Inhibition of ZNF703 alleviates the progression of gastric cancer through inhibition of HDAC activity

Inhibition of ZNF703 alleviates the progression of gastric cancer through inhibition of HDAC activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of ZNF703 alleviates the progression of gastric cancer through inhibition of HDAC activity Hao Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1945136/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: ZNF703 is identified as a therapeutic target in a variety of human cancer. Although ZNF703 overexpresses in gastric cancer frequently, the effects and mechanism of ZNF703 in the progression of gastric cancer is unclear. Methods: Therefore, ZNF703, Ki-67 and BCL-2 expression was measured by histology in clinical cases. We used gastric cells line models to explore the role of ZNF703 in vitro . ZNF703 expression intervention was employed to investigate the role of ZNF703 in proliferation and apoptosis. The relationship between ZNF703 intervention and resistance of chemotherapy was analyzed by using oxaliplatin treatment. Results: In this study, we found that ZNF703 expression in the area of gastric cancer was substantially higher than adjacent normal area. Gastric cancer tissue with ZNF703 high expression level substantially increased Ki-67 and BCL-2 expression. Inhibition of ZNF703 attenuated the gastric cancer cell proliferation and induced apoptosis in SGC7901 and BGC823 cells, while overexpression of ZNF703 in GES-1 cells resulted in the reverse effects. ZNF703 might mediate the viability of gastric cancer cells through down-regulation of HDAC1/2. In addition, after transfected with siRNA-ZNF703, down-regulation of TopoII and P-gp was observed in SGC7901 and BGC823 cells. Further, we showed that inhibition of ZNF703 enhanced the resistance to chemotherapy in vitro . Conclusions: Our study demonstrated that in gastric cancer cells, ZNF703 promoted the proliferation, inhibited apoptosis, and improved their resistance to chemotherapy, suggesting it may be a potential target for the gastric cancer. ZNF703 gastric cancer proliferation resistance of chemotherapy HDAC1/2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Gastric cancer causes the second most frequent cancer-related mortality all over the world. Over 400,000 new cases of gastric cancer in China are definitely diagnosed every year, accounting for 40% of total number in the global [ 1 ]. Despite recent advances in diagnosis and treatment, the majority of GC patients are diagnosed at the advanced stage with only 1-year expected survival. Even though targeted therapies against HER2 and VEGFR2 monoclonal antibodies have been approved for advanced gastric cancer, outcome of these patients is still depressing as targeted therapies only prolong life for 2 months [ 2 , 3 ]. The prominent characteristics of advanced gastric cancer are high-frequency metastasis and proliferation. However, the underlying mechanisms that lead to the aggressiveness of gastric cancer remains unexplored. Zinc finger genes (ZNFs), an important gene family in human, are considered to interact with regulatory proteins and DNA sequences, then regulating downstream gene expression [ 4 ]. ZNF703, located on chromosomes 8 (8p11.23), is identified as an oncogene in various human cancer, such as Breast Cancer [ 5 – 7 ], Non-Small Cell Lung Cancer [ 8 ], Head and Neck Cancer [ 9 , 10 ] and so on. As a member of zinc finger protein subfamily, ZNF703 protein can combine with Zn2 + by several conserved amino acid residues to form a tetrahedral structure. This structure forms a transcription inhibitor complex with Groucho family protein, HDAC1/2 and zinc structure. As such complex binds to the target gene promoter and amplifies the regulatory effect on the expression of target gene [ 5 ]. An investigation shows that the gene of the 8p11-12 chromosomal region is amplified in human breast cancer [ 11 , 12 ]. Several studies in human breast cancer suggest that ZNF703 has a positive impact on cells proliferation and metastasis by altering fundamental functions in physiological processes and pathways [ 6 , 7 ]. Interestingly, an elegant study finds that ZNF703 is overexpressed in gastric cancer, suggesting it may be involved in the progression of the gastric cancer [ 13 ]. However, the underlying mechanisms of ZNF703 have not yet been fully characterized. In this study, we investigated the expression of ZNF703 on human gastric cancer tissue, normal gastric epithelial cell line (GES-1) and gastric cancer cell lines (SGC7901 and BGC823). The overexpression of ZNF703 was verified in human gastric cancer tissue. In vitro experiment indicated that ZNF703 facilitated gastric cancer cells proliferation and migration and inhibited their apoptosis, which were associated with related gene expression bcl2 and bax. In addition, transfecting SGC7901 and BGC823 cells with siRNA-ZNF703 resulted in down-regulation of TopoII and P-gp. Inhibition of ZNF703 was involved in improvement of chemotherapy resistance, which was positively correlated with down-regulation of HDAC1/2. These data indicated that overexpression of ZNF703 in gastric cancer facilitated the gastric cancer progression, ZNF703 may be a potential target for gastric cancer therapy. Materials And Methods Patients and samples Accordance to histological diagnostic criteria of gastric cancer, tumor tissues were obtained from thirty gastric cancer patients. All patients signed the informed consent, and the collection of these tissue samples were approved by the Ethics Committee of the School of Medicine Fudan University. All the tissue specimens were collected from patients diagnosed with gastric cancer at Huashan Hospital. Cell lines and culture Human gastric cancer cell line BGC823 and the normal gastric epithelial cell line GES-1 were purchased from American type culture collection (ATCC, Manassas, VA). Human gastric cancer cell line SGC7901 was purchased from the China center for type culture collection (CCTCC, Wuhan, China). Cells were cultured in Dulbecco’ s modified Eagle’ s medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% FBS at 37℃ in 5% CO 2 . Cell proliferation assays MTT (Amresco, China) assay was used to measure cell proliferation. Briefly, cells were dispensed into 96-well plates. MTT was added to each plate at a final concentration of 0.5 mg/ml for 4 h incubation at 37℃. The supernatant medium was then removed. 150 µl dimethylsulfoxide (Sigma, St. Louis, MO, USA) was added into each plate, and incubated 10 min at room temperature. The absorbance value was measure at 490 nm by a microplate reader. Cell apoptosis assays Cells were harvested and washed three times with cold PBS, followed by staining with PI and Annexin V-FITC in an Annexin-V-FITC/PI apoptosis detection kit (Vazyme Biotech Co.,Ltd, China). The percentage of apoptotic cells was measured by a BD flow cytometer. Cell transfection For overexpressing ZNF703 in GES-1 cells, Lipofectamine 2000 (Life Technologies, USA) was employed for transfection. Following the manufacturer’s protocol, when cells developed 70%-90% confluent, they were transfected with plasmids pCDH-CMV-MCS-EF1-PURO or pCDH-CMV-MCS-EF1-PURO-HA-ZNF70 for 6 hours. Then cells were harvested or the medium was replaced with normal fresh medium for further experiments. For Small interfering RNA-mediated gene silencing of ZNF703 in gastric cancer cells, BGC823 Cells and SGC7901 were cultured in a 6-well plate. When cells developed at the density of 70%-90% confluent, siRNA-ZNF703 or siRNA-NC were transfected using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer’s protocol. The sequences of siRNA are shown as followed: siRNA-ZNF703 forward, GCUUGACCCUGCCGGGAUU reverse, CUUACAAGCUGGGAAUAUA siRNA-NC forward, UUCUCCGAACGUGUCACGUTT reverse, ACGUGACACGUUCGGAGAATT Short hairpins, plasmids, and lentiviral transduction and infection ZNF703-specific shRNAs was designed and developed by the TRCat the Broad Institute of MIT. One