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Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2 Lin Geng*, Xing Chen, Meng Zhang, Zhenkai Luo Department of Urology, China-Japan Friendship Hospital, No. 2 Yinghua Dongjie, Chaoyang District, Beijing, 100029, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2019 Accepted 24 December 2019 Available online xxx
Activating Transcription Factor 2 (ATF2) is a member of the ATF/CREB bZIP family of transcription factors and an oncogene in prostate cancer. ATF2 has been reported to be a critical substrate of the CUL3-SPOPRBX1 E3 ubiquitin ligase complex and the recurrent somatic mutation of SPOP has been believed to be a key feature of prostate cancer. However, the deubiquitinating enzyme required for ATF2 stabilization is still unknown. Here, we show that ATF2 is associated with ubiquitin-specific protease 14 (USP14), which increased the protein abundance and transcriptional activity of ATF2. Pharmacologic inhibition or siRNAmediated depletion of USP14 resulted in the decline and inactivation of ATF2. USP14 deubiquitinates and activates ATF2, resulting in enhanced prostate cancer cells proliferation both in vitro and in vivo. Importantly, silencing of ATF2 largely attuned USP14-mediated prostate cancer cells proliferation. Thus, our data revealed a critical role of USP1-ATF2 axis in the progress of prostate cancer and the inhibition of USP14 might be a promising strategy against prostate cancer. © 2020 Elsevier Inc. All rights reserved.
Keywords: ATF2 USP14 Deubiquitination Prostate cancer
1. Introduction Prostate cancer is the second most common cancer among men worldwide and is the third primary cause of male cancer-related mortality in United States. Prostate cancer accounts for 19% of estimated new cancer cases in men with more than 250,000 deaths each year [1]. Epidemiological studies have shown that the multifactorial aetiology of prostate cancer is related to factors such as age, ethnicity, genomic alterations, geographical location and diet and inflammation [2,3]. Activating Transcription Factor 2 (ATF2) is a member of the ATF/ CREB bZIP family of transcription factors, and shares a common feature, the b ZIP domain of this family [4]. The b ZIP domain contains two subdomains: a leucine zipper substructure region and an alkaline substructure region, which contains an N-terminal nuclear export sequence [5]. The transcriptional function of ATF2 protein is activated in two ways: to form a homodimer by binding to the b ZIP domain of another ATF2 protein or a heterodimer with other b ZIP contained proteins [6,7]. The resulting dimer is then associated with a specific DNA transcript sequence to promote genes transcription [8]. In response to stress stimuli, ATF2 is
subjected to phosphorylation by JNK and p38, leading to its transcriptional activation and the transcription of a variety of targets including cyclin A, cyclin D, SOX9, MMP9, and TGFB2 [9e11]. Interestingly, ATF2 plays a dual role in both tumor suppression and cancer promotion, in a context-dependent manner. For example, ATF2 acts as an oncogene in prostate cancer, melanoma, non-small cell lung carcinoma and pancreatic cancer, while a tumor suppressor in breast cancer [5,12e14]. In prostate cancer, ATF2 has been reported to be a critical substrate of a functional CUL3-SPOP-RBX1 E3 ubiquitin ligase complex and the recurrent somatic mutation of SPOP has been believed to be a key feature of prostate cancer [15,16]. Thus, the fine tune of ATF2 protein might contribute to facilitating prostate cancer progress. Although most ubiquitination processes can be antagonized by deubiquitinating enzymes, the deubiquitinating enzyme that required for ATF2 stabilization is still unknown. Here, we show that ATF2 is associated with ubiquitin-specific protease 14 (USP14), which deubiquitinates and activates ATF2, and results in enhanced cell proliferation of prostate cancer cells. 2. Material and methods 2.1. Cell culture and reagents
* Corresponding author. E-mail address: [email protected] (L. Geng).
HEK293, prostate cancer cell lines LNCaP and PC3 were obtained
https://doi.org/10.1016/j.bbrc.2019.12.128 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone), 100 IU/ml penicillin and 100 mg/ml streptomycin and maintained at 37 C in a humidified atmosphere containing 5% CO2. Cycloheximide (239764) was purchased from Calbiochem (San Diego, CA, USA). The proteasome inhibitor MG132 was purchased from SigmaAldrich (St. Louis, MO, USA). The USP14 specific inhibitor IU1 was purchased from BioVision (1845e25). 2.2. Cell counting Kit-8 (CCK-8) assay Cells were grown in 96-well plates and incubated for about five days. Cell viability was measured with CCK-8 kit according to the manufacturer’s protocol. At each time, 10 ml CCK-8 was added into each well and incubated at 37 C for 2 h. The optical density (OD) value was measured with Microplate Reader at 450 nm.
spectrometry analysis. For western blotting, equivalent amounts of protein sample was denatured in the loading buffer, and resolved by 10e12% SDS-PAGE and transferred onto NC membranes. Membranes were blocked in 5% non-fat milk for 1 h before incubation with primary antibody overnight at 4 C. The following primary antibodies were used: anti-ATF2 antibody at 1:2000 (#sc-242, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-USP14 antibody at 1:2000 (#11931, Cell Signaling Technology), anti-GAPDH antibody at 1:5000 (#sc-32233, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HA antibody at 1:2000 (#3724, Cell Signaling Technology), anti-Flag M2 antibody (#F1804, Sigma-Aldrich St. Louis, MO, USA), anti-GST antibody at 1:5000 (#sc-138, Santa Cruz Biotechnology, Santa Cruz, CA, USA). GAPDH served as an internal control. 2.7. Deubiquitylation assay
Cells were grown in 12-well plates at a density of 5000 cells and incubated for about 10 days. The culture medium was changed every four days. Cells were then fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 10 min. The cell colonies were photographed with a microscope. For the quantification of colonies, 30% sodium acetate was added to test the crystal violet OD value for each well. The statistical analysis was analyzed with three replicates.
