Application of acetylation site of HDAC3 protein as new target for treating non-small cell lung cancer with CBP mutation

Abstract

The invention belongs to the technical field of biological medicines, and discloses an application of HDAC3K3 site acetylation as a target in screening / preparing a medicine for treating non-small cell lung cancer, and an application of a reagent for promoting HDAC3K3 site acetylation in preparing a medicine for treating non-small cell lung cancer. The invention also discloses application of the reagent for detecting the acetylation level of the HDAC3K3 site in preparation of a kit for diagnosing non-small cell lung cancer or predicting prognosis. Experiments prove that up-regulation of the acetylation level of the HDAC3K3 site can promote transcription of a cancer suppressor gene PTEN and inhibit growth of the non-small cell lung cancer, and a new target is provided for treatment of the non-small cell lung cancer. When the non-small cell lung cancer is cured, the acetylation level of the HDAC3K3 site is increased, and the deacetylase activity of HDAC3 can be inhibited, so that the transcription of PTEN is promoted, and the purpose of relieving or treating the non-small cell lung cancer is achieved. The invention provides a new direction and idea for diagnosis and treatment of non-small cell lung cancer, and is expected to be applied to research and development of new drugs and detection kits for diagnosis and treatment of non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of the acetylation site of HDAC3 protein as a new target for treating non-small cell lung cancer with CBP mutation. Background Art

[0002] The "Cancer Statistics 2020" shows that lung cancer is the malignant tumor with the highest mortality rate in the world. As a negative regulator of PI3K / mTOR / Akt, PTEN plays an important role in maintaining chromosome integrity and DNA damage repair. Unlike other tumor suppressor genes, even a partial reduction in PTEN can significantly increase cancer susceptibility. The loss of the tumor suppressor PTEN is considered to be a key driver of cancer progression, and its reduced expression is closely related to the poor prognosis of lung cancer.

[0003] About 56% of lung cancer patients have PTEN expression loss, especially non-small cell lung cancer. However, the loss and mutation of the PTEN gene itself rarely occur in non-small cell lung cancer, and unlike breast cancer and melanoma, DNA methylation is not an important regulator of PTEN expression in non-small cell lung cancer. The transcription of PTEN is regulated by multiple transcription factors, such as PPAR, EGR1 and p53, as well as many potential epigenetic modifications, such as DNA methylation and histone modifications. The stability of PTEN protein is also affected by post-translational modifications. It has been reported that microRNAs such as miRNA21 and miRNA130a are involved in the post-transcriptional regulation of PTEN. Due to the large number of factors affecting PTEN expression, the mechanism of PTEN expression loss in non-small cell lung cancer remains unclear.

[0004] Previous studies have shown that histone acetylation modification is associated with chromatin remodeling into a transcriptionally active state. CREB binding protein (CREB [cyclic adenosine monophosphate response element binding protein] binding protein, CREBBP or CBP) is a major member of the acetyltransferase family, which promotes transcription by regulating the acetylation of histones and non-histone proteins. CBP is a large molecular protein with approximately 2,400 amino acids, containing multiple functional domains, mediating interactions between multiple proteins. This feature makes CBP a key coactivator that regulates the expression of thousands of genes in almost all types of cells. Studies have shown that approximately 10%-15% of non-small cell lung cancers have CBP loss-of-function (LOF) mutations. Recent whole-genome sequencing studies have shown that missense mutations occur in clusters in the region encoding the HAT domain of CBP, which is essential for its HAT and / or transcriptional coactivation activity, indicating that there is selective pressure for changes in this activity in non-small cell lung cancer.

[0005] HDAC3 is a histone deacetylase that forms a unique complex with nuclear receptor corepressor 1 (NCoR1), retinoic acid, and silencing mediator of thyroid hormone receptor (SMRT). HDAC3 binding to the deacetylase activation domain (DAD) of NCoR1 / SMRT and phosphorylation of HDAC3-S424 significantly enhance its deacetylase activity. However, the molecular mechanism of HDAC3-S424 phosphorylation regulation and whether there are other types of post-translational modifications (PTMs) in HDAC3 remain to be fully revealed and require further investigation.

[0006] In this study, we found that HDAC3 itself is acetylated by CBP on an unreported lysine residue, which is required for inhibition of HDAC3 deacetylase activity. We demonstrated a causal relationship between reduced histone deacetylase 3 (HDAC3) acetylation and loss of PTEN expression in CBP LOF mutant lung cancer cells. Our results suggest that acetylated HDAC3 (Ac-HDAC3) can reduce the deacetylase activity of HDAC3, thereby promoting PTEN transcription. In CBP LOF mutant lung cancer, impaired HDAC3 acetylation leads to loss of PTEN expression, thereby promoting tumorigenesis and tumor resistance to chemotherapeutic drugs. Our results reveal a new regulatory mechanism of PTEN expression and provide new directions for the treatment and prevention of CBP LOF mutant cancers. Summary of the invention

[0007] The present invention analyzes clinical CBP mutation non-small cell lung cancer samples, uses immunohistochemistry (IHC) technology to detect HDAC3-lysine (Lys, K for short) 3 site acetylation and PTEN expression levels in cancer tissues and paracancerous tissues, and combines IHC technology with clinical survival data to analyze the significance of HDAC3-K3 site acetylation in predicting the prognosis of patients with non-small cell lung cancer, providing an important theoretical basis for the diagnosis and prognosis of non-small cell lung cancer. At the same time, by combining cell experiments and animal experiments, the molecular mechanism of CBP mutation inactivation inhibiting HDAC3 K3 site acetylation modification, antagonizing tumor suppressor gene PTEN expression, and promoting the occurrence and development of non-small cell lung cancer is revealed, and based on this, a new type of specific binding agent targeting HDAC3, an agonist and a corresponding detection kit are designed and discovered for individualized precision treatment of CBP mutation non-small cell lung cancer.