ZNF703 hairpin that showed high-efficacy knockdown was selected. High-titer virus-containing supernatants of HEK293FT cells after transient co-transfection of lentiviral vectors with pMD2.G and psPAX2 packaging vectors were used for lentiviral-mediated transduction of cancer cells. Immunohistochemistry analysis Gastric cancer samples from patients were fixed with 4% paraformaldehyde, embedded in paraffin. Then the samples were cut into 4 μm slides, followed by dewaxing and rehydrating. Slides were incubated with 5% BSA for blocking at room temperature for 0.5 h and then probed with an optimal dilution of a primary rabbit antibody against human ZNF703, Ki-67 or BCL-2 overnight at 4°C. After incubated with a biotinylated goat anti-rabbit immunoglobulin G antibody and washed, the slides were visualized using diaminobenzidine (DAB) and counterstained with hematoxylin. ZNF703 expression was evaluated in tumor cells with the semi-quantitation method in accordance with the area of positive (AP) and the intensity of staining (IS). AP was scored as follows: 0 (0-5%), 1 (6-25%), 2 (26-50%), 3 (51-75%) and 4 (>75%). IS was graded as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The final expression level of ZNF703 was determined based on the equation: Intensity distribution (ID) = AP × IS. RNA extraction and quantitative real-time PCR (RT-qPCR) assays Total RNA was extracted from gastric cancer cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. And each RNA sample was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Promega, USA) in a 50 µl reaction system. According to the manufacturer's instructions, quantitative real-time PCR was performed with a conventional SYBR green-based (Life Technologies, USA) quantification method. The RT-qPCR primers were used as follows: ZFN703-Forward, 5'-GATCAGGGTCCTGAAGATGC-3, ZFN703--Reverse, 5'-CCGAGTTGAGTTTGGAGGAG-3'; GAPDH-Forward, 5′-AGGTGAAGGTCGGAGTCAAC-3′, GAPDH-Reverse, 5′-GGGTGGAATCATATTGGAACA-3′. ACY-957 and oxaliplatin treatment For inhibition of HDAC1/2 in gastric cancer cells, a selective inhibitor of HDAC1/2 (10 mM in DMSO) ACY-957 was added into the plate with the final concentration of 2 μM. The cells treated with equivalent DMSO were served as control. Cells were incubated for 24 h before subsequent assay. For oxaliplatin treatment, oxaliplatin was added into the plate with the final concentration of 25 ug/ml. Cells were incubated for 24 h before subsequent assay. Western blot analysis Total protein of cells was extracted using RIPA lysis buffer containing phenylmethane-sulfonyl fluoride (PMSF). After boiling, equal amounts of proteins (20 μg) were loaded into 10% SDS-PAGE gels for separation. And the separated proteins were transferred to PVDF membranes. The membranes were blocked with 10% non-fat dried milk dissolved in TBS containing 0.5% Tween 20 for 1.5 h, and then incubated with primary antibodies at 4℃ overnight. After incubated with appropriate secondary antibodies at room temperature for 1 h and washed, immunoreactivity was visualized using enhanced chemiluminescence (ECL) system (Image Quant LAS 4000 mini, Pittsburgh, PA, USA) according to the instructions of the manufacturer. All experiments were replicated three times for statistical analysis. Statistical analysis Results are shown as the mean ± SD. Asterisks presented significant difference. Statistical analysis was performed using Mann-Whitney U test with Graph-Pad Software. P<0.05 was considered as significant difference for all experiments. Results ZNF703 is highly expressed in clinical gastric cancer tissues and gastric cancer cell. To explore the characteristics of ZNF703 expression in gastric cancer patients, the sections of gastric cancer tissues from gastric cancer patients were stained with ZNF703. The immunohistochemical (IHC) staining showed that the expression of ZNF703 in gastric cancer area was even higher compared with that in adjacent normal area (Figure.1A and 1B). To determine the clinical utility of strategies targeting the ZNF703, the analysis for ZFN703 expression (n=619, the number of tumor tissue and No-tumor tissue was 408 and 201, respectively) in The Cancer Genome Atlas (TCGA) cohort were explored, indicating that high ZFN703 expression was observed in tumor tissue compared with no-tumor tissue (Figure. 1C). In addition, using western blotting and RT-qPCR assay, we found that the expression of ZNF703 was significant higher in gastric cancer cell line SGC7901 and BGC823 than that in the normal human gastric epithelial cell line GES-1 (Figure.1D-F). To ascertain the relationship between ZFN703 expression and survival in patients with gastric cancer, patients with gastric cancer survival data from TCGA cohort was analyzed, indicated that patients with higher expression of ZNF703 had a significant association with poor survival (P=0.031; hazard ratio (HR)=1.7; Figure.1G). These data suggested that overexpression of ZNF703 may be associated with gastric cancer. ZNF703 promotes the cell proliferation of gastric cancer cells. To assess the influence of ZNF703 on the development of gastric cancer, IHC was performed to determine the proliferation function by targeting Ki-67 in gastric cancer tissues. The result showed that ZNF703 high-expression level was associated with an increased Ki-67 expression level (Figure. 2A and B). TCGA datasets was analyzed to ascertain the relationship between ki67 and ZNF703 expression in patients with gastric cancer, the result showed that ZNF703 expression was related to tumor cell. We then analyzed the several GC cells lines proliferation via intervening the expression of ZNF703. The treatment regimens were utilized: the experiments of overexpression ZNF703 in GES-1 cells was performed by using a plasmid encoding CMV-ZNF703. The siRNA transfection assay was used to inhibit ZNF703 expression in gastric cancer cell lines SGC7901 and BGC823. The result of MTT assay showed that a significant reduction in the cell viability was observed in ZNF703 siRNA-transfected SGC7901 and BGC823 cells compared with that in control cells (Figure. 2D and E). To some extent, overexpression of ZNF703 in GES-1 cells promoted cell proliferation (Figure. 2C). Hence, our data indicated that ZNF703 promoted cell proliferation of gastric cancer cells. ZNF703 might affect the cell proliferation of gastric cancer cells through up-regulation of HDAC1/2. As a study suggests that ZNF703 can interact with HDAC1/2 to regulate downstream gene expression [5], we asked the relationship between ZNF703 and HDAC1/2 in the gastric cancer cells. Here, GES-1 cells were transfected with plasmid CMV-ZNF703 or plasmid CMV control. Using western blotting analysis, we found that overexpression of ZNF703 in GES-1 cells significantly increased the expression of HDAC1/2 compared with that in plasmid CMV control (Figure.3A and B). In addition, after being transfected with siRNA-ZNF703 or siRNA-NC, a substantial decrease was observed in the expression of HDAC1/2 in SGC7901 and BGC823 cells compared with that in control cells (Figure.3C-F). To confirm the relevance of ZNF703 and HDAC1/2 in gastric cancer cells, we analyzed the proliferation of SGC7901 and BGC823 cells that be treated with a HDAC1/2 inhibitor ACY-957. As expected, the results showed that inhibition of HDAC1/2 in SGC7901 and BGC823 cells inhibited the cell proliferation of SGC7901 and BGC823 cells compared with control group (Figure.3G and H). Thus, these results suggested that ZNF703 might be interacted with HDAC1/2 to regulate proliferation of gastric cancer cells. Inhibition of ZNF703 induces apoptosis of gastric cancer cells We then analyzed the relationship between ZNF703 expression level and anti-apoptosis function in gastric cancer, IHC assay showed that gastric cancer tissue with ZNF703 high expression level substantially increased the BCL-2 expression level (Figure. 