hERG1/Kv11.1 activation stimulates transcription of p21waf/cip in breast cancer cells via a calcineurin-dependent mechanism

In addition, our experiments have also revealed that, the inhibitory effect of NS1643 on cell proliferation was completely removed by suppressing p21waf/cip function, suggesting that even though NS1643 treatment leads to upregulation of both p21waf/cip and 16INK4A, the role of p21waf/cip appears to predominate. However, we cannot rule out that 16INK4A can still play a major role in NS1643-driven senescence by acting as a downstream effector of p21waf/cip and better dedicated experiments need to be designed.

In conclusion, we think that, we have discovered a novel mechanism through which stimulation of hERG1 potassium channel leads to inhibition of breast cancer cell proliferation via activation of NFAT1. In addition, our results offers a compelling opportunity to study Ca2+-dependent mechanism in cancer biology which can provide basis for anticancer therapeutic strategies [38].

SKBr3 were cultured in RPMI-1640 (Thermo Scientific). MDA-MB-231, T47D and MCF7 were cultured in DMEM (Corning). All media contained 10% FBS (Serum Source International) and 100 μl/ml penicillin/streptomycin (Thermo Scientific) with the exception of T47D that also contained 1x NEAA (Thermo Scientific). All cells were grown at 37°C in 5% CO2.

SKBr3 cells were harvested for western blot analysis by trypsinization with 0.25 % trypsin-EDTA (Thermo Scientific). Cells were washed with 1x PBS (Thermo Scientific) and resuspended in cold radioimmunoprecipitation assay buffer (RIPA) 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% Tergitol (Sigma-Aldrich, St. Louis, MO, USA), 0.5% Na-deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4, and 1 × Halt Protease Inhibitor Cocktail (Thermo Scientific). Protein concentration was determined by BCA assay (Thermo Scientific). 4x Laemmli buffer containing 10% 2-mercaptoethanol was added to lysates and boiled at 95 °C for 5 minutes. Lysates were loaded on 8% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad).

Nuclear and cytosol extracts from SKBR3 cells following various treatments were prepared as described below. Cells were washed with 1X PBS and cell pellets were lysed with a hypotonic buffer (10 mmol/L HEPES buffer pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 10% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 1 mmol/L dithiothreitol (DTT)). The lysates were kept on ice for 15 min, 5μl of 10% NP 40 was added to each lysate, vortexed briefly followed by centrifugation at 13200 rpm for 10min to pellet the nuclear fractions. The supernatant was collected as the cytosol extract. The nuclear pellet were lysed with buffer solution containing 20 mmol/L HEPES buffer pH 7.9, 1.5 mmol/L MgCl2, 400 mmol/L NaCl, 0.2 mmol/L EDTA, 20% glycerol, 0.5 mmol/L PMSF, and 1 mmol/L DTT. The nuclear lysates were clarified by centrifugation at 13200 rpm for 10 min at 4°C.

SKBR3 cells were grown on poly D-Lysine coated coverslips and treated with vehicle alone, 50 μM NS1643 or 80nM PMA and 2μM Ionomycin in RPMI 1640 media. Following treatment, the cells were washed with 1X PBS, fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton X-100 for 15 min at room temperature. Nonspecific binding was blocked with 5% bovine serum albumin (BSA) for 1h at room temperature and then incubated with 1:100 primary rabbit monoclonal anti-NFAT-1 antibody (Cell signaling Technology, Danvers, MA) diluted in 1% BSA at 4°C, overnight, while cells incubated with 1% BSA alone, served as negative controls. Following incubation with the primary antibody, the cells were washed with 1X PBS and incubated with Alexa fluor 594 conjugated anti-rabbit secondary antibody (Life technoloiges, Carlsbad, CA). Following washes, cells were incubated with 50ng/mL DAPI to stain the nuclei. The coverslips were mounted on VECTASHIELD reagent (Vector Laboratories, Burlingame, CA) and fluorescent images were taken using confocal microscopy (Carl Zeiss Meditec, Inc., Thornwood, NY). Nuclear translocation of NFAT-1 across groups was analyzed and quantified using the analyze particle tool from Image J.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using iScript (Biorad, Life Science Research, Hercules, CA, USA). For p21 amplification, primers p21-F (5′-TGA GCC GCG ACT GTG ATG-3′) and p21-R (5′-GTC TCG GTG ACA AAG TCG AAG TT-3′) were used.

For HSRP amplification, primers HSRP-F (5′-AGC CTC AAG ATC ATC AGC AAT G-3′) and GAPDH-R (5′-ATG GAC TGT GGT CAT GAG TCC TT-3′) were used.

To determine p21 mRNA levels and stability, quantitative RT-PCR was performed on a Mastercycler® ep Realpex (Eppendorf, Hauppauye, NY, USA) using a total volume of 20 μl containing 200 nM primers and 1X Absolute™ Blue QPCR SYBR® Green Mix (ABgene, Thermo Fisher Scientific, Rockford, IL, USA).

Dual-luciferase reporter assay was done according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, cells (4 × 104) were plated on a 24-well plate, for 24 hr.ells in triplicate wells were co-transfected with 5 μg plasmid DNA encoding firefly luciferase driven by the NFAT or p21 promoter and 1μg of DNA encoding a constitutively expressed Renilla luciferase gene.

The p21-promoter luciferase reporter luciferase plasmid was generated by Toren Finkel group. This construct was a gift from Toren Finkel (Addgene plasmid # 10959)

The luciferase reporter gene for the studies of NFAT activity contains three tandem repeats of a 30-bp fragment (GGAGG AAAAA CTGTT TCATA CAGAA GGCGT) of the IL-2. This construct was a gift from Wafik El-Deiry (Addgene plasmid # 21723).

The firefly luciferase plasmid contains a luciferase reporter gene driven by a tandem repeat of NFAT binding elements and served as reporter of NFAT activation. Renilla luciferase serves as the control reporter to normalize the activity of the experimental reporter based on transfection efficiency and cell viability. Two days after transfection, the cells were exposed to drugs as indicated. Cells were harvested, and lysates were tested for luciferase activity using a dual-luciferase assay system. Y axis indicates fold change of relative luminescence which denotes the ratio of firefly luciferase activity (NFAT-dependent) to Renilla luciferase (control) activity to standardize for transfection efficiency.