Cell Culture and stable knockdown cell line generation
Breast cancer cell lines: MDA MB 231, and HCC1937 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI1640 medium supplemented with 10% FBS and 1X penicillin and streptomycin. Mouse basal type cell line, 4T1, was cultured in DMEM with the supplements mentioned above. Scramble control and pSUPER-Retro-sh-MUC16 were transfected into phoenix cells using Lipofectamine 2000 to generate viral particles (Invitrogen, Carlsbad, CA, USA). After 48 h, supernatant (viral particles) was collected, centrifuged, and used to infect the MDA MB 231 and HCC1806. Similarly, mouse-specific pSUPER-Retro-sh-Muc16 were used for 4T1 cells. The pooled population of MUC16 knockdown cells was obtained using antibiotic selection (Puromycin 4μg/ml) and was further expanded to confluent levels to obtain stably transfected cells. Then, MUC16 knockdown and scramble cells were used for microarray analysis.
Tissue Microarray, immunohistochemistry, and immunofluorescence
Immunohistochemistry was performed in the commercially available tissue microarray (TMA)- A202V (Accumax array), BR1503 (US Biomax), which included 75 cases/150 cores and normal lung tissues. Briefly, the TMAs or slides were baked overnight at 56°C to remove the excess paraffin and hydrated in the graded alcohol (100 to 20%, 5 min each). Antigen was retrieved in the 0.01M citrate buffer for 15 min in the microwave, endogenous peroxidase activity was quenched by 0.3% H2O2 (1 h/dark) followed by blocking with 2.5% horse serum (Impress reagent kit, Vector Laboratories) at room temperature, 1 h. The slides were incubated overnight with MUC16 (M11 clone, Dako) and HuR at 4°C, washed with PBST, and further incubated with ImmPRESS Universal anti-mouse Ig/rabbit Ig for 30 min. The brown color was developed by using DAB, counterstained with hematoxylin, followed by dehydration with graded alcohol (20 to 100%), air dried, and mounted with PerMount . MUC16 immunostaining was evaluated by a trained pathologist, who was blinded to the clinical information (3). A quantitative assessment of MUC16 protein expression in the xenograft tissues was performed using Fiji-Image J software .
For immunofluorescence studies, the tissues were processed according to the above-mentioned antigen retrieval step. The tissues were then blocked with 10% goat serum (Jackson Immunoresearch Labs, Inc., West Grove, PA, USA) for 30 min followed by incubation with primary antibodies:- MUC16 (Mouse, 1:750) and HuR (rabbit, 1:500) for overnight at 4°C. The next day, the cells were washed with PBS (5 min, 3X) and incubated with fluorescein isothiocyanate-conjugated anti-mouse and Texas red-conjugated anti-rabbit secondary antibodies (Jackson Immunoresearch Labs, Inc.) for 30 min at room temperature in the dark. Next, the tissues were washed with PBS (5 min, 3X) with gentle shaking and mounted with an anti-fade vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Images were acquired with an LSM710 microscope (Carl Zeiss GmbH, Jena, Germany).
Quantitative real-time PCR
Quantitative real-time PCR was performed as previously described . Total RNA was isolated using Qiagen Kit (Germantown, MD, USA). Total RNA (two micrograms) was used for cDNA synthesis using reverse transcriptase SuperScript®II (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed using SYBER Green, and β-actin was used as an internal control.
Total protein was isolated using the RIPA buffer (50mM Tris-HCl, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors (1mM phenylmethyl sulphonyl fluoride, 1mg/ml aprotinin, 1mg/ml leupeptin). About 20-40 μg protein was run in 10% SDS-PAGE gel for the signaling studies. While for a very high molecular weight glycoprotein, MUC16, 2% agarose gel was run. The gels were transferred to PVDF membrane, blocked with 5% skimmed milk, and probed with respective primary antibodies- MUC16 (M11 clone, Mouse 1: 1000, Fisher Scientific), HuR (Rabbit, 1:1000, Abcam), MMP1 (Rabbit, 1:1000, Cell Signaling), pSrcY-416 (rabbit, 1:1000, Cell Signaling), Src (rabbit, 1:1000, Cell Signaling), c-MYC (Mouse 1:1000, Santa Cruz biotechnology), β-catenin (rabbit, 1:2000, Cell Signaling), Twist 1 (rabbit, 1:1500, Santa Cruz biotechnology), YBX1 (rabbit, 1:1000, Cell Signaling), and anti-β-actin for overnight at 4˚C. The membranes were then washed (3X, 10min) in PBST at room temperature, probed with the appropriate secondary antibodies (1:5000 dilutions) for 1 h, and washed (3X, 10min) with PBST. The signal was detected with the ECL chemiluminescence kit (Amersham Bioscience, Buckinghamshire, UK).
Invasion and cell motility assay
For invasion and migration assays, about 1 million scramble and MUC16 knockdown cancer cells (MDAMB231, HCC1806, and 4T1) were seeded (serum-free condition) in the 8 µm pore size six-well inserts (Becton Dickinson, Franklin Lakes, NJ, USA). 10-20% serum was used as an attractant in the bottom chamber. After 24h, the cancer cells that migrated to the lower chamber were stained with the Quick-Diff kit staining solution and then counted in eight different random fields, and the average number of motile cells per representative field was calculated.
Tail vein injection
About 1 million viable GFP-labeled MDAMB-231(SCR. and shMUC16) in 50 µl PBS was injected via the tail vein of nude mice (n=6/group). The mice were monitored every week for metastasis by IVIS imaging. After 30 days, the mice were sacrificed, and tissues were collected for further investigation. The mouse studies were performed in accordance with the US Public Health Service ‘Guidelines for the Care and Use of Laboratory Animals’ under an approved protocol by the Institutional Animal Care and Use Committee, University of Nebraska Medical Center.
RNA Immunoprecipitation (RIP) and PCR array
RIP was performed using an anti-HuR antibody (1, 2). HCC1806-SCR and HCC1806-shMUC16 were cultured and lysed using polysome lysis buffer (1000 mM KCl, 50 mM MgCl2, 100 mM HEPES-NaOH pH 7, 5 % NP40) supplemented with RNase and protease inhibitors. Lysates were pre-cleared by adding 30 μg of IgG1 (BD Bioscience) and 50 μl of Protein-A/G Sepharose beads swollen in NT2 buffer with 5% BSA. Beads were coated by adding either IgG1 (BD Biosciences, San Diego, CA) as control or anti-HuR antibody and incubated overnight at 4 °C. After extensive washes of pre-coated Protein-A/G sepharose beads, the pre-cleared lysate was added and incubated for 4 h at 4 °C, and then 30 μg of proteinase K was added to digest protein by incubation at 55 °C for 30 min. The extracted RNA was reverse transcribed into cDNA and performed cDNA array (PAHS-028Z, QIAGEN) to identify the HuR target mRNAs.
MS-444 and CMLD treatment and cell viability assays
MDA MB 231 and HCC1806 cells were seeded at subconfluent levels in 96-well cell culture plates and treated with increasing concentrations of MS-444 and CMLD for 48 h at 37°C [24, 25]. The cell viability of MS-444 and CMLD were determined using MTT assays . Based on the IC50 concentration of MS-444 and CMLD, MDA MB 231 and HCC1806 cells were treated for 48 h, and lysates were collected for western blot analysis.
Statistical significance was evaluated with the Student t-test using GraphPad Prism 8.1.2 software. P values less than 0.05 were considered statistically significant. All experiments were performed in triplicates.