[0008] Based on this, the present invention provides the following technical solution: the use of K3 site acetylation of HDAC3 as a target in the preparation of a drug for the treatment of non-small cell lung cancer, regulating the acetylation level of the K3 site of HDAC3, and increasing the acetylation level of the K3 site of HDAC3 to alleviate or inhibit the development of non-small cell lung cancer.

[0009] In the above-mentioned application technology scheme, by regulating the acetylation of the K3 site of HDAC3 in non-small cell lung cancer with CBP mutation, the acetylation of this site promotes the expression of the tumor suppressor gene PTEN, thereby achieving the purpose of alleviating or inhibiting the development of non-small cell lung cancer.

[0010] The present invention also provides the use of a reagent for promoting acetylation of the K3 site of HDAC3 in treating non-small cell lung cancer.

[0011] In the above-mentioned application technology scheme, the reagent that promotes the acetylation of the K3 site of HDAC3 in non-small cell lung cancer with CBP mutation promotes the acetylation of this site, inhibits the activity of HDAC3 deacetylase, thereby promoting the expression of the tumor suppressor gene PTEN, thereby achieving the purpose of alleviating or inhibiting the development of non-small cell lung cancer.

[0012] In the above application technology scheme, the reagent that promotes acetylation of the K3 site of HDAC3 is a small molecule or low molecular weight compound, an HDAC3 protein specific binder, and the HDAC3 protein K3 site specific binder includes an agonist and a binder for the site.

[0013] The present invention also provides use of a reagent for detecting CBP mutation and the acetylation level of the K3 site of HDAC3 in the preparation of a reagent for diagnosis or prognosis of non-small cell lung cancer.

[0014] In the above-mentioned technical scheme of use, the mutation of CBP and the low acetylation level of the K3 site of HDAC3 indicate a poor prognosis for patients with non-small cell lung cancer.

[0015] The beneficial effects of the present invention are as follows: experiments have shown that promoting the acetylation level of the K3 site of HDAC3 can inhibit the activity of HDAC3 deacetylase, promote the expression of PTEN, and inhibit the growth of non-small cell lung cancer with CBP mutations, providing a new target for the treatment of non-small cell lung cancer, and developing drugs targeting the acetylation of the K3 site of HDAC3, which can promote the expression of PTEN by promoting the acetylation of the K3 site of HDAC3 in non-small cell lung cancer with CBP mutations, thereby achieving the purpose of alleviating or treating non-small cell lung cancer. It provides a new direction and idea for the treatment of non-small cell lung cancer, and is expected to be applied in the research and development of new drugs for the treatment of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1.CBP promotes acetylation of HDAC3 K3 site: A. After HDAC3 plasmid was transfected into HEK293T cells and H1299 cells, the cell lysate was subjected to IP experiment with HA magnetic beads, and the acetylation level of HDAC3 was detected with acetylation antibody; B. HEK293T cells transfected with HDAC3 plasmid were treated with nicotinamide (NAM), an inhibitor of deacetylase family, or trichostatin A (TSA), an inhibitor of histone deacetylase I, II and IV, to detect the acetylation level of HDAC3; C. Multiple acetyltransferases (PCAF, CBP, P300, MOF, Tip60 and GCN5) were detected with HDAC3 to detect the acetylation level of HDAC3; DE. After knocking down or knocking out CBP in H1299 cells, the acetylation level of HDAC3 was detected; F. The sites of CBP-mediated HDAC3 acetylation in Hela cells were detected by LC-MS / MS method; G. After H1299 cells were transfected with HDAC3-WT and HDAC3-K3R plasmids, the acetylation level of HDAC3 was detected; HI. The direct interaction between HDAC3 and CBP was analyzed by performing IP experiments on HEK293T cells transfected with HDAC3 and CBP. Figure 2 .HDAC3-K3 acetylation inhibits its enzyme activity and stability: A. - / - HDAC3-WT, HDAC3-K3R and HDAC3-K3Q were recombinantly expressed in H1299 cells, and the phosphorylation of HDAC3S424 site and acetylation of K3 site were detected by IP experiment; B. - / - After restoring CBP expression in H1299 cells, the phosphorylation of HDAC3 S424 site and the acetylation of K3 site were detected by IP experiment; CE. The deacetylase activity of HDAC3 immunoprecipitated from H1299 cells was detected by HDAC fluorescence method; FG. CHX was used to inhibit protein synthesis and the effect of HDAC3K3 site acetylation level on its protein stability was detected.

