Cell culture and reagents
TNBC cell lines (MDA-MB-231, Hs578T, and MDA-MB-157) were maintained using Dulbecco’s Modified Eagle’s Medium including penicillin/streptomycin and 10% FBS (ATCC, Manassas, VA, USA). Non-TNBC cell lines (BT474, MCF7, and T47D) were cultured in RPMI-1640 supplemented with antibiotics and 10% FBS at 37°C in a 5% CO2 incubator. Cell lines were periodically checked for mycoplasma contamination with PCR-based protocol. Antibodies used for immunoblotting and immunoprecipitation are listed in Supplementary Table S1. Recombinant CK2α protein was obtained from Origene (TP760181, Rockville, MD, USA). Benzamidine (12072) and cycloheximide (CHX, C7698) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silmitasertib (CX-4945, HY-50855) was obtained from MedChemExpress Biotech Co., Ltd. (Monmouth Junction, NJ, USA).
RNA preparation and real-time RT-PCR
Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer's instructions. Then, RNA was transformed to cDNA with reverse transcription using a PrimeScript II RT Reagent Kit (Takara, Kyoto, Japan). Quantification of specific gene expression was measured with SYBR premix Ex Taq™ (Takara) and a Prism 7900HT sequence detection system (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturers' protocols. Relative gene expression was determined using the 2ΔCt (internal control) - ΔCt (gene) method and normalized to cyclophilin [34]. Data was acquired from at least triplicate experiments and denoted as mean ± standard deviation (SD). Used primer sequences are listed in Supplementary Table S2.
Immunoblotting and immunoprecipitation
Cells were harvested with lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, and protease inhibitors) for 30 min at 4°C. After removal of cell debris, protein concentrations were measured with the bicinchoninic acid method (Thermo Fisher Scientific). Equal amounts of protein samples were then separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, followed transfer to Immobilon PVDF membrane (Millipore, Bedford, MA, USA), and incubated with appropriate primary antibodies. Target proteins were detected using chemiluminescent reagents (Thermo Fisher Scientific) according to the manufacturer’s protocols [35]. For immunoprecipitation, protein samples were mixed with appropriate antibodies for overnight at 4°C and collected with addition of protein A/G-agarose beads (Santa Cruz, Dallas, TX, USA) after incubation at 4°C for another 1 hr. Interacted protein complexes were harvested with 2 × SDS sample buffer and analyzed with immunoblotting method.
Wound healing and transwell invasion assay
To assess migration ability of breast cancer cells, cells were seeded into 12-well plates and allowed to reach confluency for approximately 24 hr. After scratch was created using a pipette tip, migration ability was analyzed under a microscope observation. To evaluate the invasion ability, same number of cells (5 × 104) were seeded into the upper chamber (8-µm inserts; Thermo Scientific) with serum-free media while the lower chamber was filled with 10% FBS medium. After 24–36 hr, cells in the upper chamber were removed and fixed with 4% paraformaldehyde (PFA) and visualized with 0.05% crystal violet (Sigma Aldrich). Representative images were captured using a confocal microscopy (LSM710, Carl Zeiss, Oberkochen, Germany). Total number of invaded cells was measured with ImageJ program (National Institutes of Health, Bethesda, MD, USA).
Immunofluorescence and immunohistochemistry staining protocol
For immunofluorescence staining, cells were seeded into 4-well chamber slides. After 24 hr of incubation, cells were fixed with 4% PFA for 15 min at room temperature. Then, stained cells were washed with PBS three times and permeabilized with 1% Triton X-100/PBS. After additional washing steps, cells were treated with blocking buffer (1% BSA in PBS) for 1 hr. Primary antibodies (1:100) were then applied followed by incubation at 4°C overnight. Following removal of the primary antibodies and subsequently cells were treated with Alexa Fluor 594-conjugated (A32740, Invitrogen) and Alexa Fluor 488-conjugated (A32723, Invitrogen) secondary antibodies for 1 hr at room temperature. Then, nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 1 min. Fluorescence images were analyzed with a confocal microscope and quantified using ImageJ software [36].
For immunohistochemistry (IHC) staining, lung tissues were fixed in 4% neutral-buffered formalin, embedded, and sectioned as descried previously [37]. Slides were treated with 0.03% hydrogen peroxide, microwaved in a 10 mM citrate buffer (pH 6.0) containing 0.01% Tween 20 for 10 min, and then incubated with appropriate antibodies. Antibodies were analyzed with diaminobenzidine (DAB) reagent, while the nuclei were counterstained with hematoxylin QS (H-3404; Vector Laboratories, Burlingame, CA, USA). Random images of stained slides were acquired with Motic Easyscan Digital Slide Scanner (Motic Hong Kong Limited, Hong Kong, China)
PCR-based site-directed mutagenesis
To generate CK2α-K68M (kinase dead) and GRP94-S306A (dephosphorylation mutant of GRP94, serine at position 306 mutated to alanine) or S306D (phosphor-mimetic mutation of GRP94, serine at position 306 mutated to aspartic acid), PCR-based site-directed mutagenesis method was used as described previously [38]. Point mutations were inserted into each DNA construct with overlapping extension PCR protocol and the mutated PCR product was cloned into the plasmid pCAG-Flag vector. Then, mutation results were confirmed by sequencing. To remove potential phosphorylation site of T786 residue in GRP94, the plasmid expressing the T786 deletion mutant was generated by PCR-mediated amplification of pCS4-Flag-GRP94 wild type construct with appropriate primer. PCR products were digested with restriction enzymes and cloned into pCAG vector. Primer sets used for mutagenesis are listed in Supplementary Table S3.
