduced recurrence-free survival acute liver injury. (Fig. 6d; Supplementary Tables 5 and 6). Consistently, the As a nucleocytoplasmic shuttling transcription factor, Cancer Genome Atlas (TCGA) data also revealed that STAT3 is acetylated by p300 in the nucleus ; however, HCC patients with high HDAC3 expression have lower the details regarding deacetylation of STAT3 remain overall survival rates (Fig. 6e). unclear. Our data show that HDAC3 deficiency remark- ably increases the p300-dependent STAT3 acetylation in Inhibition of HDAC3 expression decreases HCC xenografts the cytoplasm of hepatocytes. It has been suggested that growth ac-STAT3 is likely to be deacetylated by class I HDAC To elucidate the effect of elevated HDAC3 on HCC members in the nucleus . Unlike HDAC1 or HDAC2, growth, SMMC-7721 cells were injected subcutaneously which predominantly function in the nucleus, HDAC3 is a into nude mice for xenograft assay. We treated xenograft unique Class I HDAC subfamily member that regulates with panobinostat, a pan-HDAC-inhibitor for clinical acetylation status of non-histone proteins in the cyto- 12 33,34 treatment of lymphoma and multiple myeloma ,to plasm . Our results revealed that HDAC1 and/or reduce HDAC3 expression in SMMC-7721 cells. HDAC3 HDAC2 deficiency does not change the acetylation status inhibition significantly reduced tumor growth and of STAT3 in the liver, while loss of HDAC3 results in a decreased the expression of Ki67 in HCC xenograft substantial cytoplasmic accumulation of ac-STAT3, tumors (Fig. 7a–d). Consistently, the expression of p- indicating that the deacetylation of ac-STAT3 might be STAT3(Y705) in panobinostat-treated xenograft tumors selectively dependent on HDAC3 in the cytoplasm rather was significantly reduced (Fig. 7d, e). In addition, HDAC3 than in the nucleus. knockdown remarkably suppressed cell cycle progression After IL6 stimulation, STAT3 that resides in the cyto- and inhibited the phosphorylation of STAT3(Y705) plasm rapidly becomes phosphorylated and translocates (Supplementary Fig. 8). Therefore, increased HDAC3 into the nucleus to induce target genes expression . Our enhanced tumor growth that may occur through data show that HDAC3 deficiency significantly increases STAT3 signaling in liver cancer cells. ac-STAT3 level and decreases p-STAT3(Y705) level together with a decrease in STAT3 nucleus entry, sug- Discussion gesting that HDAC3-mediated deacetylation of ac-STAT3 HDAC3 is a crucial deacetylase component of the is essential for STAT3(Y705) phosphorylation and STAT3 NCOR1/SMRT complexes, which is involved in histone reactivation cycle. It has been shown that the conserved modifications, chromatin remodeling, and transcriptional lysine residues for acetylation in STAT3 are all near the 5,37 29 regulation . In addition to the important role of Y705 residual of STAT3 , supporting the possibility that HDAC3 in DNA repair and in the maintenance of the STAT3 acetylation induced by HDAC3 deficiency could circadian rhythm of metabolism homeostasis, the essen- prevent STAT3(Y705) phosphorylation and block tial non-transcriptional function of HDAC3 during STAT3 signaling activation. However, the detail mitosis is still largely unknown. Here, we have established mechanisms of HDAC3-mediated modifications of for the first time the importance of HDAC3 for liver STAT3 deacetylation and phosphorylation in hepatocytes regeneration together with liver cancer cells proliferation. remain further studies. Regardless of other mechanisms Two types of transmembrane enzyme-linked receptor- that might be regulated by HDAC3 during the whole mediated signaling cascades, namely, the cytokine- process of hepatocyte proliferation, HDAC3 is undoubt- dependent pathway and the growth factor-dependent edly critical to the initiation of the cell cycle progression pathway, which transduce extracellular growth stimuli through the proper transition of ac-STAT3 to p-STAT3. into the nucleus, play crucial roles in priming liver Therefore, HDAC3 may act as a molecular switch in the 1,23 regeneration . Our previous study showed that loss of STAT3 signaling cascade that transmits growth stimuli the stimulatory G protein α subunit (G α), which activates from the cytoplasm to the nucleus. the cAMP-dependent pathway, leads to a breakdown in Multiple studies have shown that HDAC3 is aberrantly growth factor signaling and dramatically arrests the expressed in various human cancers and that the aberrant 38 37 regenerating hepatocytes in the G1 phase extension . expression is often associated with a poor prognosis , Lack of HDAC3 results in a similar proliferative defect at making HDAC3 inhibition a potential strategy for cancer the cell cycle entrance, which indicates a breakdown in therapy. The HDAC3 mRNA level is lacked in one-sixth the cytokine-dependent or growth factor-dependent sig- of human HCC tissues . The inactivation of HDAC3 may naling cascade. Using a microarray assay analysis and contribute to HCC development by increasing global LPS/PH model, we showed that HDAC3 deficiency dra- histone acetylation, defective DNA damage repair, and matically impairs the STAT3 signaling pathway, which is mutation accumulation . HDAC3 is also upregulated in a Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 12 of 14 Fig. 7 (See legend on next page.) Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 13 of 14 (see figure on previous page) Fig. 7 Inhibition of HDAC3 expression decreases HCC xenografts growth. a–c HCC growth in mouse xenograft model. SMMC-7721 cells (2 × 10 ) were injected subcutaneously into nude mice for xenograft assay. Tumor volume and average weight of HCC xenografts in each group were shown (n = 4) (scale bar: 1 cm). d Immunohistochemical analysis of control and panobinostat-treated tumors with p-STAT3(Y705), Ki-67, and γ-H2A.X antibodies (scale bar: 50 μm). Quantitative analysis of p-STAT3(Y705), Ki-67, and γ-H2A.X-positive rates in each group of tumors were shown. e Western blot analysis demonstrates that p-STAT3(Y705) is inhibited in panobinostat-treated tumors. Histone H3 were used as the loading controls. f Diagram of the HDAC3–STAT3 axis in the IL6/STAT3 signaling pathway and the signaling cascades that regulate hepatocyte proliferation and tumorigenesis. All data represent the mean ± SD; *p < 0.05; **p < 0.01; n.s. not significant 12,13 specific subset of HCCs ; however, the role of HDAC3 For inducible deletion of HDAC3, tamoxifen was dis- in the pathogenesis of HCC remains unknown. The solved in corn oil containing 10% (vol/vol) ethanol to 20 upregulated expression of HDAC3 in human HCC mg/ml and shaken overnight at 37 °C . Six-week-old ERT2 loxP/loxP prompts us to determine whether the HDAC3–STAT3 male Alb-Cre ;HDAC3 mice were injected pathway enhances HCC tumor growth. Notably, IL6, a intraperitoneally with tamoxifen (2 mg per mouse) for five cytokine that is essential for activating STAT3 signaling in consecutive days. Experiments on mice were performed liver inflammation, enhances HCC development .We 5 days after the last injection. Then, these mice were found that overexpression of HDAC3 is significantly treated with either classical 2/3 PH surgery or intraper- associated with an increased p-STAT3(Y705) level and itoneal injection of CCl (10 ml/kg body weight) . BrdU Ki67 index in HCC. Moreover, our xenograft assay shows (1 mg/kg body weight) was intraperitoneally injected 60 that a low dose of panobinostat could significantly min before sacrifice. decrease HCC xenografts growth along with a significant reduction in p-STAT3(Y705) level. Our data suggest that Statistical analysis the drug panobinostat approved by FDA might also pre- Data were expressed as the mean ± standard deviation vent liver cancer cells proliferation through (SD). Statistical comparisons were assessed with an HDAC3–STAT3 signaling pathway in HDAC3- unpaired, one-tailed Student’s t-test with Welch correc- overexpressed HCC, while the detail effects and tion. A p-value < 0.05 was considered significant. mechanisms of panobinostat for the clinical treatment of Acknowledgements HCC remain further studies. We thank Dr. Qing Richard Lu for kindly providing transgenic mice. We also In summary, we have demonstrated that HDAC3 acts thank Li Li, Fei Chen and Lei Zhang for pathology technique assistance. This work was supported by grants from the Natural Science Foundation of China an essential role in the enhancement of the (No. 81472303), the National Key Clinical Project, and the Major Science and STAT3 signaling pathway by catalyzing the deacetylation Technology Project of Science & Technology Department of Sichuan Province of ac-STAT3 in the cytoplasm, thereby, promoting liver (2017SZ0003). regeneration and tumor growth, as illustrated in Fig. 7f. Author details Our findings highlight the importance of the Laboratory of Pathology, Key Laboratory of Transplant Engineering and HDAC3–STAT3 signaling cascade in promoting cell cycle Immunology, NHFPC; West China Hospital, Sichuan University, Chengdu progression and support HDAC3 as a potential ther- 610041, China. Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China. Department of Pediatrics, Division of apeutic target for the treatment of HCC as well as other Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati solid cancers whose HDAC3 is overexpressed. 4 Children’s Hospital Medical Center, Cincinnati, OH 25229, USA. Department of Liver and Vascular Surgery, West China Hospital, Sichuan University, Chengdu 610041, China Materials and methods Mice Conflict of interest All mouse experimental procedures were approved by The authors declare that they have no conflict of interest. the Animal Care and Use Committee of Sichuan Uni- loxP/loxP versity. HDAC3 frozen embryos were purchased Publisher's note from the European Mouse Mutant Archive (EMMA), Springer Nature remains neutral with regard to jurisdictional claims in University of Veterinary Medicine, Vienna. Alb-Cre and published maps and institutional affiliations. ERT2 Alb-Cre transgenic mice were purchased from Supplementary Information accompanies this paper at https://doi.org/ Shanghai Biomodel Organism Science & Technology 10.1038/s41419-018-0428-x. Development Co., Ltd, China. The genotypes of Alb-Cre; loxP/loxP ERT2 loxP/loxP HDAC3 and Alb-Cre ;HDAC3 mice Received: 30 October 2017 Revised: 4 January 2018 Accepted: 12 January were determined using PCR amplification of tail DNA. The sequences of genotyping primers are presented in Supplementary Table 1. Official journal of the Cell Death Differentiation Association Lu et al. Cell Death and Disease (2018) 9:398 Page 14 of 14 References 21. Vidal-Laliena, M. et al. Histone deacetylase 3 regulates cyclin A stability. J. Biol. 1. Taub, R. Liver regeneration: from myth to mechanism. Nat. Rev. Mol. Cell Biol. 5, Chem. 288,21096–21104 (2013). 836–847 (2004). 22. Berridge, M. J. Module 2: cell signalling pathways. Cell Signal. Biol. 6,1–138 2. Michalopoulos, G. K. Liver regeneration. J. 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