[0017] Figure 3 .HDAC3-K3 acetylation inhibits proliferation and migration of non-small cell lung cancer cell lines: AD. - / - Flag-HDAC3-WT, Flag-HDAC3-K3R, Flag-HDAC3-K3Q and Flag-HDAC3-K3R-S424A were expressed in H1299 cells, and the cell proliferation ability was detected by CCK8 assay and clone formation assay; E. The HDAC3 expressing HDAC3-WT and HDAC3-K3Q under low glucose condition was detected by clone formation assay - / - Proliferation ability of H1299 cells; FG.- / - Flag-HDAC3-WT, Flag-HDAC3-K3R, Flag-HDAC3-K3Q and Flag-HDAC3-K3R-S424A were expressed in H1299 cells, and the migration ability was detected by transwell assay; HJ. - / - H1299 cells were subcutaneously injected into 8-week-old BALB / c nude mice, and the tumor volume was measured to draw a tumor growth curve (H). The tumors were removed 25 days after injection and the tumor volume and weight of each group were calculated (I&J); K. The phosphorylation level of HDAC3-S424 in the tumors of the above groups was detected by western blot.

[0018] Figure 4 .Acetylation of HDAC3 at the K3 site regulates PTEN gene transcription: AB. After stably overexpressing HDAC3-K3R, HDAC3-WT or empty vector plasmid in H1299 cells, RNA sequencing was used to analyze the changes in gene transcription profiles of each group (A), and the genes regulated by HDAC3 and its acetylation were obtained by analyzing the intersection of differentially expressed genes between the HDAC3-WT group and the empty vector group, and the intersection of differentially expressed genes between the HDAC3-WT group and the HDAC3-K3R group (B); C. Gene set enrichment analysis (GSEA) was used to detect the correlation between HDAC3 and PTEN target gene sets; DF. After knocking out HDAC3 in H1299, A549 and H1703 cells, realtime-PCR was used to detect PTEN mRNA expression; G. After knocking out HDAC3 in H1299 cells, western blot was used to detect the phosphorylation levels of PTEN and genes related to the PTEN downstream signaling pathway; HI. After knocking out HDAC3 in A549 and H1703 cells, western blot was used to detect the phosphorylation levels of PTEN and genes related to the PTEN downstream signaling pathway. Blot analysis of PTEN protein expression; JK. - / - After H1299 cells were transfected with HDAC3-WT, HDAC3-K3R, HDAC3-K3Q and HDAC3-K3R-S424A, PTEN protein and AKT phosphorylation levels were detected by western blot (J), and PTEN mRNA expression was detected by real-time PCR (K).

[0019] Figure 5.PTEN inhibits lung cancer cell proliferation in vivo and in vitro: AF. HDAC3 and PTEN were knocked out in H1299 and A549 cells, and the cell proliferation ability was detected by CCK8 proliferation assay and clone formation assay (B&C, E&F), and the knockout efficiency was detected by western blot (A&D); GI. - / - After overexpressing HDAC3-WT and HDAC3-K3R in cells, PTEN was knocked down. The cell proliferation ability was detected by CCK8 proliferation assay and clone formation assay (H&I), and the knockdown efficiency was detected by western blot (K); JL. - / - H1299 cells were subcutaneously injected into 8-week-old BALB / c nude mice, and the tumor volume was measured to draw the tumor growth curve (J). The tumors were removed 25 days after injection and the tumor volume and weight of each group were calculated (K&L).

[0020] Figure 6 .HDAC3 acetylation inhibits the expression of PTEN and activates the AKT signaling pathway: AB. After H1299 cells were transfected with HDAC3-WT, HDAC3-K3R or HDAC3 was knocked out, EGF treatment amplified the phosphorylation signal, and the phosphorylation levels of molecules related to the PTEN downstream signaling pathway were detected by western blot; C. PTEN was knocked down in HDAC3-deficient cells, and the AKT phosphorylation level was detected by western blot.

[0021] Figure 7 .CBP-HDAC3-PTEN axis regulates the resistance of non-small cell lung cancer to chemotherapeutic drugs: A&B. After knocking out CBP in H1299 cells, the expression of PTEN protein and mRNA was detected by western blot and real-time-PCR; C&D. After knocking down HDAC3 in CBP-knocked-out H1299 cells, the expression of PTEN protein and mRNA was detected by western blot and real-time-PCR; EH. After knocking out HDAC3 or overexpressing HDAC3-WT, HDAC3-K3Q, HDAC3-K3R and HDAC3-K3R-S424A plasmids in H1299 and A549 cells, the expression of PTEN protein and mRNA was detected by IC 50 The experiment detected the sensitivity of cells to the chemotherapy drugs cisplatin and paclitaxel; IK. H1299 cells expressing HDAC3-WT and HDAC3-K3R were subcutaneously injected into 8-week-old BALB / c nude mice. Cisplatin and HDAC3 inhibitor RGFP966 were injected on the 13th day after injection. The tumor volume was measured and the tumor growth curve was drawn (I). The tumor was removed 25 days after cell injection and the tumor volume and weight of each group were calculated (J&K).

