Dual inhibition of AKT ‐m TOR and AR signaling by targeting HDAC 3 in PTEN ‐ or SPOP ‐mutated prostate cancer
2018; Springer Nature; Volume: 10; Issue: 4 Linguagem: Inglês
10.15252/emmm.201708478
ISSN1757-4684
AutoresYuqian Yan, Jian An, Yinhui Yang, Di Wu, Yang Bai, William Cao, Linlin Ma, Junhui Chen, Zhendong Yu, Yundong He, Xin Jin, Yunqian Pan, Tao Ma, Shangqian Wang, Xiaonan Hou, Saravut J. Weroha, R. Jeffrey Karnes, Jun Zhang, Jennifer J. Westendorf, Liguo Wang, Yu Chen, Wanhai Xu, Runzhi Zhu, Dejie Wang, Haojie Huang,
Tópico(s)Ubiquitin and proteasome pathways
ResumoResearch Article9 March 2018Open Access Source DataTransparent process Dual inhibition of AKT-mTOR and AR signaling by targeting HDAC3 in PTEN- or SPOP-mutated prostate cancer Yuqian Yan Yuqian Yan Department of Gastroenterology, Jiangxi Institute of Gastroenterology and Hepatology, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jian An Jian An Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yinhui Yang Yinhui Yang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China Search for more papers by this author Di Wu Di Wu Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yang Bai Yang Bai Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China Search for more papers by this author William Cao William Cao Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Linlin Ma Linlin Ma Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China Search for more papers by this author Junhui Chen Junhui Chen Department of Minimally Invasive Intervention, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China Search for more papers by this author Zhendong Yu Zhendong Yu Central Laboratory, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China Search for more papers by this author Yundong He Yundong He Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Xin Jin Xin Jin Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yunqian Pan Yunqian Pan Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Tao Ma Tao Ma Department of Biomedical Statistics and Informatics, Mayo Clinic Cancer Center, Rochester, MN, USA Search for more papers by this author Shangqian Wang Shangqian Wang Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Xiaonan Hou Xiaonan Hou Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Saravut John Weroha Saravut John Weroha Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author R Jeffrey Karnes R Jeffrey Karnes Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jun Zhang Jun Zhang Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jennifer J Westendorf Jennifer J Westendorf Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Liguo Wang Liguo Wang Department of Biomedical Statistics and Informatics, Mayo Clinic Cancer Center, Rochester, MN, USA Search for more papers by this author Yu Chen Yu Chen Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Wanhai Xu Wanhai Xu Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China Search for more papers by this author Runzhi Zhu Corresponding Author Runzhi Zhu [email protected] orcid.org/0000-0002-9565-7261 Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China Search for more papers by this author Dejie Wang Corresponding Author Dejie Wang [email protected] orcid.org/0000-0003-3870-2385 Department of Gastroenterology, Jiangxi Institute of Gastroenterology and Hepatology, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Haojie Huang Corresponding Author Haojie Huang [email protected] orcid.org/0000-0003-2751-6413 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yuqian Yan Yuqian Yan Department of Gastroenterology, Jiangxi Institute of Gastroenterology and Hepatology, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jian An Jian An Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yinhui Yang Yinhui Yang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China Search for more papers by this author Di Wu Di Wu Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yang Bai Yang Bai Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China Search for more papers by this author William Cao William Cao Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Linlin Ma Linlin Ma Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China Search for more papers by this author Junhui Chen Junhui Chen Department of Minimally Invasive Intervention, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China Search for more papers by this author Zhendong Yu Zhendong Yu Central Laboratory, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China Search for more papers by this author Yundong He Yundong He Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Xin Jin Xin Jin Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yunqian Pan Yunqian Pan Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Tao Ma Tao Ma Department of Biomedical Statistics and Informatics, Mayo Clinic Cancer Center, Rochester, MN, USA Search for more papers by this author Shangqian Wang Shangqian Wang Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Xiaonan