HDAC6 promotes cell proliferation and confers

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Logan, uT, uSA) supplemented with 10% fetal bovine serum. (FBS) and 1% ..... problem that continues to puzzle clinicians since the majority of patients who ...
ONCOLOGY REPORTS 36: 589-597, 2016

HDAC6 promotes cell proliferation and confers resistance to gefitinib in lung adenocarcinoma Zhihao Wang1,2, Fang Tang1,2, Pengchao Hu3, Ying Wang3, Jun Gong1,2, Shaoxing Sun1,2 and Conghua Xie1,2 1

Department of Radiation and Medical Oncology, and 2Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University; 3Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Wuhan University, Wuchang, Wuhan 430071, P.R. China Received January 15, 2016; Accepted March 3, 2016 DOI: 10.3892/or.2016.4811

Abstract. Histone deacetylases (HDACs) are promising targets for cancer therapy, and first-generation HDAC inhibitors are currently in clinical trials for the treatment of cancer patients. HDAC6, which is a key regulator of many signaling pathways that are linked to cancer, has recently emerged as an attractive target for the treatment of cancer. In the present study, HDAC6 was found to be overexpressed in lung adenocarcinoma cell lines and was negatively correlated with the prognosis of patients with lung adenocarcinoma. Overexpression of HDAC6 promoted the proliferation of lung adenocarcinoma cells in a deacetylase activity-dependent manner. HDAC6 overexpression conferred resistance to gefitinib via the stabilization of epidermal growth factor receptor (EGFR). The inhibition of HDAC6 by CAY10603, a potent and selective inhibitor of HDAC6, inhibited the proliferation of lung adenocarcinoma cells and induced apoptosis. CAY10603 downregulated the levels of EGFR protein, which in turn inhibited activation of the EGFR signaling pathway. Moreover, CAY10603 synergized with gefitinib to induce apoptosis of the lung adenocarcinoma cell lines via the destabilization of EGFR. Taken together, our results suggest that the inhibition of HDAC6 may be a promising strategy for the treatment of lung adenocarcinoma.

Correspondence to: Dr Conghua Xie, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang, Wuhan 430071, P.R. China E-mail: [email protected] Abbreviations: SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; HDACs, histone deacetylases; EGFR, epidermal growth factor receptor; TKIs, tyrosine kinase inhibitors; ERK, extracellular signal-regulated kinase; Hsp90, heat shock protein 90; PARP, poly(ADP-ribose) polymerase; SIRT, silent mating type information regulation 2 homolog Key words: HDAC6, lung adenocarcinoma, EGFR, CAY10603, gefitinib

Introduction Lung cancer is the leading cause of cancer-related death worldwide and accounts for more than one million deaths per year (1). The morbidity of lung cancer has continued to increase worldwide, particularly in developing countries, which is partly due to air pollution. Lung cancer can be divided into two histological types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC can be further subdivided into: lung adenocarcinoma, large cell carcinoma and squamous cell carcinoma. These cancer subtypes have distinct morphologies and molecular profiles, and arise from distinct locations within the lung. Approximately 40% of lung cancers are adenocarcinomas. Despite advances in the diagnosis and treatment of lung adenocarcinoma, the 5-year overall survival rate of patients with lung adenocarcinoma is still extremely low (2). In recent years, great achievements have been made in our understanding of lung adenocarcinoma driver gene mutations and rearrangements, which play critical roles in tumor development and progression (3). Among them, somatic mutations in the gene that encodes the epidermal growth factor receptor (EGFR) and rearrangements that involve the gene that encodes anaplastic lymphoma kinase (ALK), are the most well-characterized examples (4,5). Large, prospective randomized trials have shown that molecularly selected patients with advanced lung adenocarcinoma can benefit from treatment with EGFR and ALK tyrosine kinase inhibitors (TKIs). However, despite the success of such agents in the treatment of genetically defined subsets of lung cancer, mutations such as those in EGFR and rearrangements within the ALK gene are identified in only a limited number of patients (6). Moreover, the majority of patients who initially respond to EGFR-TKI therapy develop acquired resistance within 6-12 months (5). Therefore, further research is needed to identify new therapeutic targets and tools for the treatment of lung adenocarcinoma. Histone deacetylases (HDACs) are enzymes that modulate the acetylation status of histones and other important cellular proteins (7). Eighteen HDACs have been identified in humans, and these enzymes are divided into four classes, as follows, based on sequence phylogeny and function: class I (HDAC1, 2, 3 and 8), class II (HDAC4, 5, 6, 7, 9 and 10), class III

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wang et al: HDAC6 EXERTS ONCOGENIC ACTIVITY IN LUNG ADENOCARCINOMA