[0022] Figure 8 . Reduced HDAC3 acetylation levels in clinical tissue samples predict poor prognosis of patients: AD. Histochemical staining of human non-small cell lung cancer tissue chips with paired adjacent tissues was performed to detect the levels of HDAC3-K3 acetylation and PTEN; E. The HDAC3-K3 acetylation level of non-small cell lung cancer tissue was scored according to AJCC pathological stage; FH. The expression of HDAC3 and PTEN in lung cancer in the TCGA dataset (https: / / xenabrowser.net / ) was analyzed; I. The correlation between HDAC3 and PTEN was analyzed in four independent GEO datasets (GSE19804, GSE19188, GSE18842, GSE3141); J&L. The patients were divided into high expression and low expression groups according to the expression levels of Ac-HDAC3-K3 or PTEN, and the survival curves of the two groups were compared. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified; the chemical and biological materials and reagents used are all conventional materials and reagents in the art and are commercially available unless otherwise specified.

[0025] 1. Materials and Methods

[0026] 1. Cell Culture and Transfection

[0027] Non-small cell lung cancer cell lines H1299, A549, H460 and H1703 were purchased from ATCC and cultured in RPMI-1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Human embryonic kidney HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were cultured in a humidified incubator at 37°C and 5% CO2. Cells were transfected using RNAiMAX, Lipofectamine 2000 (Invitrogen) or PEI (Polyscience) according to the manufacturer's protocol.

[0028] 2. CRISPR-Cas9 knockout and mutant cell lines

[0029] The sgRNA sequence was connected to the LentiCRISPR V2 plasmid and then co-transfected with viral packaging plasmids (PAX2 and VSVG) into 293T cells. The retroviral supernatant was harvested 36 hours after plasmid transfection and mixed with polystyrene (8 μg / ml) to improve the infection efficiency. Target cells were infected with 10 ml of viral supernatant and 10 ml of fresh culture medium for 48 hours, followed by selection with 2 μg / ml puromycin for 2 weeks to obtain CBP and HDAC3 knockout cell lines.

[0030] For HDAC3 mutant cell lines, FLAG-tagged HDAC3-WT and various mutant cDNAs were cloned into pLV-EF1a-IRES-Hygro lentiviral vector (CAT#85134; Addgene). The above plasmids were then co-transfected with viral packaging plasmids PAX2 and VSVG into 293T cells, respectively. Viral supernatants were harvested 48 hours after transfection and infected with HDAC3 knockout cell lines. Infected cells were then selected with 150 μg / mL hygromycin for 2 weeks to obtain HDAC3 mutant cell lines.

[0031] 3. Immunofluorescence

[0032] After the cells adhered to the wall or were treated accordingly, the slides were washed twice with PBS, fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 5% BSA at room temperature for 1 h, and then incubated with the corresponding primary antibody at 4°C overnight, and incubated with the secondary antibody coupled with Alexaflu488 or 568 (Invitrogen) at room temperature for 1 h. DAPI staining was used to show the cell nucleus.

[0033] 4. Real-time RT-PCR and RNA-seq

[0034] Total RNA was extracted using the RNeasy Mini Kit (CAT#74106; Qiagen) according to the manufacturer's instructions and subsequently purified using iScript TM cDNA was obtained by reverse transcription using a cDNA synthesis kit (CAT#1708891; Bio-Rad). TM Universal Real-time PCR was performed using Bio-Rad Green Supermix (CAT#1725121; Bio-Rad) and the program was run on a Bio-Rad CFX real-time PCR detection system. The primers used are listed in the Supplementary Table, and GAPDH was used as an internal reference gene to standardize and correct the experimental results.

[0035] Total RNA from H1299-HDAC3 stable overexpressing cells, H1299-HDAC3-K3R stable overexpressing cells, and control cells was extracted as described above. After passing quality control, RNA sequencing was performed by Novogene Corporation, and differential gene expression analysis was performed using DEseq2.

[0036] 5. Immunoprecipitation and Immunoblotting

[0037] The cells were collected, washed with PBS to remove cell debris, and then washed with cOmplete TM Protease inhibitor cocktail (CAT#4693116001;

[0038] Cells were lysed on ice using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM EDTA, 10 mM sodium butyrate) and centrifuged to obtain the total cell protein. The corresponding primary antibody, Pierce TM Anti-DYKDDDDK magnetic agarose (CAT#A36797; Thermo Scientific), Pierce TM Anti-c-Myc magnetic beads (CAT#88843; Thermo Scientific) or Pierce TM Anti-HA magnetic beads (CAT#88837; Thermo Scientific) were used for immunoprecipitation overnight at 4°C, and then the beads were washed 3 to 5 times with RIPA lysis buffer, and the protein complexes were separated by SDS-PAGE gel. The PVDF membrane was incubated with the corresponding primary and secondary antibodies. The PVDF membrane was purified by Li-COR CLx Imaging System or BIO-RAD ChemiDoc with SuperSignal TM Chemiluminescent detection was performed using West Pico PLUS ChemiluMinescent Substrate (CAT#34578; ThermoScientific).

[0039] 6. LC-MS / MS analysis to identify acetylation sites

[0040] HDAC3 acetylation sites were analyzed by mass spectrometry. Ten 10 cm culture dishes of HeLa cells were prepared for each group. When the cell confluence reached 80%, each dish of cells was transiently transfected with 6 μg Myc-HDAC3, 6 μg HA-CBP or 6 μg empty vector using PEI. After 24 h of transfection, the cells were treated with 5 μm TSA plus5.

