BCAS2, a protein enriched in advanced prostate cancer, interacts with NBS1 to enhance DNA double-strand break repair

Background Breast cancer amplified sequence 2 (BCAS2) plays crucial roles in pre-mRNA splicing and androgen receptor transcription. Previous studies suggested that BCAS2 is involved in double-strand breaks (DSB); therefore, we aimed to characterise its mechanism and role in prostate cancer (PCa). Methods Western blotting and immunofluorescence microscopy were used to assay the roles of BCAS2 in the DSBs of PCa cells and apoptosis in Drosophila, respectively. The effect of BCAS2 dosage on non-homologous end joining (NHEJ) and homologous recombination (HR) were assayed by precise end-joining assay and flow cytometry, respectively. Glutathione-S-transferase pulldown and co-immunoprecipitation assays were used to determine whether and how BCAS2 interacts with NBS1. The expression of BCAS2 and other proteins in human PCa was determined by immunohistochemistry. Results BCAS2 helped repair radiation-induced DSBs efficiently in both human PCa cells and Drosophila. BCAS2 enhanced both NHEJ and HR, possibly by interacting with NBS1, which involved the BCAS2 N-terminus as well as both the NBS1 N- and C-termini. The overexpression of BCAS2 was significantly associated with higher Gleason and pathology grades and shorter survival in patients with PCa. Conclusion BCAS2 promotes two DSB repair pathways by interacting with NBS1, and it may affect PCa progression.


Cell cycle analysis
HEK 293T cells transfected with the indicated lentiviral shBCAS2 plasmids were trypsinized into single cells, stained with propidium iodide (50 µg/mL), and subjected to cell cycle analysis using a flow cytometer (BD FACSCalibur).

Western blotting
Cells were lysed with RIPA buffer (50 mM pH 8.0 Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and protease inhibitors) and the supernatant was collected after centrifugation. Cell lysates were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes (Millipore), blotted with the indicated primary and horseradish peroxidase-conjugated secondary antibodies, and visualised using an enhanced chemiluminescence western blotting detection system (GE Healthcare). All antibodies used are listed in Supplementary Table 1.

Immunofluorescence microscopy
U2OS cells were seeded on glass coverslips before harvesting. They were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 solutions, followed by blocking in 5% blocking solution. The cells were then incubated in the properly diluted antibodies (Supplementary Table 1) in a humidified chamber overnight at 4°C.
Drosophila wing imaginal discs were dissected from the wandering third instar larvae of specific BCAS2-expressing genotypes. Samples were processed according to the method described previously. 2 Briefly, they were incubated with anti-cleaved caspase 3 antibody overnight at 4°C, followed by the fluorescent secondary antibody, and then observed by confocal microscopy.

Colocalisation study and confocal microscopy
Double immunofluorescence microscopy was performed as described above to detect BCAS2 and NBS1 in U2OS cells and fluorescent images were captured with a CCD camera (LAS-4000, Fujifilm, Japan). Composite images were created by superposition of fluorescence images and subjected to colocalisation analysis. The overall Manders' overlap coefficient of BCAS2 and NBS1 at each time point was calculated from 50-60 cell nuclei per 20 high-power field images per coverslip (original magnification: 1000 ×) using a colocalisation tool implemented in the ImageJ via the JACoP plugin (https://imagej.nih.gov/ij/plugins/track/jacop.html). 3 The colocalisation study using confocal microscopy was performed with a confocal microscope (TCS SP5, Leica, German). Z-stack images (XZ and YZ) were generated by combining a series of images (total 15 images) with incremental focuses. The intensity profile along the line through the region of interest was generated using the Zen microscope software (Zeiss).

Recombinant protein purification and in vitro pull-down assay
The glutathione-S-transferase (GST) fusion proteins were expressed in E. coli BL21 (DE3) and Rosetta cells (Novagen). Further purifications were performed using glutathione-Sepharose 4B beads (GE Healthcare), according to the manufacturer's protocols. His-tagged proteins were purified using Ni-NTA agarose beads (Qiagen).
We obtained the nuclear extract from HEK 293T cells using a nuclear extraction kit (Abcam) and following the manufacturer's instructions. Nuclear extracts and Histagged proteins were incubated with the GST-fused protein beads. The interacting proteins were eluted and resolved by SDS-polyacrylamide gel electrophoresis and detected by western blotting with the indicated antibodies (Supplementary Table 1). immunoprecipitation.

Supplementary table 2.
The list of BCAS2-interacting proteins. BCAS2 interaction partners were identified and characterised using glutathione S-transferase (GST) pulldown assay followed by mass spectrometry analysis using the extracts of MCF-7 cells. 4 The list is available through the following web link: https://drive.google.com/file/d/115_ZOcRYEF1OQsfJIe6ookyvVNmIGU9u/view?usp =sharing. were infected with lentiviruses carrying the shRNA against BCAS2 (shBCAS2 #1 and #2) or a scrambled shRNA (shscramble), or they were mock infected. Cells were selected using puromycin for 4 days and the stable pools were collected for experiments. The cells were exposed to 10Gy γ-radiation, followed by an 8h recovery period. RNA was extracted from irradiated and non-irradiated cells with TRIzol reagent, followed by Q-RT-PCR using the SYBR ® Green qPCR system and specific primer pairs. The expression level of BCAS2 was normalised to that of internal control ACTB gene and to the control groups, and presented as means plus SD (n = 3; knockdown vs mock or shscramble, *p < 0.05; Mann-Whitney U test). The experiments in Fig. 1a