(SIRT1-7) and class IV (HDAC11) (8). HDACs have been recognized as potentially useful therapeutic targets for a broad range of human disorders including cancer (8,9). HDACs are considered to be among the most promising targets in drug development for cancer therapy. Two HDAC inhibitors, SAHA (vorinostat) and romidepsin (FK228), have been approved by the US Food and Drug Administration for the treatment of cutaneous T cell lymphoma (10,11). However, these inhibitors elicit profound side-effects since they target several HDAC isoforms (12,13). Isoform-selective HDAC inhibitors may offer a therapeutic advantage due to minimal toxicity. Among the 18 HDACs, HDAC6 has recently sparked great interest due to its functional role in tumor progression. HDAC6 is a key regulator of many signaling pathways that have been linked to cancer (14,15). A diverse set of HDAC6 substrates is involved in tumorigenesis, such as Hsp90, α-tubulin and cortactin. Deacetylation of the Hsp90 core component by HDAC6 activates the chaperone activity of Hsp90 and stabilizes Hsp90‑associated molecules including EGFR, Akt, c-Raf, FLT3 and mutant p53 (16). HDAC6 can promote cell migration through the deacetylation of α-tubulin (17). HDAC6 has been shown to regulate the cell cycle, apoptosis, and metastasis, among other cellular processes (18,19). However, unlike the inhibition of other HDACs, the inhibition of HDAC6 is not believed to be associated with severe toxicity, which makes HDAC6 a possible target for the treatment of cancer. However, few studies have been conducted in regards to the role of HDAC6 in lung adenocarcinoma. In the present study, HDAC6 was found to be overexpressed in lung adenocarcinoma cell lines, and the overexpression of HDAC6 promoted lung adenocarcinoma proliferation and conferred resistance to gefitinib, a widely used EGFR-TKI, in a deacetylase activity-dependent manner. The inhibition of the deacetylase activity of HDAC6 by CAY10603 (20), a potent and selective HDAC6 inhibitor, inhibited the proliferation and induced th apoptosis of lung adenocarcinoma cell lines. CAY10603 synergized with gefitinib to induce the apoptosis of lung adenocarcinoma cells via the destabilization of EGFR. Our results suggest that inhibition of HDAC6 may be a promising strategy for the treatment of lung adenocarcinoma. Materials and methods Cell culture. The human lung adenocarcinoma cell lines A549, HCC827 and H1975 were obtained directly from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cell lines were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Antibodies and reagents. CAY10603 and gefitinib were purchased from Selleck (Houston, TX, USA). The antiPARP (9542) and anti-BID (2002) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti‑caspase 3 (EAP0893) antibody was purchased from Elabscience (Wuhan, Hubei, China). The anti-p53 (ab179477), anti-Bax (ab32503), anti-EGFR (ab52894) and anti-p-ERK (ab76299) antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-HDAC6 (12834-1-AP), Bcl2 (12789-1-AP), Bcl-xL (10783-1-AP), Mcl1 (16225-1-AP),

p21 (10355-1-AP) and ERK1/2 (16443-1-AP) antibodies were obtained from Proteintech (Chicago, IL, USA). The anti-β actin mouse monoclonal antibody was purchased from Abgent (San Diego, CA, USA). Cell proliferation and colony formation assays. In regards to the cell proliferation assay, cells that were cultured in 96-well plates were treated with the indicated compounds, and cell proliferation was measured at the indicated times by the Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) assay. In regards to the colony formation assay, cells were seeded at 1,000/well into 6-well plates and were cultured over a 14-day period. Colonies were fixed in 4% paraformaldehyde, washed with H2O and stained with 0.1% crystal violet. Annexin V assay of cell apoptosis. Cells were cultured in 6-well plates and were treated with the indicated compounds, trypsinized and collected. The collected cells were washed with PBS, resuspended in binding buffer, and stained with Annexin V-PE and 7-AAD for 15 min according to the manufacturer's protocol from Becton-Dickinson (San Jose, CA, USA). Fluorescence was estimated with a Becton-Dickinson flow cytometer. Plasmids and transfection. The plasmids that express wild‑type HDAC6 (HDAC6 WT) and deacetylase-deficient HDAC6 (HDAC6 MT) were kindly provided by Professor Jun Zhou of Nankai University (21,22). The plasmids were transfected into cells with TurboFect DNA transfection reagent (Thermo Scientific, Waltham, MA, USA). Control siRNA and siRNAs targeting human HDAC6 were purchased from RiboBio (Guangzhou, China). siRNA was transfected into cells using Lipofectamine RNAiMAX from Invitrogen (Waltham, MA, USA). Western blotting. Cells were lysed in lysis buffer (Beyotime, Jiangsu, China). The samples were electrophoresed by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Roche). The membranes were blocked with 5% dried skim milk and then incubated with the indicated primary antibody followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech). Finally, the bands were visualized with WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA) and developed with Kodak film. Statistical analysis. Statistical analysis was performed using the Student's t-test. Data are expressed as the mean ± standard deviation (SD). P