Overexpressed histone acetyltransferase 1 regulates cancer immunity by increasing programmed death-ligand 1 expression in pancreatic cancer
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Pancreatic ductal adenocarcinoma is one of the leading causes of cancer-related death worldwide. Immune checkpoint blockade therapy, including anti-PD-1 and anti-PD-L1, is a new therapeutic strategy for cancer treatment but the monotherapy with PD-L1 inhibitors for pancreatic cancer is almost ineffective for pancreatic cancer. Thus, exploring the regulatory mechanism of PD-L1 in cancer cells, especially in pancreatic cancer cells, is one of the key strategies to improving cancer patient response to PD-L1 blockade therapy. Histone acetyltransferase 1(HAT1) is a classic type B histone acetyltransferase and the biological role of HAT1 in pancreatic cancer is unclear.
The clinical relevance of HAT1 was examined by the GEPIA web tool, Western blotting and immunohistochemistry of pancreatic cancer tissue microarray slides. Tumor cell motility was investigated by MTS assay, colony formation assay and xenografts. The relationship between HAT1 and PD-L1 was examined by Western blot analysis, RT-qPCR and immunohistochemistry.
HAT1 was upregulated in PDAC and associated with poor prognosis in PDAC patients. The knockdown of HAT1 decreased the proliferation of pancreatic cancer cells in vivo and in vitro. Strikingly, we showed that HAT1 transcriptionally regulated PD-L1, and this process was mainly mediated by BRD4 in pancreatic cancer. The knockdown of HAT1 improved the efficacy of immune checkpoint blockade by decreasing the PD-L1.
The recognition of HAT1 in regulating tumor cell proliferation and cancer immunity indicated that HAT1 might be employed as a new diagnostic and prognostic marker and a predictive marker for pancreatic cancer therapy, especially in immune checkpoint blockade therapy. Targeting HAT1 highlights a novel therapeutic approach to overcome immune evasion by tumor cells.
KeywordsHistone acetyltransferase 1 Pancreatic ductal adenocarcinoma PD-L1
Histone acetyltransferase 1
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; TMA: tissue microarray
Pancreatic ductal adenocarcinoma
Programmed death-ligand 1
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide . Resistance to chemotherapy and radiotherapy results in the poor prognosis of PDAC . Immunotherapy is a new therapeutic strategy for cancer treatment and has made a profound progress in prolonging the survival time patients with of various types of tumors . However, the immunotherapy is almost ineffective for pancreatic cancer . Therefore, exploring the underlying mechanisms is urgently needed to overcome the resistance to immunotherapy in pancreatic cancer.
Tumors evade immune surveillance by the aberrant activation of inhibitory pathways that regulate the function of T lymphocytes, known as immune checkpoints . Programmed death-ligand 1 (PD-L1, B7-H1) is a member of the B7 family of cell surface ligands on cancer cells surfaces, which binds the programmed death-1 protein (PD-1) receptor to induce T cell apoptosis and inhibit cytotoxic T-cell activation within cancer tissues [6, 7, 8, 9]. Given that the blockade of the PD-1/PD-L1 interaction can reactivate T-cell responses, a few anti-PD-1 and anti-PD-L1 antibodies have been approved for the treatment of human cancers in the clinic . However, monotherapy with PD-L1 inhibitors for pancreatic cancer has resulted in disappointing outcomes in clinical trials . A growing body of evidence suggests that the expression level of PD-L1 in cancer cells is highly associated with the response to immune checkpoint therapies . Thus, exploring the regulatory mechanism of PD-L1 in cancer cells, especially in pancreatic cancer cells, is one of the key strategies to improve cancer patient response to PD-L1 blockade therapy.
Histone acetyltransferase 1 (HAT1) is a classic type B histone acetyltransferase, and it can only acetylate newly synthesized histone H4 and not nucleosomal histone .HAT1 was the first histone acetyltransferase identified and is one of the most poorly understood members of this family . HAT1 is overexpressed in multiple types of solid tumors, including esophageal , lung cancer  and liver cancer , and acts as an oncoprotein to promote tumorigenesis. It has been reported that HAT1 functions as a transcription factor to regulate the expression of various genes, such as Bcl2L12  and Fas , and modulates cancer cell proliferation , apoptosis  and metabolism .
