Journal: The Journal of Clinical Investigation

Article Title: Androgen deprivation–induced NCoA2 promotes metastatic and castration-resistant prostate cancer

doi: 10.1172/JCI76412

Figure Lengend Snippet: (A) Scheme of conditional overexpression of NCoA2 (ROSA26 Lox-STOP-Lox; Flag-NCoA2OE/+). (B) IF and Western blot analysis of NCoA2 overexpression in prostate epithelium from wild-type and NCoA2PCOE/+ mice at 2 months of age. (C) Top: Western blot analysis of NCoA2 expression in the prostates from wild-type and NCoA2PCOE/+ mice, immortalized prostate epithelial cells, RWPE1, and prostate cancer cells including LNCaP, LNCaP-Abl, DU145, PC3, and 22Rv1. Bottom: NCoA2 expression in NCoA2PCOE/+ and wild-type mice. (D) qRT-PCR analysis of Ncoa2 expression in the prostates from NCoA2PCOE/+ mice and the intact or castrated wild-type mice. (E) H&E-stained sections of representative anterior prostate (AP), dorsolateral prostate (DLP), and ventral prostate (VP) at 14 months of age in wild-type and NCoA2PCOE/+ mice. Arrows indicate PIN (bottom). Scale bars: 20 μm (B); 50 μm (E).

Article Snippet: All primary antibodies used in this study were purchased from Cell Signaling, except for AR (Santa Cruz), CK8 (Covance), α-SMA (Sigma-Aldrich), NCoA2 (Bethyl Laboratories), FKBP5 (Abcam), PHLPP1 (Millipore), Rac-GEF (Millipore), E-cadherin (BD), Vimentin (BD), and β-actin (Sigma-Aldrich).

Techniques: Over Expression, Western Blot, Expressing, Quantitative RT-PCR, Staining

Journal: The Journal of Clinical Investigation

Article Title: Androgen deprivation–induced NCoA2 promotes metastatic and castration-resistant prostate cancer

doi: 10.1172/JCI76412

Figure Lengend Snippet: (A) Immunostaining (left) and qRT-PCR (right) analysis of NCoA2 expression in wild-type and PTEN-null prostate with or without castration. n = 10. (B) MTT analysis of cell growth in CTRL and NCoA2-depleted LNCaP-Abl cells cultured in charcoal-stripped media. n = 3. (C) Quantitative results of Ki67-positive staining in prostate tumors from PTENPC–/– and PTENPC–/– NCoA2PC–/– mice after 1 or 6 months of castration. n = 6. (D–H) H&E staining (D); IHC analyses of α-SMA (F), E-cadherin (G), and vimentin (H) expression; and quantitative results (E) of prostate tumor progression in prostate tumors from PTENPC–/– and PTENPC–/– NCoA2PC–/– mice with or without castration (mice were castrated at 4 months of age, and the samples were isolated after 6 months of castration). P < 0.001 (invasive tumor and LGPIN), P = 0.3218 (HGPIN), castrated PTENPC–/– vs. castrated PTENPC–/– NCoA2PC–/–, Fisher’s exact test. (I) Lymph node metastasis of prostate tumors in PTENPC–/– and PTENPC–/– NCoA2PC–/– mice with or without castration are shown as above. qRT-PCR analysis of Ck8 expression in lymph node was used to quantify metastasis. n = 8. Scale bars: 100 μm (A and I), 50 μm (D, F–H). *P < 0.05; **P < 0.01.

Article Snippet: All primary antibodies used in this study were purchased from Cell Signaling, except for AR (Santa Cruz), CK8 (Covance), α-SMA (Sigma-Aldrich), NCoA2 (Bethyl Laboratories), FKBP5 (Abcam), PHLPP1 (Millipore), Rac-GEF (Millipore), E-cadherin (BD), Vimentin (BD), and β-actin (Sigma-Aldrich).

Techniques: Immunostaining, Quantitative RT-PCR, Expressing, Cell Culture, Staining, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Androgen deprivation–induced NCoA2 promotes metastatic and castration-resistant prostate cancer