[0041] Wash cells twice with PBS and add 10 mM sodium butyrate and cOmplete TM Lyse in RIPA lysis buffer with protease inhibitor cocktail for 30 minutes. Add anti-C-MYC magnetic beads to the cell lysate and incubate overnight at 4°C. Wash 4 times with RIPA lysis buffer and wash twice with 50mM ammonium bicarbonate (ABC) buffer. Then digest directly on the magnetic beads, add 0.4μg trypsin in 20μL ABC buffer and incubate overnight at 37°C. Add 100μL 3% formic acid to each sample to stop digestion, transfer the supernatant to a clean EP tube and dry.

[0042] The treated proteins were analyzed by LC-MS / MS on an UltiMate 3000RSLC system and a Q Exactive Orbitrap mass spectrometer (ThermoFisher Scientific).

[0043] 7. HDAC activity detection

[0044] Using FLUOR DE -Green HDAC fluorescent activity assay kit (#BML-AK530; Enzo LifeSciences) was used to detect HDAC activity. Total protein extracted from FLAG-HDAC3-WT and various mutant transfected cells was immunoprecipitated with anti-FLAG M2 magnetic beads and eluted with 3×FLAG Peptide. The samples were then incubated with HDAC3 substrate and HDAC3 assay buffer at room temperature with constant stirring for the specified time. 50 μL of analytical developer was added to terminate the reaction and incubated at room temperature for 15 min. The signal was then detected at EM / EX=540 / 480 using a fluorescent multi-wall plate reader.

[0045] 8.CCK8 proliferation assay

[0046] CCK-8 kit (#96992; Sigma) was used to detect the effects of different HDAC3 and CBP mutants on cell proliferation. 3 Cells were seeded on a 96-well plate at a density of 100 cells / well, with three replicates per day per group. From day 2, 10% CCK-8 medium was added to each well and incubated at 37°C for 1 h. The absorbance was then measured at 450 nm using an ELISA reader.

[0047] 9. Cell migration and invasion

[0048] Digest the cells, count them, and resuspend them in serum-free medium to obtain a concentration of 1×10 5100 μL of cell suspension was added to the upper chamber of the transwell chamber, and 600 μL of RPMI1640 containing 10% FBS was added to the lower chamber. Then the cells were migrated in a 37°C cell culture incubator for 6 hours. The chamber was removed, and the residual culture medium and floating cells were gently washed with PBS. The unmigrated cells in the upper chamber were removed with a cotton swab. Then 4% paraformaldehyde was added to fix the cells for 15 minutes, crystal violet was stained for 30 minutes, and the cells were washed with PBS and photographed and counted under a microscope.

[0049] For cell invasion assay, 100 μl BioCoat Matrigel was plated in the upper chamber of the transwell chamber. TM (CAT#354480; Corning), the matrix gel was stabilized in a 37°C cell incubator for 1 h, and then the cell suspension resuspended in serum-free medium was added as described above, and RPMI1640 containing 10% FBS was added to the lower chamber. After 48 h, the chamber was removed, non-invaded cells were removed with a cotton swab, and the cells that invaded the lower surface were stained with crystal violet, photographed and counted.

[0050] 10. Colony formation experiment

[0051] Crystal violet staining was used to count and analyze the proliferation of plate clones. 400 cells were inoculated in 6-well plates with three parallel replicates per group. After 10-14 days of growth, the culture medium was removed, washed 3 times with PBS, fixed with 4% paraformaldehyde solution for 15 minutes, and stained with 0.5% crystal violet for 15 minutes at room temperature. The plates were photographed with a Canon G9 digital camera, and the number of clones formed in each group was counted and analyzed. In order to conduct soft agar cloning experiments, 5000 cells were inoculated three times in a 6 cm dish with 0.6% RPMI 1640 low-temperature agar as the bottom layer and 0.4% RPMI 1640 agar as the top layer. After 4 weeks of growth, 1 mg / ml iodonitrotetrazolium chloride (Sigma) was used for staining overnight at 37°C. Cell colonies with a diameter greater than 100 μm were counted and analyzed.

[0052] 11. Gene Set Enrichment Analysis (GSEA)

[0053] Gene set enrichment analysis (GSEA) was used to rank 7199 genes in H1299 cells (expressing HDAC3 mutants or control plasmids). GSEA analysis was performed using GSEA V4.1.0. The significant condition was set to FDR less than 0.25.

[0054] 12. Construction of mouse xenograft tumor model

[0055] Resuspend the cells in RPMI 1640 to a concentration of 3 × 10 7100 μL of cell suspension was then subcutaneously injected into 8-week-old NU / J male mice. Tumor size was measured with a vernier caliper at designated time points, and the volume was calculated using the formula V = L × W 2 ×0.5, where L is the longest diameter and W is the shortest diameter. Mice were killed after 25 days, and tumors were obtained by dissection and photographed. Cisplatin (5 mg / kg) was injected intraperitoneally 3 times a week starting from the 14th day after cell inoculation. RGFP966 was dissolved in DMSO, diluted with 30% (wt / vol) hydroxypropyl-β-cyclodextrin and 100 mM sodium acetate (pH 5.4), and injected subcutaneously at a dose of 25 mg / kg, 3 times a week.