To date, the biological effect and clinical relevance of HAT1 in pancreatic cancer is poorly understood. In this study, we sought to determine the specific role of HAT1 in pancreatic cancer. First of all, we demonstrated that HAT1 was overexpressed in pancreatic cancer and linked with poor prognosis in PDAC patients. Then, our data showed that HAT1 acted as a tumor growth promoting protein in pancreatic cancer cells. Strikingly, HAT1 was involved in the cancer immunity response by regulating the PD-L1 expression, and this process was mainly mediated by BRD4. Taken together, our results demonstrate that aberrant expression of HAT1 promotes tumorigenesis by modulating the cancer cell growth and the immune response in pancreatic cancer.
Materials and methods
All pancreatic cancer cell lines including PANC-1, BxPC-3 and MIA PaCa-2 were purchased from the Chinese Academy of Science Cell Bank, and the Panc 02 cells were obtained from Tong Pai Technology (Shanghai, China). These cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, USA). All cell lines were routinely maintained at 37 °C in a 5% CO2 incubator.
Plasmids, antibodies and chemicals
Mammalian expression vectors for Flag-HAT1 recombinant proteins were generated using the pcDNA3.1 backbone vector. The HAT1 antibody (ab194296) was purchased from Abcam (working dilution 1:2000); beta-tubulin (2128S) was from Cell Signaling Technology - (working dilution 1:5000); BRD4 (ab128874) was from Abcam (working dilution 1:1000); PD-L1 (13684S) was from Cell Signaling Technology (working dilution 1:1000); and H4K5ac (ab17343) was from Abcam (working dilution 1:1000). Ascorbate was purchased from Sigma-Aldrich (Shanghai, China).
Western blot of cells and tissue specimens
The ethics of using human tissue (12 pairs of matched pancreatic cancer/adjacent noncancerous tissues) was approved by the local ethics committee (Tongji Medical College, China), and written informed consent was obtained from patients prior to surgery exactly as described previously . The cells or the tissue specimens were lysed with lysis buffer (Beyotime, China) containing 1% protease and phosphatase inhibitors. The protein concentration was determined with a protein assay kit (Pierce Biotechnology, USA). Equal amounts of protein for each sample were separated using SDS-PAGE gels and transferred onto PVDF membranes (Pierce Biotechnology, USA). The membranes were subsequently blocked in 5% not-fat milk for 1 h at room temperature, followed by incubation with primary antibody overnight at 4 °C. The membranes were then washed with 1x TBST and incubated with a secondary antibody for 1 h. Finally, the membranes were treated with ECL detection reagents and exposed to X-ray films.
Total RNA was extracted from the cells using Trizol reagent (Thermo Fisher Scientific, USA). First strand cDNA was synthesized from 2 μg RNA using a cDNA Reverse Transcription kit (PrimeScript™ RT reagent Kit, Code No. RR037A), and real-time PCR analysis was carried out with a PCR kit (TB Green™ Fast qPCR Mix, Code No. RR430A) according to the manufacturer’s protocols. The two kits were purchased from Takara Bio Inc. (Shigo, Japan). All the values were normalized to actin, and the 2-ΔCt method was used to quantify the fold change. The primers used for RT-qPCR are provided in Additional file 1: Table S1.
Chromatin immunoprecipitation (ChIP) and ChIP-qPCR
ChIP was performed following the manufacturer’s instructions for the Chromatin Extraction Kit (Abcam, ab117152, USA) and ChIP Kit Magnetic - One Step (Abcam, ab156907, USA) . BRD4 (Cell Signaling Technology, 13,440, dilution 1:50) was used for the ChIP assay. The purified DNA was analyzed by real-time PCR with a PCR kit (Takara Bio Inc., Japan) according to the manufacturer’s protocols . The primers for ChIP-qPCR are provided in Additional file 1: Table S2.
