Cell models of glucose-stimulated proliferation
An established model of glucose-stimulated proliferation was utilized as previously described [22, 23, 46]. Briefly, in the presence of serum, cells were incubated in glucose-free medium for 3–8 hours to deplete glucose and then in serum-free medium with or without glucose for 1–4 hours. WST1 was used to monitor cell viability/proliferation. BrdU or EdU were used to label glucose-induced DNA synthesis. BrdU/EdU-DNA levels were analyzed using ELISA or fluorescence flow cytometry. CRISPR-Cas9 gene editing systems were used to interrupt the FAK gene. Standard molecular biological techniques were used to make WT/mutant FAK constructs. All mutations such as FAKH58A/E were verified by sequencing. Cells expressing WT/mutant (His58A/E) FAK were obtained using flow cytometry sorting, G418 selection and colony expansion.
Cell Line, Cell Models And Reagents
The Cellosaurus database ExPASy Bioinformatics Resource Portal was searched for RRID numbers to verify the authenticity of each cell line. Human esophageal squamous cell carcinoma, KYSE70 (RRID: CVCL_1356), KYSE180 (RRID: CVCL_1349), KYSE520 (RRID: CVCL_1355), and TE10 (RRID: CVCL_1760) were originally obtained from Takayoshi Tobe lab (KYSE) and the Takashi SASAKI lab (TE). Human esophageal epithelial cell line (Het-1A, RRID: CVCL_3702, ATCC# CRL-2692) was purchased from ATCC. Esophageal adenocarcinoma cell lines (FLO1, RRID: CVCL_2045, SK-GT-4, RRID: CVCL_2195, and KYAE1, RRID: CVCL_1825) were purchased from Sigma-Aldrich. Cancer cell lines were maintained in recommended medium in the presence of 10% FBS and 1% antibiotics: KYSE70/KYSE520/TE10, RPMI-1640; KYAE1, F-12K + RPMI1640; SK-GT-4, RPMI-1640; and FLO1, DMEM. The human esophageal epithelial cell line was cultured in BEGM.
Antibodies: Anti-N3-pHis (Millipore, MABS1352), anti-pY397-FAK (Cell Signaling, #3283), anti-FAK (Cell Signaling, #3285), anti-FAK (Millipore, 05-537), anti-NME1 (Cell Signaling, #3345), anti-NME2 (Santa Cruz Biotechnology, SC-100400), anti-HDAC2 (Cell Signaling, #57156), anti-PPP2R2A (Abcam, ab18136), anti-RB1 (Cell Signaling, #9309), anti-phospho-RB1 (S780) (Cell Signaling, #8180), anti-E2F (Cell Signaling, #3742), anti-CDK2 (Cell Signaling, #2546), anti-β-actin (Sigma, A5441), and anti-GAPDH (Cell Signaling, #2118).
WT/H118F NME1, PPP2R2A, WT/H58E/H58A FAK and WT/RB1 site-mutated FAK: All mutations such as FAKH58A/E were verified by sequencing. Cells expressing WT/mutant (His58A/E) FAK were obtained by using flow cytometry sorting, G418 selection, and colony expansion.
NME1, PPP2R2A, and control siRNAs were purchased from Santo Cruz Biotechnology. CRISPR-Cas9 gene editing system [1 × sgRNA/Cas9 all-in-one expression clone targeting NME1 (NM_001018136.2), HCP309878-SG01-3] was purchased from GeneCopoeia. Lipofectamine® 3000 Transfection Reagent was purchased from Life Sciences. Chemicals were purchased from Sigma-Aldrich.
