Histone deacetylase inhibitors inhibit lung adenocarcinoma metastasis via HDAC2/YY1 mediated downregulation of Cdh1

Metastasis is a leading cause of mortality in patients with lung adenocarcinoma. Histone deacetylases have emerged as promising targets for anti-tumor drugs, with histone deacetylase inhibitors (HDACi) being an active area of research. However, the precise mechanisms by which HDACi inhibits lung cancer metastasis remain incompletely understood. In this study, we employed a range of techniques, including qPCR, immunoblotting, co-immunoprecipitation, chromatin-immunoprecipitation, and cell migration assays, in conjunction with online database analysis, to investigate the role of HDACi and HDAC2/YY1 in the process of lung adenocarcinoma migration. The present study has demonstrated that both trichostatin A (TSA) and sodium butyrate (NaBu) significantly inhibit the invasion and migration of lung cancer cells via Histone deacetylase 2 (HDAC2). Overexpression of HDAC2 promotes lung cancer cell migration, whereas shHDAC2 effectively inhibits it. Further investigation revealed that HDAC2 interacts with YY1 and deacetylates Lysine 27 and Lysine9 of Histone 3, thereby inhibiting Cdh1 transcriptional activity and promoting cell migration. These findings have shed light on a novel functional mechanism of HDAC2/YY1 in lung adenocarcinoma cell migration.


