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A New Regulatory Mechanism Between P53 And YAP Crosstalk By SIRT1 Mediated Deacetylation To Regulate Cell Cycle And Apoptosis In A549 Cell Lines

Authors Yuan F, Wang J, Li R, Zhao X, Zhang Y, Liu B, Lei Y, Hu Y

Received 7 May 2019

Accepted for publication 14 August 2019

Published 23 September 2019 Volume 2019:11 Pages 8619—8633

DOI https://doi.org/10.2147/CMAR.S214826

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Antonella D'Anneo



Fang Yuan,1,* Jinliang Wang,1,* Ruixin Li,1,* Xiao Zhao,1 Yuxuan Zhang,2 Biao Liu,3 Yonghong Lei,4 Yi Hu1

1Department of Oncology, Chinese PLA General Hospital, Beijing 100853, People’s Republic of China; 2Princeton International School of Mathematics and Science, Princeton, NJ 08540, USA; 3Department of Biological Analysis, Explore (Beijing) Biotech Co, Ltd, Beijing 100091, People’s Republic of China; 4Department of Plastic Surgery, Chinese PLA General Hospital, Beijing 100853, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yonghong Lei
Department of Plastic Surgery, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, People’s Republic of China
Tel +86 10 13161281016
Email [email protected]
Yi Hu
Department of Oncology, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, People’s Republic of China
Tel +86 10 13911031186
Email [email protected]

Background: Yes-associated protein (YAP) is downstream of the Hippo signaling pathway, which regulates several cellular processes. P53 is a key transcriptional regulator that responds to a variety of cellular stresses and regulates key cellular processes such as DNA repair, cell-cycle progression, angiogenesis, and apoptosis. Overexpression of YAP antagonizes P53 activity and targets its expression. However, the mechanism that underlies the post-transcriptional crosstalk between P53 and YAP has not been well dissected.
Methods: We performed an integrated analysis and found that SIRT1 is a key candidate that connects YAP and P53 by modulating their acetylation.
Results: We found that YAP promotes P53 deacetylation, promotes cell survival by inhibiting P53-induced G0/G1 arrest and apoptosis in A549 cells. Conversely, P53 enhances YAP acetylation, and decreases A549 cell survival by strengthening YAP acetylation-induced G0/G1 arrest and apoptosis both in vitro and in vivo.
Conclusion: Our results demonstrate that SIRT1 is responsible for YAP and P53 deacetylation of specific residues, and reveal for the first time, a new regulatory mechanism of P53 and YAP crosstalk by SIRT1-mediated deacetylation, which may be involved in lung tumorigenesis.

Keywords: P53, YAP, SIRT1, crosstalk

Introduction

Yes-associated protein (YAP) is a downstream effector molecule of a newly emerging pathway called the Hippo pathway.1 YAP and TAZ, two closely related transcription co-activators, are mediated by Hippo kinases and adaptor proteins.2 The Hippo pathway is evolutionarily conserved and a central regulator of organ size and tissue homeostasis. It responds to a variety of extracellular and intracellular signals, reading a broad range of mechanical cues, from shear stress to cell shape and extracellular matrix rigidity, which it then translates into cell-specific transcriptional programs.3 It is heavily involved in the control of cell proliferation, organ size and shape during development, stem cell maintenance, metastasis, tissue regeneration, apoptosis, senescence, and differentiation.4 Other factors also regulate it, such as cell density and polarity, metabolism and DNA damage.57 Hippo crosstalk with other signaling players so that it resembles a network rather than a linear pathway, such as JAK-STAT3 and fat signaling.8,9

P53 protein is a well-known tumor suppressor factor that regulates cellular homeostasis, as well as several signaling pathways involved in a cell’s response to stress, and regulates cellular homeostasis.10 Moreover, it becomes activated through several post-translational modifications such as, phosphorylation, sumoylation, acetylation and prolyl-isomerization.11 P53 is a modular protein harboring with four functionally different domains: the N-terminal transactivation domain, which is essential for binding to transcription factors and regulators of P53 activity;12 the core DBD domain, which allows the binding to DNA; the oligomerization domain (OLD), which is relevant for the tetramerization of P53, and the C-terminal domain or regulatory domain (RD), which is involved in post-translational modifications (phosphorylation, acetylation, ubiquitination and sumoylation).12

Recent studies have shown that the P53 and Hippo pathways are physically and functionally connected. They have been shown to modulate common transcriptional programs and pathways that preserve cellular and tissue homeostasis in healthy conditions. ChIP assay results indicate that YAP binds directly to the p53 promoter to improve its expression, which results in P53-dependent cycle arrest and apoptosis.13 The nuclear YAP induces p21, Bax and Caspase 3 expression and inhibits the anti-apoptotic factors Bcl-2 and Bcl-xL.13 Besides this transcriptional crosstalk between P53 and YAP, recent studies have revealed another mechanism that underlies the crosstalk between P53 and YAP. Central to the Hippo pathway is a core kinase cascade of the tumor suppressors MST1/2 and LATS1/2, and the adaptor proteins SAV1 and MOB1/2.14 These proteins form a conserved kinase cassette “Hippo”, which typically functions by phosphorylating and inactivating the transcriptional co-activators YAP and TAZ.15 Recently, LATS2 and its paralog LATS1, have been shown to contribute to tumor suppressive features of P53, also under basal conditions.16 These findings suggest the complexity of the various mechanisms underlying P53 and YAP crosstalk.

