Citation
- Authors: Nelson JK. et al.
- Year: 2022
- Journal: Nat Commun 13 2070
- Applications: in vitro / DNA / jetPRIME
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
HEK293T cells were seeded on poly-L-Lysine treated plates. Cells at 70% confluency were co-transfected using JetPRIME (Polyplus) with pLKO.1-shRNA-puro constructs, pMD2.6 G, and psPAX2 at a 1:1:3 DNA concentration ratio. Cells were incubated with transfected mixture in OptiMEM minus antibiotics for 5-6 h. Medium was then replaced with DMEM + GlutaMAX supplemented with 10% FCS and 20 mM HEPES (Gibco). 48 h post transfections, the viral supernatant was collected and filtered twice using 0.45 µm filter. Virus titre was calculated using Quicktiter Lentivirus Quantification kit.
Hek293T cells were seeded for 70% confluency at the time of transfection on poly-L-Lysine treated plates. Indicated plasmids were transfected using Jetprime, and a GFP expressing plasmid was used a transfection efficiency control (over 85-95% efficiency).
Abstract
Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited.