Materials

Dulbecco’s modified minimum essential medium (DMEM) and fetal bovine serum (FBS) were purchased from Sigma (St. Louis, MO). RT-qPCR, ChIP-qPCR primers were synthesized at Thermo Fisher Scientific (Waltham, MA) and are listed in Supplementary Table 1 and Supplementary Table 2. The pMSCVpuro-KAT2B vector (#63,705), pMSCVpuro empty control vector (#12,570), pGEX-GST-SP1 (#27,264) and pGEX-GST (#129,572) were purchased from Addgeene (Cambridge, MA). The siRNA duplexes were synthesized at Integrated DNA Technologies (Coraville, IA). The sequences of siRNAs are listed in Supplementary Table 3. Lipofectamine™ 3000 reagent, glutamine and antibiotics were obtained from Invitrogen (Carlsbad, CA). RIPA Lysis Buffer (#89,901), BL21(DE3) Competent Cells (#EC0114), Subcellular Protein Fractionation Kit (#78,840), Pierce™ GST Protein Interaction Pull-Down Kit (#21,516) and Isopropyl β-D-1-thiogalactopyranoside (IPTG) Solution (#R1171) were purchased from Thermo Fisher Scientific (Waltham, MA). The EpiQuik Nuclear Extraction Kit (#OP-0002-1) was purchased from Epigentek (Farmingdale, NY). Recombinant KAT2B-Flag protein was purchased from Active Motif (Carlsbad, CA). Rabbit polyclonal antibodies against KAT2B, GST, YAP and NF2 were purchased from Proteintech (Rosemont, IL). Rabbit polyclonal antibody against NFYB was purchased from GeneTex (Irvine, CA). Rabbit monoclonal antibodies against KAT2B, YY1, Flag and mouse monoclonal antibodies against Ki67/α-Tubulin were purchased from Cell Signaling Technology (Danvers, MA). Rabbit monoclonal antibody against SP1 and Rabbit polyclonal antibodies against NFYA/NFYC were purchased from ABclonal (Woburn, MA). Mouse monoclonal antibody against β-actin was purchased from Sigma (St. Louis, MO). IRDye goat anti-mouse/rabbit IgG secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE).

Cell culture and transfections

Human CCA cell lines (SG231, HuCCT1) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Sigma-Aldrich) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) in a humidified 5% CO2 incubator at 37 °C.

For transfections, NF2 and SP1 siRNAs and their respective scramble controls were transfected into CCA cells using Lipofectamine™ 3000 reagent. Following transfections, the cells were analyzed for proliferation and other parameters as described in the manuscript.

For establishment of CCA cells with stable overexpression of KAT2B, HuCCT1 and SG231 cells were transfected with pMSCV-KAT2B vector or empty control vector. After 48 h of transfection, the cells were cultured in DMEM medium containing 1 µg/ml Puromycin (Calbiochem, San Diego, CA). The selection medium was replaced every 3 days for the next 4 weeks. Subsequently, distinct colonies of surviving cells were transferred onto 6-well plates and the cultures were maintained under the same selection medium. Following transfections, the cells were analyzed for proliferation, invasion, and specific protein levels.

Cell proliferation WST-1 assay

Cell proliferation WST-1 assay was performed according to the manufacturer’s instruction. For cells with overexpression of KAT2B, 2 × 103 cells were seeded onto each well of 96-well plates and cultured for 5 days. For NF2 siRNA transfection, 2 × 103 cells with stable overexpression of KAT2B were seeded onto each well of 96-well plates and the cells were transfected with siRNAs in the presence of Lipofectamine™ 2000 reagent for 4 h; the cells were then continued cultured in fresh DMEM medium for additional 5 days. To determine cell proliferation, 10 µl WST-1 reagent was added to each well and the cells were incubated for 1 h at 37 °C and 5% CO2. A450 nm was measured using an automatic ELISA plate reader.

Clonogenicity assay

For anchorage-dependent colony formation assay, CCA cells transfected with or without KAT2B overexpression were seeded onto conventional 6-well plates (500 cells/ well). After culture for 14 days, the cells were fixed with methanol and stained with 0.1% crystal violet. For anchorage-independent colony formation assay, cells were cultured in ultra-low attachment plates (Corning, NY) as previously described [19,20,21]. 1 × 103 SG231cells/well or 2 × 103 HuCCT1 cells/well were seeded onto 24-well ultra-low attachment plates in DMEM medium containing 1% FBS (for SG231 cells) or 5% FBS (for HuCCT1 cells). Following culture for 10 days (for SG231) or 14 days (for HuCCT1), the cell spheroids were imaged by using an inverted Olympus microscope.

Protein extraction and Western blotting

For whole cell protein extraction, the cells were washed twice with ice-cold PBS and lysed in RIPA buffer. After sonication on ice, the cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were collected for Western blotting. For nuclear protein extraction, we followed the manufacture’s instruction using EpiQuik Nuclear Extraction Kit. Briefly, the cells were scraped with 1× buffer NE1 (plus Protease Inhibitor Cocktail (PIC)) and left on ice for 10 min and centrifuged for 10 min at 12,000 rpm (4 °C). The pellets were then washed with ice-cold PBS and resuspended in 1× buffer NE2 (containing DTT and PIC). The samples were then homogenized and left on ice for 15 min with vortex (5 s) every 3 min. After centrifugation at 14,000 rpm for 10 min, the supernatants were collected as nuclear proteins. The insoluble chromatin fraction was extracted according to the manufacturer’s instructions provided by Subcellular Protein Fractionation Kit (Thermo Fisher Scientific, Waltham, MA). The protein concentrations were measured using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA).

For Western blotting analysis, samples were boiled for 5 min in protein loading buffer with 2-mercaptoethanol and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto the nitrocellulose membrane (BioRad). Non-specific binding was blocked by incubating the membranes in PBST (0.1% Tween 20 in PBS) containing 5% nonfat milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with individual primary antibodies at the dilutions recommended by the manufacturers in PBST containing 5% nonfat milk. Following four washes with PBST, the membranes were incubated with the IRDye secondary antibodies at 1: 5000 dilutions in PBST containing 5% nonfat milk for 1 h at room temperature. After four washes with PBST, the ODYSSEY Infrared Imaging System (Licor, Lincoln, NE) was used to visualize protein bands.

Real-time quantitative PCR (qRT-PCR)

Total RNA was extracted using Tri-zol Reagents (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Reverse transcription was performed with iScript Supermix (Bio-Rad, Hercules, CA). Quantitative PCR was performed with the Bio-Rad SYBR® Green Supermix in a C1000 thermal cycler (Bio-Rad). The PCR conditions were 15 min at 95 °C, followed by 35 cycles of 15s at 94 °C, 30s at 55 °C and 30s at 72 °C, and finally 10 min at 72 °C. The PCR primer sequences are listed in Supplementary Table 1. β-actin was used as the internal control. Results were analyzed by using CFX Manager Software version 3.1 (Bio-Rad). The expression level of mRNA was normalized to the internal control gene and relative change was calculated by using the 2−ΔΔCT method.

Chromatin immunoprecipitation (ChIP)

Cells were cross-linked by 1% formaldehyde for 10 min. Chromosome DNA was extracted according to the manufacturer’s instructions provided by SimpleChIP ® Plus Enzymatic Chromatin IP Kit (Cell signaling, Danvers, MA) and precipitated by using specific anti-KAT2B rabbit antibody or anti-SP1 rabbit antibody. Rabbit IgG was used as negative control. The PCR conditions were 15 min at 95 °C, followed by 35 cycles of 15s at 94 °C, 30s at 55 °C and 30s at 72 °C, and then 10 min at 72 °C. The ChIP-PCR primer sequences are listed in Supplementary Table 2.

Protein immunoprecipitation

CCA cells (1 × 107) were lysed in 1 mL Pierce IP Lysis Buffer (Thermo Fisher Scientific) containing phosphatase and protease inhibitors. Then, 500 µl cell lysates were used for immunoprecipitation with specific antibodies. In brief, cell lysate were incubated with 10 µg antibodies by rotation at 4 °C overnight and then with the addition of 50 µL PureProteome Protein A/G Mix Magnetic Beads (MilliporeSigma #LSKMAGAG10) at 4 °C for 4 more hours. The samples were collected by magnetic stand, followed by washing five times with a beads wash solution (50 mmol/L Tris-HCl [pH 7.6], 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.1% NP-40) and washing three times with phosphate-buffered saline. After boiling for 5 min in protein loading buffer, the samples were used for Western blotting analysis with specific antibodies.

GST pull-down assay

To produce glutathione S-transferase (GST) and GST-SP1 fusion protein, pGEX-GST control vector and pGEX-GST-SP1 were transfected into BL21 (DE3) competent cells respectively. Protein expression was induced by 0.5mM ispropylb-D-1-thiogalactopyranoside (IPTG). GST pull-down assays were performed according to the manufacturer’s protocol provided by Pierce™ GST Protein Interaction Pull-Down Kit (Thermo Scientific, Waltham, MA). Briefly, the recombinant GST-SP1 or GST protein was incubated with Glutathione Agarose at 4 °C for 2 h. To minimize the possibility of nonspecific binding, we increased the washing process to 8 times using the wash buffer recommended by the manufacturer’s instructions (1:1 wash solution of TBS: Pull-Down Lysis Buffer). After washing eight times, the Glutathione Agarose was incubated with FLAG-tagged KAT2B recombinant protein (Active Motif, Carlsbad, CA) at 4 °C for 2 h, followed by washing for additional eight times with the above-indicated wash buffer. The samples were then analyzed by Western blotting.

