Journal: Cancer Gene Therapy

Article Title: NrCAM secreted by endometrial stromal cells enhances the progestin sensitivity of endometrial cancer cells through epigenetic modulation of PRB

doi: 10.1038/s41417-022-00467-0

Figure Lengend Snippet: A Cluster analysis of MPA-regulated genes encoding secretory proteins in ESCs. ESCs were treated with 10 μM MPA or ethanol (EtOH) for 6 h before RNA sequencing. B mRNA levels of NrCAM , BMP2 , WISP1 , and ITGA10 were significantly upregulated in ESCs after MPA treatment. Silencing PR expression with si PGR in ESCs weakened MPA-induced upregulation of these four proteins. ESCs or ESCs-si PGR were treated with or without 10 μM MPA for 6 h. Eleven candidate gene mRNA levels were reevaluated by real-time PCR. C Exogenous NrCAM inhibited EC cell proliferation in a dose-dependent manner. Ishikawa and ECC-1 cells were treated with 0, 1, 10, 100, and 1000 ng/mL NrCAM for 48 h before CCK-8 assays. D Exogenous NrCAM inhibited EC cell proliferation in a time-dependent manner. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM for 24, 48, and 72 h before CCK-8 assays. E MPA promoted NrCAM protein expression in ESCs in a dose-dependent manner. NrCAM expression was detected by western blotting. ESCs were treated with 0, 5, 10, and 20 μM MPA for 48 h. F MPA promoted NrCAM secretion in ESCs by ELISA. ESCs were treated with MPA at the indicated dose for 48 h (left) or 10 μM MPA for 24, 48, or 72 h (right). The CM extracted from ESCs was collected to measure NrCAM concentration by ELISA. G MPA-induced NrCAM protein expression was attenuated by silencing PGR in ESCs. ESCs or ESCs-si PGR were treated with 10 μM MPA for 48 h before western blotting analysis. H NrCAM and MPA cotreatment had a stronger inhibitory effect on EC cell proliferation than MPA or NrCAM alone. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM and/or 10 μM MPA for 48 h before CCK-8 assays. I Transfection efficiency of siRNAs targeting NrCAM was confirmed by real-time PCR and western blotting. J The inhibitory effect of ESCs on EC cell proliferation was blocked by silencing NrCAM expression in ESCs. After transfection with si NrCAM or siCtrl for 8 h, ESCs were treated with or without 10 μM MPA for 48 h, then for CM collection. Ishikawa and ECC-1 cells were treated with 10 μM MPA, CM (ESCs-si NrCAM -2) and CM (ESCs-si NrCAM -2 + MPA) for 48 h before CCK-8 assays. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s. not significant.

Article Snippet: Drugs used in this study included MPA (Sigma-Aldrich, St. Louis, MO, USA), recombinant human NrCAM protein (CC04, NovoProtein, Shanghai, China), recombinant human BMP2 protein (C012, NovoProtein), recombinant human WISP1 protein (10442-H08H, Sino Biological, Beijing, China), and recombinant human ITGA10 protein (5895-AB-050, R&D Systems, Minneapolis, MN, USA) at the indicated doses for the indicated periods.

Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, CCK-8 Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 3. Knockdown of WPMY-1 suppresses LIF-driven neuroendocrine differentiation (NED) and malignant progression in prostate cancer (PCa) cells (A) Relative protein levels of LIF, LIFR, WISP1, SOCS3, and PDL1 were measured in AR-positive PCa cell lines (LNCaP, C4-2, and 22Rv1), an AR-negative PCa cell line (PC3), and an NEPC cell line (LASCPC-01). (B) Relative protein levels of WISP1, phosphorylated (p)-STAT3, STAT3, PDL1, and SOCS3 were measured in LNCaP cells treated with PBS or 100 ng/mL of the LIF recombinant protein for a duration ranging 0 to 96 h. (C) Relative mRNA levels of WISP1, neuroendocrine (CHGA, SYP, and ENO2), stem cell (SOX2 and NANOG), and anti-inflammatory (SOCS3 and PDL1) markers in LNCaP cells treated with PBS or 100 ng/mL of the LIF recombinant protein or combined treatment with DMSO or 35 nM EC330 for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (D) Relative protein levels of WISP1, p-STAT3, STAT3, PDL1, and SOCS3 in LNCaP and C4-2 cells cultured in PBS or 100 ng/mL of the LIF recombinant protein, followed by DMSO or 35 nM EC330 treatment for 48 h. (E) Relative mRNA expression levels of WISP1, neuroendocrine markers, stem cell markers, and anti-inflammatory markers (SOCS3 and PDL1) were measured in LNCaP cells expressing the non-target control (NC) or WISP1 siRNA, followed by treatment with PBS or 100 ng/mL of the LIF recombinant protein for 48 h * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. (F and G) Relative cell proliferation (F) and sphere formation (G) were measured in LNCaP cells expressing either NC or WISP1 siRNA, followed by treatment with either PBS or 100 ng/mL of LIF recombinant protein for 5 days (F) or 1 week (G). Scale bars represent 100 mm (G). * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. (H and I) Relative cell migration (H) and invasion through Matrigel (I) were measured in LNCaP cells expressing the NC or WISP1 siRNA, followed by treatment with PBS or 100 ng/mL of LIF recombinant protein for 12 h. Scale bars representing 20 mm are shown. * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. Quantification of relative mRNA levels, cell proliferation, sphere formation, and cell invasion through Matrigel is presented as the mean G SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.005. (J) A GSEA of the TCGA PCa dataset revealed significant associations between high WISP1 expression in prostate tissues and a gene signature representing NEPC-responsive signaling. NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Knockdown, Recombinant, Cell Culture, Expressing, Control, Migration

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 4. Crosstalk between prostate cancer (PCa) cells and prostate stromal cells promotes neuroendocrine differentiation (NED) and expressions of immunosuppressive cytokines in the tumor microenvironment (TME) through the activation of LIF/LIFR signaling (A) Relative mRNA expression levels of WISP1, neuroendocrine markers (CHGA, SYP, and ENO2), stem cell markers (SOX2 and NANOG), anti-inflammatory markers (SOCS3 and PDL1), LIF, CXCR2, and CXCL5 in LNCaP cells. These cells were cultured with conditioned medium (CM) collected from human WPMY-1 stromal cells, at concentrations of 0%, 15%, or 50%, for a duration of 48 h * vs. 0%, as determined by a one-way ANOVA. (B) Relative mRNA expression levels of WISP1, neuroendocrine markers, stem cell markers, anti-inflammatory markers, LIF, CXCR2, and CXCL5 were measured in LNCaP cells. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMSO or 35 nM EC330 for 48 h * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO, as determined by a one-way ANOVA. (C) Relative mRNA expression levels of WISP1, neuroendocrine, stem cell, anti-inflammatory markers, LIF, CXCR2, and CXCL5 in LNCaP cells cultured with CM collected from the non-target control (Luc) or LIFR shRNA-expressing WPMY-1 cells for 48 h * vs. Veh; # vs. WPMY-1/shLuc CM, as determined by a one-way ANOVA. (D) Relative mRNA expression levels of LIFR, LIF, CXCL5, WISP1, and anti-inflammatory markers (IL10, IL4, IL1RN, TGFB1, VEGFA, IFNA17, and SOCS3) in WPMY-1 cells stably expressing the shLuc or LIFR shRNA. * vs. shLuc, as determined by a one-way ANOVA. (E) Relative mRNA expression levels of LIFR, LIF, CXCL5, WISP1, and anti-inflammatory markers in WPMY-1 cells treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by DMSO or 35 nM EC330 treatment for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (F and G) Cell proliferation (F) and sphere formation (G) of LNCaP cells were evaluated. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMOS or 35 nM EC330 for 48 h. Scale bars in (G) represent 100 mm. Statistical comparisons were performed using a one-way ANOVA. * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO. (H and I) Relative cell migration (H) and invasion through Matrigel (I) were measured in LNCaP cells. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMSO or 35 nM EC330 for 12 h. Scale bars representing 20 mm are shown. * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO, as determined by a one-way ANOVA. Quantification of relative mRNA levels, cell proliferation, sphere formation, and cell invasion through Matrigel is presented as the mean G SEM, based on three biological replicates. Significance levels are denoted as *p < 0.05, **p < 0.01, and ***p < 0.005.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Cell Culture, Control, shRNA, Stable Transfection, Recombinant, Migration