[0056] 13. Tissue Samples and Immunohistochemical Staining

[0057] Human non-small cell lung cancer (NSCLC) tissue array (#LC03) was purchased from Reveal Biosciences, which includes NSCLC tissue samples of different stages from 75 patients and paired adjacent non-cancerous tissues. Tissue sections were dewaxed with xylene and then hydrated in ethanol. Endogenous peroxidase was removed using dual endogenous enzyme blocking reagent (#S2003; Agilent). Antigen retrieval was performed using pH 6.0 or pH 9.0 sodium citrate solution. Anti-PTEN and Ac-HDAC3-K3 were incubated overnight at 4°C. After washing in PBS buffer, sections were stained with Elite ABC-HRP Kit Peroxidase (#PK-6101; Vector Laboratories) was used for incubation. Finally, the signal was detected using Liquid DAB+ two-component system (#K346811; Agilent). All immunohistochemical staining was independently reviewed by two pathologists. The samples were divided into 4 groups according to the evaluation of HDAC3-K3 acetylation antibody or PTEN antibody staining: low reactivity was marked as 1+, weak reactivity was marked as 2+, moderate reactivity was marked as 3+, and strong reactivity was marked as 4+. We defined 3+ and 4+ reactivity as high expression, and the rest as low expression.

[0058] 14. Statistical Analysis

[0059] Data are expressed as mean ± SD. Two-tailed Student's test was used for comparison between two groups. One-way analysis of variance was used to compare differences among multiple groups. When variances were unequal, pairwise comparisons were evaluated using the Student-Newman-Keuls procedure or Dunnett's T3 procedure. All statistical calculations were performed using SPSS13.0 software. P < 0.05 was considered statistically significant.

[0060] result

[0061] 1. Acetyltransferase CBP acetylates the K3 site of HDAC3

[0062] like Figure 1 As shown in A, endogenous HDAC3 is a protein that can be modified by acetylation. After 293T cells were treated with nicotinamide (NAM), an inhibitor of the sirtuin (SIRT) deacetylase family, or trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) I, II, and IV, the acetylation level of endogenous HDAC3 increased, especially after NAM treatment, the acetylation level of HDAC3 increased significantly ( Figure 1 B). Figure 1 C further demonstrated that HDAC3 is mainly acetylated by the acetyltransferase CBP compared with acetyltransferases such as PCAF, P300, Tip60, GCN5, or MOF. In addition, we found that the acetylation level of HDAC3 was significantly suppressed in cells lacking CBP ( Figure 1 D&E), these data indicate that CBP is the only acetyltransferase that can directly regulate HDAC3 acetylation.

[0063] Next, we used LC-MS / MS to identify the sites of CBP-mediated HDAC3 acetylation. Figure 1 As shown in F, the K3 site of HDAC3 protein isolated from HeLa cells was identified as a potential acetylation site. To further verify the accuracy of the mass spectrometry analysis results, we generated an HDAC3 mutant with a K-to-R mutation at the K3 site. Figure 1 G shows that compared with WT HDAC3, the acetylation level of HDAC-K3R mutant is significantly reduced, indicating that K3 is the main acetylation site of HDAC3. We then used co-immunoprecipitation (co-IP) experiments to prove that CBP-mediated HDAC3 acetylation depends on the direct interaction between the two. ( Figure 1 H&I).

[0064] 2. HDAC3-K3 acetylation inhibits its enzyme activity and stability

[0065] Previous studies have shown that HDAC3 S424 phosphorylation is essential for its deacetylase function. To test whether CBP-mediated HDAC3 acetylation affects its phosphorylation, we used anti-FLAG M2 magnetic beads to detect whether CBP-mediated HDAC3 acetylation affects its phosphorylation. - / - Total proteins from H1299 cells were immunoprecipitated, and the phosphorylation levels of these mutants were detected using anti-HDAC3-phos-S424 antibody. Figure 2As shown in A, compared with HDAC3-WT, the phosphorylation level of HDAC3-K3R at S424 was significantly increased, while the phosphorylation level of HDAC3-K3Q, which mimics acetylation, was significantly decreased. - / - The phosphorylation level was significantly increased in H1299 cells, while recombinant expression of wild-type HA-CBP in these cells decreased the phosphorylation level ( Figure 2 B). The above data suggest that acetylation of HDAC3-K3 may block its phosphorylation at the S424 site.

[0066] Given that phosphorylation of the S424 site leads to HDAC3 activation, we further investigated whether acetylation of the K3 site would alter HDAC3 enzyme activity. We immunoprecipitated HDAC3 from H1299 cells and detected HDAC3 enzyme activity using the HDAC fluorescence method. We found that the HDAC3-K3R mutant exhibited enhanced acetylase activity compared to HDAC3-WT, an effect that depended on S424 phosphorylation, while the S424A mutation completely abolished S424 phosphorylation ( Figure 2 CE). Next, we investigated the effect of HDAC3 acetylation on its protein stability. Figure 2 As shown in F&G, acetylation of HDAC3 at the K3 site significantly promotes the degradation of HDAC3. The above data indicate that HDAC3-K3 acetylation inhibits its enzymatic activity and destroys its protein stability.