Immunohistochemistry (IHC)

The CCA tumor tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections of 4 μm thickness were deparaffinized and processed for hematoxylin and eosin (H&E) staining and immunohistochemistry. Primary antibodies against KAT2B, YAP, NF2, or Ki67 were diluted in 1× PBS containing 4% horse serum, 0.4 mg/ml methiolate and 0.2% Triton-X100. After blocking with Peroxidazed 1 (Biocare Medical, Pike Lane Concord, CA) for 5 min, the slides were incubated with primary antibodies at room temperature for 1 h. The slides were then washed with TBS and incubated with horseradish peroxidase-conjugated second antibody at room temperature for 1 h. After washing with TBS, the slides were incubated for 5 min at room temperature with 3,3′-diaminobenzidine (DAB) for chromogenic development.

RNA-sequencing (RNA‐seq)

Total RNA was extracted from HuCCT1 cells using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. Library preparation, high-throughput sequencing and data analysis were done by LC Sciences (Houston, TX). Briefly, RNA sequencing library was prepared following Illumina’s TruSeq-stranded-mRNA sample preparation protocol. RNA integrity was checked with Agilent Technologies 2100 Bioanalyzer. Poly(A) tail-containing mRNAs were purified using oligo-(dT) magnetic beads with two rounds of purification. After purification, poly(A) RNA was fragmented using divalent cation buffer in elevated temperature, followed by DNA library construction. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illumina’s NovaSeq 6000 sequencing system.

Bioinformatics analyses

The gene expression profiles of human CCA and non-cancerous liver tissues were downloaded from the GEO datasets (GSE26566, GSE107943, GSE76297, GSE119336) and TCGA (The Cancer Genome Atlas) database. GSE26566 consisted of 104 CCA samples and 59 non-tumor liver samples. GSE107943 consisted of 30 CCA samples and 27 non-CCA samples. GSE76297 consisted of 90 CCA samples and 90 paired non-CCA samples. GSE119336 consisted of 15 CCA samples and 15 paired non-CCA samples. TCGA database consisted of 36 CCA samples and 9 non-CCA samples. Analysis of the TCGA-CHOL survival data was performed by using the online tool GEPIA (http://gepia.cancer-pku.cn/detail.php) with a standard processing pipeline [22]. The correlation between KAT2B expression and the overall survival of CCA patients was further assessed by using GSE244807 dataset.

To compare KAT2B expression levels between CCA and bile duct, we first analyzed the GSE32225 dataset which includes 104 CCA versus 6 normal bile duct samples. In addition, we analyzed the GSE32225 dataset which includes 149 CCA tissue samples versus 6 cases of benign biliary epithelia. For KAT2B gene expression analysis in cultured biliary epithelial and cancer cells from the GSE77984 dataset, the analysis includes 8 normal human biliary epithelial cell samples (NHC2#1–4, N_shC#1–4) and 6 CCA cell samples (EGI#2–4, E_pWPI#1–3). For KAT2B gene expression analysis in cultured biliary epithelial and cancer cells from the GSE144521 dataset, the analysis includes 6 normal human biliary epithelial cell samples (H69WCE_1–3, NHCWCE_1–3) and 6 CCA cell samples (EGIWCE_1–3, TFKWCE_1–3).

CCA xenograft studies in SCID mice

The athymic nude NOD CB17-Prkdc/SCID mice were obtained from Jackson Laboratory (Bar Harbor, ME). For all animal studies, the procedures were carried out in strict accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The handling of the mice and all experimental procedures were approved for this study by the Institutional Animal Care and Use Committee of Tulane University (Protocol #: 4159). To develop CCA xenografts with KAT2B overexpression, 1 × 106 SG231 or HuCCT1 cells stably transfected with the pMSCV-KAT2B vector or control vector were inoculated into the left or right flank areas of the SCID mice. The animals were then monitored for tumor growth. After 7 weeks of inoculation, the mice were sacrificed and the tumors were collected.

Statistics

Results are presented as mean ± standard error (SE) from a minimum of 3 replicates. Difference between groups was evaluated by SPSS 13.0 statistical software with one-way analysis of variance (ANOVA), Student’s t-test, and Log-rank test. Statistical graphs were plotted by GraphPad Prism 7.0 software and SigmaPlot statistical software. P value < 0.05 was considered as statistically significant.