Hou Xiaonan Hou Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Saravut John Weroha Saravut John Weroha Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author R Jeffrey Karnes R Jeffrey Karnes Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jun Zhang Jun Zhang Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jennifer J Westendorf Jennifer J Westendorf Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Liguo Wang Liguo Wang Department of Biomedical Statistics and Informatics, Mayo Clinic Cancer Center, Rochester, MN, USA Search for more papers by this author Yu Chen Yu Chen Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Wanhai Xu Wanhai Xu Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China Search for more papers by this author Runzhi Zhu Corresponding Author Runzhi Zhu [email protected] orcid.org/0000-0002-9565-7261 Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China Search for more papers by this author Dejie Wang Corresponding Author Dejie Wang [email protected] orcid.org/0000-0003-3870-2385 Department of Gastroenterology, Jiangxi Institute of Gastroenterology and Hepatology, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Haojie Huang Corresponding Author Haojie Huang [email protected] orcid.org/0000-0003-2751-6413 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Author Information Yuqian Yan1,2,‡, Jian An2,13,‡, Yinhui Yang2,3,‡, Di Wu2, Yang Bai2,3, William Cao2, Linlin Ma2,4, Junhui Chen5, Zhendong Yu6, Yundong He2, Xin Jin2, Yunqian Pan2, Tao Ma7, Shangqian Wang8, Xiaonan Hou9, Saravut John Weroha9, R Jeffrey Karnes10, Jun Zhang11, Jennifer J Westendorf2, Liguo Wang7, Yu Chen8, Wanhai Xu3, Runzhi Zhu *,4, Dejie Wang *,1,2 and Haojie Huang *,2,10,12 1Department of Gastroenterology, Jiangxi Institute of Gastroenterology and Hepatology, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China 2Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA 3Department of Urology, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, China 4Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China 5Department of Minimally Invasive Intervention, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China 6Central Laboratory, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China 7Department of Biomedical Statistics and Informatics, Mayo Clinic Cancer Center, Rochester, MN, USA 8Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 9Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA 10Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA 11Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA 12Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA 13Present address: Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 511-84405370; E-mail: [email protected] *Corresponding author. Tel: +86 79188692507, E-mail: [email protected] *Corresponding author. Tel: +1 507-293-1712, E-mail: [email protected] EMBO Mol Med (2018)10:e8478https://doi.org/10.15252/emmm.201708478 See also: A Zoubeidi & ME Gleave (April 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract AKT-mTOR and androgen receptor (AR) signaling pathways are aberrantly activated in prostate cancer due to frequent PTEN deletions or SPOP mutations. A clinical barrier is that targeting one of them often activates the other. Here, we demonstrate that HDAC3 augments AKT phosphorylation in prostate cancer cells and its overexpression correlates with AKT phosphorylation in patient samples. HDAC3 facilitates lysine-63-chain polyubiquitination and phosphorylation of AKT, and this effect is mediated by AKT deacetylation at lysine 14 and 20 residues and HDAC3 interaction with the scaffold protein APPL1. Conditional homozygous deletion of Hdac3 suppresses prostate tumorigenesis and progression by concomitant blockade of AKT and AR signaling in the Pten knockout mouse model. Pharmacological inhibition of HDAC3 using a selective HDAC3 inhibitor RGFP966 inhibits growth of both PTEN-deficient and SPOP-mutated prostate cancer cells in culture, patient-derived organoids and xenografts in mice. Our study identifies HDAC3 as a common upstream activator of AKT and AR signaling and reveals that dual inhibition of AKT and AR pathways is achievable by single-agent targeting of HDAC3 in prostate cancer. Synopsis Both AKT and AR signaling pathways are activated in PTEN-deficient or SPOP-mutated prostate cancers and inhibition of one pathway often activates the other. AKT and AR pathways in PTEN-deficient or SPOP-mutated prostate cancers are both inhibited by the same HDAC3 inhibitor RGFP966. HDAC3 is the only member of the class I/II HDAC family that regulates AKT phosphorylation and its expression correlates with AKT phosphorylation in prostate cancer patient specimens. HDAC3 regulation of AKT phosphorylation is mediated by deacetylation of K14 and K20 residues on AKT and the function of HDAC3 in the cytoplasm. Conditional knockout of Hdac3 gene blocks Pten deletion-induced Akt activation, prostate tumorigenesis and progression, and overall survival of mice. The selective HDAC3 inhibitor