Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Osteoblast‐derived ECM1 induces PCa cell resistance to ENZ. A) Transwell system model (top) of C4‐2B cells with mouse‐derived osteoblasts (MC3T3‐E1), and cell proliferation of C4‐2B cells in Transwell system, cultured alone or co‐cultured with osteoblasts in the presence of ENZ (10 µM) on day 7, compared to untreated cells (bottom). B) Schematic diagram of CM from mouse‐derived osteoblasts treated with ENZ for 72 hours added to C4‐2B cells (top); Cell proliferation of C4‐2B cells cultured alone or with CM from ENZ‐treated osteoblasts in the presence of ENZ (10 µM) on day 7, compared to untreated cells (bottom). Medium Control: DMEM. C) Cell proliferation of C4‐2B cells on day 7 supplemented with either control or heat‐inactivated CM (left), and with the addition of CM treated with either proteinase K (200 µg mL −1 ) or inactivated proteinase K (right). D) Schematic diagram showing the identification of highly abundant, secreted proteins ECM1, LGALS3BP, and HMGB1 from the supernatant of human or mouse osteoblasts treated with ENZ (10 µM) or Veh (DMSO) for 72 h, followed by LC‐MS/MS analysis. E) Representative images (left) and quantification (right) of surviving colonies formed by C4‐2B and LNCaP cells treated with ENZ (10 µM) with the addition of either PBS, ECM1 (200 ng mL −1 ), LGALS3BP (200 ng mL −1 ), or HMGB1 (200 ng mL −1 ) protein, compared to untreated cells. F) Flow cytometry analysis showing representative images (left) and quantification (right) of apoptosis in C4‐2B and LNCaP cells treated as grouped in E. G) Cell proliferation on day 7 of either the indicated C4‐2B cells cultured with osteoblasts in the Transwell system, or C4‐2B cells cultured alone, in the presence of ENZ (10 µM), compared to untreated cells. H) Cell proliferation on day 7 of C4‐2B cells cultured alone or cultured with osteoblasts in the Transwell system in the presence of ENZ (10 µM), with the addition of either IgG or ECM1 antibody, compared to untreated cells. I) Representative BLI of intratibial tumors in mice treated daily with oral Veh, ENZ (20 mg kg −1 ), ENZ (20 mg kg −1 ) combined with tail vein injection of either IgG or ECM1 pAb (50 mg kg −1 ) twice weekly at 4 weeks before treatment, and at weeks 0, 6, and 8 during treatment (n = 6 per group). J) Quantification of BLI signals in intratibial tumors of mice before and after Tx as grouped in I (n = 6 per group). K) Representative micro‐CT images of intratibial lesions from mice after 8 weeks of treatment grouped as shown in I (n = 6 per group). L) Representative H&E images of intratibial tumors grouped as shown in I (T, tumor; N, the adjacent non‐tumor tissues. Scale bars, 500 µm and 100 µm). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Derivative Assay, Cell Culture, Control, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry, Injection, Micro-CT

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: ECM1 activates the MAPK signaling pathway in PCa cells. A) Waterfall plot showing differentially expressed genes (p < 0.05, log2 fold change>1.5) in C4‐2B cells treated with ENZ (10 µM, 48 h) combined with ECM1 protein (200 ng mL −1 , 48 h) or ENZ (10 µM, 48 h) alone. The highlighted genes were related to proliferation and apoptosis. B) KEGG analysis of pathways enriched in ENZ combined with ECM1 protein treatment group compared to the ENZ treatment group. C) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated groups of C4‐2B cells. D) IHC staining and quantification of p‐ERK1/2 expression in mice intratibial and subcutaneous tumors grouped as indicated (Scale bars, 500 µm and 100 µm, n = 6 per group). E) WB analysis and quantification of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in C4‐2B cells stimulated with ENZ (10 µM) combined with ECM1 (200 ng mL −1 ) in the presence of inhibitors for either RAS (MCP110, 10 µM), MEK (U0126, 10 µM), ERK1/2 (Ulixertinib, 10 µM), EGFR (Lapatinib, 10 µM), FGFR1 (Fexagratinib, 10 µM), IGF1R (Linsitinib, 10 µM) or Veh (DMSO), compared to ENZ‐treated alone or untreated C4‐2B cells. F) C4‐2B cell proliferation on day 7 of groups as shown in E. G) WB analysis and quantification of EGFR, p‐EGFR, FGFR1, p‐FGFR1, IGF1R and p‐IGF1R expression in groups as indicated. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Expressing, Immunohistochemistry