HDAC2 is involved in HDAC inhibitors suppressed TGF-β-induced EMT of lung adenocarcinoma cells.
In order to investigate the potential involvement of HDACs in the migration of lung cancer cells, we conducted an examination of the effects of two HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaBu), which are structurally unrelated. Specifically, we sought to determine the impact of these inhibitors on TGF-β-induced EMT in A549 and H441 lung adenocarcinoma cells. Our findings indicate that both HDAC inhibitors effectively prevented TGF-β-induced cell migration ( Given that TSA and NaBu are pan-HDAC inhibitors, we further sought to identify the most relevant HDACs involved in this process by examining the expression of different HDACs in lung adenocarcinoma cells. We found that HDAC2 exhibits the highest level of expression among all HDACs (Fig. 1D,E), and its expression is positively correlated with the dosage of TGF-β (Fig. 1F,G). We also found that TSA and NaBu have no obvious effects on the mRNA change of TGF-β induced the expression of HDACs (Fig. S1B). Furthermore, our findings indicate that HDAC2 knockdown effectively inhibits H441 cell migration with or without TGF-β treatment ( Fig. 1H and Fig. S1C, D), implying that HDAC2 may be involved in the process of TGF-β-induced EMT. In conclusion, our study highlights the significant role of HDAC2 in TGF-β-induced EMT and suggests that HDAC2 could be a promising therapeutic target for the inhibition of lung cancer cell migration.
HDAC2 is highly expressed in lung adenocarcinomas cells. In order to examine the potential impact of HDAC2 on the survival of individuals with lung cancer, an analysis of the TCGA database was conducted utilizing GEPAI. The results indicated that the expression of HDAC2 was not significantly associated with the overall survival of lung cancer patients ( Fig. 2A). Nevertheless, a negative correlation was observed between the expression of HDAC2 and the overall survival of lung adenocarcinoma patients (Fig. 2B), which suggests that HDAC2 may play a role in the advancement of lung adenocarcinoma.
Further analysis of the TCGA database demonstrated a marked elevation of HDAC2 expression in lung adenocarcinomas (Fig. 2C). Our additional scrutiny of HDAC2 expression in clinical lung adenocarcinomas corroborated this observation (Fig. 2D). These findings provide evidence that HDAC2 is significantly upregulated in lung adenocarcinoma cells and is closely associated with the overall survival of lung adenocarcinoma patients, suggesting that HDAC2 may exert a pivotal role in the progression of lung adenocarcinoma and could represent a promising therapeutic target.
HDAC2 promotes lung adenocarcinomas migration. In order to examine the involvement of HDAC2 in the migration of lung adenocarcinoma cells, we conducted an overexpression of HDAC2 in A549 and H441 cells, both are well used KRAS-mutant lung adenocarcinoma. Our results indicate that HDAC2 facilitates cell migration (Fig. 3A,B and Fig. S2A) and metastasis (Fig. S2B). To further validate the role of HDAC2, we also assessed the impact of HDAC2 knockdown on cell migration ( Fig. 3C and Fig. S2C). Additionally, we conducted a thorough analysis of the TCGA database, which revealed a positive correlation between HDAC2 and key EMT-related transcription factors, such as snail1 and snail2, but not other transcriptional factors, in lung cancer tissues (Fig. 3D,E and Fig. S2C). These findings suggest that HDAC2 plays a crucial role in promoting lung adenocarcinoma metastasis.
HDAC2 interacts with YY1 in lung adenocarcinoma cells. In order to explicate the functional mechanism of HDAC2 in the cellular migration process, an analysis of its interaction proteins was conducted using STRING. This analysis revealed the presence of a well-known tumor inducer, Yin Yang 1 (YY-1) (Fig. 4A). Further analysis of YY-1's interaction proteins indicated that HDAC2 was among its top 10 binding proteins (Fig. 4B). To delve deeper into this interaction in the context of lung adenocarcinoma, the interaction of endogenous HDAC2 and YY1 was examined in A549 cells, revealing that YY1 and HDAC2 can indeed interact with one another (Fig. 4C and Fig. S3). These findings suggest that HDAC2 has the potential to interact with YY1. YY1 functions as a collaborative partner with HDAC2 to facilitate cellular migration. The  www.nature.com/scientificreports/ involvement of YY1 in lung adenocarcinoma migration. Our findings indicate that TGF-β induced YY1 expression within 2 h of treatment, followed by a gradual decrease until 24 h (Fig. 5A,B and Fig S4A, B). Moreover, the overexpression of YY1 significantly facilitated cell migration (Fig. 5C-E and Fig. S4C) and lung metastasis (Fig.  S4D). Further analysis revealed a positive correlation between YY1 and key EMT-related transcription factors, such as snail1 and snail2, but not other transcriptional factors (Fig. 5F).
In order to validate the crucial involvement of YY1 in HDAC2-mediated cellular migration, we conducted a deletion of YY1 in HDAC2 overexpressed lung adenocarcinoma cells (Fig. 5G). Our findings demonstrate that the removal of YY1 resulted in the elimination of HDAC2-induced cellular migration ( Fig. 5H-J). These outcomes provide evidence that YY1 serves as a functional collaborator with HDAC2 in the migration of lung www.nature.com/scientificreports/ adenocarcinoma cells, and emphasize the potential for targeting HDAC2-YY1 interactions as a means of developing innovative therapeutic approaches.
YY1 and HDAC2 is highly expressed in lung adenocarcinoma tissues. In order to examine the clinical significance of YY1 and HDAC2 in lung adenocarcinoma, we conducted an analysis of their expression levels in clinical patient samples. Our findings indicate that both YY1 and HDAC2 were significantly upregulated in cancerous samples when compared to their corresponding adjacent tissues (Fig. 6A,B). Furthermore, our examination of the TCGA database demonstrated a positive association between YY1 and HDAC2 expression in both lung (Fig. 6C,D) and adenocarcinoma tissues (Fig. 6E). These observations suggest that YY1 and HDAC2 may have crucial roles in the initiation and progression of lung adenocarcinoma. www.nature.com/scientificreports/ Magnification is 200-fold, and scale bar is 50 μm. Data are presented as mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group. All experiments were performed at least three times. www.nature.com/scientificreports/ www.nature.com/scientificreports/ HDAC2 inhibits YY1 induced Cdh1 transcription. YY1, a well-known transcription factor, plays a crucial role in regulating gene expression in diverse cellular processes, with its canonical binding sites being CCAT (Fig. 7A). Upon analyzing the promoter sequence of EMT-related genes, we identified two binding sites on the Cdh1 promoter and subsequently designed three mutated promoters with altered binding sites (Fig. 7B).The ChIP assay yielded evidence that YY1 interacts with the Cdh1 promoter, as depicted in Fig. 7C. Furthermore, YY1 was found to enhance Cdh1 promoter activity, which was impeded by mutations in the binding sites on the promoter (Fig. 7D). Our investigation also involved an analysis of the acetylation level of the canonical lysine sites of histone 3, which revealed that HDAC2 deacetylated K27 and K9 acetylation ( Fig. 7E and Fig. S5). Finally, the graphic abstract of this study is presented in Fig. 7F.