RGFP966 inhibits growth of both PTEN-deficient and SPOP-mutated prostate cancer cells by suppressing phosphorylation of AKT and its downstream effector kinases and AR expression. Introduction The majority of prostate cancers are dependent on androgens and activation of the androgen receptor (AR) for growth and survival, and androgen deprivation therapy remains the mainstay of treatment for advanced prostate cancer (Watson et al, 2015). The PI3K/AKT pathway is often aberrantly activated due to frequent deletion or mutation of the PTEN tumor suppressor gene and activation mutations in PIK3CA and AKT1 genes during prostate tumorigenesis and progression (Cancer Genome Atlas Research Network, 2015, Robinson et al, 2015), representing another key actionable target. However, both patient data analysis and pre-clinical animal model studies invariably show that loss of PTEN promotes enhanced AKT activity and reduced AR signaling and that inhibition of AKT results in AR activation while blockade of AR function increases AKT activities (Carver et al, 2011; Mulholland et al, 2011), stressing the requirement of co-targeting of both pathways for effective treatment of prostate cancer. HDACs belong to a super family of proteins. In humans, there are 18 HDAC proteins categorized into four distinct classes (I, II, III, and IV) according to their homology to yeast proteins, subcellular location, and enzymatic activities (de Ruijter et al, 2003; Gallinari et al, 2007). The HDAC family was originally found to be involved in deacetylating the histone core of nucleosomes to configure chromosomal structure and regulate gene expression (Taunton et al, 1996). Also, it is implicated in regulating deacetylation and phosphorylation of non-histone proteins (Kouzarides, 2000; Kramer et al, 2009). In fact, HDAC inhibitors (HDACIs) have been developed for cancer therapy (Dokmanovic et al, 2007), supporting the critical oncogenic role of HDACs in tumorigenesis. HDAC3, a class I HDAC, plays critical roles in S phase progression, DNA damage control, maintenance of genomic stability, and T-cell development (Bhaskara et al, 2008, 2010; Wang et al, 2015). HDAC3 is overexpressed in a majority of prostate cancers (Weichert et al, 2008), implying a role of HDAC3 in prostate tumorigenesis. Depletion of HDAC3 or other HDACs suppresses expression of AR and its downstream target genes in prostate cancer cells, although the underlying mechanism remains poorly understood (Welsbie et al, 2009). Additionally, it has been shown that HDAC3 knockdown dramatically reduces leukemia and lymphoma cell proliferation (Matthews et al, 2015). These data suggest that HDAC3 could be a therapeutic target for cancers such as those in the prostate. Speckle-type POZ protein (SPOP) is the substrate-binding adaptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex (Zhuang et al, 2009). The gene encoding SPOP is the most frequently mutated gene in human primary prostate cancers (Barbieri et al, 2012; Cancer Genome Atlas Research Network, 2015). Functional studies show that ectopic expression of the most frequent SPOP-mutant F133V in human prostatic cells or knock-in in the mouse prostate results in aberrant activation of AR and AKT-mTORC1 signaling (An et al, 2014, 2015; Geng et al, 2014; Blattner et al, 2017; Zhang et al, 2017). Most importantly, the Cancer Genome Atlas (TCGA) data demonstrate that SPOP-mutated prostate cancers exhibit the highest AR activity among all molecular subtypes of prostate cancer examined (Cancer Genome Atlas Research Network, 2015). Thus, it is important to identify a common target to inhibit both AKT-mTORC1 and AR signaling in SPOP-mutated prostate cancer. In the present study, we demonstrated that HDAC3 is required for AKT phosphorylation in prostate cancer cells. Prostate-specific knockout of Hdac3 decreased Akt phosphorylation, alleviated the tumor burden, and ultimately prolonged survival of Pten knockout mice. In human prostate cancer organoids and xenograft models, we further showed that a selective HDAC3 inhibitor is efficacious in inhibition of AKT and AR signaling in both PTEN- and SPOP-mutant background. Results HDAC3 is the only class I/II HDAC protein that regulates AKT phosphorylation It has been shown previously that different pan class I/II HDACIs have differential effects on AKT phosphorylation at both threonine 308 (T308) and serine 473 (S473) in AR-negative prostate cancer PC-3 cells (Chen et al, 2005a). By treating AR-positive prostate cancer C4-2 cells with the commonly used pan class I/II HDACIs trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), panobinostat (LBH589), and HDAC6-selective inhibitor tubastatin A, we demonstrated that these pan class I/II HDACIs, but not tubastatin A completely inhibited AKT phosphorylation at S473 and T308 (Fig 1A). To further clarify whether this was due to the possibility that these HDACIs potentially regulate the expression of upstream regulators (e.g., CXCR7 or PHLPP1) (Wang et al, 2008; Luan et al, 2009; Bradley et al, 2013), and