Fig. 2: LCMT1 loss activates AR signaling and promotes castration-independent tumor growth. | Nature Communications

Fig. 2: LCMT1 loss activates AR signaling and promotes castration-independent tumor growth.

From: Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance

Fig. 2

a Methyl-C reduction upon LCMT1 silencing results in a concomitant increase in p-AR, p-MED1, and MYC expression. Representative immunoblots in a panel of prostate cancer cells stably expressing shNTC or shLCMT1 showing indicated proteins. Vinculin was used as a loading control. b Reduced AB56αCme and AB55αCme (PP2A) heterotrimers in LCMT1 silenced LNCaP cells. Coimmunoprecipitation (IP) analysis using lysates from the indicated cells with V5-PP2A-Aα and EGFP-specific antibodies, demonstrating the interaction between PP2A-Aα, methyl-PP2A-C, B56-α, B55-α. PR130 and STRN are used as methylation insensitive controls and EGFP as a negative IP control. Total lysate was used as input control. c LCMT1 loss disrupts canonical AB56αCme heterotrimer association with chromatin. Chromatin and soluble nuclear fractions from control and LCMT1 silenced LNCaP cells were subjected to immunoblotting for the indicated proteins. Lamin and H3 served as controls for the nuclear and chromatin fractions, respectively. d LCMT1 abrogation resists androgen-ablation arbitrated suppression of AR and MED1 recruitment to the chromatin. Chromatin and soluble nuclear fractions extracted from the cells grown in charcoal-stripped fetal bovine serum (CSS) medium for six days were subject to immunoblotting for the indicated proteins. Lamin and H3 served as controls for nuclear and chromatin fractions, respectively. e Continuous AR transcriptional activity under androgen deprivation. qRT-PCR for AR-regulated genes in cells grown in CSS-containing medium. f Colony formation assay demonstrates the increased proliferation of LCMT1 depleted cells in hormone-deprived media. Cells were cultured in CSS media for 10 days, followed by crystal violet staining (top), and quantification (bottom), error bars mean ± s.d., n = 3 biologically independent replicates. Students two-tailed t-test, p value: 0.006. g Castration-independent tumor growth in vivo upon LCMT1 silencing. Castrated and non-castrated mice bearing shLCMT1- or shNTC-LNCaP were evaluated for tumor growth. The tumor volume is plotted (left) and the percentage of tumors first detected is shown (right). Fisher’s exact test of relative risk. The data in a–d are representative of n = 3 independent experiments.

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