SIRT1 is a protein involved in the deacetylation of key histone lysine residues, including histone H3 lysine 9 (H3K9) and histone H4 lysine 16 (H4K16), thus regulating gene expression that governs cell fate.17 Besides SIRT1’s epigenetic role being implicated in the regulation of the chromatin state for the expression of specific genes, it also deacetylates many transcriptional factors in a NAD+-dependent manner, including P53.18 Many key cellular events are regulated through the SIRT1-P53 interaction.19 Elevated SIRT1 deacetylates activated P53, allowing cells with damaged DNA to proliferate and thus, promoting tumor development.19 One recent study showed that, SIRT1 deacetylates YAP2 protein in hepatocellular carcinoma (HCC) cells, and SIRT1-mediated deacetylation increases the YAP2/TEAD4 association. This led to YAP2/TEAD4 transcriptional activation and upregulated cell growth in HCC cells.20 Although a acetylation/deacetylation cycle of nuclear YAP exists downstream of the Hippo signaling pathway,21 the mechanism that underlies this post-translational crosstalk between P53 and YAP, and the role of this P53-SIRT1-YAP axis controlling of cell cycle transition and apoptosis, is still unknown.

We utilized A549 cell lines and examined the importance of SIRT1’s involvement in the post-translational crosstalk between P53 and YAP. We then disected the feedback loop between these two signaling pathways in maintaining cell cycle arrest and apoptosis. Our study clarifies the effect of SIRT1-induced deacetylation of P53 and YAP on cell growth and identifies the mechanisms responsible for these effects in A549 cells, while shedding new light on the post-translational interaction between P53 and YAP, which may be involved in lung tumorigenesis.

Materials And Methods

Cell Culture And Reagents

The human A549 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained at 37°C with 5% CO2 in a humidified atmosphere and grown in Dulbecco’s modified Eagle’s medium (HyClone Laboratories; GE Healthcare, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Methyl methane sulfonate (MMS) and 2, 6-diisopropylaniline (DIPA) were purchased from Sigma.

Plasmid Construction And Transfection

The pIRES2-EGFP-p53 WT Plasmid, pFLAG-YAP1 Plasmid, Flag-SIRT1 Plasmid were purchased from Addgene (#49242, #66853, #1791). All the Plasmids were confirmed by DNA sequencing and transfected into A549 cells using Lipofectamine 2000 reagent (Invitrogen, CA, USA). The cells were transfected when they reached 70–80% confluence and were harvested after 48 hrs.

RNA Interference

A549 cells were transfected with siRNA using Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. siRNAs specific for P53, YAP and SIRT1 were purchased from Santa Cruz (sc-29435, sc-38637, sc-40986, America). Control siRNA of P53, YAP and SIRT1 were purchased from Santa Cruz (sc-37007).

Real-time Quantitative Polymerase Chain Reaction

Total RNA was extracted by Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). Briefly, 1 μg RNA was used to synthesize cDNA using a kit (Thermo Fisher Scientific, Rockford, IL, USA). The primer sequences were as follows: P21, F, 5ʹ-TGTCCGTCAGAACCCATGC-3ʹ, R, 5ʹ-AAAGTCGAAGTTCCATCGCTC-3ʹ; P27, F, 5ʹ-AACGTGCGAGTGTCTAACGG-3ʹ, R, 5ʹ-CCCTCTAGGGGTTTGTGAT TCT-3ʹ; BIM, F, 5ʹ-TAAGTTCTGAGTGTGACCGAGA-3ʹ, R, 5ʹ-GCTCTGTCTGT AGGGAGGTAGG-3ʹ; PUMA, F, 5ʹ-GCCAGATTTGTGAGACAAGAGG-3ʹ, R, 5ʹ-CAGGCACCTAATTGGGCTC-3ʹ; Cyclin E, F, 5ʹ-AAGGAGCGGGACACCATG A-3ʹ, R, 5ʹ-ACGGTCACGTTTGCCTTCC-3ʹ; Connective Tissue Growth Factor (CTGF), F, 5ʹ-CAGCATGGACGTTCGTCTG-3ʹ, R, 5ʹ-AACCACGGTTTGGT CCTTGG-3ʹ; MYC, F, 5ʹ-GGCTCCTGGCAAAAGGTCA-3ʹ, R, 5ʹ-CTGCGTAGTTG TGCTGATGT-3ʹ; DIPA, F, 5ʹ-GAATCGGCGGGCCATTCAT-3ʹ, R, 5ʹ-CTGAGC TTTTATTGTGTGGGAGA-3ʹ; GAPDH, F, 5ʹ-TTTGTCAAGCTCATTTCCTG-3ʹ, R, 5ʹ-TGGTCCAGGGTTTCTTACTC-3ʹ. GAPDH was employed as the internal control. The expression of candidate genes was measured by SYBR Green (Takara Biotechnology Co., Ltd., Dalian, China), and real-time PCR assays were performed using ABI-7300 (Applied Biosystems, Shanghai, China). The relative gene expression was calculated by the 2−ΔΔCt.

Cell Cycle Progression Assay

Cell cycle analysis was performed by flow cytometry. A549 cells were harvested, fixed, treated with RNase A (50 μg/mL) and stained with propidium iodide (10 μg/mL). Cellular DNA content was analyzed using flow cytometry (FACS Canto II, BD Biosciences, USA). ~10,000 cells were acquired for each analysis, and results were analyzed using ModFit LTTM software (version2) and displayed as a histogram.

Apoptosis Assay

Cell apoptosis was detected by Annexin V-FITC/PI double-staining. Annexin V-FITC/PI staining was used for the quantitation of early and late apoptotic cells. A549 cells were stained with annexin V- FITC (0.2 mg/mL) and PI (0.05 mg/mL) for 20 mins and were examined by flow cytometry (FACS Calibur, BD Biosciences) using Cell Quest pro software at an excitation with 488 nm laser and emission at 530 nm. A minimum of 10,000 cells was analyzed per sample and illustrated as a dot plot using Flowing software.

Western Blotting

The total protein was extracted from cells after transfection using RIPA buffer. The extract was centrifugated for 15 mins at 4 °C at 1,4000 g. The upper supernatant was then collected, and protein concentration was assessed by the bicinchoninic acid (BCA) method. After, the protein was electrophoresed in SDS–polyacrylamide gels (Invitrogen, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk and incubated with the primary antibody overnight. TBST buffer was used to wash the membrane three times. The membrane was then incubated with the corresponding secondary antibody conjugated by horseradish peroxidase (HRP) for 1 hr. Images were captured by ChemiDoc XRS (Bio-Rad, CA, USA). The antibodies used in the present study were: anti-p21 (1:2000, Abcam, ab109520), anti-Bim (1:2000, Abcam, ab32158), anti-SIRT1 (1:1000, Abcam, ab110304), anti-PUMA (1:5000, Abcam, ab33906), anti-YAP1 (1:20000, Abcam, ab52771), anti-CTGF (1:500, Santa, sc-365970), anti-cyclin E (1:500, Santa, sc-247), anti-c-Myc (1:2000, Abcam, ab32), anti-DIPA (1:500, ABGENT, AP9120c), anti-pan acetyl-lysine antibody (1:1000, Cell Signaling Technology, #9441), anti-P53 (acetyl K382) (1:1000, Abcam, ab75754), anti-P53 (acetyl K373) (1:5000, Abcam, ab62376), anti-P53 (acetyl K370) (1:1000, Abcam, ab183544), anti-P53 (acetyl K120) (1:100, Abcam, ab78316), anti-YAP (acetyl K494) (1:500, in-house), anti-YAP (acetyl K497) (1:500, in-house), anti-GAPDH (1:5000, Abcam, ab8245), anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350) and anti-acetyl Lysine (1:500, Abcam, ab80178).

Immunofluorescence

A549 cells were cultured on a glass coverslip and fixed with 4% paraformaldehyde in PBS at room temperature. After treatment with 0.2% Triton X-100 in PBS, the cells were incubated with blocking solution (5% bovine serum albumin in TBS) before incubation with primary Abs for 1 hr at room temperature. Cells were washed with PBS and incubated for 1 hr with Alexa 488- or 546-conjugated secondary Absolute. After PBS washes, coverslips were mounted and viewed on a Carl Zeiss confocal microscope equipped with LSM510 software.

Ingenuity Pathway Analysis

Protein–protein interaction networks were built by Core Analysis and Network Analysis of the online IPA@ software package (Version 8.7). (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis). The network size parameters were set in order to optimize visualization and analyze the biologically relevant background. Networks analysis can provide a quick solution to assess the data of interest in regulatory networks. Usngi IPA Core Analysis and Network Analysis, we built a P53 Acetylation network, YAP Acetylation network, P53 Singling pathway and YAP Singling pathway. We used the Cytoscape software to visualize the interaction of these networks.22

In Vivo Tumorigenesis In Nude Mice

Animal experiments were approved by the Ethical Committee of Animal Research at Chinese PLA General Hospital. The experimental protocol was established according to the associated national guidelines from Ministry of Science and Technology of China. The effects of YAP on the in vivo tumorigenic ability was investigated by tumor xenograft experiment. A total of 1 × 106 A549 cells with different treatment (WT, YAP or P53 OE and YAP or P53 KD) in 0.2 mL RPMI 1640 medium were subcutaneously injected into the dorsal flanks of 4–6-week-old male BALB/c nu/nu mice. The mice were maintained in a barrier facility on HEPA-filtered racks and fed with an autoclaved laboratory rodent diet. Each experimental group contained 10 mice. Tumor size was monitored using a calliper in the process of tumor growth and measured every 3 days. After 5 weeks, mice were killed and tumors were excised and weighed. Tumor volumes were calculated as follows: volume = (D × d2)/2, where D is the longest diameter and d is the shortest diameter.

Statistical Analysis

All the quantitative data are presented as means ± standard deviation (SD). The statistical significance levels for all tests were set as *P < 0.05, **P < 0.01 and ***P < 0.001. For multiple group comparisons, ANOVA with post hoc Dunnett’s test was used. Student’s t test was used to perform comparisons between two groups. All analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc., LaJolla, CA, USA). All experiments were repeated three times.21

Results

Integrated Analysis Identified Crosstalk Between The Acetylation Of YAP And P53

To dissect the crosstalk between the acetylation of YAP and P53 signaling, we performed an integrated analysis on the basis of Ingenuity Pathway Analysis (IPA) and content-analysis-based literature reviews, and constructed the “P53 Acetylation network” (based on 289 published papers retrieved from a search using the key word, “P53 Acetylation”), the “YAP Acetylation network” (based on 22 from doing a search for “YAP Acetylation”), the “P53 Signaling pathway” and the “YAP Signaling pathway” (based on IPA) (Figure 1AC). In the P53 Acetylation network, histone acetyltransferase P300 and CBP can acetylate P53 and increase its activation. In contrast, several histone deacetylase (HDAC) family members, including SIRT1, HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8, can deacetylate P53 and inhibit its activation.21 Interestingly, P53 can also suppress SIRT1 via HIC1 and Myc, forming a negative feedback loop (Figure 1A). In the YAP Acetylation network, histone acetyltransferase P300 and CBP can acetylate YAP. On the contrary, several HDAC family members, including SIRT1 and HDAC1 can deacetylate YAP and inhibit its activation23 (Figure 1B). Interestingly, YAP can also enhance SIRT1 via Myc, forming a positive feedback loop. Together, these results suggest that the common acetylases and deacetylases between P53 and YAP may form a steady-state network, which is maintained primarily by the crosstalk between P53 and YAP (Figure 1C).

