Overexpressed Histone Acetyltransferase 1 Regulates
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Fan et al. Journal of Experimental & Clinical Cancer Research (2019) 38:47 https://doi.org/10.1186/s13046-019-1044-z RESEARCH Open Access Overexpressed histone acetyltransferase 1 regulates cancer immunity by increasing programmed death-ligand 1 expression in pancreatic cancer Ping Fan1†, Jingyuan Zhao1†, Zibo Meng1, Heyu Wu2, Bo Wang1, Heshui Wu1* and Xin Jin1* Abstracts Background: 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. Methods: 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. Results: 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. Conclusions: 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. Keywords: Histone acetyltransferase 1, Pancreatic ductal adenocarcinoma, PD-L1 Background patients with of various types of tumors [3]. However, Pancreatic ductal adenocarcinoma (PDAC) is one of the the immunotherapy is almost ineffective for pancreatic leading causes of cancer-related death worldwide [1]. Re- cancer [4]. Therefore, exploring the underlying mecha- sistance to chemotherapy and radiotherapy results in the nisms is urgently needed to overcome the resistance to poor prognosis of PDAC [2]. Immunotherapy is a new immunotherapy in pancreatic cancer. therapeutic strategy for cancer treatment and has made Tumors evade immune surveillance by the aberrant a profound progress in prolonging the survival time activation of inhibitory pathways that regulate the func- tion of T lymphocytes, known as immune checkpoints [5]. Programmed death-ligand 1 (PD-L1, B7-H1) is a * Correspondence: [email protected]; [email protected] † member of the B7 family of cell surface ligands on can- Ping Fan and Jingyuan Zhao contributed equally to this work. 1Department of Pancreatic Surgery, Union Hospital, Tongji Medical College, cer cells surfaces, which binds the programmed death-1 Huazhong University of Science and Technology, Wuhan 430022, China protein (PD-1) receptor to induce T cell apoptosis and Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fan et al. Journal of Experimental & Clinical Cancer Research (2019) 38:47 Page 2 of 12 inhibit cytotoxic T-cell activation within cancer tissues Plasmids, antibodies and chemicals [6–9]. Given that the blockade of the PD-1/PD-L1 inter- Mammalian expression vectors for Flag-HAT1 recom- action can reactivate T-cell responses, a few anti-PD-1 binant proteins were generated using the pcDNA3.1 and anti-PD-L1 antibodies have been approved for the backbone vector. The HAT1 antibody (ab194296) was treatment of human cancers in the clinic [10]. However, purchased from Abcam (working dilution 1:2000); monotherapy with PD-L1 inhibitors for pancreatic can- beta-tubulin (2128S) was from Cell Signaling Technol- cer has resulted in disappointing outcomes in clinical ogy - (working dilution 1:5000); BRD4 (ab128874) was trials [11]. A growing body of evidence suggests that the from Abcam (working dilution 1:1000); PD-L1 (13684S) expression level of PD-L1 in cancer cells is highly associ- was from Cell Signaling Technology (working dilution ated with the response to immune checkpoint therapies 1:1000); and H4K5ac (ab17343) was from Abcam (work- [12]. Thus, exploring the regulatory mechanism of PD- ing dilution 1:1000). Ascorbate was purchased from L1 in cancer cells, especially in pancreatic cancer cells, is Sigma-Aldrich (Shanghai, China). one of the key strategies to improve cancer patient response to PD-L1 blockade therapy. Western blot of cells and tissue specimens Histone acetyltransferase 1 (HAT1) is a classic type B The ethics of using human tissue (12 pairs of matched histone acetyltransferase, and it can only acetylate newly pancreatic cancer/adjacent noncancerous tissues) was synthesized histone H4 and not nucleosomal histone approved by the local ethics committee (Tongji Medical [13].HAT1 was the first histone acetyltransferase identi- College, China), and written informed consent was