Materials and methods
Cell culture. The HEK293T, A549, and H441 cell lines were procured from the American Type Culture Col-   Tables 1 and 2. HEK293T cells were employed to package the expressed lentivirus of pCDH-hHDAC2, pCDH-hYY1, shHDAC2, and shYY1 via calcium chloride transfection, followed by virus concentra- Data are presented as mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group. All experiments were performed at least three times. RNA isolation and real-time PCR. RNA extraction was carried out by following the manufacturer's instructions 23 using RNAiso Plus (Takara, Japan). Subsequently, the mRNA was reverse transcribed at 37 °C for 15 min using PrimeScript™ RT Master Mix (Takara, Japan). Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, USA). The relative expression levels were determined using the ΔΔCt method of relative quantitation and normalized to human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression. Unless otherwise specified, the data presented are derived from three independent biological replicates, each of which was assayed in triplicate. The primers used for real-time PCR are listed in Table 3.  www.nature.com/scientificreports/ Luciferase reporter assay. The Homo Cdh1 wt, mut1, mut2, and mut1-2 promoter sequences were cloned into the pGL3 basic firefly luciferase vector (Thermo Scientific) and subsequently transfected into 293T cells. Following a 24-h incubation period, cell lysates were prepared in reporter lysis buffer (Promega, Madison, WI, USA) and luciferase substrate was introduced. The resulting luciferase activity was measured using a luminometer (Veritas, Promega).

Stable transfection.
The lentivirus containing HDAC2, YY1, shHDAC2, and shYY1 was produced in HEK293T cells through calcium chloride transfection. The viral supernatants were collected thrice at 24-h intervals post-transfection, followed by centrifugation, filtration, and infection of lung cancer cells. The cells were then subjected to selection with puromycin for a minimum of 1-2 weeks. The efficacy of gene overexpression or knockdown was assessed through qPCR or western blotting.

Cell lysates preparation and immunoblotting. The preparation of cell lysates and immunoblotting
was conducted in accordance with previously described methods 24 . Specifically, cells were lysed using RIPA lysis buffer (Beyotime, Shanghai) supplemented with protease and phosphotase inhibitors and PMSF. The resulting lysates were collected and subjected to centrifugation at 12,000 rpm for 15 min at 4 °C. The soluble protein was then quantified using a BCA quantification kit (Beyotime, Shanghai), and protein samples were subsequently electrophoresed on SDS-PAGE and transferred onto nitrocellulose membranes (Pall, Amersham). The membranes were blocked using 5% defatted milk in TBST buffer supplemented with 0.1% Tween-20. Following this, the membranes were subjected to an overnight incubation with the suitable primary antibodies at 4 °C. The membranes were then rinsed with TBST and exposed to secondary antibodies. Protein bands were detected using super signal reagents. Antibodies were diluted in accordance with the manufacturer's instructions. β-actin was utilized as a loading control.
Co-Immunoprecipitation. The present study conducted an assay in accordance with a prior report 25 . Specifically, cellular lysis was carried out in ice-cold RIPA buffer supplemented with 1 mM PMSF and 10uM Protein Kinase Inhibitor on ice for 30 min. Subsequently, the insoluble fraction was removed via centrifugation at 4℃ for 15 min at 12,000 rpm, and the soluble protein was quantified using a BCA quantification kit and pre-cleared using protein-A/G sepharose. Immunoprecipitation was performed by incubating the aforementioned lysates with either anti-HDAC2 or anti-YY1 primary antibody at 4 °C for 8-12 h, with normal IgG serving as the negative control. The mixture of antibody-lysates was supplemented with protein-A/G sepharose and incubated at 4 °C for 2-4 h. The resulting antigen-antibody-sepharose complex was washed with PBS at 4 °C every 5 min. Subsequently, the complex was collected and heated in 1 × loading buffer at 95 °C for 5 min, followed by immunoblotting of the eluted proteins. The manufacturer's instructions were followed to determine the appropriate quantity of specific antibody for this assay.
Wound healing assays. 5 × 10 5 cells were seeded in 35-mm culture dish. 24 h later, wounds were incised in the middle area of the confluent cell culture, followed by addition of fresh medium after carefully washing off the detached cells. Images were taken of the wounded area using Nikon digital camera at 4 × magnification at 0, 24, and 48 h.
Transwell assays. The present study conducted an assay in accordance with a prior report 26 . Specifically, 5 × 10 4 cells were suspended in culture medium containing 0.5% FBS and subsequently seeded into the upper well of a transwell chamber (Corning Costar, Thermo fisher, NY), while the lower well was filled with culture medium containing 10% FBS as a chemoattractant. Following incubation for 16 h at 37 °C in the presence of 5% CO 2 , non-migrated cells were removed from the upper surface. The migrated cells were then fixed with alcohol and stained with H/E. The total number of migrated cells was determined by counting the cells using a Nikon digital camera at 200× magnification.
Xenograft mouse model. Female athymic BALB/c nude mice (4 weeks) were purchased from Gempharmatech (Nanjing, China). All animal experiments were approved by Animal Ethics Committee of Zhengzhou University (Approval Number: ZZU-LAC20230616 18 ). All methods were performed in accordance with the relevant guidelines and regulations. Also all methods are reported in accordance with ARRIVE guidelines. Mice were allocated www.nature.com/scientificreports/ to experimental groups randomly. 5 × 10 6 cells were injected into mice through tail vein. 4-6 weeks later, animals were euthanized and tissues were collected for tumor counting and HE staining.
STRING analysis. The analysis of the interaction among proteins are performed by STRING online software, the website is https:// string-db. org/.
Online database. The present study assessed the expression levels of HDACs in lung tumor and adjacent tissues, which were categorized as "high" or "low" based on their expression levels relative to the median value of all samples. The survival rate of the "high" and "low" expression groups was analyzed using the log-rank test. The correlation between gene expression and survival was evaluated using Kaplan-Meier plots generated by Gene Expression Profiling Interactive Analysis (GEPIA) in the http:// gepia. cancer-pku. cn/ 27 .
Ethical approval. The lung cancer tissue chip (Hlug-Ade 060PG-01) was purchased from Shanghai Outdo Biotechnology Company and the clinical information was listed in Table 4.
Statistical analysis. The statistical analysis was from more than three independent experiments performed in duplicates or up to three parallel controls. For experiments with two groups, statistical signifificance was determined by Student's t-test. For experiments with more than three groups, statistical analyses were performed with analysis of variance followed by post hoc pairwise comparisons. The data shown are means ± SEM. The P-value of less than 0.05 was considered statistically significant. The P-values were designated as *, P < 0.05, **, P < 0.01, ***, P < 0.001. ns, non significant.