Figure 1 Crosstalk network between acetylation of YAP and P53. (A) P53 acetylation network by content-analysis-based literature reviews. The pink node indicated the protein interacting with P53 acetylation. (B) YAP acetylation network by content-analysis-based literature reviews. The pink node indicated the protein interacting with YAP acetylation. (C) Crosstalk between the YAP acetylation network and the P53 acetylation network. The pink node indicated the proteins medicated p53 and YAP acetylation network, including SIRT1, HDAC1, P300 and SBP. → and ┤stood for promoting and inhibiting P53 and YAP acetylation.

YAP Promotes P53 Deacetylation

To investigate the regulation of P53 acetylation in response to DNA damage, we treated A549 cells with the SN2 alkylating agent methyl methane sulfonate (MMS) and used anti-P53 Ab to immunoprecipitated endogenous P53 from lysates of MMS-treated A549 cells, the control group was set as the one with IgG antibody. We performed immunoblotting using anti-pan-AcK Ab, which specifically recognizes acetylated lysine residues. Other DNA damage agents, such as H2O2, Cisplatin, N-methyl-N’-nitro-N-nitrosoguanidine, and nitrosomethylurea may not lead to the acetylation of YAP (data not shown). The time- and dose- dependent kinetics of P53 acetylation assays, as well as the γH2AX levels showed that MMS treatment induced marked increases in levels of acetylated P53; MMS treatment with 1mM for 2 hrs was the optimal condition for subsequent experiments (Figure 2A). These results show that endogenous P53 is acetylated in response to MMS treatment.

Figure 2 YAP inhibits P53 acetylation. (A) Time-dependent (left) and dose-dependent (right) kinetics of P53 acetylation assays and γH2AX expression after MMS treatment in A549 cells. (B) A549 cells were transfected respectively with oeYAP, siYAP, oeSIRT1, and siSIRT1 plasmid using Lipo2000 reagent, Western blotting examined the expression level of YAP and SIRT1. (C) Effects of overexpression or knockdown of YAP on P53 acetylation and γH2AX expression in A549 cells with or without MMS treatment (1mM for 2 hrs). (D) Effects of overexpression or knockdown of YAP on SIRT1 protein expression in A549 cells with or without MMS treatment (1mM for 2 hrs). (E) Effects of overexpression or knockdown of SIRT1 and YAP on P53 deacetylation in A549 cells. (*P<0.05, **P<0.01 and ***P<0.001).

To confirm the crosstalk between acetylation of YAP and P53, YAP was then overexpressed in A549 cells under MMS treatment. Interestingly, overexpression of YAP induced marked decreases of acetylated P53 and γH2AX levels with MMS treatment (Figure 2C). Conversely, when YAP was knocked down by short hairpin RNAs (shRNAs) in A549 cells, the P53 acetylation levels were enhanced in both A549 cells with, or without MMS treatment (Figure 2C). The expression level of YAP was also examined by Western blotting (Figure 2B). Collectively, these findings suggest that YAP can inhibit P53 acetylation.

To further determine the modulating role of YAP on P53 acetylation, we screened the acetylase and deacetylase with P53 as a substrate (Figure 1). We found that P300, CBP, and PCF are the major acetylases for P53, and HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and SIRT1 are the major deacetylases for P53. We also found that, SIRT1 is the major candidate for further analysis. SIRT1 is an important responder to MMS, which is upregulated after MMS treatment.24 It has been well established that SIRT1 can decrease P53 acetylation and transcriptional activity.19 More importantly, it has been found that YAP induced SIRT1 activation through the pro-proliferation effector MYC.25 Therefore, we hypothesized that the suppressive effect of YAP1 on P53 acetylation is dependent on SIRT1. To confirm this, we examined the regulation of YAP on SIRT1 under MMS treatment. As expected, the MMS treatment induced upregulation of SIRT1, and overexpression of YAP led to a further enhancement (Figure 2D). In contrast, YAP depletion decreased the level of SIRT1 (Figure 2D). We also knocked down SIRT1 by siRNA and analyzed the expression level of SIRT1 by Western blotting (Figure 2B). Results indicate that knockdown of SIRT1 can enhance the acetylation of P53 and the knockdown of YAP displayed further enhancement. In contrast, overexpression of SIRT1 can reduced the acetylation of P53 (Figure 2E). Together, our results indicate that, YAP induces deacetylation of P53 by activating SIRT1.

YAP Increases The Deacetylation Of P53 And Promotes Cell Survival

Deacetylation of P53 has a profoundly negative impact on the capacity of P53 to induce the expression of target genes involved in the cell cycle and apoptosis. We then investigated whether YAP-induced deacetylation of P53 can affect P53’s function on the cell cycle and apoptosis. First, we checked the protein levels of P53 targets and downstream effectors involved in cell cycle modulation (P21 and P27) and apoptosis (BIM and PUMA) after overexpression or knockdown of YAP under MMS treatment. Consistent with the above findings, we found that MMS treatment enhanced the expression of P53 targets, and YAP inhibition induced further enhancement (Figure 3A). In contrast, overexpression of YAP attenuated the MMS-induced promoting effect on the expression of P53 targets (Figure 3A). Second, we tested the role of YAP in the regulation of mRNA expression of P53 targets. Consistent with the results of proteins expression, P21, P27, BIM, and PUMA were mostly upregulated under MMS treatment, and showed even more elevation after YAP depletion, while they were downregulated after the overexpression of YAP (Figure 3B). Third, we assessed the effect of YAP-induced P53 deacetylation on cell phenotypes, especially cell cycle modulation and apoptosis. As expected, MMS treatment induced G0/G1 arrest in A549 cells. YAP deletion enhanced this effect, while YAP overexpression attenuated this effect (Figure 3C). In addition, the apoptosis rate of A549 cells increased after MMS treatment and was further enhanced by YAP depletion, while weakened by YAP overexpression (Figure 3D). We further evaluated the in vivo effectiveness of YAP in mice bearing tumors originating from A549 cells. As expected, YAP OE promoted tumor growth, and P53 KD further enhanced this effect (Figure 3E and F). These results demonstrate that, YAP decreases P53 transcriptional activation and promotes cell survival by inhibiting P53-induced G0/G1 arrest and apoptosis.