Tissue microarray and immunohistochemistry (IHC)
The tissue microarray slides were purchased from Outdo Biobank (Shanghai, China) (HPan-Ade060CD-01). The tissue microarray specimens were immunostained with PD-L1 (Cell Signaling Technology, 13,684, dilution 1: 1000) and HAT1 antibodies (Abcam, ab194296, dilution 1:3000) as described previously. Staining intensity was scored in a blinded fashion: 1 = weak staining at 100× magnification but little or no staining at 40× magnification; 2 = medium staining at 40× magnification; 3 = strong staining at 40× magnification . The degree of immunostaining was reviewed and scored by two independent pathologists who were blinded to the clinical details. The scores were determined by the percentage of positive cells multiplied by the staining intensity.
The lentivirus-based control and gene-specific shRNAs were purchased from Sigma-Aldrich. Lipofectamine 2000 was used to transfect 293 T cells with shRNA plasmids and viral packaging plasmids (pVSV-G and pEXQV). Twenty-four hours after transfection, the medium was replaced with fresh DMEM, containing 10% FBS and 1 mM of sodium pyruvate. Next, 48 h post transfection, the virus culture medium was collected and added to the PANC-1, MIA PaCa-2 and BxPC-3 cells supplemented with 12 μg/ml of polybrene. Twenty-four hours after infection, the infected cells were selected with 10 μg /ml of puromycin. The shRNA sequence information is provided in Additional file 1: Table S3.
The pTsin lentiviral expression vector was used to generate lentiviral plasmids for pTsin-Flag-HAT1. Lipofectamine 2000 was used to transfect 293 T cells with the pTsin expression plasmid and viral packaging plasmids (pHR’ CMVδ 9.8 and pVSV-G). Twenty-four hours after transfection, the medium was replaced with fresh DMEM, containing 10% FBS and 1 mM of sodium pyruvate. Next, 48 h post transfection, the virus culture medium was collected and added to PANC-1 cells supplemented with 12 μg/ml of polybrene. Twenty-four hours after infection, the infected cells were selected with 10 μg/ml of puromycin.
Cell proliferation assay
Cell viability was evaluated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay according to the manufacturer’s instructions (Abcam, USA). Briefly, the pancreatic cancer cells (1 × 103 cells) were seeded in 96-well plates with 100 μl of culture medium. The cells were treated with serial concentrations of small molecular inhibitors. After 72 h, 20 μl of MTS reagent (Abcam, USA) was added to each well of the cells and incubated for 1 h at 37 °C in standard culture conditions. The absorbance was measured in a microplate reader at 490 nm.
Generation of PDAC xenografts in nude mice
The BALB/c-nu mice (4–5 weeks of age, 18–20 g) were purchased from Vitalriver (Beijing, China) and randomly divided into two groups (n = 7/group) for the subcutaneous inoculation with 5 × 106 of PANC-1 cells infected with shControl or shHAT1 lentivirus in the left dorsal flank of the mice. The tumors were examined every other day for 21 days; the length and width measurements were obtained with calipers to calculate the tumor volumes by using the eq. (L x W2)/2. On day 21, the animals were euthanized, and the tumors were excised and weighed. All the animal experimental procedures were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.
Survival analysis and correlation analysis using the GEPIA web tool
The online database Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancerpku.cn/index.html.)  was used to analyze the RNA sequencing expression data related to our project based on The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects. GEPIA performs survival analyses based on gene expression levels and uses a log-rank test for hypothesis evaluation. GEPIA performs a pairwise gene correlation analysis for any given set of TCGA and/or GTEx expression data using Pearson correlation statistics.