4A). As inhibition of cancer cell apoptosis may be associated with induction of cell proliferation, we explored the relationship between ZNF703 expression and gastric cancer cells apoptosis. Using the Annexin-V-FITC/PI staining, the apoptotic cells were identified as Annexin V+ cells. After being transfecting with siRNA-ZNF703 in SGC7901 and BGC823 cells, a significant increase was observed in the percentages of apoptotic cells as compared with these cells transfected with siRNA-NC (Figure. 4 B). In comparison, the adverse results were observed in GES-1 cells that transfected with plasmid CMV-ZNF703 (Figure.4D). In addition, the apoptosis-associated proteins were measured by using western blotting assay. The results showed that transfecting SGC7901 and BGC823 cells with siRNA-ZNF703 reduced the protein levels of bcl2 and increased the expression of bax (Figure.4 C), while overexpression of ZNF703 in GES-1 showed reverse expressions of above proteins (Figure. 4 E). Taken together, these data demonstrated that inhibition of ZNF703 was involved in the apoptosis of gastric cancer cells. Inhibition of ZNF703 is involved in improvement of chemotherapy resistance and Next, we investigated the role of ZNF703 expression on the improvement of chemotherapy resistance of gastric cancer cells. After transfecting with siRNA-ZNF703 or siRNA-NC in SGC7901 and BGC823 cells, these cells were treated with a kind of chemotherapeutic drug, oxaliplatin. MTT assay was performed to evaluate the effect of oxaliplatin. The results showed that inhibition of ZNF703 significantly reduced the cell viability of the SGC7901 and BGC823 cells to chemotherapy (Figure. 5A and B). In addition, the chemotherapeutic resistance-associated proteins were detected by western blotting assay. After transfected with siRNA-ZNF703, we found that SGC7901 and BGC823 cells that dramatically reduced the protein levels of TopoII and P-gp (Figure. 5 C-F). As overexpression of ZNF703 contributed to the increase of HDAC1/2 expression, we queried the relevance of HDAC1/2 expression and chemotherapy resistance. SGC7901 cells were treated with a HDAC1/2 selective inhibitor ACY-957. The result indicated that inhibition of HDAC1/2 significantly sensitize the SGC7901 and BGC823 cells to chemotherapy (Figure. 5G). Taken together, these data suggested that inhibition of ZNF703 might sensitized the SGC7901 and BGC823 cells to chemotherapy through HDAC1/2 expression. Moreover, xenograft mice models were established by subcutaneously injecting scrambled- or ZNF703-shRNA-infected SGC7901 cells. Compared with the control group, the xenograft volume decreased in the shRNA-ZNF703 groups (Figure. 5H-K). Taken together, these data demonstrated that inhibition of ZNF703 may delay the development of gastric cancer. Discussion Chromosomal breaks and rearrangements generally result in altering oncogenes and tumor suppressor genes expression, which contributes to cancer progression and oncogenesis [ 14 , 15 ]. The gene sequence for the ZNF703 transcription factor is one of the frequently amplified genes in cancer [ 16 , 17 ]. An investigation in human breast cancer indicates that the amplification of the chromosome 8p11-12 region contains tumor metastasis-promoting genes [ 18 ]. As a result of investigations to identify the genes which lie in that region, ZNF703 has been identified as the driver oncogene within this region [ 16 ]. A recent study demonstrates a compelling case for amplification of ZNF703 as an important contributor to aggressive tumor behavior in a subset of estrogen receptor-positive breast cancers [ 12 ]. Emerging literatures shows that the ZNF703 gene is identified act as an oncogene to promote the development of luminal B breast cancers [ 19 ]. In accordance with a previous report in non-small cell lung cancer [ 8 ], our study also suggested that ZFN703 gene was highly expressed in gastric cancer tissues and gastric cancer cell. In vitro experiment indicated that ZNF703 facilitated gastric cancer cells proliferation. An evident observation indicates that the amplification of the chromosome 8p11-12 and 11q13 region induces ZNF703 expression and regulates cancer stem cell proliferation [ 12 ]. Furthermore, ZNF703 can activates the E2F1, RB, and CCNE1 network and induces a short G1 phase, which aggravates cancer proliferation [ 20 , 21 ]. In the present study, although overexpression of ZNF703 were associated with the proliferative function in gastric cancer cells, whether it induced the expressions of E2F1, RB, and CCNE1 need to be further study. Interestingly, we found that ZNF703 inhibition resulted in down-regulation of HDAC1/2 and inhibition of HDAC1/2 impaired the viability of gastric cancer cells. Belonging to HDAC family, HDAC1/2 can remove acetyl groups, inhibit gene transcription and interact with some transcription factors to regulate gene transcription [ 22 , 23 ]. As DNA of almost human cancer cells are mostly in low histones acetylation state, HDAC1/2 are identified as important oncogene in cancer progression especially proliferation and anti-apoptosis [ 24 , 25 ]. Moreover, the HDAC inhibitors including vorinostat and panobinostat were used to in clinical cancer therapy [ 26 , 27 ]. Hence, these data suggested that ZNF703 might exhibit the pro-proliferative activity by HDAC1/2 expression. The effect of ZNF703 on inhibiting cell apoptotic is investigated in a variety of human cancer [ 8 , 9 ]. Consistent with previous studies, ZNF703 inhibition induced apoptosis in gastric cancer cells, and resulted in less level expression of bcl2 and increased bax expression, in turn overexpression of ZNF703 in GES-1 showed reverse expressions of above proteins. A recent study suggests that expression of ZNF703 gene induces apoptotic cell death in breast cancer-derived cell lines [ 28 ]. In addition to impacting the proliferation and apoptosis of gastric cancer cells, ZNF703 also has the multiple influence on mediation of tumor invasion, migration, and the resistance of chemotherapy [ 13 , 19 , 29 ]. A recent observation demonstrates that ZNF703 can inhibit the expression of E-cadherin and regulate the localization of Catenin protein family in breast cancer cell, resulting in promoting the adhesion, migration and invasion of tumor cells [ 29 ]. Consistent with clinical investigation involved in the relationship between ZNF703 expression and chemotherapy resistance of breast cancer [ 30 ], our study also suggested that overexpression of ZNF703 in gastric cancer cells showed the function to mediate the resistance of chemotherapy, which was involved in decreased expression of TopoII and P-gp, which might be associated with HDAC1/2 expression. Overexpression of HDAC1/2 in various malignant tumor can promote tumor progression [ 27 ]. Our study also indicated ZNF703 was closely associated with HDAC1/2 expression. Hence, in our future study, we will focus on the detail of the relationship between ZNF703 and HDAC1/2 and explore the underlying mechanism. Conclusion In summary, our study expanded the insight of inhibition of ZNF703 in gastric cancer progression, and highlighted that influence of ZNF703 on promote gastric cancer cells proliferation, anti-apoptosis and resistance of chemotherapy, which may facilitate the development of gastric cancer and implied that ZNF703 may be a potential target for gastric cancer therapy. Declarations Acknowledgements No Funding This study was supported by Baoshan district Science and Technology Innovation Fund supported project (No. 18-E-23). Availability of data and materials All data generated or analyzed during this study are included in this published article. Authors' contributions Hao Lin supervised, designed and performed this study. Ethics approval and consent to participate Written informed consent was obtained from patients with approval by the Institutional Review Board in Huashan hospital, The Ethics Committee of the School of Medicine Fudan University. Patient consent for publication All patients were informed about this study and gave consent before specimen collection Competing interests The author reports no conflicts of interest. References Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66(2):115-132. Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric cancer. Lancet. 2016;388(10060):2654-2664. Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial .Lancet.2010;376(9742):687-697. Nakamura M, Choe SK, Runko AP, Gardner PD, Sagerström CG. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1945136","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":127837118,"identity":"b02fcfc1-275e-40c0-9cde-8c797a867748","order_by":0,"name":"Hao Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACPmYGNiBlI8fPzHzgwIcfRGhhg2hJM5Zsb0s8OLOHGC1gxHA4ccOZM8aHOdiI0cLOe+zBjwrmxA03cj4cZuBhkOcXO0DIYXzphj1n2Ixn3sjdcLjAgsFw5uwEQlp4zCR423hk+0BaZvAwJBjcJkKL5N82CcaGGzkPDvOwEalFmrfNQHHCmTMMJGiROZMACmQDYCBLEPYLP/8ZM8k3Ff9BUfn4w4cfNvL80gS0oAMJ0pSPglEwCkbBKMAOAJBuQHQWM/6vAAAAAElFTkSuQmCC","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2022-08-09 12:29:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1945136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1945136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25158227,"identity":"79b01f95-e567-4d55-850e-358963e7c8f2","added_by":"auto","created_at":"2022-08-12 20:01:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2070369,"visible":true,"origin":"","legend":"\u003cp\u003eZNF703 expression in clinical gastric cancer tissue and gastric cancer cell lines. (A) Gastric cancer samples were collected and performed Immunochemical staining assay to mark ZNF703. (B) According to immunohistochemical analysis, the score of ZNF703 expression was calculated in gastric cancer tissue and adjacent normal area. (C) The ZNF703 expression was analyzed in GC-tumor and no-tumor tissue from TCGA data set. The mRNA (D) and protein (E and F) levels of ZNF703 were analyzed by quantitative RT-qPCR and western blotting assay in GES-1, SGC7901, and BGC823 cells. (G) Kaplan–Meier curves for Overall survival in GC patients with high or low TLS density based on TCGA data set. Data are expressed as mean±SD. All indicated P values were tested using Mann-Whitney U test. *P\u0026lt;0.05, **P\u0026lt;0.01, and ***P\u0026lt;0.001 were indicated as a significant difference.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1945136/v1/eb6923b608cad043455d4e07.png"},{"id":25158226,"identity":"e385a55a-1538-49f5-9973-c9143e9aba46","added_by":"auto","created_at":"2022-08-12 20:01:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eZNF703 promotes the gastric cancer cells proliferation. (A and B) according to ZNF703 expression level, gastric cancer tissue sample was divided into ZNF703 high group (n=17) and ZNF703 low group (n=13). Immunochemical staining assay was performed to target Ki-67. (C) GES-1 cells were transfected with plasmid encoding CMV-ZFN703 or CMV control and the cell proliferation was detected by MTT assay in different time points. After being transfected with siRNA-ZNF703 and siRNA-NC control in SGC7901 and BGC823 cells, the proliferation of SGC7901 (D) and BGC823 (E) cells were measured by MTT assay in different time point. Data are expressed as mean±SD. All indicated P values were tested using Mann-Whitney U test. *P\u0026lt;0.05 were indicated as a significant difference.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-1945136/v1/39a351d7284e6b2a5d2dbb02.png"},{"id":25158228,"identity":"329e9049-d326-49f6-a822-2d54ade38dca","added_by":"auto","created_at":"2022-08-12 20:01:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":244486,"visible":true,"origin":"","legend":"\u003cp\u003eZNF703 facilitates the gastric cancer cells proliferation through down-regulation of HDAC1/2. (A and B) GES-1 cells were transfected with plasmid encoding CMV-ZFN703 or CMV control, and then expression of HDAC1/2 was detected by western blotting assay. After being transfected with siRNA-ZNF703 and siRNA-NC control in SGC7901 and BGC823 cells, expression of HDAC1/2 in SGC7901 and BGC823 cells were measured by western blotting assay (C and E) and then quantified (D and F). SGC7901 and BGC823 cells were treated with a HDAC1/2 inhibitor ACY-957, and these cells were incubated for 24 h. And then the proliferation of SGC7901 (G) and BGC823 (H) cells were measured by MTT assay in different time points. Data are expressed as mean±SD. All indicated P values were tested using Mann-Whitney U test. *P\u0026lt;0.05, **P\u0026lt;0.01, and ***P\u0026lt;0.001 were indicated as a significant difference.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1945136/v1/05518c9d4dde7b2d0e48264c.png"},{"id":25158230,"identity":"3aa5fb93-3143-46bd-a1a0-e1033eb928b6","added_by":"auto","created_at":"2022-08-12 20:01:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2397219,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of ZNF703 induces the gastric cancer cells apoptosis. According to ZNF703 expression level, gastric cancer tissue sample was divided into ZNF703 high group (n=17) and ZNF703 low group (n=13). Immunochemical staining assay was performed to target BCL-2 (A). After being transfected with siRNA-ZNF703 and siRNA-NC control in SGC7901 and BGC823 cells, the percentage of apoptosis in SGC7901 and BGC823 (B) cells was measured by Annexin-V-FITC/PI assay. The expression of bax and bcl2 was analyzed by western blotting assay and then quantified (C). GES-1 cells were transfected with plasmid encoding CMV-ZFN703 or CMV control and then the percentage of apoptosis in these cells was detected by Annexin-V-FITC/PI assay (D). The expression of BAX and BCL2 was analyzed by western blotting assay and then quantified (E). Data are expressed as mean±SD. All indicated P values were tested using Mann-Whitney U test. *P\u0026lt;0.05, **P\u0026lt;0.01, and ***P\u0026lt;0.001 were indicated as a significant difference.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1945136/v1/0ff9176733a1a1ae52c53825.png"},{"id":25158229,"identity":"01013dbf-6fc6-4195-94ff-4e5c630a074f","added_by":"auto","created_at":"2022-08-12 20:01:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":549508,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of ZNF703 might sensitized the gastric cancer cells to chemotherapy through HDAC1/2 expression. SGC7901 and BGC823 cells were transfected with siRNA-ZNF703 and siRNA-NC control. These cells were treated with oxaliplatin or not, and then the proliferation of SGC7901 and BGC823 cells detected by MTT assay in different time points (A and B). The expression of TopoII and P-gp was analyzed by western blotting assay (C and E) and then quantified (D and F). Before being treated with oxaliplatin, SGC7901 was cultured in plate cell with ACY-957 for 24h. And then the proliferation of SGC7901 cells were measured by MTT assay in different time points (G). Scramble or sh-PKM2 was stably infected into SGC7901 cells, which were injected into nude mice. Tumor volumes were calculated after injection every 3 days (H-I). Tumor volumes are represented as the means of tumor volumes ± SEM (n = 5) (J), and the each tumor volumes are shown (K). Data are expressed as mean±SD. All indicated P values were tested using Mann-Whitney U test. *P\u0026lt;0.05, **P\u0026lt;0.01, and ***P\u0026lt;0.001 were indicated as a significant difference.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1945136/v1/abf91248009345c3136934f3.png"},{"id":27252118,"identity":"d73663b5-bcc0-4142-90fe-6bf955224c6b","added_by":"auto","created_at":"2022-10-03 01:29:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2109086,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1945136/v1/e9baa4be-4e0d-47c0-bfd9-b3bbbb34bd7d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of ZNF703 alleviates the progression of gastric cancer through inhibition of HDAC activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer causes the second most frequent cancer-related mortality all over the world. Over 400,000 new cases of gastric cancer in China are definitely diagnosed every year, accounting for 40% of total number in the global [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite recent advances in diagnosis and treatment, the majority of GC patients are diagnosed at the advanced stage with only 1-year expected survival. Even though targeted therapies against HER2 and VEGFR2 monoclonal antibodies have been approved for advanced gastric cancer, outcome of these patients is still depressing as targeted therapies only prolong life for 2 months [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The prominent characteristics of advanced gastric cancer are high-frequency metastasis and proliferation. However, the underlying mechanisms that lead to the aggressiveness of gastric cancer remains unexplored.