thereby indirectly affect AKT phosphorylation, we treated C4-2 cells with cycloheximide (CHX) to block de novo protein synthesis. To our surprise, CHX treatment only had very minimal effect on pan HDACI-induced inhibition of AKT phosphorylation (Fig 1A), suggesting that decreased AKT phosphorylation by pan class I/II HDACIs was not primarily mediated by their effect on expression of AKT upstream regulators. Figure 1. HDAC3 regulates AKT phosphorylation HDACIs inhibited AKT phosphorylation. C4-2 cells were pre-treated with 20 μM of CHX for 30 min followed by treatment with pan HDACIs TSA (1 μM), SAHA (5 μM), LBH589 (0.1 μM), or a HDAC6 selective inhibitor Tuba (5 μM) for 24 h prior to Western blot analysis with indicated antibodies. The efficacy of CHX was evident by blockade of induction of FBP1 expression by HDACIs as reported (Yang et al, 2017). The knockdown efficiency of each member of class I/II HDACs by shRNAs. C4-2 cells were stably infected with control or gene-specific shRNAs for 4 days and harvested for RT–qPCR. Expression of each gene was first normalized to the level of GAPDH, and then the expression level of each gene in gene knockdown cells was normalized by that in control knockdown cells. The shRNA knockdown efficiency was determined by subtracting the normalized value from 100%. Data represents means ± SEM. The RT–qPCR was performed in triplicate for each sample. Control or gene-specific knockdown C4-2 cells were serum starved for 24 h and then cultured in regular culture medium for 12 h followed by Western blots for indicated proteins. Western blot bands for total and phosphorylated AKT were quantified and normalized to the quantified values of β-Tubulin (loading control). The normalized values were further normalized to the value of shC-infected cells without serum stimulation. C4-2 cells were treated with vehicle (DMSO) or HDAC3 inhibitor RGFP966, and at different time points, cells were harvested for Western blots with the indicated antibodies. The representatives of IHC staining for HDAC3 and AKT S473 phosphorylation in prostate cancer patient specimens; scale bar: 50 μm; scale bar for the inset: 20 μm. Correlation between expression of AKT S473 phosphorylation and HDAC3 was shown, n = 55, ***P = 1.87e-07 was performed by Pearson's product-moment correlation test. Source data are available online for this figure. Source Data for Figure 1 [emmm201708478-sup-0003-SDataFig1.tif] Download figure Download PowerPoint To identify which member(s) in the class I/II HDAC family is the major modulator of AKT phosphorylation, all 11 members in these subfamilies were knocked down individually by two independent small hairpin RNAs (shRNAs). Each HDAC gene was effectively knocked down to 40% or more at mRNA level (Fig 1B). Notably, only HDAC3 knockdown substantially decreased AKT phosphorylation at both T308 and S473 residues in a similar degree in C4-2 cells (Fig 1C). We also examined the effect of a selective HDAC3 inhibitor, RGFP966, on AKT phosphorylation. We demonstrated that RGFP966 inhibited AKT phosphorylation as early as 0.5 h post-treatment (Fig 1D), further suggesting a direct effect of HDAC3 inhibition of AKT phosphorylation. Together, these data indicate that HDAC3, but not other class I/II HDACs, is primarily required for AKT phosphorylation in this cell line. Consistent with a previous finding that class I HDAC members (HDAC1, 2, 3, and 8) are highly expressed in prostate cancers (Weichert et al, 2008), analysis of TCGA data also showed that expression of these four HDAC genes was upregulated at the mRNA level in tumors compared to normal tissues (Fig EV1A). Specifically, comparison of 52 paired normal and tumor samples showed that the majority of them [approximately 56% (29 out of 52)] exhibited an increased expression of HDAC3 at the mRNA level in tumors (Fig EV1B), suggesting that HDAC3 is a highly relevant protein in prostate cancer. We further examined the correlation between HDAC3 protein expression and AKT phosphorylation by performing immunohistochemistry (IHC) on a tissue microarray (TMA) containing 55 prostate cancer samples. We demonstrated that increased expression of HDAC3 correlated with higher levels of AKT phosphorylation (S473) in this cohort of patients (Fig 1E and F). Therefore, HDAC3 might be an essential upstream regulator of AKT phosphorylation in prostate cancer cells in culture and in patients. Click here to expand this figure. Figure EV1. HDAC3 is overexpressed in prostate cancer patient specimens The mRNA level of 11 HDAC gene family members was compared between normal and tumor tissues (the mRNA expression data were extracted from the TCGA project). P-values were performed between normal tissues (n = 52) with tumor tissues (n = 497) for each gene by Wilcoxon rank sum test with continuity correction and shown alongside the graph. The description for box plot is the same as the figure legend in Fig 7H. The mRNA level of HDAC3 gene was compared between paired normal and cancer tissues for individual patient. Normal/tumor