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: ECM1 recruits GRB2 and SOS1 to the membrane to activate the MAPK signaling pathway. A) Affinity purification MS analysis of ECM1 interaction complexes in C4‐2B cells (left). Representative mass spectra of GRB2 and SOS1 peptides (right). B) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in C4‐2B cells with ECM1 (200 ng mL −1 ) treatment. C) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with Veh (PBS) or ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). D,E) WB analysis of GRB2 and SOS1 protein expression in whole lysis (WL) and membrane proteins from C4‐2B cells treated with increasing concentrations of ECM1 (0, 200, 400, 800 ng mL −1 ), or with the addition of either DMEM or CM. GAPDH was used as a loading control for whole lysis, and PMCA1 for membrane proteins. F) IP detection of the interaction between ECM1 and GRB2 as well as SOS1 in C4‐2B cells treated with CM. G) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with DMEM or CM. Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). H,I) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated C4‐2B cells treated with or without ECM1 (200 ng mL −1 ). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Membrane, Affinity Purification, Staining, Expressing, Lysis, Control

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Phosphorylated ENO1 bridges ECM1 with GRB2 and SOS1 at the membrane. A) Representative mass spectra of ENO1 peptide. B) IP analysis detected the interaction between ECM1 and ENO1 in C4‐2B cells. C) IF staining and quantification of ENO1 and ECM1 in C4‐2B cells. Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). D) Proximity ligation assay (PLA) analysis of the interaction between ECM1‐Flag and ENO1 (Scale bar, 10 µm). E) IP assays of the interaction between ENO1 and GRB2 as well as SOS1 in C4‐2B cells in the presence or absence of ECM1 (200 ng mL −1 ). F) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in the indicated C4‐2B cells treated with ECM1 (200 ng mL −1 ). G) Expression of GRB2 and SOS1 protein in whole lysis and membrane proteins from the indicated C4‐2B cells with ECM1 (200 ng mL −1 ) treatment by WB analysis. H) IF staining and quantification of GRB2, SOS1, and DiI in the indicated C4‐2B cells supplemented with ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). I) Representative peptides showing phosphorylation of ENO1 at the Y189 site by affinity purification MS analysis of protein modifications in ECM1‐treated C4‐2B cells. J) Tyr phosphorylation detection of the immunoprecipitated ENO1 in indicated C4‐2B and LNCap cells with ECM1 treatment (200 ng mL −1 ). K) Tyr phosphorylation levels of HA‐ENO1‐WT and ‐Y189F in C4‐2B cells with or without ECM1 (200 ng mL −1 ) treatment. L) IF staining and quantification of GRB2, SOS1, and DiI in the indicated C4‐2B cells supplemented with ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). M,N) IP assays of the interaction between ECM1 and ENO1, GRB2 as well as SOS1 in the indicated C4‐2B cells in the presence of ECM1 (200 ng mL −1 ). O) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated C4‐2B cells treated with or without ECM1 (200 ng mL −1 ). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Membrane, Staining, Proximity Ligation Assay, Expressing, Lysis, Phospho-proteomics, Affinity Purification, Immunoprecipitation

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: The phosphorylation of ENO1 is indispensable for ENZ resistance. A) Representative images (left) and quantification (right) of surviving colonies formed by the indicated C4‐2B and LNCaP cells treated with ENZ (10 µM) and ECM1 (200 ng mL −1 ). B) Flow cytometry analysis showing representative images (left) and quantification (right) of apoptosis in indicated C4‐2B and LNCaP cells after ENZ (10 µM) and ECM1 (200 ng mL −1 ) treatment. C,D) Representative BLI and quantification of intratibial tumors formed by the indicated C4‐2B cells in mice treated daily oral treatment with ENZ (20 mg kg −1 ) at 4 weeks before treatment, and at weeks 0, 6, and 8 during treatment (n = 6 per group). E) Representative micro‐CT images of intratibial lesions from mice after 8 weeks of treatment grouped as shown in C (arrows and circles indicate osteoblastic lesions, n = 6 per group). F) Representative H&E images of intratibial tumors grouped as shown in C (T, tumor; N, the adjacent non‐tumor tissues. Scale bars, 500 µm and 100 µm). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Phospho-proteomics, Flow Cytometry, Micro-CT