\u003c/p\u003e \u003cp\u003eZinc finger genes (ZNFs), an important gene family in human, are considered to interact with regulatory proteins and DNA sequences, then regulating downstream gene expression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ZNF703, located on chromosomes 8 (8p11.23), is identified as an oncogene in various human cancer, such as Breast Cancer [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Non-Small Cell Lung Cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Head and Neck Cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and so on. As a member of zinc finger protein subfamily, ZNF703 protein can combine with Zn2\u0026thinsp;+\u0026thinsp;by several conserved amino acid residues to form a tetrahedral structure. This structure forms a transcription inhibitor complex with Groucho family protein, HDAC1/2 and zinc structure. As such complex binds to the target gene promoter and amplifies the regulatory effect on the expression of target gene [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. An investigation shows that the gene of the 8p11-12 chromosomal region is amplified in human breast cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Several studies in human breast cancer suggest that ZNF703 has a positive impact on cells proliferation and metastasis by altering fundamental functions in physiological processes and pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Interestingly, an elegant study finds that ZNF703 is overexpressed in gastric cancer, suggesting it may be involved in the progression of the gastric cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the underlying mechanisms of ZNF703 have not yet been fully characterized.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the expression of ZNF703 on human gastric cancer tissue, normal gastric epithelial cell line (GES-1) and gastric cancer cell lines (SGC7901 and BGC823). The overexpression of ZNF703 was verified in human gastric cancer tissue. \u003cem\u003eIn vitro\u003c/em\u003e experiment indicated that ZNF703 facilitated gastric cancer cells proliferation and migration and inhibited their apoptosis, which were associated with related gene expression bcl2 and bax. In addition, transfecting SGC7901 and BGC823 cells with siRNA-ZNF703 resulted in down-regulation of TopoII and P-gp. Inhibition of ZNF703 was involved in improvement of chemotherapy resistance, which was positively correlated with down-regulation of HDAC1/2. These data indicated that overexpression of ZNF703 in gastric cancer facilitated the gastric cancer progression, ZNF703 may be a potential target for gastric cancer therapy.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccordance to histological diagnostic criteria of gastric cancer, tumor tissues were obtained from thirty gastric cancer patients. All patients signed the informed consent, and the collection of these tissue samples were approved by the Ethics Committee of the School of Medicine Fudan University. All the tissue specimens were collected from patients diagnosed with gastric cancer at Huashan Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman gastric cancer cell line BGC823 and the normal gastric epithelial cell line GES-1 were purchased from American type culture collection (ATCC, Manassas, VA). Human gastric cancer cell line SGC7901 was purchased from the China center for type culture collection (CCTCC, Wuhan, China). Cells were cultured in Dulbecco\u0026rsquo; s modified Eagle\u0026rsquo; s medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% FBS at 37℃\u0026nbsp;in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTT (Amresco, China) assay was used to measure cell proliferation. Briefly, cells were dispensed into 96-well plates. MTT was added to each plate at a final concentration of 0.5 mg/ml for 4 h incubation at 37℃. The supernatant medium was then removed. 150 \u0026micro;l dimethylsulfoxide (Sigma, St. Louis, MO, USA) was added into each plate, and incubated 10 min at room temperature. The absorbance value was measure at 490 nm by a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were harvested and washed three times with cold PBS, followed by staining with PI and Annexin V-FITC in an Annexin-V-FITC/PI apoptosis detection kit (Vazyme Biotech Co.,Ltd, China). The percentage of apoptotic cells was measured by a BD flow cytometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor overexpressing ZNF703 in GES-1 cells, Lipofectamine 2000 (Life Technologies, USA) was employed for transfection. Following the manufacturer\u0026rsquo;s protocol, when cells developed 70%-90% confluent, they were transfected with plasmids pCDH-CMV-MCS-EF1-PURO or pCDH-CMV-MCS-EF1-PURO-HA-ZNF70 for 6 hours. Then cells were harvested or the medium was replaced with normal fresh medium for further experiments. For Small interfering RNA-mediated gene silencing of ZNF703 in gastric cancer cells, BGC823 Cells and SGC7901 were cultured in a 6-well plate. When cells developed at the density of 70%-90% confluent, siRNA-ZNF703 or siRNA-NC were transfected using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer\u0026rsquo;s protocol. The sequences of siRNA are shown as followed:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003esiRNA-ZNF703 forward, GCUUGACCCUGCCGGGAUU\u003c/p\u003e\n\u003cp\u003ereverse, CUUACAAGCUGGGAAUAUA\u003c/p\u003e\n\u003cp\u003esiRNA-NC \u0026nbsp; \u0026nbsp; forward, UUCUCCGAACGUGUCACGUTT\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; reverse, ACGUGACACGUUCGGAGAATT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShort hairpins, plasmids, and lentiviral transduction and infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZNF703-specific shRNAs was designed and developed by the TRCat the Broad Institute of MIT. One ZNF703 hairpin that showed high-efficacy knockdown was selected. High-titer virus-containing supernatants of HEK293FT cells after transient co-transfection of lentiviral vectors with pMD2.G and psPAX2 packaging vectors were used for lentiviral-mediated transduction of cancer cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGastric cancer samples from patients were fixed with 4% paraformaldehyde, embedded in\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;paraffin. Then the samples were cut into 4 \u0026mu;m slides, followed by dewaxing and rehydrating. Slides were incubated with 5% BSA for blocking at room temperature for 0.5 h and then probed with an optimal dilution of a primary rabbit antibody against human ZNF703, Ki-67 or BCL-2 overnight at 4\u0026deg;C. After incubated with a biotinylated goat anti-rabbit immunoglobulin G antibody and washed, the slides were visualized using diaminobenzidine (DAB) and counterstained with hematoxylin. ZNF703 expression was evaluated in tumor cells with the semi-quantitation method in accordance with the area of positive (AP) and the intensity of staining (IS). AP was scored as follows: 0 (0-5%), 1 (6-25%), 2 (26-50%), 3 (51-75%) and 4 (\u0026gt;75%). IS was graded as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The final expression level of ZNF703 was determined based on the equation: Intensity distribution (ID) = AP\u0026nbsp;\u0026times;\u0026nbsp;IS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative real-time PCR (RT-qPCR) assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from gastric cancer cells using TRIzol reagent (Invitrogen) according to the manufacturer\u0026apos;s instructions. And each RNA sample was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Promega, USA) in a 50 \u0026micro;l reaction system. According to the manufacturer\u0026apos;s instructions, quantitative real-time PCR was performed with a conventional SYBR green-based (Life Technologies, USA) quantification method. The RT-qPCR primers were used as follows: ZFN703-Forward, 5\u0026apos;-GATCAGGGTCCTGAAGATGC-3, ZFN703--Reverse, 5\u0026apos;-CCGAGTTGAGTTTGGAGGAG-3\u0026apos;; GAPDH-Forward, 5\u0026prime;-AGGTGAAGGTCGGAGTCAAC-3\u0026prime;, GAPDH-Reverse, 5\u0026prime;-GGGTGGAATCATATTGGAACA-3\u0026prime;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACY-957 and oxaliplatin treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor inhibition of HDAC1/2 in gastric cancer cells, a selective inhibitor of HDAC1/2 (10 mM in DMSO) ACY-957 was added into the plate with the final concentration of 2 \u0026mu;M. The cells treated with equivalent DMSO were served as control. Cells were incubated for 24 h before subsequent assay. For oxaliplatin treatment, oxaliplatin was added into the plate with the final concentration of 25 ug/ml. Cells were incubated for 24 h before subsequent assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein of cells was extracted using RIPA lysis buffer containing phenylmethane-sulfonyl fluoride (PMSF). After boiling, equal amounts of proteins (20 \u0026mu;g) were loaded into 10% SDS-PAGE gels for separation. And the separated proteins were transferred to PVDF membranes. The membranes were blocked with 10% non-fat dried milk dissolved in TBS containing 0.5% Tween 20 for 1.5 h, and then incubated with primary antibodies at 4℃\u0026nbsp;overnight. After incubated with appropriate secondary antibodies at room temperature for 1 h and washed, immunoreactivity was visualized using enhanced chemiluminescence (ECL) system (Image Quant LAS 4000 mini, Pittsburgh, PA, USA) according to the instructions of the manufacturer. All experiments were replicated three times for statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are shown as the mean \u0026plusmn; SD. Asterisks presented significant difference. Statistical analysis was performed using Mann-Whitney U test with Graph-Pad Software. P\u0026lt;0.05 was considered as significant difference for all experiments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eZNF703 is highly expressed in clinical gastric cancer tissues and gastric cancer cell.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the characteristics of ZNF703 expression in gastric cancer patients, the sections of gastric cancer tissues from gastric cancer patients were stained with ZNF703. The immunohistochemical (IHC) staining showed that the expression of ZNF703 in gastric cancer area was even higher compared with that in adjacent normal area (Figure.1A and 1B). To determine the clinical utility of strategies targeting the ZNF703, the analysis for ZFN703 expression (n=619, the number of tumor tissue and No-tumor tissue was 408 and 201, respectively) in The Cancer Genome Atlas (TCGA) cohort were explored, indicating that high ZFN703 expression was observed in tumor tissue compared with no-tumor tissue (Figure. 1C). In addition, using western blotting and RT-qPCR\u0026nbsp;assay, we found that the expression of ZNF703 was significant higher in gastric cancer cell line SGC7901 and BGC823 than that in the normal human gastric epithelial cell line GES-1 (Figure.1D-F). To ascertain the relationship between ZFN703 expression and survival in patients with gastric cancer, patients with gastric cancer survival data from TCGA cohort was analyzed, indicated that patients with higher expression of ZNF703 had a significant association with poor survival (P=0.031; hazard ratio (HR)=1.7; Figure.1G). These data suggested that overexpression of ZNF703 may be associated with gastric cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZNF703 promotes the cell proliferation of gastric cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the influence of ZNF703 on the development of gastric cancer, IHC was performed to determine the proliferation function by targeting Ki-67 in gastric cancer tissues. The result showed that ZNF703 high-expression level was associated with an increased Ki-67 expression level (Figure. 2A and B). TCGA datasets was analyzed to ascertain the relationship between ki67 and ZNF703 expression in patients with gastric cancer, the result showed that ZNF703 expression was related to tumor cell. We then analyzed the several GC cells lines proliferation via intervening the expression of ZNF703. The treatment regimens were utilized: the experiments of overexpression\u0026nbsp;ZNF703\u0026nbsp;in GES-1 cells was performed by using a plasmid encoding CMV-ZNF703. The siRNA transfection assay was used to inhibit ZNF703 expression in gastric cancer cell lines SGC7901 and BGC823. The result of MTT assay showed that a significant reduction in the cell viability was observed in ZNF703 siRNA-transfected SGC7901 and BGC823 cells compared with that in control cells (Figure. 2D and E). To some extent, overexpression of ZNF703 in GES-1 cells promoted cell proliferation (Figure. 2C). Hence, our data indicated that ZNF703 promoted cell proliferation of gastric cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZNF703 might affect the cell proliferation of gastric cancer cells through up-regulation of HDAC1/2.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a study suggests that ZNF703 can interact with HDAC1/2 to regulate downstream gene expression [5], we asked the relationship between ZNF703 and HDAC1/2 in the gastric cancer cells. Here, GES-1 cells were transfected with plasmid CMV-ZNF703 or plasmid CMV control. Using western blotting analysis, we found that overexpression of ZNF703 in GES-1 cells significantly increased the expression of HDAC1/2 compared with that in plasmid CMV control (Figure.3A and B). In addition, after being transfected with siRNA-ZNF703 or siRNA-NC, a substantial decrease was observed in the expression of HDAC1/2 in SGC7901 and BGC823 cells compared with that in control cells (Figure.3C-F). To confirm the relevance of ZNF703 and HDAC1/2 in gastric cancer cells, we analyzed the proliferation of SGC7901 and BGC823 cells that be treated with a HDAC1/2 inhibitor ACY-957. As expected, the results showed that inhibition of HDAC1/2 in SGC7901 and BGC823 cells inhibited the cell proliferation of SGC7901 and BGC823 cells compared with control group (Figure.3G and H). Thus, these results suggested that ZNF703 might be interacted with HDAC1/2 to regulate proliferation of gastric cancer cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of ZNF703 induces apoptosis of gastric cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then analyzed the relationship between ZNF703 expression level and anti-apoptosis function in gastric cancer, IHC assay showed that gastric cancer tissue with ZNF703 high expression level substantially increased the BCL-2 expression level (Figure. 4A). As inhibition of cancer cell apoptosis may be associated with induction of cell proliferation, we explored the relationship between ZNF703 expression and gastric cancer cells apoptosis. Using the Annexin-V-FITC/PI staining, the apoptotic cells were identified as Annexin V+ cells. After being transfecting with siRNA-ZNF703 in SGC7901 and BGC823 cells, a significant increase was observed in the percentages of apoptotic cells\u0026nbsp;as compared with these cells transfected with siRNA-NC\u0026nbsp;(Figure. 4 B). In comparison, the adverse results were observed in GES-1 cells that transfected with plasmid CMV-ZNF703 (Figure.4D). In addition, the apoptosis-associated proteins were measured by using western blotting assay. The results showed that transfecting SGC7901 and BGC823 cells with siRNA-ZNF703 reduced the protein levels of bcl2 and increased the expression of bax (Figure.4 C), while overexpression of ZNF703 in GES-1 showed reverse expressions of above proteins (Figure. 