Journal: The Journal of Clinical Investigation

Article Title: Cancer-associated fibroblast-secreted glucosamine alters the androgen biosynthesis program in prostate cancer via HSD3B1 upregulation

doi: 10.1172/JCI161913

Figure Lengend Snippet: ( A ) Protein expression of ELK1 in LNCaP and C42 cells treated with CAF–conditioned medium (CAF-CM) (left) and increasing concentrations of glucosamine (right) for 48 hours. ( B ) ELK1 protein expression in LNCaP cells expressing control or OGT shRNA (left) and sgRNA (right) treated with CAF-CM or 10 mM glucosamine for 48 hours. ( C ) Pearson correlation analysis of ELK1 and OGA mRNA expression in prostate cancer (GSE21032). ( D ) Left: Gene expression of HSD3B1 in control (sgControl) or ELK1-KO (sgELK1) LNCaP cells treated with CAF-CM or 10 mM glucosamine for 48 hours. Right: Luciferase assay of LNCaP control and ELK1-KO cells cotransfected with an HSD3B1 promoter-firefly luciferase and Renilla luciferase plasmid constructs, which were treated with CAF-CM or 10 mM glucosamine 48 hours. ( E ) ChIP assay of Elk1. C4-2 cells were treated with 10 mM glucosamine for 24 hours. ( F ) Protein expression of ELK1 and 3βHSD1 in sgControl and ELK1 -KO LNCaP cells treated with CAF-CM or glucosamine. ( G ) HPLC analysis of steroids in media of LNCaP cells expressing sgControl and ELK1 KO (left) or siRNA and shRNA knockdown of ELK1 (right). Cells were treated with CAF-CM or glucosamine for 48 hours, followed by [ 3 H]-DHEA (100 nM) for 48 hours. ( H ) Gene expression of PSA , TMPRSS2 , and FKBP5 in sgControl and ELK1 -KO LNCaP cells treated with 10 nM DHEA along with CAF-CM or glucosamine for 48 hours. ( I ) Pearson correlation analysis of ELK1 and HSD3B1 mRNA expression in prostate cancer (GSE21032). Significance was calculated using 2-tailed t tests. * P < 0.05, ** P < 0.01.

Article Snippet: Primary antibodies used were anti-3βHSD1 antibody (Abcam, 55268; 1:1,000), anti-OGT antibody (Proteintech, 11576; 1:4,000), anti-OGA antibody (Proteintech, 14711; 1:4,000), anti-Elk1 antibody (Proteintech, 27420; 1:4,000), and anti-O-GlcNac antibody (Invitrogen, MA1-072; 1:2,000).

Techniques: Expressing, Control, shRNA, Gene Expression, Luciferase, Plasmid Preparation, Construct, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: Cancer-associated fibroblast-secreted glucosamine alters the androgen biosynthesis program in prostate cancer via HSD3B1 upregulation

doi: 10.1172/JCI161913

Figure Lengend Snippet: ( A ) Cell viability of sgControl and ELK1 -KO LNCaP cells treated with DMSO (control) or 10 nM DHEA for 48 hours. ( B ) Xenograft tumor growth of orchiectomized mice subcutaneously injected with control or ELK1 -KO C42 cells in the absence or presence of CAFs. A 2-tailed paired t test was performed between control and ELK1 -KO tumors coinjected with CAFs at day 21. ( C ) A log-rank test was used to compare progression-free survival between control and ELK1 -KO and C4-2 cells grown with CAFs. ( D ) Mass spectrometry analysis of intratumoral and serum DHEA, AD, and testosterone (T) in control or ELK1 -KO C42 cells. ( E ) Representative multiplexed fluorescence image of a patient with prostate cancer (Gleason 4+4). 3βHSD1, orange; Elk1, green; CAF, α-SMA, purple), and DAPI, blue. Scale bar: 50 μm. Pearson correlation analysis of gene expression in tissues from patients with primary prostate cancer (3βHSD1 and CAF [α-SMA], n = 22; 3βHSD1 and Elk1, n = 14). ( F ) Pearson correlation analysis of HSD3B1 and FAP mRNA (CAF) in human CRPC metastases (GSE77930). Unless otherwise noted, data are shown as mean ± SEM. Significance was calculated using a 2-tailed t test or 1-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: Primary antibodies used were anti-3βHSD1 antibody (Abcam, 55268; 1:1,000), anti-OGT antibody (Proteintech, 11576; 1:4,000), anti-OGA antibody (Proteintech, 14711; 1:4,000), anti-Elk1 antibody (Proteintech, 27420; 1:4,000), and anti-O-GlcNac antibody (Invitrogen, MA1-072; 1:2,000).