The in vivo deubiquitylation assays were performed under denaturing conditions. PC3 cells were grown in 10 cm plates to 80% confluence and then were transiently transfected with empty vector (EV) or Flag-USP14 for 36 h using lipo2000 transfection reagent. Cells were harvested, lysed in denaturing lysis buffer [1% SDS, 50 mM Tris, 10 mM DTT (pH 7.5)], and boiled at 95 C for 5 min. The denatured proteins were diluted in RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1% NP40 and 0.1% SDS and immunoprecipitated by Agarose TUBE1 resin (UM401, Agarose-TUBE1, Nacalai Tesque) for ubiquitin chain enrichment and immunoblotted as indicated.
2.4. RNA isolation and quantitative real-time PCR
2.8. Cycloheximide (CHX) assay
Total RNAs from cells were extracted by using Trizol reagents (Invitrogen). The mRNAs were then reversed transcripted into cDNA using the Promega Reverse Transcription System (Madison, WI, USA). Oligo dT was used to prime cDNA synthesis. Quantitative real-time PCR was performed using a SYBR Green Premix Ex Taq (Takara, Japan) on Light Cycler 480 (Roche, Switzerland). The GAPDH mRNA levels were used as internal control. Primers used for qPCR analysis were available upon request.
PC3 cells were seeded overnight in complete medium in 6-well plates to 80% confluence and then transfected with control-shRNA or USP14-shRNA for 36 h followed by the addition of 20 mM cycloheximide (CHX). Samples at the indicated times were harvested for immunoblot analysis.
2.3. Colony formation assay
2.5. Luciferase reporter assays To monitor the transfection activity of ATF2, the phRL-3CRE-TK plasmid which contains three cyclic AMP-responsive elements (CREs) was used. The phRL-3CRE-TK and the pRL-null plasmid encoding Renilla luciferase were co-transfected with other plasmids for 36 h. Luciferase activity was measured using the Dual Luciferase Reporter Assay System. The firefly luciferase luminescence data were normalized by dividing the Renilla luciferase luminescence data. Results are expressed relative to the activity in vector control. 2.6. Immunoprecipitation (IP), mass spectrometry and western blotting Cells were lysed in RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1% NP40 and 0.1% sodium dodecyl sulfate (SDS). Lysates were cleared by centrifugation at 15,000 g for 20 min at 4 C to remove cell debris. The resulting lysates were subjected to IP with either anti-USP14 or anti-ATF2 antibodies overnight at 4 C. Protein G beads were then added for additional 4 h. For affinity purification, 293T cells transfected with control or Flag-ATF2 plasmids were subjected to Flag M2 beads purification. Bound proteins were resolved by SDS-PAGE and stained with Coomassie blue staining, followed by mass
2.9. Xenograft models This study was conducted in compliance with Institutional Animal Care and Use Committee of institutional ethical guidelines in China-Japan Friendship Hospital. All 4e8 weeks old BALB/c nude mice were purchased from the Shanghai Laboratory Animal Company (SLAC) and housed at specific facility. The mice were divided into groups randomly and the indicated cells were implanted subcutaneously on the axilla of mice with DMEM medium with FBS. Four weeks after injection, mice were sacrificed, and the weight and volume of tumors were measured. 2.10. Statistical analysis All in vitro experiments were repeated for three times. Data are expressed as the means ± SD. The differences between groups were calculated using the Student’s t-test or one-way ANOVA using a Tukey post-hoc test. Statistical analyses were performed using Graphpad 6.0. A value of P < 0.05 was considered to indicate a statistically significant difference. *p < 0.05,**p < 0.01, ***p < 0.001. 3. Results 3.1. USP14 is associated with ATF2 To investigate how ATF2 was regulated, we first analyzed the interacting protein complex of ATF2 by immunoprecipitation combined with mass spectrometry (MS) assay. Flag-con or Flag-
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Fig. 1. USP14 is associated with ATF2. A. A partial list of proteins identified by mass spectrometry analysis. 293T cells transfected with Flag-control or Flag-ATF2 plasmids were subjected to Flag M2 beads purification. Bound proteins were resolved by SDS-PAGE and stained with Coomassie blue staining, followed by mass spectrometry analysis. B. 293T cells transfected with Flag-control or Flag-ATF2 plasmids were subjected to Flag M2 beads purification and immunoblotting with antibodies against Flag and USP14. C. The cell lysates of PC3 cells were subjected to immunoprecipitation with anti-ATF2 or IgG antibodies. The bound proteins were detected by immunoblotting with antibodies against USP14 and ATF2. D. The cell lysates of LNCaP cells were subjected to immunoprecipitation with anti-USP14 or IgG antibodies. The bound proteins were detected by immunoblotting with antibody against USP14 and ATF2. E. 293T cells transfected Flag-ATF2 plasmid were lysed and the lysate was incubated with GST-USP14 immobilized on GST-Sepharose beads, and bound ATF2 was detected by immunoblotting with antibody against Flag.