4 E). Taken together, these data demonstrated that inhibition of ZNF703 was involved in the apoptosis of gastric cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of ZNF703 is involved in improvement of chemotherapy resistance and\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated the role of ZNF703 expression on the improvement of chemotherapy resistance of gastric cancer cells. After transfecting with siRNA-ZNF703 or siRNA-NC in SGC7901 and BGC823 cells, these cells were treated with a kind of chemotherapeutic drug, oxaliplatin. MTT assay was performed to evaluate the effect of oxaliplatin. The results showed that inhibition of ZNF703 significantly reduced the cell viability of the SGC7901 and BGC823 cells to chemotherapy (Figure. 5A and B). In addition, the chemotherapeutic resistance-associated proteins were detected by western blotting assay. After transfected with siRNA-ZNF703, we found that SGC7901 and BGC823 cells that dramatically reduced the protein levels of TopoII and P-gp (Figure. 5 C-F). As overexpression of ZNF703 contributed to the increase of HDAC1/2 expression, we queried the relevance of HDAC1/2 expression and chemotherapy resistance. SGC7901 cells were treated with a HDAC1/2 selective inhibitor ACY-957. The result indicated that inhibition of HDAC1/2 significantly sensitize the SGC7901 and BGC823 cells to chemotherapy (Figure. 5G). Taken together, these data suggested that inhibition of ZNF703 might sensitized the SGC7901 and BGC823 cells to chemotherapy through HDAC1/2 expression. Moreover, xenograft mice models were established by subcutaneously injecting scrambled- or ZNF703-shRNA-infected SGC7901 cells. Compared with the control group, the xenograft volume decreased in the shRNA-ZNF703 groups (Figure. 5H-K). Taken together, these data demonstrated that inhibition of ZNF703 may delay the development of gastric cancer.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChromosomal breaks and rearrangements generally result in altering oncogenes and tumor suppressor genes expression, which contributes to cancer progression and oncogenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The gene sequence for the ZNF703 transcription factor is one of the frequently amplified genes in cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. An investigation in human breast cancer indicates that the amplification of the chromosome 8p11-12 region contains tumor metastasis-promoting genes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As a result of investigations to identify the genes which lie in that region, ZNF703 has been identified as the driver oncogene within this region [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A recent study demonstrates a compelling case for amplification of ZNF703 as an important contributor to aggressive tumor behavior in a subset of estrogen receptor-positive breast cancers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Emerging literatures shows that the ZNF703 gene is identified act as an oncogene to promote the development of luminal B breast cancers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In accordance with a previous report in non-small cell lung cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], our study also suggested that ZFN703 gene was highly expressed in gastric cancer tissues and gastric cancer cell. \u003cem\u003eIn vitro\u003c/em\u003e experiment indicated that ZNF703 facilitated gastric cancer cells proliferation. An evident observation indicates that the amplification of the chromosome 8p11-12 and 11q13 region induces ZNF703 expression and regulates cancer stem cell proliferation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, ZNF703 can activates the E2F1, RB, and CCNE1 network and induces a short G1 phase, which aggravates cancer proliferation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, although overexpression of ZNF703 were associated with the proliferative function in gastric cancer cells, whether it induced the expressions of E2F1, RB, and CCNE1 need to be further study. Interestingly, we found that ZNF703 inhibition resulted in down-regulation of HDAC1/2 and inhibition of HDAC1/2 impaired the viability of gastric cancer cells. Belonging to HDAC family, HDAC1/2 can remove acetyl groups, inhibit gene transcription and interact with some transcription factors to regulate gene transcription [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. As DNA of almost human cancer cells are mostly in low histones acetylation state, HDAC1/2 are identified as important oncogene in cancer progression especially proliferation and anti-apoptosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, the HDAC inhibitors including vorinostat and panobinostat were used to in clinical cancer therapy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Hence, these data suggested that ZNF703 might exhibit the pro-proliferative activity by HDAC1/2 expression.\u003c/p\u003e \u003cp\u003eThe effect of ZNF703 on inhibiting cell apoptotic is investigated in a variety of human cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consistent with previous studies, ZNF703 inhibition induced apoptosis in gastric cancer cells, and resulted in less level expression of bcl2 and increased bax expression, in turn overexpression of ZNF703 in GES-1 showed reverse expressions of above proteins. A recent study suggests that expression of ZNF703 gene induces apoptotic cell death in breast cancer-derived cell lines [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition to impacting the proliferation and apoptosis of gastric cancer cells, ZNF703 also has the multiple influence on mediation of tumor invasion, migration, and the resistance of chemotherapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A recent observation demonstrates that ZNF703 can inhibit the expression of E-cadherin and regulate the localization of Catenin protein family in breast cancer cell, resulting in promoting the adhesion, migration and invasion of tumor cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Consistent with clinical investigation involved in the relationship between ZNF703 expression and chemotherapy resistance of breast cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], our study also suggested that overexpression of ZNF703 in gastric cancer cells showed the function to mediate the resistance of chemotherapy, which was involved in decreased expression of TopoII and P-gp, which might be associated with HDAC1/2 expression. Overexpression of HDAC1/2 in various malignant tumor can promote tumor progression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our study also indicated ZNF703 was closely associated with HDAC1/2 expression. Hence, in our future study, we will focus on the detail of the relationship between ZNF703 and HDAC1/2 and explore the underlying mechanism.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study expanded the insight of inhibition of ZNF703 in gastric cancer progression, and highlighted that influence of ZNF703 on promote gastric cancer cells proliferation, anti-apoptosis and resistance of chemotherapy, which may facilitate the development of gastric cancer and implied that ZNF703 may be a potential target for gastric cancer therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Baoshan district Science and Technology Innovation Fund supported project (No. 18-E-23).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHao Lin supervised, designed and performed this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from patients with approval by the Institutional Review Board in\u0026nbsp;Huashan hospital, The Ethics Committee of the School of Medicine Fudan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients were informed about this study and gave consent before specimen collection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author reports no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen W, Zheng R, Baade PD, et al. 