CXC receptor-1 silencing inhibits androgen-independent prostate cancer.
The CXC receptor-1 (CXCR1) is a coreceptor for interleukin-8 (IL-8) and is expressed on both normal and tumor cells. The function of CXCR1 in prostate cancer was investigated by silencing its expression, using RNA interference. We established stable cell colonies of PC-3 cells, depleted of CXCR1, using lentiviral plasmids (pLK0.1puro) generating small hairpin RNA (shRNA) against CXCR1 mRNA. Stable shRNA transfectants (PLK1-PLK5) that express significantly reduced CXCR1 mRNA (>or=90% down) and protein (>or=43% down) or vector-only transfectants (PC-3V) were characterized. PLK cells showed reduced cell proliferation (down, >or=66%), due to cell cycle arrest at G(1)-S phase, decreases in Cyclin D1, CDK4, phosphorylated Rb, and extracellular signal-regulated kinase 1/2 levels compared with those in PC-3V cells. CXCR1 depletion lead to increases in spontaneous apoptosis by mitochondria-mediated intrinsic mechanism and increases in proapoptotic proteins (BAD, 40%; BAX, 12%), but decreases in antiapoptotic proteins (BCL2, down 38%; BCL(xL), 20%). PLK2 cells grew as slow-growing tumors (decrease of 54%), compared with that of PC3V tumors in athymic mice. Ex vivo analyses of PLK2 tumor tissues showed reduced expression of Cyclin D1 and vascular endothelial growth factor, and increased apoptosis activity. Other IL-8-expressing prostate cancer cell lines also exhibited similar phenotypes when CXCR1 was depleted by CXCR1 shRNA transfection. In contrast to these cells, CXCR1 depletion had little effect on IL-8 ligand-deficient LNCaP cells. RNA interference rescue using mutated CXCR1 plasmids reversed the silencing effect of PLK2, thus demonstrating the specificity of phenotypic alteration by CXCR1 shRNA. These studies establish that CXCR1 promotes IL-8-mediated tumor growth.
Departments of Urology, and Sylvester Cancer Center, University of Miami Miller School of Medicine, VA Medical Center, Miami, Florida 33101, USA.
Nagarajarao
N
Shamaladevi
Dominic A
DA
Lyn
Diogo O
DO
Escudero
Bal L
BL
Lokeshwar
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D020868:Gene Silencing; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D016209:Interleukin-8 / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009376:Neoplasms, Hormone-Dependent / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D023062:Receptors, Interleukin-8A / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D016209:Interleukin-8; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D023062:Receptors, Interleukin-8A; D048052:Mitogen-Activated Protein Kinase 3