[0067] 3. HDAC3 acetylation inhibits proliferation and migration of non-small cell lung cancer

[0068] To reveal the potential biological function of K3 site acetylation of HDAC3, we constructed HDAC3 vectors using Flag-HDAC3-WT, Flag-HDAC3-K3R, Flag-HDAC3-K3Q, Flag-HDAC3-K3R-s424a and empty vector. - / - H1299 cells ( Figure 3 A). Compared with cells expressing HDAC3-WT, HDAC3 deficiency significantly inhibited monoclonal growth, while the acetylation-deficient mutant HDAC3-K3R significantly promoted the growth of lung cancer cells, and the expression of the acetylation-mimicking mutant HDAC3-K3Q significantly inhibited cell proliferation ( Figure 3 BD). In addition, cells expressing HDAC3-K3Q were more sensitive to low glucose than cells expressing HDAC3-WT ( Figure 3 E), indicating that downregulation of HDAC3 acetylation is critical for NSCLC growth under energy-deficient conditions.

[0069] Next, we examined the effects of HDAC3 acetylation on the migration and invasion of lung cancer cells. Figure 4 As shown in F&G, HDAC3-KO significantly inhibited cell migration and invasion. Compared with HDAC3-WT, HDAC3-K3R promoted the migration and invasion of lung cancer cells, while HDAC3-K3Q inhibited their migration and invasion.

[0070] To investigate the effect of HDAC3-K3 acetylation on tumor growth in vivo, H1299 HDAC3 overexpressed with Flag-HDAC3-WT, Flag-HDAC3-K3R, Flag-HDAC3-K3Q, or a control plasmid were transfected into - / - The cells were injected subcutaneously into 8-week-old BALB / c nude mice. Figure 3 As shown in HJ, compared with HDAC3-WT, HDAC3-K3R significantly promoted tumor growth in xenograft models, while HDAC3-K3Q inhibited tumor growth and reduced tumor volume. At the same time, the phosphorylation level of HDAC3-S424 was significantly increased in tumor tissues generated by HDAC3-K3R cells ( Figure 3 K), HDAC3-S424 phosphorylation level is consistent with the tumor growth ability in vivo. In summary, K3 acetylation of HDAC3 inhibits the proliferation and migration of lung cancer cells in vitro and the growth of mouse tumors.

[0071] 4. Acetylation of HDAC3 at the K3 site regulates PTEN gene transcription

[0072] In order to clarify the molecular mechanism of HDAC3 acetylation regulating tumor growth and cell migration, transcriptome sequencing was performed on H1299 cells stably overexpressing HDAC3-K3R, HDAC3-WT or empty vector. First, the genes regulated by HDAC3 were obtained by analyzing the transcriptome results of the HDAC3-WT overexpression group and the empty vector group. Then, the transcriptome results of the HDAC3-K3R group and the HDAC3-WT group were analyzed, and the genes regulated by HDAC3 acetylation were obtained ( Figure 4 A). The intersection of the two RNA-seq datasets above yielded 389 overlapping genes (p<0.05, log2FC<-0.2 or >0.2), which may be regulated by HDAC3 and its acetylation ( Figure 4 B). Further analysis of the above genes showed that in the intersection of differentially expressed genes in transcriptome sequencing between HDAC3-K3R and HDAC3-WT, and between HDAC3-WT and control plasmid, 203 genes were down-regulated and 60 genes were up-regulated. The regulation of these 263 genes may depend on HDAC3 and its acetylation.

[0073] Among these 263 differentially expressed genes, PTEN is a well-known gene that regulates tumorigenesis. Gene set enrichment analysis (GSEA) further demonstrated a negative correlation between HDAC3 activation and the PTEN target gene set ( Figure 4 C). Therefore, we selected PTEN as a downstream molecule to study HDAC3 acetylation-dependent transcriptional regulation in non-small cell lung cancer. Real-time qPCR experiments showed that HDAC3 deficiency could upregulate PTEN mRNA levels in three non-small cell lung cancer cell lines (H1299, A549, H1703) ( Figure 4 DF) and protein level ( Figure 4 GI). HDAC3 expressing wild-type HDAC3 - / - Compared with H1299 cells, the PTEN mRNA level in HDAC3-K3R mutant-overexpressing cells was significantly downregulated ( Figure 4 J). In addition, signals such as AKT (S473 / T308), S6K (T389), mTOR, GSK3, and 4E-BP-1 phosphorylation, which are negatively regulated by PTEN, are expressed in HDAC3 - / - The corresponding decrease in H1299 cells ( Figure 4 D). Compared with HDAC3-WT, HDAC3-K3R significantly reduced PTEN protein levels in a manner dependent on S424 phosphorylation and activated the signaling that was inhibited by it ( Figure 4 J).

[0074] We further evaluated whether HDAC3 maintained tumor growth by inhibiting PTEN by knocking down PTEN in H1299 and A549 cells, and the results showed that it completely eliminated the growth advantage conferred by HDAC3-KO to wild-type cells ( Figure 5 AF). In vitro and in vivo experiments showed that re-expression of PTEN can also inhibit the proliferation and cloning ability of tumor cells conferred by HDAC3-K3R ( Figure 5 The above results suggest that HDAC3 and its acetylation may regulate tumor proliferation by regulating PTEN transcription.

[0075] We further examined the role of PTEN in the regulation of AKT signaling mediated by HDAC3 and its acetylation. Figure 6 As shown in A&B, HDAC3-K3R significantly enhanced AKT signaling compared with HDAC3-WT, and knockdown of PTEN in HDAC3-deficient cells reversed the impaired AKT signaling activation (6C). The above data indicate that in non-small cell lung cancer, HDAC3 acetylation inhibits PTEN expression, thereby activating the AKT signaling pathway and promoting cancer cell proliferation.