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: PhAH attenuates ENO1‐mediated PCa cell resistance to ENZ. A) Chemical structure of PhAH. B) Ligand interaction diagram (left) and binding amino acid residue sites (right) of the highest‐scoring PhAH and ENO1 protein molecular docking complex (RMSD = 1.9977; E_score = −3.9538). C) WB detection of the interaction between ECM1 and ENO1, as well as Tyr phosphorylation of ENO1 following immunoprecipitating ENO1 in C4‐2B cells in the presence of ECM1 (200 ng mL −1 ) with or without PhAH (1 µM). D) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). E) Representative images (top) and quantification (bottom) of surviving colonies formed by C4‐2B and LNCaP cells treated with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM). F) Flow cytometry analysis showing representative images (top) and quantification (bottom) of apoptosis in C4‐2B and LNCaP cells with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM) treatment. G) Representative BLI of intratibial tumors in mice treated daily oral treatment with Veh, ENZ (20 mg kg −1 ), ENZ (20 mg kg −1 ) combined with intraperitoneal (i.p.) injection of either Veh (DMSO) or PhAH (5 mg kg −1 ) twice weekly at 4 weeks before treatment, and at weeks 0, 6, and 8 during treatment (n = 6 per group). H) Quantification of BLI signals in intratibial tumors of mice before and after Tx as grouped in G (n = 6 per group). I) Representative micro‐CT images of intratibial lesions from mice after 8 weeks of treatment grouped as shown in G (arrows and circles indicate osteoblastic lesions, n = 6 per group). J) Representative H&E images of intratibial tumors grouped as shown in G (T, tumor; N, the adjacent non‐tumor tissues. Scale bars, 500 µm and 100 µm). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Binding Assay, Residue, Phospho-proteomics, Staining, Flow Cytometry, Injection, Micro-CT

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Validation of ECM1/ENO1/MAPK signaling axis in patients with bmCRPC. A) Representative images (left) of PCa PDOs treated with Veh (PBS), ECM1 (200 ng mL −1 ), or ECM1 (200 ng mL −1 ) combined with either DMSO or PhAH (1 µM) in the presence of ENZ (10 µM). B) Assessment of PDOs growth by changes in organoid diameter (right; Scale bar, 50 µm). C) IHC staining of ALP, ECM1, ENO1, and p‐ERK1/2 in ENZ‐untreated (n = 12) or ENZ‐treated (n = 22) bmCRPC patients tissue (Scale bars, 250 µm and 50 µm). D‐G) Staining index quantification of ALP, ECM1, p‐ERK1/2 expression and relative ENO1 membrane/cytoplasm cell number as indicated in C. H) Percentage of specimens showing ENZ‐untreated or treated in relation to the expression levels of ALP, ECM1, p‐ERK1/2 and the membrane or cytoplasm level of ENO1. I‐K) Spearman correlation analysis between the expression levels of ALP, relative ENO1 membrane/cytoplasm cell number, and p‐ERK1/2 with ECM1 expression. L) Representative mIHC staining images of ALP (red, osteoblast marker), EPCAM (yellow, PCa marker), and ENO1 (green) in human bmCRPC tissues (Scale bars, 100 µm and 25 µm). M) Quantification of ALP, EPCAM, and relative ENO1 membrane/cytoplasm intensity per cell in L). N) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in prostate cancer cells extracted from human bmCRPC tissue with or without ECM1 treatment (200 ng mL −1 ). GAPDH was used as the loading control. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Biomarker Discovery, Immunohistochemistry, Staining, Expressing, Membrane, Marker, Control

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Schematic diagram illustrating that increased osteoblast‐derived ECM1 from the bone microenvironment of BMPC patients induced by ENZ treatment, interacts with the ENO1 receptor on the prostate cancer cell membrane, further recruiting adapter proteins including GRB2 and SOS1, which activates the downstream MAPK signaling pathway to promote the proliferation of PCa cells and induce anti‐androgen resistance.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Derivative Assay, Membrane

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Osteoblast-Derived ECM1 Promotes Anti-Androgen Resistance in Bone Metastatic Prostate Cancer.