Generation and treatment of Panc 02 xenografts in mice
Six-week-old C57BL/6 mice were purchased from Charles River Laboratories (Wuhan, China). All the animal experimental procedures were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Panc 02 cells (5 × 106 in 100 μl 1 × PBS) infected with shControl or shHAT1 lentivirus were injected s.c. into the right flank of mice. The volume of xenografts was measured every other day and calculated using the formula LxW2x0.5. After the xenografts reached a size of approximately 50 mm3, mice carrying similar types of tumors were randomized into different groups and treated with anti-PD-1 (BioXcell, Clone RMP1–14)/IgG (BioXcell, Clone 2A3) (200 μg, i.p., given at days 0, 3, 6); or anti-PD-L1 (BioXcell, Clone 10F.9G2)/IgG (BioXcell, Clone MPC-11) (200 μg, i.p., given at days 0, 3, 6). Mice were euthanized and tumors were collected from all animals once the tumors reached a volume of 200 mm3.
Flow cytometry analysis
The PANC-1, MIA PaCa-2 and BxPC-3 cells infected with control or HAT1-specific shRNAs were harvested and washed with PBS. Cells were fixed in 4% paraformaldehyde for 15 min. After washing with PBS, cells were incubated with ice-cold 100% methanol for 30 min on ice. Cells were washed with PBS and incubated with PD-L1 antibody (Biolegend, APC anti-human CD274, clone 29E.2A3) or isotype IgG (Biolegend,APC anti-human IgG Fc Antibody, clone HP6017) for 15 min at room temperature. After washing three times with PBS, the cells were resuspended in PBS and analyzed by flow cytometry.
For flow cytometry analysis of the mouse tissue samples, the tumors were cut into small pieces and digested with 2 mg/ml collagenase (Sigma, USA) in DMEM for 1 h at 37 °C. Cells were filtered through a 70 μm nylon strainer and resuspended in red blood cell lysis buffer (Biolegend) for 3 min at room temperature. The cells were then suspended in PBS with 2% BSA and costained with the following antibodies: CD45 (Biolegend, 103,112, APC conjugated); CD4 (Biolegend, 100,510, FITC conjugated); CD8 (Biolegend, 100,708, PE conjugated); CD11b (Biolegend,101,212, APC conjugated); and Gr1 ((Biolegend, 108,406, FITC conjugated)). After incubation with antibody for 15 min, the cells were washed with PBS and analyzed by flow cytometry.
Statistical analyses were performed with one-sided or two-sided paired Student’s t-test for single comparison and one-way ANOVA with a post hoc test for multiple comparisons. A P value < 0.05 was considered statistically significant. All the values are expressed as the mean ± SD.
HAT1 is up-regulated in PDAC and associated with poor prognosis in PDAC patients
HAT1 promotes cell proliferation in pancreatic cancer in vivo and in vitro
HAT1 transcriptionally increases PD-L1 expression in pancreatic cancer cells
PD-L1 is positively correlated with HAT1 in PDAC patient specimens
Knockdown of HAT1 improves the efficacy of immune checkpoint blockade by decreasing the PD-L1 expression in vivo
HAT1 increases PD-L1 expression through BRD4 in pancreatic cancer cells
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) influence DNA transcription through the balance between histone acetylation and deacetylation . HAT1 acetylates newly synthesized histone H4 but not nucleosomal histones and regulates the genes involved in cell differentiation, proliferation, cell metabolism and apoptosis [14, 15, 16]. HAT1 plays a critical role in the tumorigenesis of digestive system cancer. It has been well documented that HAT1 is an important determinant in the regulation of the proliferation of esophageal and liver cancer cells in vivo and in vitro [14, 16]. Moreover, HAT1 is overexpressed in multiple types of cancer and associated with poor prognosis . In this study, we demonstrate that HAT1 is upregulated and highly correlated with poor prognosis in pancreatic cancer specimens. The aberrant expression of HAT1 participates in promoting tumor cell growth in pancreatic cancer.