ATF2 plasmids were transfected into 293T cells which were harvested and lysed 48 h later. The cell lysate was incubated with Flag M2 beads for immunoprecipitation. The immunoprecipitate was then subjected to MS analysis. Our MS data revealed several known ATF2 interacting proteins, including ATF1, MAPK1, MAPK14 and CREBBP [17e19], which demonstrated the reliability of our data (Fig. 1A). Meanwhile, we also identified a new ATF2 interacting protein, USP14. To confirm the MS data, the Flag-ATF2 immunoprecipitate was subjected to immunoblot with anti-USP14
antibody. We found that endogenous USP14 was readily detected in Flag-ATF2 immunoprecipitate (Fig. 1B). Furthermore, we performed immunoprecipitation assay by using anti-ATF2 or -USP14 antibodies to enrich the endogenous ATF2 or USP14 proteins in PC3 cells or LNCaP cells, respectively. The interaction between endogenous ATF2 and USP14 was clearly observed (Fig. 1C and D). Furthermore, through GST-pull-down assay, we found that USP14 bound directly to ATF2 protein (Fig. 1E). Therefore, these results indicate that USP14 is an endogenous ATF2 interacting protein.
Fig. 2. USP14 deubiquitinates and stabilizes ATF2. A. Lysates of PC3 cells transduced with empty vector (EV) or increased doses of Flag-USP14 were immunoblotted with indicated antibodies. B. Lysates of PC3 cells transduced with empty vector (EV) or Flag-USP14 wild type (WT) or Flag-USP14 catalytic inactivated mutant (C114A) were immunoblotted with indicated antibodies. C. The mRNA levels of ATF2 in PC3 cells transduced with empty vector (EV) or Flag-USP14 wild type (WT) or Flag-USP14 mutant (C114A) were analyzed by realtime PCR. D. PC3 cells were treated with DMSO control or 50 mM IU1 for 12 h, and the cell lysates were immunoblotted with indicated antibodies. E. PC3 cells were transfected with shRNA control or USP14-shRNA for 36 h, and the cell lysates were immunoblotted with indicated antibodies. F. PC3 cells were transfected with shRNA control or USP14-shRNA for 36 h, 20 mM cycloheximide (CHX) was added for the indicated time. The cell lysates were immunoblotted with indicated antibodies (n ¼ 3 for each group). G. PC3 cells were transfected with empty vector (EV) or Flag-USP14 for 36 h, cell lysates were immunoprecipitated by Agarose TUBE1 resin for ubiquitin chain enrichment, and immunoblotted as indicated.
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Fig. 3. USP14 regulates the transcriptional activity of ATF2. A. 293T cells were co-transfected empty vector (EV) or Flag-USP14 wild type (WT) or Flag-USP14 mutant (C114A) with luciferase reporter plasmid containing the binding site of ATF2 for 36 h. Luciferase activity was measured using the Dual Luciferase Reporter Assay System. B. 293T cells were cotransfected shRNA control or USP14-shRNA with luciferase reporter plasmid containing the binding site of ATF2 for 36 h. Luciferase activity was then measured. C. The mRNAs expression of three ATF2 transcriptional targets including Cyclin D1, SOX9 and TGFB2 in PC3 cells were analyzed by real-time PCR. Data are presented as mean ± SD of three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001.
3.2. USP14 deubiquitinates and stabilizes ATF2 USP14 belongs to the deubiquitinating enzyme family and could interact with and stabilize its substrate proteins by removing the ubiquitin chain from them [20]. The interaction between USP14 and ATF2 prompted us to ask whether USP14 could regulate the protein level of ATF2. To this end, we overexpressed USP14 into PC3 cells and found that USP14 induced the expression of ATF2 in a dose-dependent manner (Fig. 2A). This regulation is closely related to the deubiquitination activity of USP14, because overexpression of the catalytic inactivated (CI) USP14 C114A mutant failed to induce ATF2 expression (Fig. 2B). However, the mRNA levels of ATF2 were not affected by overexpression of USP14 wild type (WT) or USP14 C114A mutant (Fig. 2C), suggesting that the regulation of ATF2 by USP14 occurs at the post-translational level. Consistently, ATF2 expression was declined by the addition of the USP14-specific small molecule inhibitor IU1 [21] or the transfection of shRNA specifically targeting USP14 (Fig. 2D and E). Moreover, the half-life of endogenous ATF2 protein was significantly reduced in USP14 knockdown (KD) PC3 cells (Fig. 2F), suggesting that the degradation of ATF2 protein was accelerated in the absent of USP14. Indeed, overexpression of USP14 decreased the ubiquitination of ATF2 protein
(Fig. 2G). Therefore, these data indicated that USP14 stabilized ATF2 by removing the ubiquitin chain of ATF2 protein. 3.3. USP14 regulates the transcriptional activity of ATF2 ATF2 is a transcription factor, the biological functions of which depend on the regulation of a series of downstream target genes [5]. We found that USP14 can regulate the expression of ATF2, but it is unclear whether it can also regulate the transcriptional activity of ATF2. Therefore, the regulation role of USP14 on the transcriptional activity of ATF2 was investigated by using a luciferase reporter assay. We found that overexpression of USP14WT, but not USP14 C114A mutant, significantly enhanced the transcriptional activity of ATF2 (Fig. 3A). In contrast, the transcriptional activity of ATF2 was significantly reduced by the addition of a USP14-specific small molecule inhibitor IU1(Fig. 3B). ATF2 transcriptionally activates the expression of a series of downstream target genes, including Cyclin D1, SOX9 and TGFB2 [9,10]. By performing real-time PCR assay, we found that overexpression of USP14 increased the expression of all these genes (Fig. 3C). However, the regulation of these genes by USP14 overexpression was significantly reversed by simultaneous knockdown of ATF2 (Fig. 3C), suggesting that USP14 induced the transcription of these genes in an ATF2-dependent manner.