Techniques: Control, Injection, Mass Spectrometry, Fluorescence, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Cancer-associated fibroblast-secreted glucosamine alters the androgen biosynthesis program in prostate cancer via HSD3B1 upregulation

doi: 10.1172/JCI161913

Figure Lengend Snippet: Cancer-associated fibroblasts synthesize and secrete glucosamine in the tumor microenvironment. In the prostate cancer tumor cell, glucosamine induces an increase in O-GlcNAc, which in turn elicits Elk1-dependent transcription of HSD3B1 . HSD3B1 is translated to its corresponding enzyme, 3βHSD1, which is the rate-limiting step for prostate cancer to convert adrenal DHEA to the potent androgen, DHT, to promote progression to castration-resistant prostate cancer (CRPC).

Article Snippet: Primary antibodies used were anti-3βHSD1 antibody (Abcam, 55268; 1:1,000), anti-OGT antibody (Proteintech, 11576; 1:4,000), anti-OGA antibody (Proteintech, 14711; 1:4,000), anti-Elk1 antibody (Proteintech, 27420; 1:4,000), and anti-O-GlcNac antibody (Invitrogen, MA1-072; 1:2,000).

Techniques:

Journal: Journal of Clinical Investigation

Article Title: Cancer-associated fibroblast-secreted glucosamine alters the androgen biosynthesis program in prostate cancer via HSD3B1 upregulation

doi: 10.1172/jci161913

Figure Lengend Snippet: Figure 4. High O-GlcNAcylation increases Elk1 to induce 3βHSD1 expression and enzyme activity. (A) Protein expression of ELK1 in LNCaP and C42 cells treat- ed with CAF–conditioned medium (CAF-CM) (left) and increasing concentrations of glucosamine (right) for 48 hours. (B) ELK1 protein expression in LNCaP cells expressing control or OGT shRNA (left) and sgRNA (right) treated with CAF-CM or 10 mM glucosamine for 48 hours. (C) Pearson correlation analysis of ELK1 and OGA mRNA expression in prostate cancer (GSE21032). (D) Left: Gene expression of HSD3B1 in control (sgControl) or ELK1-KO (sgELK1) LNCaP cells treated with CAF-CM or 10 mM glucosamine for 48 hours. Right: Luciferase assay of LNCaP control and ELK1-KO cells cotransfected with an HSD3B1 promoter-fire- fly luciferase and Renilla luciferase plasmid constructs, which were treated with CAF-CM or 10 mM glucosamine 48 hours. (E) ChIP assay of Elk1. C4-2 cells were treated with 10 mM glucosamine for 24 hours. (F) Protein expression of ELK1 and 3βHSD1 in sgControl and ELK1-KO LNCaP cells treated with CAF-CM or glucosamine. (G) HPLC analysis of steroids in media of LNCaP cells expressing sgControl and ELK1 KO (left) or siRNA and shRNA knockdown of ELK1 (right). Cells were treated with CAF-CM or glucosamine for 48 hours, followed by [3H]-DHEA (100 nM) for 48 hours. (H) Gene expression of PSA, TMPRSS2, and FKBP5 in sgControl and ELK1-KO LNCaP cells treated with 10 nM DHEA along with CAF-CM or glucosamine for 48 hours. (I) Pearson correlation analysis of ELK1 and HSD3B1 mRNA expression in prostate cancer (GSE21032). Significance was calculated using 2-tailed t tests. *P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies used were anti-3βHSD1 antibody (Abcam, 55268; 1:1,000), anti-OGT antibody (Proteintech, 11576; 1:4,000), anti-OGA antibody (Proteintech, 14711; 1:4,000), anti-Elk1 antibody (Proteintech, 27420; 1:4,000), and anti-O-GlcNac antibody (Invitrogen, MA1-072; 1:2,000).

Techniques: Expressing, Activity Assay, Control, shRNA, Gene Expression, Luciferase, Plasmid Preparation, Construct, Knockdown