Fig 1: PRODH2-mediated Hyp promotes bone metastasis by enhancing YY1 acetylation. A, Schematic of deactivated Cas9 (dCas9)–mediated capture of the SLC7A11 or IL8 promoter (left) and mass spectrometry analysis of transregulatory factors targeting the SLC7A11 or IL8 promoter (right). gRNA, guide RNA. B, Relative expression of SLC7A11 or IL8 in the indicated siRNA-transfected cells as quantified by qRT-PCR analysis with treatment of Hyp. C, ChIP analyses of the enrichment of the indicated transregulatory factors on the SLC7A11 or IL8 promoter with treatment of Hyp. D, Left, volcano plot of differential metabolites between the PRODH2-overexpressing cells and vector cells with treatment of Hyp. Middle, PRODH2 cytoplasmic metabolic pathway. Right, relative intracellular acetyl-CoA and pyruvic acid levels in cells with PRODH2 overexpression (OE) or knockout with treatment of Hyp. FC, fold change. E, PRODH2 proteins were immunoprecipitated from HEK293T cells with treatment of Hyp and subsequently analyzed by LC/MS to identify acetyltransferases associated with PRODH2. F, MDA-MB-231 cells were harvested, and YY1 proteins were immunoprecipitated. G, Representative immunofluorescence staining showing the interaction of YY1 and KAT7 assessed using the Duolink proximity assay (DAPI, blue) with treatment of Hyp. Scale bar, 20 μm. H, Left, the YY1-FLAG plasmid was transfected into MDA-MB-231 PRODH2_OE cells with treatment of Hyp. YY1-FLAG was then immunoprecipitated using an anti-FLAG antibody, and the acetylation status of the immunoprecipitated YY1-FLAG was detected using anti–Pan-Ac and Ac-K230 antibodies. Right, the K230 acetylation site was identified on immunoprecipitated YY1-FLAG using mass spectrometry. I, Impact of overexpressing PRODH2 or the K230 mutation of YY1 on the half-life of the YY1 protein with treatment of Hyp. CHX, chlorhexidine. J, Effects of the proteasome inhibitor MG132 on YY1 degradation with treatment of Hyp. K, HEK293T cells were cotransfected with HA-Ub, siNC, siKAT7, YY1 WT, and YY1 Mut. Proteins were then pulled down using anti-FLAG beads. L, After Hyp treatment, SLC7A11 or IL8 promoter activity was measured in YY1 WT, YY1 K230R, and YY1 K230Q cells following YY1 depletion, using a dual-luciferase reporter assay. Each experiment was performed in triplicate and independently repeated three times. Data are presented as the mean ± SD of n = 3 biologically independent samples. *, P < 0.05; **, P < 0.01. Mut, mutant; NC, negative control; WT, wild type.
Fig 2: PRODH2 suppresses ferroptosis and regulates osteoclast differentiation. A, Untargeted metabolomics analysis in PRODH2-overexpressing cells vs. vector cells with treatment of Hyp. B, Changes in intracellular GSH levels upon PRODH2 overexpression (OE) or knockout in SCP2 cells with treatment of Hyp. C, Western blot analysis of ferroptosis-related proteins in indicated cells following Hyp treatment. Band intensities were normalized to β-actin and expressed as fold change relative to control. D, mRNA expression levels of SLC7A11 in different PRODH2 expression conditions with treatment of Hyp. E, Relative expression levels of intracellular GSH in breast cancer indicated cells with treatment of Hyp. F, Representative images and quantitative analysis of C11-BODIPY oxidation (green)/reduction (red) ratio in cells. Scale bar, 100 μm. G, Representative staining images and quantification of osteoclast differentiation in the presence of CM from indicated cells with treatment of Hyp. Black arrows, multinuclear TRAP+ osteoclasts. Scale bar, 100 μm. H, Left, μCT, hematoxylin and eosin (H&E), and TRAP staining images of bone metastasis in mice after intracardiac injection. Right, quantification of the positive area of Sirius red staining, BLI signals, μCT osteolytic lesion sites, and TRAP+ osteoclasts along the bone–tumor interface of metastases. n = 6 mice per group. Each experiment was performed in triplicate and independently repeated three times. Data are presented as the mean ± SD of n = 3 biologically independent samples. Scale bar, 100 μm. *, P < 0.05; **, P < 0.01. B, bone, T, tumor; M, marrow; NC, negative control.