Discussion
HDAC inhibitors have demonstrated potential in regulating the progression of cancer cells. A multitude of HDAC inhibitors, such as vorinostat(SAHA) 28 , romidepsin 29 , belinostat, panobinostat, and entinostat, have been associated with the management of cancer cell progression 30 . Prior studies have indicated that HDACi (histone deacetylase inhibitors) impede cancer progression by influencing diverse cellular processes, including tumor growth, programmed cell death, metastasis, and angiogenesis 31 . Further investigation is necessary to comprehensively elucidate the precise molecular mechanisms underlying the control of cancer progression by HDAC inhibitors. Although more than ten HDACs are present in mammalian cells, there is currently no evidence indicating which one is pivotal in the migration of lung adenocarcinoma. This study has provided insight into the heightened expression and induction of HDAC2 during the process of lung adenocarcinoma, which has captured our attention. Furthermore, we have identified that two HDAC inhibitors with distinct structures, TSA and NaBu 32 , impede lung adenocarcinoma migration via HDAC2 by interacting with YY1, a transcription factor, and deacetylating Cdh1, a tumor suppressor gene 33 . The downregulation of Cdh1 is often observed in cancer cells undergoing epithelial-mesenchymal transition (EMT), a process that enables cancer cells to acquire a more motile and invasive phenotype 34 . It is noteworthy that the involvement of HDAC2 in tumorigenesis varies across different cancer types and is often complex and even contradictory. For instance, in glioblastoma tumorigenesis, HDAC2 knockdown has been shown to impede tumor-sphere formation and proliferation by upregulating miR-3189-mediated GLUT3 35 .In contrast, HDAC2 functions as a metastasis suppressor in colorectal cancer by impeding EMT and the expression of H19 and MMP14 36 . These observations indicate that HDAC2 exhibits disparate roles in various cancer types. Nevertheless, the precise functional mechanism of HDAC2 in lung adenocarcinoma migration remains elusive, necessitating a comprehensive comprehension of its role in lung adenocarcinoma patients.
Multiple studies have reported on the interplay between HDAC2 and YY1, but this complex exhibits divergent functions in different tissues. For example, the physical interaction between FKBP25 and histone deacetylases HDAC1 and HDAC2, as well as the HDAC-binding transcriptional regulator YY1, results in the modification of YY1's DNA-binding activity 37 . In clear cell renal cell carcinoma, the YY1/HDAC2 complex reduces the expression of YTHDC1, which modulates the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway 38 . Additionally, the YY1/HDAC2 signaling pathway is crucial in regulating cell proliferation in human colorectal cancer 39 . Furthermore, a recent investigation has uncovered the indispensable role of the HDAC2/YY1 complex in lung adenocarcinoma metastasis.
This study illuminates the intricate interplay among diverse molecular pathways in cancer metastasis and emphasizes the significance of devising targeted therapies that can impede these pathways and forestall cancer dissemination. Nevertheless, it is crucial to acknowledge that this study was executed in vitro, and additional research is imperative to authenticate these findings in vivo and in human clinical trials.

Data availability
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