Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 expression is increased in melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. A, WISP1 mRNA expression in benign skin conditions (normal skin and benign melanocytic skin nevus) compared with primary melanoma. The original expression data set (GSE3189) was deposited by Talantov et al. (55). p values were calculated using analysis of variance with the post hoc Tukey honest significant difference test. B, representative original and deconvoluted color images derived from human normal skin and melanoma tissue microarray probed using a WISP1 antibody (HPA007121) and imaged using 3,3′-diaminobenzidine and stained using hematoxylin for normal skin (left) and two melanoma (right) tissue samples. Original tissue microarray images were obtained from www.proteinatlas.org3 (77). Deconvoluted intensity of WISP1 staining is shown in red, whereas the cellular structures stained using hematoxylin are shown in blue. Arrows, melanocytes in epidermis; arrowheads, fibroblasts in dermis (stroma). C, average WISP1 staining within normal skin and primary melanoma tissue samples. D, distributions in nonzero pixel intensity values of WISP1 staining for normal skin (black curves) and primary melanoma (red curves) tissue samples. Numbers, percentage of the distribution that has pixel intensity values greater than a normalized pixel intensity of 0.2. E, Kaplan–Meier estimate of overall survival of melanoma patients stratified by WISP1 transcript abundance. The original data set was from the Cancer Genome Atlas. Sample numbers and p values calculated using the Peto and Peto modification of the Gehan–Wilcoxon test are indicated. F, patient population characteristics of WISP1 high and WISP1 low groups. Statistical differences among categorical data and age were assessed using Fisher's exact test and Student's t test, respectively (n.s., p > 0.05).

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Expressing, Derivative Assay, Microarray, Staining, Modification

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A, 48-h 2D growth of mouse metastatic melanoma cell line B16F10 and two B16F10 Wisp1-knockout cells (-KO1 and -KO2). B, anchorage-independent growth assay of B16F10 and the two knockout cells in soft agar. Colonies were fixed and counted after 14 days. A representative staining image for each sample is shown on the left, and colony counts are plotted on the right. C, wound healing assay of B16F10 and the two knockout cells. Scratches were created on 6-well plates in biological triplicate, and the healing rate was calculated after 24 h. D, Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on the left, and relative migration efficiency is graphed on the right. E, Boyden transwell invasion assay of B16F10 and the two knockout cells. F, Boyden transwell invasion assay of human metastatic melanoma cell line RPMI-7951 and its two WISP1-knockout cells (-KO1 and -KO2). G, transwell migration assay of B16F10 and its knockout cell (-KO1) using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 migrated cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative migration efficiency and compared with other cells. H, transwell invasion assay of B16F10 and the two knockout cells using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 invaded cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative invasion efficiency and compared with other cells. Statistical significance was determined by Student's t test, where p < 0.05 was considered significant, and asterisks were used to indicate calculated range in p values. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Knock-Out, Migration, Growth Assay, Staining, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: Wisp1 knockout repressed the experimental metastasis of melanoma cell line B16F10 in immunodeficient NSG mice and immunocompetent C57BL/6Ncrl mice. Experimental metastasis assays were performed in NSG mice (A–F) and C57BL/6Ncrl mice (G–I) using B16F10 and the indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (n = 5), and only mice surviving the whole experiments were analyzed at the same time for imaging, photography, and qPCR (final n ≥ 3). These experiments were repeated, and similar results were achieved. A, bioluminescence imaging performed 1 day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B and C, tumor lung metastases (black colonies) of NSG mice as captured by photography (B) and real-time genomic qPCR (C). Quantitative tumor lung metastatic burden was assayed and presented as tumor cell number within 10,000 mouse tissue cells. D–E, tumor liver metastases (black and white nodules) of NSG mice as captured by photography (D) and real-time genomic qPCR (E). Quantitative tumor liver metastatic burden was assayed and presented as tumor cell number within 10,000 mouse tissue cells. F, tumor kidney metastases (black colonies) of NSG mice as captured by photography. G, bioluminescence imaging performed 1 day before C57BL/6Ncrl mice were euthanized. All animals were compared with the same bioluminescence scale. H–I, tumor lung metastases of C57BL/6Ncrl mice as captured by photography (H) and real-time genomic qPCR (I). Four high-resolution images for B, D, F, and H are provided as Figs. S1–S4. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Knock-Out, Injection, Imaging

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: Wisp1 knockout repressed the experimental metastasis of melanoma cell line YUMM1.7 in NSG and C57BL/6Ncrl mice. Experimental metastasis assays were performed in NSG (A–D) and C57BL/6Ncrl (E–H) mice using YUMM1.7 and the indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (n = 5), and two representative images are shown. A, bioluminescence imaging performed 1 day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B, tumor lung metastases (white nodules) of NSG mice as captured by photography. C, real-time genomic qPCR quantitatively comparing tumor lung metastatic burdens (tumor cell number within 10,000 mouse tissue cells). D, the whole-body metastasis of tumor cells in NSG mice was plotted and compared using bioluminescence intensity detected in A. Total flux is presented as photons/s (p/s). E, bioluminescence imaging performed 1 day before C57BL/6Ncrl mice were euthanized. All animals were compared with the same bioluminescence scale. F, tumor lung metastases (white nodules) of C57BL/6Ncrl mice as captured by photography. G, real-time genomic qPCR quantitatively comparing tumor lung metastatic burdens. H, whole-body metastasis of tumor cells in C57BL/6Ncrl mice was plotted and compared using bioluminescence intensity detected in E. Two high-resolution images for B and F are provided as Figs. S7 and S8. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Knock-Out, Injection, Imaging

