Citation
- Authors: Ortiz-Hernandez GL. et al.
- Year: 2021
- Journal: Cells 10 2723
- Applications: in vitro / siRNA / INTERFERin
- Cell types:
- Name: DU145
- Name: PC-3
Description: Human prostate carcinoma cells
Known as: PC3, PC 3
Method
PC3-DR and DU145-DR cells (50,000 cells per well) were cultured on 6-well plates and transfected 24 h later with either 50 nM LEDGF/p75, 100 nM JPO2, 100 nM menin, or 200 nM HRP2 siRNAs for up to 96 h. The siRNA sequences used were as follows: si-LEDGF/p75 (′5-AGACAGCAUGAGGAAGCGAUU-3′), validated previously ; si-JPO2 (pool of three siRNAs, A = ′5-UGAAAGGCUACUCGAAGACUU-3′, B = ′5-UUAUCUCGAACAGUUAU GGTT-3′, C = ′5-UUAGGCACCAAUGGUAUGCTT-3′); si-menin (′5-GAUCAUGCCUGG GUAGUGUUUG-3′), selected from a pool of 10 siRNAs validated previously; si-HRP2.
Cells were transfected using Interferin® siRNA transfection reagent (Polyplus-transfection®). Scrambled siRNA duplex was used as non-targeting negative control. Protein depletion was assessed by immunoblotting.
Abstract
Patients with prostate cancer (PCa) receiving docetaxel chemotherapy invariably develop chemoresistance. The transcription co-activator lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 and PSIP1, is upregulated in several human cancers, including PCa and promotes resistance to docetaxel and other drugs. The C-terminal region of LEDGF/p75 contains an integrase binding domain (IBD) that tethers nuclear proteins, including the HIV-1 integrase and transcription factors, to active chromatin to promote viral integration and transcription of cellular survival genes. Here, we investigated the contribution of the LEDGF/p75 IBD interactome to PCa chemoresistance. Quantitative immunoblotting revealed that LEDGF/p75 and its IBD-interacting partners are endogenously upregulated in docetaxel-resistant PCa cell lines compared to docetaxel-sensitive parental cells. Using specific human autoantibodies, we co-immunoprecipitated LEDGF/p75 with its endogenous IBD-interacting partners JPO2, menin, MLL, IWS1, ASK1, and PogZ, as well as transcription factors c-MYC and HRP2, in docetaxel-resistant cells, and confirmed their nuclear co-localization by confocal microscopy. Depletion of LEDGF/p75 and selected interacting partners robustly decreased the survival, clonogenicity, and tumorsphere formation capacity of docetaxel-resistant cells. These results implicate the LEDGF/p75 IBD interactome in PCa chemoresistance and could lead to novel therapeutic strategies targeting this protein complex for the treatment of docetaxel-resistant tumors.