Generation of knockout and overexpression of breast cancer cell lines
To generate GRP94 and CK2α knockout (KO) cell lines, the following sgRNA sequences were cloned into the LentiCRISPRv2 vector (AddGene, Watertown, MA, USA): 5’-CGGATGATGAAGTAGTACAG-3’ for GRP94 sgRNA and 5’-GAACTGACCTGACATCATAT-3’ for CK2α sgRNA [39]. For overexpression (OE), Flag-CK2α-WT, CK2α-K68M, GRP94-WT, GRP94-S306A, GRP94-S306D, GRP94-ΔT786, and GRP94-S306D/ΔT786 DNAs were cloned into the pCAG lentiviral vector. Lentivirus production was performed by transfecting HEK293T cells with lentiviral packaging plasmids, psPAX2 and pMD2.G (AddGene). After 48 hr, viral supernatants were harvested, filtered through a 0.45 µm syringe filter, and stored at -80°C for future use.
Fluorescent gelatin degradation assay
Activity of invadopodia was measured using a QCMTM gelatin degradation assay kit (ECM670, Millipore, Burlington, MA, USA) according to manufacturer's protocols [40]. Glass-bottom 8-well dishes were incubated with 0.1 mg/ml poly L-lysine and crosslinked with 0.5% glutaraldehyde. Then, dishes were coated with Oregon Green 488-conjugated gelatin at 37°C for 1 hr. After treatment with sodium borohydride and ethanol, medium was added to dishes. After plating cells followed by 48 hr of incubation, cells were immobilized with 4% PFA, permeabilized with 0.5% Triton X-100 following blocking with 3% bovine serum albumin in PBS, and incubated with primary antibody against F-actin/Phalloidin (Invitrogen) overnight at 4°C. Then, nuclei were counterstained with DAPI for 1 min before observation.
CK2 in vitro kinase assay
CK2 activity in breast cancer cells was analyzed using a Cyclex CK2 Kinase Assay/Inhibitor Screening kit (MBL International Corp., Woburn, MA, USA) following the manufacturer's protocols. MDA-MB231 cells were treated with benzamidine (10 µM or 50 µM) or silmitasertib (20 µM) for 24 hr. After lysis with kinase reaction buffer, samples were moved into 96-well plates pre-coated with a substrate of CK2α (recombinant p53 amino acids 1–55) and incubated at 30°C for 10 min in the presence of ATP. These wells were washed and treated with HRP-conjugated antibody and substrate reagent. The reaction was terminated by adding TK-4D4 and TMB. The color was quantified using a 96-well plate reader at 450 nm/540 nm. Data were acquired at least triplicate experiments and statistical significance was set at p < 0.05.
Thermal shift assay
Thermal shift binding assay (TSA) was performed with a Bio-Rad CFX real-time PCR system (Hercules, CA, USA). CK2α protein was equilibrated in a buffer (pH 7.5, 100 mM Tris-HCl, 100 mM NaCl) with SYPRO Orange dye (Invitrogen) after addition of DMSO or benzamidine. Samples were freshly prepared and dispensed into 384-well PCR plates at a final volume of 10 µL per well. Fluorescence intensity of each well was measured with a temperature gradient range from 25 to 95°C with a heating rate increase of 1°C per minute. To determine the effect of benzamidine on melting temperature (Tm) of CK2α protein, a Boltzmann model was used to generate the protein unfolding curves with GraphPad Prism (v10.0) software.
In vivo metastasis animal model
Xenograft tumor models were constructed using 6-8-week-old immunodeficient BALB/c athymic female nude mice (Orient Bio, Seongnam, Korea). Animal experiments followed the guidelines approved by Institutional Animal Care and Use Committee-approved protocols of the Lee Gil Ya Cancer and Diabetes Institute, Gachon University (#LCDI-2019-0146, Incheon, Korea). MDA-MB231 cells stably expressing luciferase and CK2αKO, CK2αKO/GRP94-S306AOE, and CK2αKO/GRP94-S306DOE or MCF7 cells stably expressing luciferase with CK2αOE, CK2α-K68MOE, and CK2α-K68MOE/GRP94-S306DOE (1 x 106 cells in 100 µl) were intravenously injected into nude mice (five animals per group).
To evaluate the effect of benzamidine, a CK2α inhibitor, on metastasis of TNBC cells, MDA-MB231 cells stably expressing luciferase were intravenously injected into nude mice (five animals per group). Mice were randomly assigned to three groups and treated with benzamidine (10 mg/kg or 50 mg/kg) or DMSO by intraperitoneal injection every other day. Metastasis intensity into lung was monitored with in vivo optical imaging system (Caliper Life Sciences, Hopkinton, MA, USA) at the Core Facility for Cell In-vivo Imaging of Gachon University weekly by intraperitoneal injection of luciferin reagent (150 mg/ml). After six weeks, mice were sacrificed and lung tissues were fixed with 10% formalin, processed, and embedded in paraffin. Prepared lung slides were analyzed with hematoxylin and eosin (H&E) and IHC staining with Vimentin and β-catenin antibodies. Metastatic areas were calculated with ImageJ software.
Statistics
All statistical results are presented as mean ± standard deviations (SDs). Student's t-test or one-way ANOVA was performed using GraphPad Prism 10.0 software. The sample size for each statistical analysis was indicated. Differences with p < 0.05 were considered statistically significant (NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).