Fig. 5: AR activates G3BP1 transcription to maintain a feed-forward amplification of AR signaling, resulting in sensitization of G3BP1high PCa cells to AR-targeted drugs.
From: G3BP1 inhibits Cul3SPOP to amplify AR signaling and promote prostate cancer
a, b Control and AR KO mPEC-derived organoids were treated with DHT at the indicated concentrations. Expression of G3BP1 (a) and the AR-target gene FKBP5 (b) was quantitatively measured by RT-qPCR. n = 3 biologically independent experiments. Error bars, ±S.E.M. p values are indicated in the figure. c Immunoblotting of WCL derived from control and AR KO organoids treated with vehicle or DHT for G3BP1, AR, FKBP5, and α-tubulin (loading control). n = 3. (d left) Schematic diagram of luciferase reporter constructs showing sequence of wild-type (in Luc-G3BP1) and mutant ARE (in Luc-G3BP1Mut) in G3BP1 promoter region. (d, middle) 22Rv1 cells were transfected with G3BP1 promoter luciferase reporter constructs and treated with DHT alone or in combination with enzalutamide (Enza) as indicated. Gaussia Luciferase activity was determined and normalized against secreted Alkaline Phosphatase activity. NT, no transfection. (d right) 22Rv1 cells were transfected with G3BP1 promoter luciferase reporter (Luc-G3BP1) and G3BP1 promoter luciferase reporter where ARE site is mutated (Luc-G3BP1Mut) constructs and treated with 100 nM DHT. Gaussia Luciferase activity was determined and normalized against secreted Alkaline Phosphatase activity. n = 3 biologically independent experiments. p value is indicated in figure. Error bars, ±S.E.M. e Immunoblots of nuclear extract (left) and whole cell lysate (middle) derived from DOX-inducible empty vector (Ctrl) and pCW57.1-FLAG G3BP1 (overexpressed) 22Rv1 cells treated with enzalutamide for 24 h with the indicated concentrations for the indicated proteins. n = 3. Detection of growth inhibition of Ctrl and G3BP1 overexpressed 22Rv1 cells (right) in the presence of enzalutamide with the indicated concentrations using Cell Titer Glow assay. n = 3 biologically independent experiments. Error bars, ±S.E.M. f Photomicrographs of representative mouse prostate organoids (scale bar, 200 µm) from the indicated mouse genotype treated with enzalutamide with the indicated concentrations following quantification of organoid number. A representative result from more than three independent experiments is shown. Source data are provided as a Source Data file. WCL whole cell lysate.