Figure 3 Effects of YAP on P53 signaling and subsequent cell survival. (A–B) Effects of overexpression or knockdown of YAP on P53 targets protein expression (A) and mRNA expression (B) in A549 cells with or without MMS treatment (1mM for 2 hrs). (C–D) Effects of overexpression or knockdown of YAP on cell cycle modulation (C) and apoptosis (D) in A549 cells with or without MMS treatment (1mM for 2 hrs). (E) The tumor volume was periodically tested for each mouse and tumor growth curve was plotted. (F) The tumor was excised and weighed after 35 days. Data were shown as the mean ± SEM. (*P<0.05, **P<0.01 and ***P<0.001).

P53 Enhances YAP Acetylation

To investigate whether YAP underwent a qualitative change in response to MMS treatment, we performed similar time- and dose- dependent kinetics of YAP acetylation assays. Findings showed that, MMS treatment also resulted in increased acetylated YAP, as well as the γH2AX levels and that, MMS treatment with 1mM for 2 hrs was the optimal conditions for subsequent experiments (Figure 4A). The control group was set as the one with IgG antibody. These results show that endogenous YAP is also acetylated in response to MMS treatment, and MMS treatment induced the DNA damage.

Figure 4 P53 promotes YAP acetylation. (A) Time-dependent (upper) and dose-dependent (bottom) kinetics of YAP acetylation assays and γH2AX expression after MMS treatment in A549 cells. (B) A549 cells were transfected respectively with oeP53 and siP53 plasmid using Lipo2000 reagent, Western blotting examined the expression level of P53. (C) Effects of overexpression or knockdown of P53 on YAP acetylation and γH2AX expression in A549 cells with or without MMS treatment (1mM for 2 hrs). (D) Immunofluorescence effects of overexpression or knockdown of P53 on YAP nuclear translocation in A549 cells with or without MMS treatment (1mM for 2 hrs). (E) Effects of overexpression or knockdown of P53 on SIRT1 protein expression in A549 cells with or without MMS treatment (1mM for 2 hrs). (F) Effects of overexpression or knockdown of P53 and SIRT1 on YAP deacetylation in A549 cells. (*P<0.05, **P<0.01 and ***P<0.001).

To confirm the crosstalk between acetylation of P53 and YAP, P53 was then overexpressed in A549 cells under MMS treatment. P53 overexpression significantly elevated the acetylated YAP and γH2AX levels with MMS treatment (Figure 4C). Conversely, P53 depletion resulted in marked decreases of YAP acetylation levels in A549 cells both with, or without MMS treatment (Figure 4C). The expression level of P53 were also examined by Western blotting (Figure 4B). Collectively, these findings suggest that P53 can enhance YAP acetylation. We also confirmed these findings by immunofluorescence assays in A549 cells. This was consistent with previous evidence that showed MMS treatment induced YAP nuclear translocation and P53 knockdown resulted in a further enhancement, while P53 overexpression decreased the YAP nuclear translocation in A549 cells both with, or without MMS treatment (Figure 4D). Those results have been demonstrated in our previous studies.20

To understand the mechanism underlying these observations, we screened the acetylase and deacetylase with YAP as a substrate (Figure 1). It is well known that, P300 and CBP are the major acetylases for YAP and SIRT1 is its major deacetylase. More importantly, previous work has shown that, P53 inhibited SIRT1 activation by suppressing the pro-proliferation effector MYC,26 and SIRT1 regulates YAP-mediated cell proliferation and chemoresistance in hepatocellular carcinoma.20 Therefore, we hypothesized that SIRT1 is also required for the promoting effect of P53 on YAP acetylation. We then assessed the regulation of P53 on SIRT1 under MMS treatment. As expected, MMS treatment induced upregulation of SIRT1, and knockdown of P53 displayed a further enhancement (Figure 4E). In contrast, P53 overexpression decreased the level of SIRT1 (Figure 4E). Given that the overexpression of P53 could enhance the level of YAP acetylation, and knockdown SIRT1, displayed further enhancement. In contrast, SIRT1 overexpression decreased the level of YAP acetylation (Figure 4F). Taken together, these results indicate that P53 induces acetylation of YAP by inactivating SIRT1.

P53 Increases The Acetylation Of YAP And Cell Death

We then investigated whether P53-induced acetylation of YAP can affect the YAP function on the cell cycle and apoptosis. First, we checked the protein levels of YAP downstream effectors involved in cell cycle modulation and apoptosis (CTGF, Cycline E, MYC, and DIPA) after overexpression or knockdown of P53 under MMS treatment (Figure 5A). Consistently, A549 cells with MMS treatment exhibited decreased protein expression of these YAP downstream effectors, and P53 overexpression led to further inhibition (Figure 5A). In contrast, P53 knockdown recovered the MMS-induced suppressive effect on the expression of these YAP downstream effectors (Figure 5A). Second, we tested the role of P53 in the regulation of mRNA expression of these YAP downstream effectors. Consistent with the results of immunoblotting, CTGF, Cyclin E, MYC, and DIPA were mostly downregulated under MMS treatment and showed greater inhibition after P53 overexpression, while they were substantially elevated after knockdown of P53 (Figure 5B). Third, we examined the effect of P53-induced YAP acetylation cell phenotypes. As expected, MMS treatment induced G0/G1 arrest in A549 cells and P53 overexpression enhanced this effect, while P53 deletion attenuated this effect compared to controls (Figure 5C). Additionally, the apoptosis rate of A549 cells increased after MMS treatment and was further enhanced by P53 overexpression, while weakened by P53 depletion (Figure 5D). We further evaluated the in vivo effectiveness of P53 in mice bearing tumors originating from A549 cells. As expected, P53 OE induced inhibited tumor growth, and YAP KD further enhanced this inhibitory effect (Figure 5E and F). These results further indicate that P53 decreases YAP transcriptional activation and inhibits cell survival by strengthening YAP acetylation induced G0/G1 arrest and apoptosis.

Figure 5 Effects of P53 on YAP signaling and subsequent cell survival. (A-B) Effects of overexpression or knockdown of P53 on YAP downstream effectors protein expression (A) and mRNA expression (B) in A549 cells with or without MMS treatment (1mM for 2 hrs). (C–D) Effects of overexpression or knockdown of P53 on cell cycle modulation (C) and apoptosis (D) in A549 cells with or without MMS treatment (1mM for 2 hrs). (E) The tumor volume was periodically tested for each mouse and tumor growth curve was plotted. (F) The tumor was excised and weighed after 35 days. Data were shown as the mean ± SEM. (*P<0.05, **P<0.01 and ***P<0.001).

SIRT1 Is Responsible For Deacetylation Of YAP And P53

SIRT1 is a class III deacetylase. Overexpression of SIRT1 could enhance the level of YAP and decrease the level of P53, while knockdown of SIRT1 displayed an opposite result (Figure 6A). This indicated that SIRT1 is responsible for YAP and P53 expression. We also confirmed these findings by immunofluorescence assays in A549 cells, showing that MMS treatment induced YAP nuclear translocation and SIRT1 overexpression resulted in further enhancement, while SIRT1 knockdown decreased the YAP nuclear translocation in A549 cells both with, or without MMS treatment (Figure 6D). P53 was the first non-histone protein found to be acetylated in which acetylation competes to modify lysine residues that are also ubiquitinated, sumoylated, and methylated. When P53 is deubiquitinated by HAUSP, P53 can be acetylated at four well-known lysines (K120, K370, K373, and K382) by CBP and P300, leading to the transactivation of various P53 transcriptional targets.27,28 Thus, we investigated whether SIRT1 deacetylates P53 at these lysines. The total P53 protein was first immunoprecipitated and then immunoblotted with acetylated lysine antibodies recognizing acetyl-P53K382, acetyl-P53K373, acetyl-P53K370, and acetyl-P53K120. After MMS treatment, there were significant increases of acetylation at K370, K373, and K382 lysines in in A549 cells, whereas no significant change was observed at K120 acetylation (Figure 6B). These results provided evidence that MMS is involved in promoting acetylation of specific residues in P53.

Figure 6 Acetylation of P53 and YAP by SIRT1. (A) Effects of overexpression or knockdown of SIRT1 on P53 and YAP in A549 cells with or without MMS treatment. (B) Effect of SIRT1 on acetylation of four well-known lysines in P53. The total P53 protein was first immunoprecipitated and then immunoblotted with acetylated lysine antibodies recognizing acetyl-P53K382, acetyl-P53K373, acetyl-P53K370, and acetyl-P53K120. (C) Effects of SIRT1 on acetylation of two well-known lysines in YAP. The total YAP protein was first immunoprecipitated and then immunoblotted with acetylated lysine antibodies recognizing acetyl-YAPK494 and acetyl-YAPK497. (D) Immunofluorescence effects of overexpression or knockdown of SIRT1 on YAP nuclear translocation in A549 cells with or without MMS treatment. (*P<0.05, **P<0.01 and ***P<0.001).

We further evaluated the impact of SIRT1 on these residues’ acetylation in P53 and found that overexpression of SIRT1 inhibited the acetylation at all four residues (Figure 6B). Conversely, SIRT1 depletion elevated acetylation at the K370, K373, and K382 residues (Figure 6B), whereas no significant changes were observed at K120. These observations demonstrate that SIRT1 is involved with P53 acetylation of specific residues, including K370, K373, and K382.

The crosstalk between YAP and P53 also encouraged us to dissect the involvement of SIRT1 in YAP acetylation. The nuclear acetyltransferases CBP and P300 are responsible for YAP acetylation that occurs on specific and highly conserved C-terminal lysine residues K494 and K497. Thus, we hypothesized that SIRT1 is also involved in YAP acetylation at these two residues. To test this, the total YAP protein was first immunoprecipitated and then immunoblotted with acetylated lysine antibodies that recognize acetyl-YAPK494 and acetyl-YAPK497. Consistent with a previous finding that, MMS-induced DNA damage causes acetylation of K494 and K497 of YAP,29 we also observed increased K494 and K497 acetylation in YAP under MMS treatment. When SIRT1 was overexpressed in A549 cells, the K494 acetylation was significantly inhibited, whereas the K497 acetylation exhibited no changes (Figure 6C). In contrast, knockdown of SIRT1 substantially enhanced K494 acetylation in A549 cells both with, or without MMS treatment. These observations indicate that SIRT1 is involved in YAP acetylation of specific residues, especially K494.

Discussion

We have demonstrated previously undescribed post-translational crosstalk between the tumor suppressor protein P53 and YAP. This regulatory mechanism provides a molecular explanation for how the cell can integrate the divergent functional consequences of P53 and YAP activation. Using bioinformatics analyses, we found putative crosstalk between acetylation of YAP and P53. Through numerous biochemical experiments, we showed YAP enhanced the P53 deacetylation and inhibited P53 transcriptional activation, which prevented cell G0/G1 arrest and apoptosis. In contrast, the acetylation of YAP was promoted by P53, which led to decreasing YAP transcriptional activation, and promoting effects on cell G0/G1 arrest and apoptosis. Of note, the deacetylase SIRT1 was shown to be responsible for this negative feedback between the P53 and YAP signaling pathways. Furthermore, we showed that SIRT1 was involved with the P53 acetylation of specific residues (K370, K373, and K382), and the YAP acetylation of K494 residue. Together, these results demonstrate competition between P53 and YAP for limiting quantities of SIRT1 and provide a new paradigm of crosstalk between P53 and YAP which may be involved in lung tumorigenesis.