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Fig. 4. USP14 promotes prostate cancer cells proliferation partially dependent on ATF2. A. The growth curve of control or USP14 stable expressing PC3 cells with or without ATF2 depletion. B. Clone formation of control or USP14 stable expressing PC3 cells with or without ATF2 depletion were determined by colony formation assay for about 10 days. Cells were stained with crystal violet and then counted. Data are presented as mean ± SD of three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001. C. Each nude mice were subcutaneously injected with 5 106 control or USP14 stable expressing PC3 cells with or without ATF2 depletion for four weeks. Then, mice were sacrificed and the weight of tumors were measured. n ¼ 5 for each group. *p < 0.05,**p < 0.01, ***p < 0.001. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
ATF2 plays diverse, even opposite roles in tumors from different sources [12]. In prostate cancer, ATF2 plays a critical role in promoting tumorigenesis by inducing the expression of a serial of downstream oncogenic genes [22,23]. USP14 exerts its biological function by targeting and stabilizing different substrate proteins. We then asked whether USP14 also promoted tumorigenesis in prostate cancer cells. We found that the cell growth and colony formation ability of prostate cancer PC3 cells can be significantly enhanced by stable expression of USP14. (Fig. 4A and B). However, these phenomenon caused by USP14 overexpression can be significantly reversed by silencing the expression of ATF2 in these cells (Fig. 4A and B, and Supplementary Fig. 1). Next, ATF2 was stable silenced in both control and USP14 overexpressing PC3 cells and these cells were injected subcutaneously into four-week-old male nude mice to monitor tumor formation. We found that PC3 cells overexpressing USP14 showed a significant increase in tumor volume and weight compared to controls (Fig. 4C and Supplementary Fig. 2). However, the cancer-promoting effects of USP14 were significantly reversed by silencing of ATF2 (Fig. 4D). Therefore, these data indicated that USP14 can promote the proliferation of prostate cancer cells by stabilizing ATF2 protein.
It is presumed that the human genome encodes approximately 100 deubiquitinating enzymes, and the deubiquitinating enzyme USP14 greatly enhanced its deubiquitination activity by reversibly binding to Rpn1 [25,26]. Recent studies have shown that USP14 plays important roles in signal transduction, neurological disease, glucose and lipid metabolism as well as tumorigenesis, which making it a promising drug target [27e29]. However, the role of USP14 and its downstream substrate in prostate cancer is not fully understood. We found that overexpression of USP14 promoting prostate cancer cells proliferation was partially depended on the function of ATF2. The proliferation of subcutaneous tumor cells induced by USP14 overexpression in nude mice can also be inhibited by silencing the expression of ATF2. Therefore, these in vitro and in vivo experiments indicate that ATF2 is a critical downstream target of USP14 in prostate cancer. Taken together, our study reveals a novel regulator of ATF2 in prostate cancer. USP14 interacted with and stabilized ATF2 to activate its downstream gene expression, leading to enhanced prostate cancer cells proliferation. Thus, our work demonstrated a critical role of USP1-ATF2 axis in the progress of prostate cancer. USP14-specific small molecule inhibitor IU1 has been used preclinically [21]. In future, the advent of more specific or potent small molecule inhibitors against USP14 will benefit the treatment of prostate cancer.
4. Discussion
Declaration of competing interest
3.4. USP14 promotes prostate cancer cells proliferation partially dependent on ATF2
The role of ATF2 in carcinogenesis is mainly related to its nuclear transcriptional function, while its cytoplasmic localization is associated with tumor suppressor function [24]. Inhibition of ATF2 expression or attenuation of its transcriptional function may be a promising strategy for cancer treatment. ATF2 acts as an oncogene in prostate cancer, and its mRNA and protein level are dysregulated in prostate cancer. It has been reported that an oncogenic long noncoding RNA UCA1 promoted ATF2 expression through functioning as a competing endogenous RNA (ceRNA) [22]. Thus, inhibition of UCA1 suppressed prostate cancer cells proliferation. The protein level of ATF2 was governed by the CUL3-SPOP E3 ligase complex and the recurrent somatic mutation of SPOP caused ATF2 accumulation and activation, which contributed to the progress of prostate cancer [15]. In the present study, we identified USP14 as a novel ATF2 interacting protein by IP/MS assay and found that USP14 promoted the transcriptional activity and expression of ATF2 downstream target genes by directly binding to and stabilizing ATF2 protein.
None. Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.128. References [1] D. Romero, Prostate cancer: genomic information improves risk prediction, Nat. Rev. Urol. 15 (2018) 68. [2] G.K. Alderton, Tumour microenvironment: obesity promotes prostate cancer invasion, Nat. Rev. Cancer 16 (2016) 70. [3] Z.T. Schug, J. Vande Voorde, E. Gottlieb, The metabolic fate of acetate in cancer,
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Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2 Lin Geng*, Xing Chen, Meng Zhang, Zhenkai Luo Department of Urology, China-Japan Friendship Hospital, No. 2 Yinghua Dongjie, Chaoyang District, Beijing, 100029, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2019 Accepted 24 December 2019 Available online xxx
Activating Transcription Factor 2 (ATF2) is a member of the ATF/CREB bZIP family of transcription factors and an oncogene in prostate cancer. ATF2 has been reported to be a critical substrate of the CUL3-SPOPRBX1 E3 ubiquitin ligase complex and the recurrent somatic mutation of SPOP has been believed to be a key feature of prostate cancer. However, the deubiquitinating enzyme required for ATF2 stabilization is still unknown. Here, we show that ATF2 is associated with ubiquitin-specific protease 14 (USP14), which increased the protein abundance and transcriptional activity of ATF2. Pharmacologic inhibition or siRNAmediated depletion of USP14 resulted in the decline and inactivation of ATF2. USP14 deubiquitinates and activates ATF2, resulting in enhanced prostate cancer cells proliferation both in vitro and in vivo. Importantly, silencing of ATF2 largely attuned USP14-mediated prostate cancer cells proliferation. Thus, our data revealed a critical role of USP1-ATF2 axis in the progress of prostate cancer and the inhibition of USP14 might be a promising strategy against prostate cancer. © 2020 Elsevier Inc. All rights reserved.
Keywords: ATF2 USP14 Deubiquitination Prostate cancer
1. Introduction Prostate cancer is the second most common cancer among men worldwide and is the third primary cause of male cancer-related mortality in United States. Prostate cancer accounts for 19% of estimated new cancer cases in men with more than 250,000 deaths each year [1]. Epidemiological studies have shown that the multifactorial aetiology of prostate cancer is related to factors such as age, ethnicity, genomic alterations, geographical location and diet and inflammation [2,3]. Activating Transcription Factor 2 (ATF2) is a member of the ATF/ CREB bZIP family of transcription factors, and shares a common feature, the b ZIP domain of this family [4]. The b ZIP domain contains two subdomains: a leucine zipper substructure region and an alkaline substructure region, which contains an N-terminal nuclear export sequence [5]. The transcriptional function of ATF2 protein is activated in two ways: to form a homodimer by binding to the b ZIP domain of another ATF2 protein or a heterodimer with other b ZIP contained proteins [6,7]. The resulting dimer is then associated with a specific DNA transcript sequence to promote genes transcription [8]. In response to stress stimuli, ATF2 is
subjected to phosphorylation by JNK and p38, leading to its transcriptional activation and the transcription of a variety of targets including cyclin A, cyclin D, SOX9, MMP9, and TGFB2 [9e11]. Interestingly, ATF2 plays a dual role in both tumor suppression and cancer promotion, in a context-dependent manner. For example, ATF2 acts as an oncogene in prostate cancer, melanoma, non-small cell lung carcinoma and pancreatic cancer, while a tumor suppressor in breast cancer [5,12e14]. In prostate cancer, ATF2 has been reported to be a critical substrate of a functional CUL3-SPOP-RBX1 E3 ubiquitin ligase complex and the recurrent somatic mutation of SPOP has been believed to be a key feature of prostate cancer [15,16]. Thus, the fine tune of ATF2 protein might contribute to facilitating prostate cancer progress. Although most ubiquitination processes can be antagonized by deubiquitinating enzymes, the deubiquitinating enzyme that required for ATF2 stabilization is still unknown. Here, we show that ATF2 is associated with ubiquitin-specific protease 14 (USP14), which deubiquitinates and activates ATF2, and results in enhanced cell proliferation of prostate cancer cells. 2. Material and methods 2.1. Cell culture and reagents
* Corresponding author. E-mail address: [email protected] (L. Geng).
HEK293, prostate cancer cell lines LNCaP and PC3 were obtained
https://doi.org/10.1016/j.bbrc.2019.12.128 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone), 100 IU/ml penicillin and 100 mg/ml streptomycin and maintained at 37 C in a humidified atmosphere containing 5% CO2. Cycloheximide (239764) was purchased from Calbiochem (San Diego, CA, USA). The proteasome inhibitor MG132 was purchased from SigmaAldrich (St. Louis, MO, USA). The USP14 specific inhibitor IU1 was purchased from BioVision (1845e25). 2.2. Cell counting Kit-8 (CCK-8) assay Cells were grown in 96-well plates and incubated for about five days. Cell viability was measured with CCK-8 kit according to the manufacturer’s protocol. At each time, 10 ml CCK-8 was added into each well and incubated at 37 C for 2 h. The optical density (OD) value was measured with Microplate Reader at 450 nm.
spectrometry analysis. For western blotting, equivalent amounts of protein sample was denatured in the loading buffer, and resolved by 10e12% SDS-PAGE and transferred onto NC membranes. Membranes were blocked in 5% non-fat milk for 1 h before incubation with primary antibody overnight at 4 C. The following primary antibodies were used: anti-ATF2 antibody at 1:2000 (#sc-242, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-USP14 antibody at 1:2000 (#11931, Cell Signaling Technology), anti-GAPDH antibody at 1:5000 (#sc-32233, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HA antibody at 1:2000 (#3724, Cell Signaling Technology), anti-Flag M2 antibody (#F1804, Sigma-Aldrich St. Louis, MO, USA), anti-GST antibody at 1:5000 (#sc-138, Santa Cruz Biotechnology, Santa Cruz, CA, USA). GAPDH served as an internal control. 2.7. Deubiquitylation assay
Cells were grown in 12-well plates at a density of 5000 cells and incubated for about 10 days. The culture medium was changed every four days. Cells were then fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 10 min. The cell colonies were photographed with a microscope. For the quantification of colonies, 30% sodium acetate was added to test the crystal violet OD value for each well. The statistical analysis was analyzed with three replicates.