Fig 3: PRODH2-mediated Hyp metabolism promotes breast cancer bone metastasis. A, Representative images and quantification of PRODH2 expression in normal breast tissues (n = 15), primary breast cancer (BC) tissues without bone metastasis (n = 80), primary breast cancer tissues with bone metastasis (n = 12), and breast cancer bone metastatic lesions (n = 9). Scale bar, 100 μm. MOD, mean optical density. B, Western blot analysis of PRODH2 expression in vector and PRODH2-overexpressing MDA-MB-231 and MCF7 cells, as well as sgNC and sgPRODH2 SCP2 cells. Band intensities were normalized to β-actin and expressed as fold change relative to control. C,In vitro osteoclastogenesis assay was assessed by TRAP staining and quantitative analysis with or without exogenous Hyp. Black arrows, multinuclear TRAP+ osteoclasts. Scale bar, 100 μm. D, Top, μCT, hematoxylin and eosin (H&E), and TRAP staining images showing bone metastases following tibial injection of indicated cells. Bottom, quantification of tibial Hyp concentration, osteolytic lesions by μCT analysis, and TRAP+ osteoclasts at the bone–tumor interface. n = 6 mice per group. Scale bar, 100 μm. E, BLI, μCT, and histologic (hematoxylin and eosin, TRAP, and Sirius red staining) images of bone metastases in BALB/c-nude mice after intracardiac injection of indicated cells. n = 6 mice per group. Scale bar, 100 μm. F, Quantification of BLI signals, Sirius red–positive area, and Hyp concentration in tibial tissue from mice intracardially injected with indicated cells. G, Quantification of TRAP+ osteoclasts, osteolytic lesions, and bone parameters obtained from μCT analysis. Each experiment was performed in triplicate and independently repeated three times. Data are presented as the mean ± SD of n = 3 biologically independent samples. ns, not significant; *, P < 0.05; **, P < 0.01. B, bone; BM, bone marrow; BS/BV, bone surface/bone volume, bone surface density; BV/TV, bone volume/total volume, bone volume fraction; M, marrow; NC, negative control; OE, overexpression; T, tumor; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; Un, untreated.
Fig 4: Pharmaceutical inhibition of PRODH2 blocks bone metastasis of breast cancer cells in vivo. A, Representative images and quantitative analysis of the effect of N-propargylglycine on osteoclast differentiation in the presence of CM from indicated cells treated with Hyp. Black arrows, multinuclear TRAP+ osteoclasts. Scale bar, 100 μm. B, Top left, μCT and histologic [hematoxylin and eosin (H&E), TRAP, and Sirius red staining] images of SCP2-injected mice treated with N-propargylglycine (50 mg/kg/mouse). Top right, quantification of BLI signals and positive area of Sirius red staining. Bottom, quantification of the TRAP+ osteoclasts along the bone–tumor interface of metastases, osteolytic lesion sites, and the bone parameters analyzed by μCT. n = 6 mice per group. Scale bar, 100 μm. B, bone, T, tumor; M, marrow. C, Representative IHC staining and quantitative analysis of PRODH2, YY1, SLC7A11, and IL8 expression in bone marrow tissues (sgNC vs. sgPRODH2). n = 6 mice per group. Scale bar, 100 μm. D, Correlation of PRODH2 expression with nuclear YY1, SLC7A11, and IL8 levels in clinical samples (n = 9). Spearman correlation analysis was used to assess the relationships of PRODH2 expression with SLC7A11 and IL8 levels. The χ2 test was used to analyze the association between PRODH2 expression and nuclear YY1 localization. Scale bar, 100 μm. E, Proposed model illustrating how PRODH2 promotes breast cancer bone metastasis through the SLC7A11–IL8 axis. Each experiment was performed in triplicate and independently repeated three times. Data are presented as the mean ± SD of n = 3 biologically independent samples. ns, not significant; *, P < 0.05; **, P < 0.01. Ac, acetyl; BS/BV, bone surface density; BV/TV, bone volume fraction; GSSG, oxidized glutathione; MOD, mean optical density; NC, negative control; OE, overexpression; ROS, reactive oxygen species; T, tumor; Tb.N, trabecular number; Tb.Sp, separation; Tb.Th, trabecular thickness; Un, untreated. E, Created in Figdraw. ID: USOTOdfeee, Home for Researchers. https://www.figdraw.com.
Fig 5: PRODH2 regulates IL8 secretion to promote osteoclast differentiation. A, Schematic representation of the establishment of a highly bone metastatic MDA-MB-231-BM4 cell line. Tumor cells were isolated from bone lesions in mice injected with MDA-MB-231 parental cells, cultured, and reinjected into mice. This procedure was repeated for four cycles. B, GSEA of RNA sequencing data revealed enriched pathways in MDA-MB-231-BM4 cells. C, mRNA levels of 30 bone-remodeling factors in PRODH2-overexpressing and vector control cells, as well as in 231-BM4 and parental 231 cells. β-Actin served as a loading control. D, Relative IL8 secretion levels derived from PRODH2 overexpression or knockout cells. E,In vitro osteoclastogenesis assay by TRAP staining and quantitative analysis in the presence of CM from indicated cells. Black arrows, multinuclear TRAP+ osteoclasts. Scale bar, 100 μm. F, μCT, hematoxylin and eosin (H&E), and TRAP staining images of bone metastases in mice following either intracardiac injection of SCP2 cells plus treatment with IgG or anti-IL8 (0.1 mg/mouse) or intracardiac injection of SCP2-shCtrl or SCP2-shIL8 cells. n = 6 mice per group. Scale bar, 100 μm. B, bone; T, tumor; M, marrow. G, Quantification of the positive area of Sirius red staining, tibial Hyp concentrations, TRAP+ osteoclasts along the bone-tumor interface of metastases, osteolytic lesion sites, and bone parameters analyzed by μCT assay. Each experiment was performed in triplicate and independently repeated three times. Data are presented as the mean ± SD of n = 3 biologically independent samples. ns, not significant; *, P < 0.05; **, P < 0.01. BV/TV, bone volume/total volume, bone volume fraction; NC, negative control; OE, overexpression; Tb.N, trabecular number.
Supplier Page from Abcam for Anti-PRODH2 antibody