The implications are numerous. It is well known that both the Hippo pathway and P53 act as tumor suppressors to induce senescence and apoptosis. The Hippo pathway is mediated by the canonical function of inhibiting YAP and TAZ oncogenic activation, while P53 functions as a tumor suppressor in response to stress conditions.30 The cooperation between the wild-type P53 protein and the Hippo components, including YAP, can induce cell cycle arrest and apoptosis, contravening tumor transformation and progression. It was recently revealed that, P53 and YAP share a common transcriptional program showing a significant overlap with gene signatures primarily involved in cell cycle regulation.31 Our results suggest that, one factor influencing cell survival is the ability of P53 to decrease YAP transactivation and thus promote the transcriptionally dependent induction of cell G0/G1 arrest apoptosis. Consistently, YAP functions in a similar way. It appears likely that the outcome of crosstalk between P53 and YAP depends on the nature of the intrinsic function of the proteins, stimuli, the growth conditions, and the cell type.

Sequestration of SIRT1 is likely to be understood as an increasingly common mechanism reducing acetylation of targets, including many transcriptional factors, including P53, E2F1, FOXO, NF-κB, c-Myc, and YAP.32 The interaction of SIRT1 with tumor-suppressor proteins and oncoproteins implicates its role in cancer development and progression.33 SIRT1 deacetylates YAP2 protein, and that deacetylation upregulates the YAP2/TEAD4 association, leading to YAP2/TEAD4 transcriptional activation and cell growth in HCC cells.20 The key role of SIRT1 is exhibited through its specific interaction with P53 via P53 deacetylation at C-terminal lysine-373 and 382 residues in the NAD+-dependent manner.34 This action decreases P53-mediated transcriptional activity and reduces the expression of its targets, such as p21 (cell cycle inhibitor) and PUMA (modulator of apoptosis).19 Therefore, SIRT1 could inhibit P53-dependent cell cycle arrest and apoptosis, which facilitates cell death mechanism, while meanwhile enhancing the DNA repair mechanism to facilitate the maintenance of genomic stability. This would have a promoting effect on cell survival and proliferation.19 Thus, both P53 and YAP are substrates of SIRT1, and function as competitors for SIRT1. This crosstalk between P53 and YAP governs the balance of cell fate decisions in normal human cells. However, the P53 coding gene is frequently mutated in human cancers (50–70% of cases), and most of these mutations are of missense type.35 Knock-in mice with P53 missense mutations provided evidence that some mutant P53 exert pro-tumorigenic activities.36 We propose that the axis of P53-SIRT1-YAP may be perturbed by the somatic mutations of P53, resulting in the transformation of a normal cell into a cancerous cell. The cell lines used in this study are adenocarcinomic human alveolar basal epithelial cells carrying wild-type P53. Thus, further work is needed to investigate the dysregulation of this newly identified regulatory axis.

Conclusion

The present study demonstrated a novel post-translational crosstalk between P53 and YAP, potentially through the competition for the deacetylase SIRT1. Notably, this P53-SIRT1-YAP axis is important for cell cycle transition and apoptosis, while dysregulation of either of P53 or YAP can lead to the other competitor-induced transcriptional activation and cell phenotypes. Given the high mutation rate of P53 in cancers, it is possible that novel lung cancer therapies based on reactivation of wild-type P53 function might benefit from cooperation with YAP to promote a beneficial outcome.

Acknowledgments

This work was supported by grants from the National Key R&D Program of China, Stem Cell and Translation Research (no.2017YFA0106200 to FY), the National Natural Science Foundation of China (no.81402319 to FY, no.81202965 to JW), and the Beijing Nova Program (no.Z161100004916133 to FY).

Author contributions

All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

References

1. Ahmed AA, Mohamed AD, Gener M, Li W, Taboada E. YAP and the Hippo pathway in pediatric cancer. Mol Cell Oncol. 2017;4(3):e1295127. doi:10.1080/23723556.2017

2. Zanconato F, Forcato M, Battilana G, et al. Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat Cell Biol. 2015;17(9):1218–1227. doi:10.1038/ncb3216

3. Panciera T, Azzolin L, Cordenonsi M, Piccolo S. Mechanobiology of YAP and TAZ in physiology and disease. Nat Rev Mol Cell Biol. 2017;18(12):758–770. doi:10.1038/nrm.2017.87

4. Maugeri-Sacca M, De Maria R. The Hippo pathway in normal development and cancer. Pharmacol Ther. 2018;186:60–72. doi:10.1016/j.pharmthera.2017.12.011

5. Piccolo S, Dupont S, Cordenonsi M. The biology of YAP/TAZ: hippo signaling and beyond. Physiol Rev. 2014;94(4):1287–1312. doi:10.1152/physrev.00005.2014

6. Sorrentino G, Ruggeri N, Specchia V, et al. Metabolic control of YAP and TAZ by the mevalonate pathway. Nat Cell Biol. 2014;16(4):357–366. doi:10.1038/ncb2936

7. Strano S, Fausti F, Di Agostino S, Sudol M, Blandino G. PML surfs into HIPPO tumor suppressor pathway. Front Oncol. 2013;3:36. doi:10.3389/fonc.2013.00036

8. Karpowicz P, Perez J, Perrimon N. The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration. Development. 2010;137(24):4135–4145. doi:10.1242/dev.060483

