We asked if a similar outcome could be achieved through pharmacological inhibition of BMI1 activity. BMI1 expression in African-Americans than Caucasians. TCGA and NIH/GEO clinical data corroborated to our findings. We show that expression (i) positively correlates to metastatic (and models. Conclusion BMI1, a major driver of metastasis, represents a promising therapeutic target for treating advanced CaP in patients (including those belonging to high-risk group). showed that PTC-209, a small molecule, has the potential to inhibit the expression of BMI1 (15). In this study, we tested the relevance of BMI1 molecule in the metastasis of CaP in African-American and Caucasian RIPK1-IN-4 patients. We tested the therapeutic efficacy of small molecule inhibitor of BMI1 against localized and metastasis models of African-American and Caucasian CaP disease using xenograft mouse and zebrafish models. Material and Methods Cell lines Cell lines were purchased from ATCC (MDA-PCa2b, PC3, WBNB26) and MD-Anderson cancer center (PC3M). E006AA and E006AA-ht cell RIPK1-IN-4 lines were obtained from Dr. Shahriar Koochekpour (Roswell Park Cancer Center, Buffalo, NY). RC77/N and RC77/T were provided by Johng S. Rhim (Uniformed Services University, Bethesda, MD). Cell lines were authenticated (for STR analysis) and the report is attached as RIPK1-IN-4 supplemental data file. PC3, PC3M-luc were cultured in RPMI-1640 medium whereas MDA-PCa2b cells were cultured in HPC1 + 20% FBS medium. WBNBA26 cells (purchased from ATCC) were grown in basic KFC medium. Patient tissues Frozen and paraffin-embedded prostatic tissues were procured from NCI-sponsored Cooperative Human Tissue Network (CHTN, Columbus, OH). The metastatic tissues and tumor-RNA of CaP patients were obtained from (i) BioNet-University of Minnesota, (ii) and the University of Washington (Seattle, WA) and the Prostate Cancer Biorepository Network (Johns Hopkins University, Baltimore, MD). The metastatic tissues (Mets) were obtained from patients who died of mCRPC and who signed written informed consent for a rapid autopsy to be performed within hours of death, under the aegis of the CaP Donor Program at the University of Washington. Chemicals, antibodies and plasmids Docetaxel, and PTC-209 were purchased from selleckchem (Houston, TX USA). The ChIP-grade anti-BMI1 antibody was obtained from Millipore (Bedford, MA, USA). We used following antibodies: anti-BCL2, anti-BMI1, anti-mitochondrial protein, anti-PCNA, anti-Cyclin D1 (Cell Signaling, Danvers, MA), anti-P16, anti-c-MYC (Abcam, Cambridge, MA) and anti-VEGF (SantCruz, CA). We used following plasmids pTK-TCF-Luc (Upstate Laboratories, Lake Placid, NY); pGL3-MMP2, pGL3-VEGF, pGL3-MYC (Addgene, Cambridge, MA) and pGL3-BMI1 (gifted by Goberdhan Dimri, George Washington University, Washington DC). assays were performed as described previously (13, 14, 16). Human CaP-associated gene microarray Total cDNA prepared from control and treated cells were used for qRT-PCR-based microarray analysis as per vendors protocol (microarray# PPAHS-135Z ; Qiagen, Germantown, MD). The data analysis was performed by using array analyzer software. The array data has been deposited to NIH-Gene bank database (accession number- “type”:”entrez-geo”,”attrs”:”text”:”GSE113309″,”term_id”:”113309″GSE113309). 3-Dimensional prostato-spheroid formation Cells were cultured in cancer stem cell medium using ultra-low binding culture plates (ScienCell Research, Carlsbad, CA ). At 5th day of post-seeding, cell spheroids were treated with therapies (PTC-209 and Docetaxel) in fresh culture medium. Therapeutic agents were added to the cells at every 48th h in fresh media, and the treatments continued for 8C12 days. At the termination, images were captured under microscope fitted with a CCD camera and the spheroid volumes and size were measured using software. Rabbit polyclonal to AIG1 Quantitative Chromatin Immunoprecipitation (ChIP) assay BMI1 occupancy on promoter region of gene was determined by using the method as described by song (17). Briefly, cells RIPK1-IN-4 were cross-linked with formaldehyde and trypsinized in lysis buffer followed by the nuclear digestion to obtain 300C800 bp DNA fragments. Equal quantity of chromatin supernatants was subjected to immunoprecipitation with anti-BMI1 and anti-IgG antibodies. Purified DNA was analyzed by real-time PCR (ABI Prism 7500) using and promoter. All ChIP assays were performed three times. The sequences of the qPCR primers are listed RIPK1-IN-4 in supplemental Table 1. Zebrafish metastasis model Cancer cells were trypsinized, counted, and labeled with Cell Tracker Orange CMTMR (Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions. Cells were resuspended in PBS containing DNase I and heparin, and 50C200 cells were microinjected in anesthetized embryos into the injection site, which varied according to the specific.