Expression of YAP1 and TAZ in Melanoma-, Bronchial- and Breast Carcinoma Brain Metastasis: Primary vs. Relapsed Tumors

Materials and Methods

Patient samples. Samples of metastasis of melanoma (n=13), breast cancer (n=20) and bronchial carcinoma (n=32) were obtained during neurosurgical resection at the Department of Neurosurgery of the University Hospital of Cologne, Germany. Immediately after surgery they were frozen in liquid nitrogen and long-time stored at −80°C. The ethics committee of the University Hospital Cologne approved the study in accordance with the Declaration of Helsinki (application no. 03-170).

PCR. Twenty-five mg of each specimen was homogenized with TissueLyser LT (Qiagen, Hilden, Germany) for RNA isolation. For RNA extraction, the RNeasy Mini Kit (#74104, Qiagen) was used, followed by the QuantiTect Reverse Transcription Kit (#20531, Qiagen) for cDNA synthesis using 1 μg of RNA. qPCR was performed with Rotor Gene SYBR Green PCR Kit (Qiagen) and 5 μl of the synthesized cDNA (concentration 1:50). We used non-template control and beta-actin for normalization; each sample was measured three times using Rotor Gene Q (Qiagen) starting with the denaturation for 5 min at 95°C followed by 50 cycles of a two-step protocol (95°C for 5 s and 60°C for 10 s). The nucleotide sequences of the primers were the following: TAZ-FW 5′-GGCTGGGAGATGACCTTCAC-3′, TAZ-RV 5′-CTGAGTGGGGTGGTTCTCGT-3′, YAP1-FW 5′-CACAGCTCAGC ATCTTCGAC-3′, YAP1-RV 5′-TATTCTGCTGCACTGGTGGA-3′ (all by Eurofins Genomics, Ebersberg, Germany).

Western blot. For protein isolation, samples of melanoma (n=13), breast cancer (n=9) and bronchial carcinoma (n=13) were suspended for one hour in RIPA buffer on ice followed by centrifugation (15 min, max. speed, 4°C). They were stored at −80°C until usage. A total of 50 μg of each sample was mixed with NuPAGE SDS Sample Buffer and NuPAGE Sample reducing agent (both from Thermo Fisher Scientific, Waltham, MA, USA). After 10 min of denaturation at 70°C, proteins were separated with Tris/Glycine/SDS Buffer and prefabricated Mini PROTEAN TGX stain-free gels using the Bio-Rad system at 200 mV for 28 min (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Precision Plus Protein™ All Blue Prestained Protein Standards (#1610373, Bio-Rad Laboratories GmbH) was used as protein standard. Gels were blotted on nitrocellulose blotting membranes (#10600002, Cytiva Europe GmbH, Freiburg, Germany) with the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories GmbH) at 25 mV for 10 min. Membranes were blocked in 5% dry non-fat milk with 3% BSA dissolved in TBST for 90 min. Melanoma samples were bleached in 10% H₂O₂ for 15 min. Membranes were incubated with the following primary antibodies: TAZ 1:500 in 5% dry non-fat milk with 3% BSA dissolved in TBST for 2 h (#83669, Cell Signaling Technology, Danvers, MA, USA), YAP1 1:1,000 in 5% dry non-fat milk with 3% BSA dissolved in TBST for 2 h (#ab39361, abcam, Cambridge, UK), beta-actin 1:10,000 in TBST for 30 min (#A1978, Sigma-Aldrich, St. Louis, MO, USA). The membranes were incubated in anti-mouse-antibody (#7076, Cell Signaling Technology, Danvers, MA, USA) for YAP1 and beta-actin and in anti-rabbit-antibody (#7074, Cell Signaling Technology) for TAZ, each in a concentration of 1:10,000 in TBST for 30 min. For visualization of bands, Clarity Western ECL Substrate (Bio-Rad Laboratories GmbH) and ChemiDoc imaging system (Bio-Rad Laboratories GmbH) was used. Bands were analyzed with ImageLab software (Bio-Rad Laboratories GmbH).

Statistical analysis. Statistical analysis was performed using Prism 9 software (GraphPad software, San Diego, CA, USA). Kruskal–Wallis test was used for comparison of expression levels of different metastases. For analysis of paired patient samples, the Wilcoxon test was performed.