The in vivo deubiquitylation assays were performed under denaturing conditions. PC3 cells were grown in 10 cm plates to 80% confluence and then were transiently transfected with empty vector (EV) or Flag-USP14 for 36 h using lipo2000 transfection reagent. Cells were harvested, lysed in denaturing lysis buffer [1% SDS, 50 mM Tris, 10 mM DTT (pH 7.5)], and boiled at 95 C for 5 min. The denatured proteins were diluted in RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1% NP40 and 0.1% SDS and immunoprecipitated by Agarose TUBE1 resin (UM401, Agarose-TUBE1, Nacalai Tesque) for ubiquitin chain enrichment and immunoblotted as indicated.
2.4. RNA isolation and quantitative real-time PCR
2.8. Cycloheximide (CHX) assay
Total RNAs from cells were extracted by using Trizol reagents (Invitrogen). The mRNAs were then reversed transcripted into cDNA using the Promega Reverse Transcription System (Madison, WI, USA). Oligo dT was used to prime cDNA synthesis. Quantitative real-time PCR was performed using a SYBR Green Premix Ex Taq (Takara, Japan) on Light Cycler 480 (Roche, Switzerland). The GAPDH mRNA levels were used as internal control. Primers used for qPCR analysis were available upon request.
PC3 cells were seeded overnight in complete medium in 6-well plates to 80% confluence and then transfected with control-shRNA or USP14-shRNA for 36 h followed by the addition of 20 mM cycloheximide (CHX). Samples at the indicated times were harvested for immunoblot analysis.
2.3. Colony formation assay
2.5. Luciferase reporter assays To monitor the transfection activity of ATF2, the phRL-3CRE-TK plasmid which contains three cyclic AMP-responsive elements (CREs) was used. The phRL-3CRE-TK and the pRL-null plasmid encoding Renilla luciferase were co-transfected with other plasmids for 36 h. Luciferase activity was measured using the Dual Luciferase Reporter Assay System. The firefly luciferase luminescence data were normalized by dividing the Renilla luciferase luminescence data. Results are expressed relative to the activity in vector control. 2.6. Immunoprecipitation (IP), mass spectrometry and western blotting Cells were lysed in RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1% NP40 and 0.1% sodium dodecyl sulfate (SDS). Lysates were cleared by centrifugation at 15,000 g for 20 min at 4 C to remove cell debris. The resulting lysates were subjected to IP with either anti-USP14 or anti-ATF2 antibodies overnight at 4 C. Protein G beads were then added for additional 4 h. For affinity purification, 293T cells transfected with control or Flag-ATF2 plasmids were subjected to Flag M2 beads purification. Bound proteins were resolved by SDS-PAGE and stained with Coomassie blue staining, followed by mass
2.9. Xenograft models This study was conducted in compliance with Institutional Animal Care and Use Committee of institutional ethical guidelines in China-Japan Friendship Hospital. All 4e8 weeks old BALB/c nude mice were purchased from the Shanghai Laboratory Animal Company (SLAC) and housed at specific facility. The mice were divided into groups randomly and the indicated cells were implanted subcutaneously on the axilla of mice with DMEM medium with FBS. Four weeks after injection, mice were sacrificed, and the weight and volume of tumors were measured. 2.10. Statistical analysis All in vitro experiments were repeated for three times. Data are expressed as the means ± SD. The differences between groups were calculated using the Student’s t-test or one-way ANOVA using a Tukey post-hoc test. Statistical analyses were performed using Graphpad 6.0. A value of P < 0.05 was considered to indicate a statistically significant difference. *p < 0.05,**p < 0.01, ***p < 0.001. 3. Results 3.1. USP14 is associated with ATF2 To investigate how ATF2 was regulated, we first analyzed the interacting protein complex of ATF2 by immunoprecipitation combined with mass spectrometry (MS) assay. Flag-con or Flag-
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Fig. 1. USP14 is associated with ATF2. A. A partial list of proteins identified by mass spectrometry analysis. 293T cells transfected with Flag-control or Flag-ATF2 plasmids were subjected to Flag M2 beads purification. Bound proteins were resolved by SDS-PAGE and stained with Coomassie blue staining, followed by mass spectrometry analysis. B. 293T cells transfected with Flag-control or Flag-ATF2 plasmids were subjected to Flag M2 beads purification and immunoblotting with antibodies against Flag and USP14. C. The cell lysates of PC3 cells were subjected to immunoprecipitation with anti-ATF2 or IgG antibodies. The bound proteins were detected by immunoblotting with antibodies against USP14 and ATF2. D. The cell lysates of LNCaP cells were subjected to immunoprecipitation with anti-USP14 or IgG antibodies. The bound proteins were detected by immunoblotting with antibody against USP14 and ATF2. E. 293T cells transfected Flag-ATF2 plasmid were lysed and the lysate was incubated with GST-USP14 immobilized on GST-Sepharose beads, and bound ATF2 was detected by immunoblotting with antibody against Flag.