paired samples were available only in 52 patients in the TCGA cohort. Download figure Download PowerPoint HDAC3 is required for growth factor-induced AKT polyubiquitination and activation Polyubiquitination is a critical step for growth factor-induced phosphorylation and activation of AKT (Yang et al, 2009). Given that acetylation and polyubiquitination can compete with each other by occurring at the same lysine residues (Yang & Seto, 2008), we investigated whether HDAC3 regulates AKT acetylation and polyubiquitination. Firstly, we showed that HDAC3 overexpression substantially decreased AKT acetylation without affecting total AKT level in C4-2 cells (Fig 2A). Decreased AKT acetylation was accompanied with increased polyubiquitination of AKT, and the effect was dose-dependent (Fig 2B). In agreement with the observation that HDAC3 knockdown decreased AKT phosphorylation (Fig 1C), overexpression of HDAC3 increased AKT phosphorylation at both T308 and S473 sites (Fig 2C). In contrast, depletion of HDAC3 by a pool of three independent siRNAs increased AKT acetylation (Fig 2D), but decreased AKT polyubiquitination and phosphorylation (Fig 2D and E). The HDAC3 inhibitor RGFP966 also undermined AKT ubiquitination (Fig 2F). Figure 2. HDAC3 is important for growth factor-induced AKT deacetylation and polyubiquitination A. 293T cells were transfected with plasmids for Flag-HDAC3 and Myc-AKT followed by IP and Western blots with the indicated antibodies. B. C4-2 cells were transfected with plasmids for Myc-AKT, HA-Ub, and Flag-HDAC3 (different doses) for 24 h followed by IP and Western blots with the indicated antibodies. C. C4-2 cells were transfected with plasmids for Myc-AKT and Flag-HDAC3 (different doses) for 24 h followed by Western blots with the indicated antibodies. D, E. C4-2 cells were transfected with a pool of control or HDAC3-specific siRNAs for 48 h followed by IP and/or Western blots with the indicated antibodies. F. C4-2 cells were transfected with HA-Ub plasmids and treated with 3 μM of RGFP966 for 24 h followed by IP and/or Western blots with the indicated antibodies. G. C4-2 cells were treated with 10 ng/ml of IGF-1 for different periods of time and harvested for IP and Western blots with the indicated antibodies. H, I. C4-2 cells were transfected with a pool of control and HDAC3-specific siRNAs for 48 h and then treated with IGF-1 (H) or EGF (I) followed by Western blots for indicated proteins. Data information: The asterisk (*) indicates the specific HDAC3 protein band. Source data are available online for this figure. Source Data for Figure 2 [emmm201708478-sup-0004-SDataFig2.tif] Download figure Download PowerPoint Growth factors, such as insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF), are the potent upstream stimulators of AKT signaling pathway (Song et al, 2005; Morgan et al, 2009). To examine whether growth factor stimulation affects AKT acetylation, we treated C4-2 cells with IGF-1 and found that AKT acetylation was inhibited by IGF-1 in a dose-dependent manner, while its phosphorylation at both T308 and S473 was concomitantly upregulated (Fig 2G). These data further confirmed that AKT phosphorylation is negatively regulated by its acetylation. Most importantly, knockdown of HDAC3 abolished IGF-1-induced elevation of AKT phosphorylation (Fig 2H). Similar to IGF-1, EGF is another well-known growth factor that activates the PI3K/AKT signaling pathway. Similarly, we found that AKT phosphorylation at T308 and S473 sites was enhanced by EGF treatment in a time-dependent manner, but the magnitude of AKT activation was substantially diminished in HDAC3 knockdown cells (Fig 2I). Our data show that HDAC3 is required for growth factor-stimulated phosphorylation of AKT. A functional AKT-binding domain is identified in HDAC3 To understand the molecular mechanisms by which HDAC3 regulates acetylation, polyubiquitination, and phosphorylation of AKT, we examined whether HDAC3 interacts with AKT. Reciprocal co-immunoprecipitation (co-IP) assays showed that both ectopically expressed and endogenous HDAC3 and AKT proteins were detectable in the same protein complex in 293T and C4-2 cell lines, respectively (Fig 3A–C). In contrast, consistent with the unbiased screening results (Fig 1B and C), endogenous AKT did not interact with endogenous HDAC1 and HDAC2 in C4-2 cells (Fig 3D). Figure 3. A region (aa 141–160) in HDAC3 is indispensable for its interaction with AKT A, B. 293T cells were transfected with indicated plasmids for 24 h followed by IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific Flag-HDAC3 protein band. C, D. C4-2 cell lysate was prepared for IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific Flag-HDAC3 protein band. E. An illustration depicts four fragments of HDAC3 protein for generation of GST recombinant proteins. F, G. C4-2 cells were transfected with Myc-tagged AKT for 24 h, and cell lysate was prepared for GST pull-down assay. Input and GST or GST-HDAC3 recom
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