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: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 expression is increased in melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. A, Comparison of WISP1 mRNA expression in benign skin conditions (normal skin and benign melanocytic skin nevus) to primary melanoma. Original expression profiles were from . P-values calculated using ANOVA with post-hoc Tukey HSD 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 a normal skin (left) and two melanoma (right) tissue samples (original tissue microarray images were obtained from www.proteinatlas.org ) . Deconvoluted intensity of WISP1 staining is shown in red while cellular structures stained using hematoxylin are shown in blue. Arrows indicate melanocytes in epidermis and arrowheads indicate fibroblasts in dermis (stroma). C, The average WISP1 staining within normal skin and primary melanoma tissue samples. D, Distributions in non-zero pixel intensity values of WISP1 staining for normal skin (black curves) and primary melanoma (red curves) tissue samples. Numbers indicate the percentage of the distribution that have normalized pixel intensity values greater than 0.2. E, Kaplan-Meier estimate of overall survival of patients diagnosed with primary melanoma stratified by WISP1 transcript abundance (data from TCGA). Sample numbers and p-values calculated using the Peto & 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. indicates p-value > 0.05).

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A, 48-hour 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 left, colony counts is 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 hours. D, Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on left, 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 concentration 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 concentration 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 a p-value < 0.05 was considered significant and asterisks was used to indicate calculated range in p-values. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; and ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Real time genomic qPCR revealed that Wisp1 knockout repressed the spontaneous metastasis of melanoma cell line B16F10 in C57BL/6Ncrl mice. Growth of tumors derived from B16F10 or its knockout cell (-KO2) were monitored following subcutaneous injection in NSG mice (A: B16F10 (n = 5) or WISP1 KO cell (n = 5)) or in C57BL/6Ncrl mice (B: B16F10 (n=6) or WISP1 KO cell (n=6)). After 21 days, remaining C57BL/6crl mice (n = 4 in each group) were euthanized and lungs and livers were assayed for B16F10 tumor cells using real time genomic qPCR, as described in Materials and Methods. (C) Representative lungs and livers from C57BL/6Ncrl mice with B16F10 or knockout cell at day 21. (D) Real time genomic qPCR results showed quantitative tumor lung and liver metastatic burden in spontaneous metastasis assays, n.d., not detected.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Derivative Assay, Injection

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

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 indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (N=5) and three representative images were shown. These experiments were repeated and similar results were achieved. A, Bioluminescence imaging performed one day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B-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 one 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 ). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Injection, Imaging

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

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 indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (N=5) and two representative images were shown. A, Bioluminescence imaging performed one 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 were plotted and compared using bioluminescence intensity detected in panel (A). Total flux is presented as photon/second (p/s). E, Bioluminescence imaging performed one 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, The whole-body metastasis of tumor cells in C57BL/6Ncrl mice were plotted and compared using bioluminescence intensity detected in panel (E). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Injection, Imaging

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

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 hours before harvested for RNA analysis or treated with 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 Boyden transwell invasion assay. B, Immunoblot analysis of Wisp1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20μg of whole cells lysate was load in each lane and P-actin was used as internal loading control. B16F10-KO1-mWisp1 cell, in which mouse Wisp1 expression was resumed with retroviral transduction, was used as a positive control. C, Immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20μg of whole cells lysate was load 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 5μg/ml) and harvested at 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. The conditioned media were pre-treated with indicated antibodies for 30 minutes before used on Wisp1-knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 hour treatment. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Snai1 overexpression in B16F10 Wisp1-knockout cell rescued the repression on 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 hours before 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 left, relative invasion efficiency is graphed on the right. D, Experimental metastasis assay in NSG mice using indicated cells. Each group contained 3-4 mice. All mice were imaged one day before the end of the assay and representative bioluminescence images were shown. E, Representative lung and liver images from NSG mice in experimental metastasis assay described in panel ( D ). Metastatic tumor colonies on lung surface from mice with (-mWisp1) or (-hSnai1) cells were pointed by arrows. F, Real time genomic qPCR for lungs and livers from experimental metastasis assay in panel ( D ). The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Wisp1 activated Akt/MAPK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis maintained for 30 minutes before cells were lysed for protein extraction while cells were treated for 3 hours prior to RNA extraction for comparing EMT marker gene expression. A, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in B16F10 cells. Cells were treated with specific phospho-Akt inhibitor MK-2206 and/or phospho-MAPK inhibitor U0126. Immunoblot for phospho-Akt and phospho-Erk1/2 inhibition was shown on the right upper corner. Pan-Akt and total Erk1/2 were also probed as loading control. B, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in YUMM1.7 cells, with DMSO as control. C, Immunoblot for phospho-Akt and phospho-Erk1/2 in indicated mouse melanoma cells with treatment of recombinant mouse Wisp1 protein (rmWisp1, final 5μg/ml). Cells grown on 6-well plates in complete DMEM for 48 hours and serum-free DMEM (SFM) for another 48 hours before rmWISP1 was added. Pan-Akt and total Erk1/2 were probed as loading control. D, Immunoblot analysis of Akt/MAPK activation in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were grown on 6-well plates in complete DMEM for 48 hours (0 hour point for SFM) and switched to SFM for 24 hour or 48 hours. Indicated cells were treated with rmWisp1 for 30 minutes following 0, 24, or 48 hours in SFM before analyzed for kinase activation. The first lane loaded with YUMM 1.7 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, Immunoblot for Akt/MAPK activation in YUMM1.7 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D). The first lane loaded with B16F10 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. F Snai1 activation and E-cadherin repression in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D) except that rmWisp1 treatment at each point maintained for 3 hours. G-H, EMT marker gene expression after Akt/MAPK activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWisp1 was blocked by Akt/MAPK inhibitors. rmWisp1 with DMSO or inhibitors was added after indicated cells were grown on 6-well plates in complete DMEM for 48 hours and in SFM for 24 hours. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Marker, Expressing, Western Blot, Protein Extraction, RNA Extraction, Inhibition, Recombinant, Activation 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