ATF2 plasmids were transfected into 293T cells which were harvested and lysed 48 h later. The cell lysate was incubated with Flag M2 beads for immunoprecipitation. The immunoprecipitate was then subjected to MS analysis. Our MS data revealed several known ATF2 interacting proteins, including ATF1, MAPK1, MAPK14 and CREBBP [17e19], which demonstrated the reliability of our data (Fig. 1A). Meanwhile, we also identified a new ATF2 interacting protein, USP14. To confirm the MS data, the Flag-ATF2 immunoprecipitate was subjected to immunoblot with anti-USP14
antibody. We found that endogenous USP14 was readily detected in Flag-ATF2 immunoprecipitate (Fig. 1B). Furthermore, we performed immunoprecipitation assay by using anti-ATF2 or -USP14 antibodies to enrich the endogenous ATF2 or USP14 proteins in PC3 cells or LNCaP cells, respectively. The interaction between endogenous ATF2 and USP14 was clearly observed (Fig. 1C and D). Furthermore, through GST-pull-down assay, we found that USP14 bound directly to ATF2 protein (Fig. 1E). Therefore, these results indicate that USP14 is an endogenous ATF2 interacting protein.
Fig. 2. USP14 deubiquitinates and stabilizes ATF2. A. Lysates of PC3 cells transduced with empty vector (EV) or increased doses of Flag-USP14 were immunoblotted with indicated antibodies. B. Lysates of PC3 cells transduced with empty vector (EV) or Flag-USP14 wild type (WT) or Flag-USP14 catalytic inactivated mutant (C114A) were immunoblotted with indicated antibodies. C. The mRNA levels of ATF2 in PC3 cells transduced with empty vector (EV) or Flag-USP14 wild type (WT) or Flag-USP14 mutant (C114A) were analyzed by realtime PCR. D. PC3 cells were treated with DMSO control or 50 mM IU1 for 12 h, and the cell lysates were immunoblotted with indicated antibodies. E. PC3 cells were transfected with shRNA control or USP14-shRNA for 36 h, and the cell lysates were immunoblotted with indicated antibodies. F. PC3 cells were transfected with shRNA control or USP14-shRNA for 36 h, 20 mM cycloheximide (CHX) was added for the indicated time. The cell lysates were immunoblotted with indicated antibodies (n ¼ 3 for each group). G. PC3 cells were transfected with empty vector (EV) or Flag-USP14 for 36 h, cell lysates were immunoprecipitated by Agarose TUBE1 resin for ubiquitin chain enrichment, and immunoblotted as indicated.
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Fig. 3. USP14 regulates the transcriptional activity of ATF2. A. 293T cells were co-transfected empty vector (EV) or Flag-USP14 wild type (WT) or Flag-USP14 mutant (C114A) with luciferase reporter plasmid containing the binding site of ATF2 for 36 h. Luciferase activity was measured using the Dual Luciferase Reporter Assay System. B. 293T cells were cotransfected shRNA control or USP14-shRNA with luciferase reporter plasmid containing the binding site of ATF2 for 36 h. Luciferase activity was then measured. C. The mRNAs expression of three ATF2 transcriptional targets including Cyclin D1, SOX9 and TGFB2 in PC3 cells were analyzed by real-time PCR. Data are presented as mean ± SD of three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001.
3.2. USP14 deubiquitinates and stabilizes ATF2 USP14 belongs to the deubiquitinating enzyme family and could interact with and stabilize its substrate proteins by removing the ubiquitin chain from them [20]. The interaction between USP14 and ATF2 prompted us to ask whether USP14 could regulate the protein level of ATF2. To this end, we overexpressed USP14 into PC3 cells and found that USP14 induced the expression of ATF2 in a dose-dependent manner (Fig. 2A). This regulation is closely related to the deubiquitination activity of USP14, because overexpression of the catalytic inactivated (CI) USP14 C114A mutant failed to induce ATF2 expression (Fig. 2B). However, the mRNA levels of ATF2 were not affected by overexpression of USP14 wild type (WT) or USP14 C114A mutant (Fig. 2C), suggesting that the regulation of ATF2 by USP14 occurs at the post-translational level. Consistently, ATF2 expression was declined by the addition of the USP14-specific small molecule inhibitor IU1 [21] or the transfection of shRNA specifically targeting USP14 (Fig. 2D and E). Moreover, the half-life of endogenous ATF2 protein was significantly reduced in USP14 knockdown (KD) PC3 cells (Fig. 2F), suggesting that the degradation of ATF2 protein was accelerated in the absent of USP14. Indeed, overexpression of USP14 decreased the ubiquitination of ATF2 protein
(Fig. 2G). Therefore, these data indicated that USP14 stabilized ATF2 by removing the ubiquitin chain of ATF2 protein. 3.3. USP14 regulates the transcriptional activity of ATF2 ATF2 is a transcription factor, the biological functions of which depend on the regulation of a series of downstream target genes [5]. We found that USP14 can regulate the expression of ATF2, but it is unclear whether it can also regulate the transcriptional activity of ATF2. Therefore, the regulation role of USP14 on the transcriptional activity of ATF2 was investigated by using a luciferase reporter assay. We found that overexpression of USP14WT, but not USP14 C114A mutant, significantly enhanced the transcriptional activity of ATF2 (Fig. 3A). In contrast, the transcriptional activity of ATF2 was significantly reduced by the addition of a USP14-specific small molecule inhibitor IU1(Fig. 3B). ATF2 transcriptionally activates the expression of a series of downstream target genes, including Cyclin D1, SOX9 and TGFB2 [9,10]. By performing real-time PCR assay, we found that overexpression of USP14 increased the expression of all these genes (Fig. 3C). However, the regulation of these genes by USP14 overexpression was significantly reversed by simultaneous knockdown of ATF2 (Fig. 3C), suggesting that USP14 induced the transcription of these genes in an ATF2-dependent manner.
Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128
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Fig. 4. USP14 promotes prostate cancer cells proliferation partially dependent on ATF2. A. The growth curve of control or USP14 stable expressing PC3 cells with or without ATF2 depletion. B. Clone formation of control or USP14 stable expressing PC3 cells with or without ATF2 depletion were determined by colony formation assay for about 10 days. Cells were stained with crystal violet and then counted. Data are presented as mean ± SD of three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001. C. Each nude mice were subcutaneously injected with 5 106 control or USP14 stable expressing PC3 cells with or without ATF2 depletion for four weeks. Then, mice were sacrificed and the weight of tumors were measured. n ¼ 5 for each group. *p < 0.05,**p < 0.01, ***p < 0.001. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
ATF2 plays diverse, even opposite roles in tumors from different sources [12]. In prostate cancer, ATF2 plays a critical role in promoting tumorigenesis by inducing the expression of a serial of downstream oncogenic genes [22,23]. USP14 exerts its biological function by targeting and stabilizing different substrate proteins. We then asked whether USP14 also promoted tumorigenesis in prostate cancer cells. We found that the cell growth and colony formation ability of prostate cancer PC3 cells can be significantly enhanced by stable expression of USP14. (Fig. 4A and B). However, these phenomenon caused by USP14 overexpression can be significantly reversed by silencing the expression of ATF2 in these cells (Fig. 4A and B, and Supplementary Fig. 1). Next, ATF2 was stable silenced in both control and USP14 overexpressing PC3 cells and these cells were injected subcutaneously into four-week-old male nude mice to monitor tumor formation. We found that PC3 cells overexpressing USP14 showed a significant increase in tumor volume and weight compared to controls (Fig. 4C and Supplementary Fig. 2). However, the cancer-promoting effects of USP14 were significantly reversed by silencing of ATF2 (Fig. 4D). Therefore, these data indicated that USP14 can promote the proliferation of prostate cancer cells by stabilizing ATF2 protein.
It is presumed that the human genome encodes approximately 100 deubiquitinating enzymes, and the deubiquitinating enzyme USP14 greatly enhanced its deubiquitination activity by reversibly binding to Rpn1 [25,26]. Recent studies have shown that USP14 plays important roles in signal transduction, neurological disease, glucose and lipid metabolism as well as tumorigenesis, which making it a promising drug target [27e29]. However, the role of USP14 and its downstream substrate in prostate cancer is not fully understood. We found that overexpression of USP14 promoting prostate cancer cells proliferation was partially depended on the function of ATF2. The proliferation of subcutaneous tumor cells induced by USP14 overexpression in nude mice can also be inhibited by silencing the expression of ATF2. Therefore, these in vitro and in vivo experiments indicate that ATF2 is a critical downstream target of USP14 in prostate cancer. Taken together, our study reveals a novel regulator of ATF2 in prostate cancer. USP14 interacted with and stabilized ATF2 to activate its downstream gene expression, leading to enhanced prostate cancer cells proliferation. Thus, our work demonstrated a critical role of USP1-ATF2 axis in the progress of prostate cancer. USP14-specific small molecule inhibitor IU1 has been used preclinically [21]. In future, the advent of more specific or potent small molecule inhibitors against USP14 will benefit the treatment of prostate cancer.
4. Discussion
Declaration of competing interest
3.4. USP14 promotes prostate cancer cells proliferation partially dependent on ATF2
The role of ATF2 in carcinogenesis is mainly related to its nuclear transcriptional function, while its cytoplasmic localization is associated with tumor suppressor function [24]. Inhibition of ATF2 expression or attenuation of its transcriptional function may be a promising strategy for cancer treatment. ATF2 acts as an oncogene in prostate cancer, and its mRNA and protein level are dysregulated in prostate cancer. It has been reported that an oncogenic long noncoding RNA UCA1 promoted ATF2 expression through functioning as a competing endogenous RNA (ceRNA) [22]. Thus, inhibition of UCA1 suppressed prostate cancer cells proliferation. The protein level of ATF2 was governed by the CUL3-SPOP E3 ligase complex and the recurrent somatic mutation of SPOP caused ATF2 accumulation and activation, which contributed to the progress of prostate cancer [15]. In the present study, we identified USP14 as a novel ATF2 interacting protein by IP/MS assay and found that USP14 promoted the transcriptional activity and expression of ATF2 downstream target genes by directly binding to and stabilizing ATF2 protein.
None. Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.128. References [1] D. Romero, Prostate cancer: genomic information improves risk prediction, Nat. Rev. Urol. 15 (2018) 68. [2] G.K. Alderton, Tumour microenvironment: obesity promotes prostate cancer invasion, Nat. Rev. Cancer 16 (2016) 70. [3] Z.T. Schug, J. Vande Voorde, E. Gottlieb, The metabolic fate of acetate in cancer,
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Please cite this article as: L. Geng et al., Ubiquitin-specific protease 14 promotes prostate cancer progression through deubiquitinating the transcriptional factor ATF2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.128