The nonimmunogenic characteristics of pancreatic cancer are responsible for the failure of immunotherapy. Only a small portion of pancreatic cancer patient specimens were positive for PD-L1, representing the best candidates for PD-L1 blockade therapy. However, monotherapy with PD-L1 blockade has no effect on the survival time of pancreatic cancer patients. Given that the expression level of PD-L1 plays a key role in determining the efficacy of anti-PD-L1 therapy, understanding the regulatory mechanism of PD-L1 in cancer cells sheds new light on the exploration of novel therapy strategies for cancer treatment. Recent studies showed that various transcriptional factors, including BRD4 , MYC , p65  and STAT3 , could directly bind to the PD-L1 promoter and initiate PD-L1 transcription. Moreover, RAS signaling is involved in regulating the mRNA stabilization of PD-L1 in cancer cells . Furthermore, beyond the mRNA level regulation, it has been documented that the E3 ligase, SPOP , β-TrCP , and the deubiquitinase CSN5  participate in modulating the stability of PD-L1 through the proteasome pathway. In addition, the transmembrane proteins, CMTM4 and CMTM6, stabilize the PD-L1 protein via the lysosome pathway [37, 38]. In our study, we demonstrate that HAT1 is a novel regulator of PD-L1 at the transcriptional level and that BRD4 might be an important mediator of this process.
Our data indicated that the knockdown of HAT1 blocked pancreatic cancer tumor growth (Fig. 2), but the overexpression of HAT1 promoted the tumor growth in vivo (Additional file 1: Figure S1). Our results further showed that HAT1 regulated PD-L1 expression in pancreatic cancer (Fig. 3), and PD-L1 has been reported to promote tumor cell growth not only via immune effects but also through tumor cell-intrinsic signals, including the regulation of autophagy and the mTOR pathway . Therefore, HAT1 might regulate cancer cell proliferation via PD-L1. It has been well documented that HAT1 could regulate the function of BRD4 or increase the acetylation level of histones to influence the expression of a number of genes involved in apoptosis and glucose metabolism [15, 16, 25], which was critical for the cell viability and not involved in the PD-L1 effect. Therefore, PD-L1 is partially responsible for promoting the pancreatic cancer cell growth induced by HAT1 in vivo, and this is confirmed by our results in Additional file 1: Figure S2.
Our data indicated that HAT1 increased the PD-L1 expression in vivo and in vitro, which identifies HAT1 as a previously unrecognized master regulator of this critical immune checkpoint. However, there is no small molecular inhibitor to specifically target HAT1. Recent studies have indicated that ascorbate represses the HAT1 expression via the TET-mediated DNA hydroxymethylation pathway . Our findings suggest that ascorbate could suppress PD-L1 expression by influencing the HAT1 level in pancreatic cancer cells. Although ascorbate is not a the specific inhibitor of HAT1, it may regulate PD-L1 expression through other pathways. These data also suggest that ascorbate might be a potential new avenue to overcome immune evasion by tumor cells.
In summary, we proposed a new understanding of the specific role of HAT1 in pancreatic cancer. We showed that HAT1 is overexpressed in pancreatic cancer specimens and highly correlated with poor prognosis in pancreatic cancer. Furthermore, our results suggested that HAT1 promoted cell proliferation in pancreatic cancer cells. In particular, we demonstrated that HAT1 functioned as an important regulator in cancer immunity via transcriptionally upregulating the PD-L1 level in tumor cells. The recognition of HAT1 in the regulation of PD-L1 expression suggests that HAT1 might be employed as a new diagnostic and prognostic marker and as a predictive marker for pancreatic cancer therapy, especially in immune checkpoint blockade therapy. Targeting HAT1 highlights a novel therapeutic target to overcome immune evasion by tumor cells.
This work was supported in part by grants from the Chinese National Natural Science Foundation Grant No. 81702374.
Availability of data and materials
Please contact the author for data requests.
PF and JZ performed the experiments and wrote the paper, ZM, HW and BW collected the data. XJ and HW wrote the paper and analyzed the data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was conducted in accordance with the principles of the Declaration of Helsinki principles. It was approved by the Animal Use and Care Committees at Tongji Medical College, Huazhong University of Science and Technology.
Consent for publication
The authors declare that they have no competing interest.
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