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Figure 4. ECM1 recruits GRB2 and SOS1 to the membrane to activate the MAPK signaling pathway. A) Affinity purification MS analysis of ECM1 interaction complexes in C4-2B cells (left). Representative mass spectra of GRB2 and SOS1 peptides (right). B) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in C4-2B cells with ECM1 (200 ng mL−1) treatment. C) IF staining and quantification of GRB2, SOS1, and DiI in C4-2B cells treated with Veh (PBS) or ECM1 (200 ng mL−1). Pearson R value greater than 0.5 indicated co-localization of the two proteins (Scale bar, 5 μm). D,E) WB analysis of GRB2 and SOS1 protein expression in whole lysis (WL) and membrane proteins from C4-2B cells treated with increasing concentrations of ECM1 (0, 200, 400, 800 ng mL−1), or with the addition of either DMEM or CM. GAPDH was used as a loading control for whole lysis, and PMCA1 for membrane proteins. F) IP detection of the interaction between ECM1 and GRB2 as well as SOS1 in C4-2B cells treated with CM. G) IF staining and quantification of GRB2, SOS1, and DiI in C4-2B cells treated with DMEM or CM. Pearson R value greater than 0.5 indicated co-localization of the two proteins (Scale bar, 5 μm). H,I) WB analysis of MEK, p-MEK, ERK1/2, and p-ERK1/2 expression in the indicated C4-2B cells treated with or without ECM1 (200 ng mL−1). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521-1-AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5-47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254-2-AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041-1-AP, 1:100).

Techniques: Membrane, Staining, Expressing, Lysis, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Osteoblast-Derived ECM1 Promotes Anti-Androgen Resistance in Bone Metastatic Prostate Cancer.

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Figure 5. Phosphorylated ENO1 bridges ECM1 with GRB2 and SOS1 at the membrane. A) Representative mass spectra of ENO1 peptide. B) IP analysis detected the interaction between ECM1 and ENO1 in C4-2B cells. C) IF staining and quantification of ENO1 and ECM1 in C4-2B cells. Pearson R value greater than 0.5 indicated co-localization of the two proteins (Scale bar, 5 μm). D) Proximity ligation assay (PLA) analysis of the interaction between ECM1-Flag and ENO1 (Scale bar, 10 μm). E) IP assays of the interaction between ENO1 and GRB2 as well as SOS1 in C4-2B cells in the presence or absence of ECM1 (200 ng mL−1). F) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in the indicated C4-2B cells

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521-1-AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5-47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254-2-AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041-1-AP, 1:100).

Techniques: Membrane, Staining, Proximity Ligation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Osteoblast-Derived ECM1 Promotes Anti-Androgen Resistance in Bone Metastatic Prostate Cancer.

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Figure 7. PhAH attenuates ENO1-mediated PCa cell resistance to ENZ. A) Chemical structure of PhAH. B) Ligand interaction diagram (left) and binding amino acid residue sites (right) of the highest-scoring PhAH and ENO1 protein molecular docking complex (RMSD = 1.9977; E_score = −3.9538). C) WB detection of the interaction between ECM1 and ENO1, as well as Tyr phosphorylation of ENO1 following immunoprecipitating ENO1 in C4-2B cells in the presence of ECM1 (200 ng mL−1) with or without PhAH (1 μM). D) IF staining and quantification of GRB2, SOS1, and DiI in C4-2B cells

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521-1-AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5-47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254-2-AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041-1-AP, 1:100).

Techniques: Binding Assay, Residue, Phospho-proteomics, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Osteoblast-Derived ECM1 Promotes Anti-Androgen Resistance in Bone Metastatic Prostate Cancer.

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Figure 9. Schematic diagram illustrating that increased osteoblast-derived ECM1 from the bone microenvironment of BMPC patients induced by ENZ treatment, interacts with the ENO1 receptor on the prostate cancer cell membrane, further recruiting adapter proteins including GRB2 and SOS1, which activates the downstream MAPK signaling pathway to promote the proliferation of PCa cells and induce anti-androgen resistance.

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521-1-AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5-47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254-2-AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041-1-AP, 1:100).

Techniques: Derivative Assay, Membrane