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis or treated with the indicated conditioned medium or recombinant protein. A, mRNA expression, revealed by real-time quantitative RT-PCR, of select EMT marker genes and Mitf in uninvaded and invaded B16F10 cells from a Boyden transwell invasion assay. B, immunoblot analysis of WISP1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and β-actin was used as an internal loading control. B16F10-KO1-mWisp1 cells, in which mouse WISP1 expression was resumed with retroviral transduction, were used as a positive control. C, immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and all cells were compared on the same gel to reveal the relative intensity of each protein. D, comparison of EMT marker gene expression in mouse melanoma B16F10 and its two Wisp1-knockout cells (-KO1 and -KO2). E, comparison of EMT marker gene expression in mouse melanoma YUMM1.7 and its two Wisp1-knockout cells (-KO1 and -KO2). F, comparison of EMT marker gene expression in human melanoma RPMI-7951 and its two WISP1-knockout cells (-KO1 and -KO2). G, stimulation of EMT marker gene expression with recombinant mouse WISP1 protein (rmWISP1). B16F10-KO1 cells were treated with rmWISP1 (final concentration 5 μg/ml) and harvested at the indicated time point for real-time quantitative RT-PCR analysis. H, stimulation of EMT marker gene expression with WISP1-overexpressed or WISP1-immunodepleted conditioned medium (CM). The conditioned media were pretreated with the indicated antibodies for 30 min before they were used on Wisp1-knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 h of treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Marker, Transwell Invasion Assay, Western Blot, Disruption, Knock-Out, Control, Retroviral, Transduction, Positive Control, Comparison, Gene Expression, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: SNAI1 overexpression in B16F10 Wisp1-knockout cell rescued the repression of tumor invasion in vitro and metastasis in vivo. A, immunoblot analysis of WISP1 and SNAI1 using B16F10-KO1 cell that were transduced with retroviral vector control (-pBabe) or retrovirus expressing either mouse WISP1 (-mWisp1) or human SNAI1 (-hSnai1). B, comparison of EMT marker gene expression after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. Cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis. C, Boyden transwell invasion assay after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. A representative staining image for each sample is shown on the left, and relative invasion efficiency is graphed on the right. D, experimental metastasis assay in NSG mice using the indicated cells. Each group contained 3–4 mice. All mice were imaged 1 day before the end of the assay, and representative bioluminescence images are shown. E, representative lung and liver images from NSG mice in the experimental metastasis assay described in D. Metastatic tumor colonies on the lung surface from mice with -mWisp1 or -hSnai1 cells are indicated by arrows. F, real-time genomic qPCR for lungs and livers from the experimental metastasis assay in D. The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. A high-resolution image for E is provided as Fig. S9. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Over Expression, Knock-Out, In Vitro, In Vivo, Western Blot, Transduction, Retroviral, Plasmid Preparation, Control, Expressing, Comparison, Marker, Gene Expression, Transwell Invasion Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 activated AKT and MEK/ERK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis was maintained for 30 min before cells were lysed for protein extraction, whereas cell treatment for comparison of EMT marker gene expression was maintained for 3 h before cells were harvested for RNA extraction. A, comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in B16F10 cells. DMSO was used for control cells. Immunoblotting for phospho-AKT (pAKT) and phospho-ERK1/2 (pERK1/2) is shown in the top right corner. Pan-AKT and total ERK1/2 were probed as loading control. B, comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in YUMM1.7 cells. C, immunoblot analysis of AKT and ERK1/2 activation in the indicated mouse melanoma cells with treatment of recombinant mouse WISP1 protein (rmWISP1; final concentration 5 μg/ml). All cells were grown on 6-well plates in complete DMEM for 48 h and SFM for another 48 h before rmWISP1 was added. D, immunoblot analysis of AKT and ERK1/2 activation in B16F10 knockout cell (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were grown on 6-well plates in complete DMEM for 48 h (0-h point for SFM) and switched to SFM for 24 or 48 h. The indicated cells were treated with rmWISP1 at the 0-, 24-, and 48-h time points (of SFM) for 30 min before they were lysed for kinase analysis. The first lane on the gels was loaded with YUMM1.7 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, immunoblot analysis of AKT and ERK1/2 activation in YUMM1.7 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D. The first lane on the gels was loaded with B16F10 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. F, comparison of SNAI11 activation and E-cadherin repression in B16F10 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D except that rmWISP1 treatment at each point was maintained for 3 h. G and H, comparison of EMT marker gene expression after AKT/ERK1/2 activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWISP1 was blocked. rmWISP1 with DMSO or inhibitors was added after the indicated cells were grown on 6-well plates in complete DMEM for 48 h and in SFM for 24 h. The relative protein levels of pAKT and AKT and of pERK1/2 and ERK1/2 in C–E were measured, and they are listed in Table S3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Marker, Gene Expression, Western Blot, Protein Extraction, Comparison, RNA Extraction, Inhibition, Control, Activation Assay, Recombinant, Concentration Assay, Knock-Out

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