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ZNF703 is a common Luminal B breast cancer oncogene that differentially regulates luminal and basal progenitors in human mammaryepithelium.EMBOMolMed.2011;3(3):167-180.\u003c/li\u003e\n\u003cli\u003eBazarov, A. V., and P. Yaswen. 2011. Who is in the driver\u0026rsquo;s seat in 8p12 amplifications? ZNF703 in luminal B breast tumors. Breast Cancer Res. 13:308.\u003c/li\u003e\n\u003cli\u003eBaykara O, Dalay N, Kaynak K, Buyru N. ZNF703 Overexpression may act as an oncogene in non-small cell lung cancer. Cancer Med. 2016;5(10):2873-2878.\u003c/li\u003e\n\u003cli\u003eOrhan C, Bakır B, Dalay N, Buyru N. ZNF703 is an important player in head and neck cancer. Clin Otolaryngol. 2019;44(6):1080-1086.\u003c/li\u003e\n\u003cli\u003eYang H, Jiang WQ, Cao Y, et al. Elevated ZNF703 Protein Expression Is an Independent Unfavorable Prognostic Factor for Survival of the Patients with Head and Neck Squamous Cell Carcinoma. Dis Markers. 2015;2015:640263.\u003c/li\u003e\n\u003cli\u003eChin K, DeVries S, Fridlyand J, et al. Genomic and transcriptional aberrations linked to breast cancer pathophysiologies. Cancer Cell. 2006;10(6):529-541.\u003c/li\u003e\n\u003cli\u003eSircoulomb F, Nicolas N, Ferrari A, et al. ZNF703 gene amplification at 8p12 specifies luminal B breast cancer. EMBO Mol Med. 2011 Mar;3(3).\u003c/li\u003e\n\u003cli\u003eYang G, Ma F, Zhong M, et al. ZNF703 acts as an oncogene that promotes progression in gastric cancer. Oncol Rep. 2014;31(4):1877-1882.\u003c/li\u003e\n\u003cli\u003ePole JC, Courtay-Cahen C, Garcia MJ, et al. High-resolution analysis of chromosome rearrangements on 8p in breast, colon and pancreatic cancer reveals a complex pattern of loss, gain and translocation. Oncogene. 2006;25(41):5693-5706.\u003c/li\u003e\n\u003cli\u003eBirnbaum D, Ad\u0026eacute;la\u0026iuml;de J, Popovici C, Charafe-Jauffret E, Mozziconacci MJ, Chaffanet M. Chromosome arm 8p and cancer: a fragile hypothesis. Lancet Oncol. 2003;4(10):639-642.\u003c/li\u003e\n\u003cli\u003eBaykara O, Bakir B, Buyru N, Kaynak K, Dalay N. Amplification of chromosome 8 genes in lung cancer. J Cancer. 2015;6(3):270-275. Published 2015 Jan 20.\u003c/li\u003e\n\u003cli\u003eBaltaci E, Karaman E, Dalay N, Buyru N. Analysıs of gene copy number changes in head and neck cancer. Clin Otolaryngol. 2018;43(4):1004-1009.\u003c/li\u003e\n\u003cli\u003eGelsi-Boyer V, Orsetti B, Cervera N, et al. Comprehensive profiling of 8p11-12 amplification in breast cancer. Mol Cancer Res. 2005;3(12):655-667. \u003c/li\u003e\n\u003cli\u003eGinestier C, Sircoulomb F, Charafe-Jauffret E, Chaffanet M, Birnbaum D. ZNF703 : un nouvel oncog\u0026egrave;ne du cancer du sein (ZNF703: a novel oncogene involved in breast cancer). Med Sci (Paris). 2011;27(4):357-359.\u003c/li\u003e\n\u003cli\u003eRasamny JJ, Allak A, Krook KA, et al. Cyclin D1 and FADD as biomarkers in head and neck squamous cell carcinoma. Otolaryngology\u0026ndash;head and neck surgery. 2012;146(6):923-931.\u003c/li\u003e\n\u003cli\u003eFreier K, Joos S, Flechtenmacher C, et al. Tissue microarray analysis reveals site-specific prevalence of oncogene amplifications in head and neck squamous cell carcinoma. Cancer Res. 2003;63(6):1179-1182.\u003c/li\u003e\n\u003cli\u003eCress WD, Seto E. Histone deacetylases, transcriptional control, and cancer. J Cell Physiol. 2000;184(1):1-16. \u003c/li\u003e\n\u003cli\u003eMarchion D, M\u0026uuml;nster P. Development of histone deacetylase inhibitors for cancer treatment. Expert Rev Anticancer Ther. 2007;7(4):583-598. doi:10.1586/14737140.7.4.583.\u003c/li\u003e\n\u003cli\u003eMariadason JM, Corner GA, Augenlicht LH. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res. 2000;60(16):4561-4572.\u003c/li\u003e\n\u003cli\u003eHalkidou K, Gaughan L, Cook S, Leung HY, Neal DE, Robson CN. Upregulation and nuclear recruitment of HDAC1 in hormone refractory prostate cancer. Prostate. 2004;59(2):177-189.\u003c/li\u003e\n\u003cli\u003eMarks PA. The clinical development of histone deacetylase inhibitors as targeted anticancer drugs. Expert Opin Investig Drugs. 2010;19(9):1049-1066. \u003c/li\u003e\n\u003cli\u003eBose P, Dai Y, Grant S. Histone deacetylase inhibitor (HDACI) mechanisms of action: emerging insights. Pharmacol Ther. 2014;143(3):323-336. \u003c/li\u003e\n\u003cli\u003eSlorach EM, Chou J, Werb Z. Zeppo1 is a novel metastasis promoter that represses E-cadherin expression and regulates p120-catenin isoform expression and localization. Genes Dev. 2011;25(5):471-484.\u003c/li\u003e\n\u003cli\u003eMollashahee-Kohkan F, Saravani R, Khalili T, Galavi H, Sargazi S. Levisticum Officinale Extract Triggers Apoptosis and Down-Regulates ZNF703 Gene Expression in Breast Cancer Cell Lines. Rep Biochem Mol Biol. 2019;8(2):119-125.\u003c/li\u003e\n\u003cli\u003eMoelans CB, van Maldegem CMG, van der Wall E, van Diest PJ. Copy number changes at 8p11-12 predict adverse clinical outcome and chemo- and radiotherapy response in breast cancer. Oncotarget. 2018;9(24):17078-17092.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ZNF703, gastric cancer, proliferation, resistance of chemotherapy, HDAC1/2","lastPublishedDoi":"10.21203/rs.3.rs-1945136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1945136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e ZNF703 is identified as a therapeutic target in a variety of human cancer. Although ZNF703 overexpresses in gastric cancer frequently, the effects and mechanism of ZNF703 in the progression of gastric cancer is unclear. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Therefore, ZNF703, Ki-67 and BCL-2 expression was measured by histology in clinical cases. We used gastric cells line models to explore the role of ZNF703 \u003cem\u003ein vitro\u003c/em\u003e. ZNF703 expression intervention was employed to investigate the role of ZNF703 in proliferation and apoptosis. The relationship between ZNF703 intervention and resistance of chemotherapy was analyzed by using oxaliplatin treatment. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this study, we found that ZNF703 expression in the area of gastric cancer was substantially higher than adjacent normal area. Gastric cancer tissue with ZNF703 high expression level substantially increased Ki-67 and BCL-2 expression. Inhibition of ZNF703 attenuated the gastric cancer cell proliferation and induced apoptosis in SGC7901 and BGC823 cells, while overexpression of ZNF703 in GES-1 cells resulted in the reverse effects. ZNF703 might mediate the viability of gastric cancer cells through down-regulation of HDAC1/2. In addition, after transfected with siRNA-ZNF703, down-regulation of TopoII and P-gp was observed in SGC7901 and BGC823 cells. Further, we showed that inhibition of ZNF703 enhanced the resistance to chemotherapy \u003cem\u003ein vitro\u003c/em\u003e. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our study demonstrated that in gastric cancer cells, ZNF703 promoted the proliferation, inhibited apoptosis, and improved their resistance to chemotherapy, suggesting it may be a potential target for the gastric cancer.\u003c/p\u003e","manuscriptTitle":"Inhibition of ZNF703 alleviates the progression of gastric cancer through inhibition of HDAC activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-12 20:01:38","doi":"10.21203/rs.3.rs-1945136/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5bddd510-7597-44c2-8b5a-1813b7af6b0e","owner":[],"postedDate":"August 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-10-03T01:29:13+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-12 20:01:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1945136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1945136","identity":"rs-1945136","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}