ob- fied and is one of the most poorly understood members tained from patients prior to surgery exactly as described of this family [13]. HAT1 is overexpressed in multiple previously [18]. The cells or the tissue specimens were types of solid tumors, including esophageal [14], lung lysed with lysis buffer (Beyotime, China) containing 1% cancer [15] and liver cancer [16], and acts as an onco- protease and phosphatase inhibitors. The protein protein to promote tumorigenesis. It has been reported concentration was determined with a protein assay kit that HAT1 functions as a transcription factor to regulate (Pierce Biotechnology, USA). Equal amounts of protein the expression of various genes, such as Bcl2L12 [17] for each sample were separated using SDS-PAGE gels and Fas [15], and modulates cancer cell proliferation and transferred onto PVDF membranes (Pierce Biotech- [16], apoptosis [15] and metabolism [16]. nology, USA). The membranes were subsequently To date, the biological effect and clinical relevance of blocked in 5% not-fat milk for 1 h at room temperature, HAT1 in pancreatic cancer is poorly understood. In this followed by incubation with primary antibody overnight study, we sought to determine the specific role of HAT1 at 4 °C. The membranes were then washed with 1x in pancreatic cancer. First of all, we demonstrated that TBST and incubated with a secondary antibody for 1 h. HAT1 was overexpressed in pancreatic cancer and Finally, the membranes were treated with ECL detection linked with poor prognosis in PDAC patients. Then, our reagents and exposed to X-ray films. data showed that HAT1 acted as a tumor growth pro- moting protein in pancreatic cancer cells. Strikingly, Real-time RT-PCR HAT1 was involved in the cancer immunity response by Total RNA was extracted from the cells using Trizol regulating the PD-L1 expression, and this process was reagent (Thermo Fisher Scientific, USA). First strand mainly mediated by BRD4. Taken together, our results cDNA was synthesized from 2 μg RNA using a cDNA demonstrate that aberrant expression of HAT1 promotes Reverse Transcription kit (PrimeScript™ RT reagent Kit, tumorigenesis by modulating the cancer cell growth and Code No. RR037A), and real-time PCR analysis was the immune response in pancreatic cancer. 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 Materials and methods Inc. (Shigo, Japan). All the values were normalized to Cell culture actin, and the 2-ΔCt method was used to quantify the All pancreatic cancer cell lines including PANC-1, fold change. The primers used for RT-qPCR are pro- BxPC-3 and MIA PaCa-2 were purchased from the vided in Additional file 1: Table S1. Chinese Academy of Science Cell Bank, and the Panc 02 cells were obtained from Tong Pai Technology (Shang- Chromatin immunoprecipitation (ChIP) and ChIP-qPCR hai, China). These cell lines were cultured in Dulbecco’s ChIP was performed following the manufacturer’s Modified Eagle Medium (DMEM) medium (Invitrogen, instructions for the Chromatin Extraction Kit (Abcam, USA) supplemented with 10% fetal bovine serum (FBS) ab117152, USA) and ChIP Kit Magnetic - One Step (HyClone, USA). All cell lines were routinely maintained (Abcam, ab156907, USA) [19]. BRD4 (Cell Signaling at 37 °C in a 5% CO2 incubator. Technology, 13,440, dilution 1:50) was used for the ChIP Fan et al. Journal of Experimental & Clinical Cancer Research (2019) 38:47 Page 3 of 12 assay. The purified DNA was analyzed by real-time PCR 96-well plates with 100 μl of culture medium. The cells with a PCR kit (Takara Bio Inc., Japan) according to the were treated with serial concentrations of small molecular manufacturer’sprotocols[20]. The primers for ChIP-qPCR inhibitors. After 72 h, 20 μl of MTS reagent (Abcam, are provided in Additional file 1:TableS2. USA) was added to each well of the cells and incubated for 1 h at 37 °C in standard culture conditions. The ab- Tissue microarray and immunohistochemistry (IHC) sorbance was measured in a microplate reader at 490 nm.