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 5. LIF/STAT3-driven transcription of WISP1 through direct binding to the gamma interferon activation site (GAS) of the regulatory sequence (A) ChIP-sequencing analysis was performed to detect the GAS for WISP1. Detected GAS sites are labeled as black boxes in the tracks. ChIP-sequencing data were downloaded from Gene Expression Omnibus (GEO) (GSM2752900) and analyzed using the Genome Browser (Genomics Institute, UCSC). (B) A schematic representation is shown for the predicted wild-type (WT) and mutant (M)-GASs in the regulatory sequence reporter constructs of the human WISP1 gene (GRCh38:8) (C and D) A ChIP assay was performed to show the binding of phosphorylated (p)-STAT3 to the predicted GAS in the WISP1 gene regulatory sequence. The assay was conducted in LNCaP cells (C) or WPMY-1 cells (D) treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by treatment with DMSO or with 10 or 35 nM EC330 for 48 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to p-STAT3 or control IgG, and predictive primers (B, indicated by black arrows) were used to quantify the precipitated DNA using a qPCR. Enrichment of each protein at each site is presented as a percentage of the total input and then normalized to IgG. * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (E) A ChIP assay was performed to demonstrate the binding of p-STAT3 to the predicted GAS in the regulatory sequence of the LIF gene in WPMY-1 cells. Cells were treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by treatment with DMSO or with 10 or 35 nM EC330 for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (F–I) The relative mean fluorescence intensity (MFI) of the GFP reporter gene containing either the WT-GAS or M-GAS from the WISP1 regulatory sequence was measured in LNCaP cells (F and G) and WPMY-1 cells (H and I) after treatment with PBS or 100 ng/mL of LIF recombinant protein. This was followed by further treatment with either DMSO or EC330 (10 or 35 nM) for 48 h (F and H). * vs. WT/PBS+DMSO (F and H) or WT + PBS (G and I); # vs. WT/LIF+DMSO (F and H) or WT + LIF (G and I), as determined by a one-way ANOVA. (J and K) The relative MFI of the GFP reporter gene containing the WT-GAS or M-GAS from the LIF regulatory sequence was measured in WPMY-1 cells treated with PBS or 100 ng/mL of LIF recombinant protein or combined treatment with DMSO or 10 or 35 nM EC330 for 48 h * vs. WT/PBS+DMSO (J) or WT + PBS (K); # vs. WT/LIF+DMSO (J) or WT + LIF (K), as determined by a one-way ANOVA. Quantification of relative p-STAT3 enrichment and MFI is presented as the mean G SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Binding Assay, Activation Assay, Sequencing, ChIP-sequencing, Labeling, Gene Expression, Mutagenesis, Construct, Recombinant, Control

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 6. WISP1 abundance in serum relative to prostate cancer (PCa) progression (A) Relative mRNA levels of CXCL5, CXCR2, LIF, WISP1, neuroendocrine (CHGA, SYP, and ENO2), and stem cell (SOX2 and NANOG) markers in C4-2 cells expressing the empty vector (EV) or CXCL5-expressing vector, followed by treatment with DMSO or 35 nM EC330 for 48 h * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a one-way ANOVA. (B and C) Tumor growth analysis was conducted by subcutaneously inoculating male nude mice with C4-2 cells expressing either the EV or a CXCL5-expressing vector. The mice were then treated bi-daily with either DMSO or 2.5 mg/kg EC330 via intraperitoneal injection and allowed to grow for 8 weeks. Tumor sizes were measured weekly (B). Tumor weights were measured upon tumor collection (C). n = 5 per group. * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a one- way ANOVA and t-test. (D and E) Immunohistochemical (IHC) staining and intensity analyses were performed to assess protein levels of CXCL5, CXCR2, LIF, WISP1, and ENO2 in subcutaneous tumors derived from (B). * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a two-tailed Student’s t-test. Scale bars, 100 mm. (F) WISP1 concentrations were measured in patient sera derived from samples of benign prostatic hyperplasia (BPH; n = 10), hormone-sensitive PCa (HSPC, n = 10), and metastatic castration-resistant PCa (mCRPC; n = 8). * vs. BPH; # vs. HSPC, analyzed by a one-way ANOVA.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Immunohistochemistry, Derivative Assay, Two Tailed Test