Site-directed Mutagenesis And Overexpression
GeneArt® Site-Directed Mutagenesis System (Invitrogen) was used for construction of pEGFP-FAK-H58A/E and pEGFP-FAK-C173A-E175A (171LxCxE → 171LxAxA): Mutagenesis primers for H58A: forward, 5’-C AGT ATT ATC AGG GCA GGA GAT GCT ACT GAT G and reverse, 5’-C ATC AGT AGC ATC TCC TGC CCT GAT AAT ACT G; for H58E: forward, 5’-C AGT ATT ATC AGG GAG GGA GAT GCT ACT GAT G and reverse, 5’-C ATC AGT AGC ATC TCC CTC CCT GAT AAT ACT G; for FAK-C173A-E175A: forward, 5’- GCT TTG AAG TTG GGT GCT TTG GCA ATT AGG CGA TC and reverse, 5’- GAT CGC CTA ATT GCC AAA GCA CCC AAC TTC AAA GC (the underlined triplets are those that are mutated). The mutation was verified by Sanger sequencing. Cells were transfected with the constructs using Lipofectamine® 3000 Transfection Reagent. G418 selection, fluorescence flow cytometry sorting and colony expansion were performed as previously described [22, 23].
Assessments Of Glucose-induced Dna Replication
BrdU ELISA and EdU coupling flow cytometry were utilized to analyze DNA synthesis as previously described [22, 23]. Briefly, cells were incubated in glucose-free medium containing 5% FBS for 3–8 hr, and then in FBS-free medium with or without glucose in the presence of BrdU for 1 hr or EdU for 4 hr. BrdU-DNA and EdU-DNA were determined using the Cell Proliferation ELISA (BrdU, colorimetric, Roche) and Click-iT® Plus EdU Pacific Blue™ Flow Cytometry Assay Kit (Life Science), respectively. Cells were trypsinized, mixed with trypan blue and counted on a TC-20 cell counter to examine the cell viability (Bio-Rad).
Tcga Database Search And Analysis
Esophageal cancer cohort of TCGA consisted of 183 patients (95 ESCC + 88 EAC). mRNA expression z-scores from RNA-seq were downloaded via cBioPortal Center (http://www.cbioportal.org/). Statistical analyses were performed using R software (http:///www.r-project.org/) and Bioconductor (http://bioconductor.org/). GSEA was performed on TCGA cohort using software provided by the Broad Institute (http://software.broadinstitute.org/gsea/index.jsp), as previously described . Patients were classified into two groups: ESCC and EAC.
Elisa Of Phis-fak
The protocol for analysis of pHis-FAK (FAKpHis) levels was modified since pHis is heat and acid labile. The wells were coated with an anti-pHis-N3 antibody  (0.98 ng/µl, rabbit, MABS1352, Millipore) in 50 µl of coating buffer (PBS, pH7.4) or coating buffer alone at 4oC overnight. After the wells were washed with washing buffer (PBS, pH 7.4, 0.03% Tween-20, 200 µl/well) for 3 times, 200 µl of 10% dry milk in washing buffer was added to the wells and kept at room temperature (R/T) for 2 hr. Then, the wells were rinsed with washing buffer X2. To detect FAKpHis, the reaction mixture containing Na2HPO4/NaH2PO4 buffer (20 mM, pH 7.6), NaCl (50 mM), MgCl2 (5 mM), EDTA (1 mM), DTT (2 mM), PEP (20 mM), and purified recombinant FAK (0.26 ng/µl, Cat#: PV3832, Invitrogen) was added to the pHis antibody-coated wells and kept at R/T for 5 hr. For the assessment of cellular FAKpHis levels, 50 µl of cell lysates (0.17 ug/µl) were added to the anti-pHis antibody-coated wells. The wells were washed with washing buffer X3 to remove any unbound materials. The first antibody [FAK4.47, mouse, 1:1000 in 50 µl of antibody buffer (PBS, pH 7.4, 0.03% Tween-20, 1% BSA)] was added to the wells and kept at 4oC overnight. After washing 3X, an anti-mouse-IgG-HRP (1:1000 in 50 µl of antibody buffer) was added to the wells and kept at R/T for 2hr. After washing 3X, the substrate TMB (50 µl /well) was used and detected at 650 nm wavelength on a microplate reader.
Western Blot Analysis And Immunoprecipitation (Ip)
Cell lysates and SDS PAGE gels were prepared as previously described . Modified protocols of Western blot analysis for the detection of pHis protein levels were utilized. The modification includes omitting the step of sample heating, using alkaline buffer systems, and blocking members with dry milk.
IP was carried out as previously described . Briefly, lysates derived from control or glucose-stimulated KYSE70 cells were pre-cleared using Agarose-IgG and then incubated with agarose-conjugated anti-FAK-AC antibody. After washing, the beads (immunoprecipitates) were directly loaded to a SDS-PAGE gel without heating to preserve pHis. After detection of FAKpHis using an anti-pHis antibody, the same membrane was stripped and then reprobed with the anti-FAK antibody.
To overcome the labile nature of the pHis phosphoramidate moiety, we utilized the reported protocols that have been established for pHis detection [22, 23]. Phosphorylated human FAK (PV3832, Invitrogen) or pHis antibody-immunoprecipitated pHis protein derived from ESCC cells was digested with trypsin as previously described [49–51]. The protein samples were processed using a surfactant-aided-precipitation/on-pellet digestion (SOD) procedure, which provides extensive cleanup to remove detergents and non-protein matrix components, deep protein denaturation (by both surfactants and precipitation) for rapid, efficient, and reproducible digestion, and thereby achieves reliable quantification of samples. Briefly, 100 µg protein was reduced with 10 mM DTT with incubation at 37°C for 30 min in Eppendorf Thermomixer (Eppendorf, Hauppauge, NY), and cysteine residues were alkylated with 20 mM iodoacetamide (IAM) at 37°C for 30 min in the dark. For protein precipitation, one volume of chilled acetone (-20°C) was gently added into each sample and mixed for 1 min to obtain a cloudy suspension. Then, another 8 volumes of chilled acetone were added to the mixture to precipitate proteins. The solution was vortexed until it became clear and stored at -20°C overnight to allow complete precipitation. Subsequently, samples were centrifuged at 20,000 g for 30 min at 4°C to obtain a protein pellet. After removing the supernatant, 500 µl chilled acetone/water mixture (85/15, v/v %) was added to wash the pellet. Samples were centrifuged for 3–5 min, acetone/water supernatant was discarded, and the sample was allowed to air-dry. The system was kept under pH = 8.5 all the time to prevent potential acid catalysis of pHis degradation.
For protein digestion, the pellet was dissolved in 100 µl Tris (pH = 8.5) buffer and sonicated in a water bath at 37°C. Then, 80 µl Tris buffer was added to 20 µg enzyme powder (Sigma) on ice for activation. The digestion procedure composed 2 steps: 1) activated trypsin was added to the samples at a ratio of 1:40 (enzyme: substrate) and incubated for 6 h at 37°C in an Eppendorf Thermomixer; and 2) a second aliquot of trypsin solution with equal volume was added to the samples and incubated overnight. After centrifugation at 20,000g for 20 min at 4°C, 2/3 of the digestion solution was carefully transferred into a tube for LC-MS analysis.
The peptide fragments were subjected to HCD MS/MS analysis as previously described [52–54]. The nano-flow reverse phase LC included a Spark Endurance autosampler (Emmen, Holland) and an ultrahigh-pressure Dionex ultimate Nano-2D Ultra capillary/nano-LC system. Peptide separation employed a long nano-LC column (75-µm ID × 100 cm) with Pepmap 3 µm C18 particles. A large-ID trap (300 µm ID × 1 cm) was packed with Zorbax 5 µm C18 materials to allow large-capacity loading and removal of hydrophobic and hydrophilic matrix components. Mobile phase A was 10 mM ammonium acetate in 2% acetonitrile (pH = 8), and mobile phase B was 10 mM ammonium acetate, pH = 8 in 88% acetonitrile. A 4 µg peptide sample was loaded onto the trap with 1% B at 10 µl/min. After the trap was washed for 3 min, a 250 nl/min flow rate was used to back-flush the samples onto the nano-LC column for further separation. The column was enclosed in a heating sheath filled with heat-conductive silicone and warmed homogeneously at 40°C, which helps improve the chromatographic resolution and reproducibility. The following was the 2.5 h separation gradient used on the column: 4% B for 15 min; 13–28% B for 110 min; 28–44% B for 5 min; 44–60% B for 5 min; 60–97% B for 1 min; 97% B for 17 min. The trap was turned offline at 45 min to flush hydrophobic components. No perceivable degradation of pHis was observed under this LC condition.
An Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA) was employed for peptide identification and quantification. Data collection was operated in a 3-second cycle using the data-dependent top-speed mode. The MS1 survey scan (m/z 400–1500) was at a resolution of 120,000, with automated gain control (AGC) target of 500,000 and a maximum injection time of 50 ms. Precursors were fragmentized in HCD activation mode at a normalized collision energy of 35% and the dynamic exclusion was set with 45s. Precursors were filtered by quadrupole using an isolation window of 1 Th. The MS2 spectra were collected at a resolution of 15,000 in the Orbitrap, with an AGC target of 50,000 and a maximum injection time of 50 ms.
The raw files (.raw) generated by LC-MS were matched to database with SEQUEST-HT searching engine embedded in Proteome Discoverer (v2.1, Thermo Scientific). The search parameters were set as follows: 1) Precursor ion tolerance: 20 ppm; 2) Fragment ion tolerance: 0.6 Da; 3) Maximum missed cleavages: 2; 4) Static modifications: carbamidomethylation/ +57.021 Da (C); 5) Dynamic modifications: oxidation/ +15.995 Da (M); phosphorylation + 79.9663Da (S, T, Y, H); ; 6) Decoy database search: target FDR 0.01; 7) Site probability threshold: 50; 9) Co-Isolation interference: 60%.
Proximity ligation assay (PLA)
Briefly, Duolink™ In Situ Detection PLA kit was used with anti-HA tag/anti-FAK antibody for FAK detection (mouse) and anti-RB1 (rabbit) antibody. Deparaffinized slides or fixed cells on a chamber slide were blocked, incubated with primary antibodies (mouse anti-HA-tag/FAK and rabbit anti-RB1 antibodies) and with PLA probes. After DNA ligation and amplification, slides were examined under a fluorescence confocal microscope using DAPI for nuclear staining. Controls such as FAK KO cells and primary antibody omission were used to verify the specificity of FAK-RB1 interaction.
ESCC cells were cultured on a 25 CM2 flask. Live cell images were acquired using a Zeiss AX10 Observer microscope with a LD APIan 20X/0, 30 Ph 1 lens.
Biospecimens or research pathology services for this study were provided by the Pathology Resource Network, which is funded by the National Cancer Institute and is a Roswell Park Comprehensive Cancer Center Support Grant shared resource. Clinical Data Delivery and Honest Broker services for this study were provided by the Clinical Data Network, which is funded by the National Cancer Institute and is a Roswell Park Cancer Center Support Grant shared resource. All protocols were approved by the Roswell Park Comprehensive Cancer Center Institutional Review Board (IRB). Frozen tumor and non-tumor tissue from esophageal adenocarcinoma and squamous cell carcinoma patients were obtained (BDR 071316). Paraffin embedded tissue samples of esophageal squamous cell carcinoma, esophageal adenocarcinoma and normal esophagus were obtained (BDR 114019).
GraphPad Prism was used for statistical analysis. Student’s t tests were used for single comparisons. For comparisons that involve multiple variables and observations, ANOVA was used. For in vitro and in vivo studies, the number of biological replicates was calculated using a statistical analysis for power determination. For all studies, we set an alpha value of 0.05, a power of 0.8, and a standard deviation of 0.25.