doi: 10.1172/JCI76412

Figure Lengend Snippet: (A) RNA profiling datasets, in which a particular pathway (AKT/PTEN, EGFR, MEK, MAPK, and AR) was activated in an experimental model, were collected from various published studies (described in Supplemental Table 1). Graphic representation of the significance of gene overlap is shown (Fisher’s exact z score), for NCoA2-regulated genes, as defined by microarray from PTENPC+/– NCoA2PCOE/+ versus PTENPC+/– mice, with the various external pathway signatures. (B) Western blot analysis of phospho-AKT, AKT, downstream targets (phospho-S6 and phospho-GSK3β), AR, phospho-ERK, and total ERK in anterior prostate from 24-week-old mice as indicated. (C) IHC analysis of phospho-AKT, phospho-S6, and phospho-ERK as indicated. Scale bars: 50 μm. (D) NCoA2 expression and NCoA2 signature in primary versus metastatic PCa specimens from patients (GSE21032; ref. 9), in which each tumor expression profile was scored for manifestation of the NCoA2 signature. Box plots represent 5%, 25%, 75%, median, and 95%. P values were examined using t test. (E) Pearson’s correlations between NCoA2 signature and PTEN loss, AKT activation, MAPK, or androgen signature as reflected by PSA levels within primary prostate tumors (Taylor dataset, GSE21032, n = 150, ref. 9; Nakagawa dataset, GSE10645, n = 596, ref. 30).

Article Snippet: All primary antibodies used in this study were purchased from Cell Signaling, except for AR (Santa Cruz), CK8 (Covance), α-SMA (Sigma-Aldrich), NCoA2 (Bethyl Laboratories), FKBP5 (Abcam), PHLPP1 (Millipore), Rac-GEF (Millipore), E-cadherin (BD), Vimentin (BD), and β-actin (Sigma-Aldrich).

Techniques: Microarray, Western Blot, Expressing, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Androgen deprivation–induced NCoA2 promotes metastatic and castration-resistant prostate cancer

doi: 10.1172/JCI76412

Figure Lengend Snippet: (A) ChIP-seq results of NCoA2 binding in the Fkbp5, Phlpp1, and Pten locus. Numbers indicate NCoA2 binding sites (top). N indicates a region without NCoA2 binding sites that served as a negative control. ChIP-PCR assays for NCoA2 binding to Fkbp5, Phlpp1, and Pten genes on pooled 7-month-old PTENPC+/– NCoA2PCOE/+ prostate samples using anti-Flag antibody (NCoA2 with Flag tag) or IgG (bottom). Bar graphs show an enrichment of DNA fragments pulled down by anti-Flag antibodies. (B) Left: ChIP-seq result of NCoA2 binding in the Map3k1 and Rac-GEF locus. Right: ChIP-PCR assays for NCoA2 binding to Map3k1 and Rac-GEF genes are as denoted. (C) Relative mRNA levels of denoted genes from PTENPC+/– and PTENPC+/– NCoA2PCOE/+ prostates (n = 6). (D–F) IHC analysis of phospho-AKT (D), FKBP5 (E) and PHLPP1 (F) expression in prostate tumors isolated from PTENPC–/– and PTENPC–/– NCoA2PC–/– mice with or without castration (6 months after castration). Student’s t test was used for all the statistical analysis. Scale bars: 50 μm (D–F). *P < 0.05; **P < 0.01.

Article Snippet: All primary antibodies used in this study were purchased from Cell Signaling, except for AR (Santa Cruz), CK8 (Covance), α-SMA (Sigma-Aldrich), NCoA2 (Bethyl Laboratories), FKBP5 (Abcam), PHLPP1 (Millipore), Rac-GEF (Millipore), E-cadherin (BD), Vimentin (BD), and β-actin (Sigma-Aldrich).

Techniques: ChIP-sequencing, Binding Assay, Negative Control, FLAG-tag, Expressing, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Androgen deprivation–induced NCoA2 promotes metastatic and castration-resistant prostate cancer

doi: 10.1172/JCI76412

Figure Lengend Snippet: (A) Kaplan-Meier plot indicates the association of NCoA2 signature with PSA recurrence and disease-specific death in patients with PCa (using dataset GSE10645; ref. 30), with the top third, middle third, and bottom third of patients representing inferred NCoA2 activities from high to low (P values by log-rank test and univariate Cox). (B) Model of NCoA2 in prostate tumorigenesis. ADT induces NCoA2 expression, which in turn activates PI3K/AKT signaling (via negative regulation of FKBP5, PHLPP1, and PTEN) and MAPK signaling (via positive regulation of MAP3K1, Rac-GEF, and others) that together promote PCa metastasis and CRPC.

Article Snippet: All primary antibodies used in this study were purchased from Cell Signaling, except for AR (Santa Cruz), CK8 (Covance), α-SMA (Sigma-Aldrich), NCoA2 (Bethyl Laboratories), FKBP5 (Abcam), PHLPP1 (Millipore), Rac-GEF (Millipore), E-cadherin (BD), Vimentin (BD), and β-actin (Sigma-Aldrich).

Techniques: Expressing