[0076] 5. CBP-HDAC3-PTEN axis regulates the resistance of non-small cell lung cancer to chemotherapy drugs. Studies have reported that both lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) have recurrent changes in the CBP gene. The above results show that CBP-regulated HDAC3 acetylation inhibits HDAC3 enzyme activity and protein stability. We further explored whether the loss of CBP in non-small cell lung cancer regulates PTEN transcription through HDAC3. First, CRISPR / Cas9 gene editing technology was used to construct a CBP knockout H1299 cell line. It can be seen that the expression level of PTEN is significantly downregulated as the expression of CBP mRNA and protein levels decreases ( Figure 7 A&B). Furthermore, the reduction in PTEN expression in CBP-deficient cells could be restored by knocking down HDAC3 ( Figure 7 C&D). These data indicate that PTEN expression in H1299 cells is dependent on CBP and HDAC3.

[0077] Reduced or absent PTEN expression, which is common in non-small cell lung cancer, can promote cancer cell proliferation and resistance to chemotherapy. We found that knocking out HDAC3 significantly increased the sensitivity of non-small cell cancer cell lines to the chemotherapeutic drugs cisplatin and paclitaxel in vivo and in vitro, while HDAC3-K3R conferred resistance to these two chemotherapeutic drugs in non-small cell lung cancer cell lines compared with HDAC3-WT ( Figure 7 EH). Moreover, cisplatin combined with the selective HDAC3 inhibitor RGFP966 had a significant inhibitory effect on the growth of tumor xenografts, especially in the HDAC3-K3R group ( Figure 7 IK).

[0078] 6. Reduced HDAC3 acetylation levels in clinical tissue samples indicate poor prognosis

[0079] We used human non-small cell lung cancer tissue microarrays with paired adjacent tissues to perform immunohistochemical staining for HDAC3-K3 acetylation and PTEN. Figure 8 As shown in AD, HDAC3-K3 acetylation and PTEN levels were significantly downregulated in non-small cell lung cancer compared with paired normal lung tissues. In addition, HDAC3-K3 acetylation levels were significantly correlated with AJCC pathological stage ( Figure 8 E). Analysis of the TCGA dataset (https: / / xenabrowser.net / ) also showed that HDAC3 was upregulated and PTEN was downregulated in lung cancer ( Figure 8 FH), and the negative correlation between HDAC3 and PTEN expression was also confirmed in four independent GEO datasets ( Figure 8I). And patients with higher PTEN expression or increased HDAC3-K3 acetylation level have better prognosis than those with lower PTEN expression or decreased HDAC3-K3 acetylation level ( Figure 8 H&I). This indicates that PTEN and HDAC3-K3 acetylation levels are associated with the prognosis of non-small cell lung cancer. The overall survival rate of patients with low HDAC3-K3 acetylation levels and low PTEN expression is much lower than that of patients with high K3 acetylation levels or high PTEN expression ( Figure 8 JL). Therefore, PTEN expression level is positively correlated with HDAC3-K3 acetylation in NSCLC, suggesting that HDAC3-K3 acetylation may be an ideal biomarker for the diagnosis of NSCLC and a potential target for treatment.

Claims

1. Application of HDAC3 gene as a target in screening / preparing drugs for treating non-small cell lung cancer, characterized in that: Regulating the protein acetylation modification of HDAC3, the increased acetylation modification level of HDAC3 protein K3 site inhibits or alleviates the development of non-small cell lung cancer.

2. The use according to claim 1, characterized in that: By upregulating the acetylation modification level of the K3 site of the HDAC3 protein, reducing the activity of HDAC3 deacetylase, and increasing the expression of the tumor suppressor gene PTEN, the proliferation of non-small cell lung cancer can be inhibited, thereby achieving the purpose of alleviating or inhibiting the development of non-small cell lung cancer.

3. The use according to claim 2, characterized in that: HDAC3 regulates the expression of PTEN through CBP-regulated K3 site acetylation modification.

4. Application of reagents that promote acetylation of the K3 site of HDAC3 protein in the preparation of drugs for the treatment of non-small cell lung cancer.

5. The use according to claim 4, characterized in that: Reagents that promote acetylation of the K3 site of the HDAC3 protein inhibit the deacetylase activity of HDAC3 and promote the expression of PTEN, thereby achieving the purpose of alleviating or inhibiting the development of non-small cell lung cancer.

6. The use according to claim 5, characterized in that: The reagent promoting acetylation of the K3 site of the HDAC3 protein is a small molecule or a low molecular weight compound, a HDAC3 protein K3 site-specific binding agent, or an acetylation-inducing chimeric small molecule targeting the site.

7. The use according to claim 6, characterized in that: The specific binding agent is a selective agonist, chelator and deacetylase inhibitor for the site.

8. Use of a reagent for detecting acetylation at the K3 site of HDAC3 protein in the preparation of a kit for diagnosis or prognosis of non-small cell lung cancer, wherein the kit can be used for diagnosis, treatment monitoring, prognosis observation and health status evaluation of non-small cell lung cancer.

9. The use according to claim 8, characterized in that: The decreased acetylation level of the K3 site of the HDCA3 protein indicates that the patient suffers from non-small cell lung cancer with CBP mutation, or predicts a poor prognosis for patients with non-small cell lung cancer.

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