9. Cho E, Feng Y, Rauskolb C, Maitra S, Fehon R, Irvine KD. Delineation of a fat tumor suppressor pathway. Nat Genet. 2006;38(10):1142–1150. doi:10.1038/ng1887

10. Ryan KM, Phillips AC, Vousden KH. Regulation and function of the p53 tumor suppressor protein. Curr Opin Cell Biol. 2001;13(3):332–337. doi:10.1016/S0955-0674(00)00216-7

11. Dai C, Gu W. p53 post-translational modification: deregulated in tumorigenesis. Trends Mol Med. 2010;16(11):528–536. doi:10.1016/j.molmed.2010.09.002

12. Harms KL, Chen X. The functional domains in p53 family proteins exhibit both common and distinct properties. Cell Death Differ. 2006;13(6):890–897. doi:10.1038/sj.cdd.4401904

13. Bai N, Zhang C, Liang N, et al. Yes-associated protein (YAP) increases chemosensitivity of hepatocellular carcinoma cells by modulation of p53. Cancer Biol Ther. 2013;14:511–520. doi:10.4161/cbt.24345

14. Meng Z, Moroishi T, Guan KL. Mechanisms of Hippo pathway regulation. Genes Dev. 2016;30(1):1–17. doi:10.1101/gad.274027.115

15. Hong W, Guan KL. The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway. Semin Cell Dev Biol. 2012;23(7):785–793. doi:10.1016/j.semcdb.2012.05.004

16. Furth N, Bossel Ben-Moshe N, Pozniak Y, et al. Down-regulation of LATS kinases alters p53 to promote cell migration. Genes Dev. 2015;29(22):2325–2330. doi:10.1101/gad.268185.115

17. Imai S, Armstrong CM, Kaeberlein M, Guarente L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature. 2000;403(6771):795–800. doi:10.1038/35001622

18. Lin Z, Fang D. The roles of SIRT1 in cancer. Genes Cancer. 2013;4:97–104. doi:10.1177/1947601912475079

19. Yi J, Luo J. SIRT1 and p53, effect on cancer, senescence and beyond. Biochim Biophys Acta. 2010;1804(8):1684–1689. doi:10.1016/j.bbapap.2010.05.002

20. Mao B, Hu F, Cheng J, et al. SIRT1 regulates YAP2-mediated cell proliferation and chemoresistance in hepatocellular carcinoma. Oncogene. 2014;33(11):1468–1474. doi:10.1038/onc.2013.88

21. Hata S, Hirayama J, Kajiho H, et al. A novel acetylation cycle of transcription co-activator yes-associated protein that is downstream of Hippo pathway is triggered in response to sn2 alkylating agents. J Biol Chem. 2012;287(26):22089–22098. doi:10.1074/jbc.M111.334714

22. Liu X, Ehmed E, Li B, et al. Breast cancer metastasis suppressor 1 modulates sirt1-dependent p53 deacetylation through interacting with dbc1. Am J Cancer Res. 2016;6(6):1441–1449.

23. Demchak B, Hull T, Reich M, et al. Cytoscape: the network visualization tool for genomespace workflows. F1000research. 2014;3:151. doi:10.12688/f1000research

24. Yamamori T, DeRicco J, Naqvi A, et al. SIRT1 deacetylates APE1 and regulates cellular base excision repair. Nucleic Acids Res. 2010;38(3):832–845. doi:10.1093/nar/gkp1039

25. Xiao W, Wang J, Ou C, et al. Mutual interaction between YAP and c-Myc is critical for carcinogenesis in liver cancer. Biochem Biophys Res Commun. 2013;439(2):167–172. doi:10.1016/j.bbrc.2013.08.071

26. Yuan F, Liu L, Lei Y, Tang P. p53 inhibits the upregulation of sirtuin 1 expression induced by c-Myc. Oncol Lett. 2017;14(4):4396–4402. doi:10.3892/ol.2017.6661

27. Liu L, Scolnick DM, Trievel RC, et al. p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage. Mol Cell Biol. 1999;19(2):1202–1209. doi:10.1128/mcb.19.2.1202

28. Li M, Chen D, Shiloh A, et al. Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature. 2002;416(6881):648–653. doi:10.1038/nature737

29. Kodaka M, Hata Y. The mammalian Hippo pathway: regulation and function of YAP1 and TAZ. Cell Mol Life Sci. 2015;72(2):285–306. doi:10.1007/s00018-014-1742-9

30. Mao B, Gao Y, Bai Y, Yuan Z. Hippo signaling in stress response and homeostasis maintenance. Acta Biochim Biophys Sin (Shanghai). 2015;47(1):2–9. doi:10.1093/abbs/gmu109

31. Di Agostino S, Sorrentino G, Ingallina E, et al. YAP enhances the pro-proliferative transcriptional activity of mutant p53 proteins. EMBO Rep. 2016;17(2):188–201. doi:10.15252/embr.201540488

32. Wang Z, Chen W. Emerging roles of SIRT1 in cancer drug resistance. Genes Cancer. 2013;4:82–90. doi:10.1177/1947601912473826

33. Song NY, Surh YJ. Janus-faced role of SIRT1 in tumorigenesis. Ann N Y Acad Sci. 2012;1271:10–19. doi:10.1111/j.1749-6632.2012.06762.x

34. Zhao Y, Lu S, Wu L, et al. Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1). Mol Cell Biol. 2006;26(7):2782–2790. doi:10.1128/MCB.26.7.2782-2790.2006

35. Rivlin N, Brosh R, Oren M, Rotter V. Mutations in the p53 tumor suppressor gene: important milestones at the various steps of tumorigenesis. Genes Cancer. 2011;2:466–474. doi:10.1177/1947601911408889

36. Freed-Pastor WA, Prives C. Mutant p53: one name, many proteins. Genes Dev. 2012;26(12):1268–1286. doi:10.1101/gad.190678.112

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