CHI3L1 results in poor outcome of ovarian cancer by promoting properties of stem-like cells

in Endocrine-Related Cancer
Correspondence should be addressed to W-F Cheng: wenfangcheng@yahoo.com
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The role of chitinase-3-like protein 1 (CHI3L1) in ovarian cancer and the possible mechanisms were elucidated. CHI3L1 is a secreted glycoprotein and associated with inflammation, fibrosis, asthma, extracellular tissue remodeling and solid tumors. Our previous study showed CHI3L1 could be a potential prognostic biomarker for epithelial ovarian cancer and could protect cancer cells from apoptosis. Therefore, clinical data and quantitation of CHI3L1 of ovarian cancer patients, tumor spheroid formation, side-population assays, Aldefluor and apoptotic assays, ELISA, RT-PCR, immunoblotting and animal experiments were performed in two ovarian cancer cells lines, OVCAR3 and CA5171, and their CHI3L1-overexpressing and -knockdown transfectants. High expression of CHI3L1 was associated with poor outcome and chemoresistance in ovarian cancer patients. The mRNA expression of CHI3L1 in CA5171 ovarian cancer stem-like cells was 3-fold higher than in CA5171 parental cells. CHI3L1 promoted the properties of ovarian cancer stem-like cells including generating more and larger tumor spheroids and a higher percentage of ALDH+ in tumor cells and promoting resistance to cytotoxic drug-induced apoptosis. CHI3L1 could induce both the Akt (essential) and Erk signaling pathways, and then enhance expression of β-catenin followed by SOX2, and finally promote tumor spheroid formation and other properties of ovarian cancer stem-like cells. OVCAR3 CHI3L1-overexpressing transfectants were more tumorigenic in vivo, whereas CA5171 CHI3L1-knockdown transfectants were not tumorigenic in vivo. CHI3L1 critically enhances the properties of ovarian cancer stem-like cells. CHI3L1 or CHI3L1-regulated signaling pathways and molecules could be potential therapeutic targets in ovarian cancer.

Downloadable materials

  • Supplementary Table 1. The representative 10 genes whose expression are elevated at least 2-fold in ovarian cancer stem-like cells compared with the maternal cells
  • Supplementary Figure 1. The molecular authentication of original OVCAR3 cell line by STRS analyses.
  • Supplementary Figure 2. The molecular authentication of present OVCAR3 cell line by STRS analyses.

 

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    Correlation between the mRNA expression levels of CHI3L1 and chemoresistance and outcome of 113 ovarian cancer patients. (A) Correlation between the mRNA expression levels of CHI3L1 and chemoresistance. Chemoresistant patients had higher levels of CHI3L1 than chemosensitive patients (18.5 vs 4.3, P = 0.001, Kruskal–Wallis test). (B) Correlation between mRNA expression levels of CHI3L1 and disease-free survival (DFS) of patients. The high CHI3L1 group had shorter DFS than low CHI3L1 group (P < 0.001, Log-rank test). (C) Correlation between mRNA expression levels of CHI3L1 and overall survival (OS) of patients. The high CHI3L1 group had shorter OS than low CHI3L1 group (P < 0.001, Log-rank test).

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    Characteristics of CA5171 cancer stem-like cells. (A) Representative images of CA5171 parental cells and spheroids in low- (40×) and high- (200×) power fields photographed using an inverted phase-contrast microscope. CA5171 parental cells could be cultured to CA5171 spheroids under stem cell conditions. (B) Side-population assays of CA5171 parental cells treated with DMSO or verapamil (100 µM), stained with Hoechst 33342 dye, and then analyzed by flow cytometry. The percentages of CA5171 parental cells pumping out Hoechst33342 decreased from 2.8 to 0.7% under verapamil treatment. (C) ALDH+ CA5171 parental cells and spheroids stained in Aldefluor assays. 3.8% of CA5171 parental cells expressed ALDH compared to 55% of CA5171 stem-like cells. (D) Cytotoxicities of CA5171 parental cells and spheroids treated with cytotoxic drugs by MTT assays. CA5171 spheroids had significantly higher IC50 concentrations compared to parental cells under either paclitaxel or cisplatin treatment. (E) In vivo tumor growth experiments with CA5171 parental cells and its spheroids. Cell numbers for in vivo tumorigenesis were fewer in CA5171 spheroid cells than in CA5171 parental cells. A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0300.

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    CHI3L1 expression promotes cancer stem-like cell properties in OVCAR3 tumor cells. (A) CHI3L1 mRNA expression levels in CA5171 parental cells and spheroids by RT-PCR. CHI3L1 RNA expression was higher in CA5171 spheroids than in CA5171 parental cells. (B) Bar graphs of CHI3L1 mRNA expression levels in CA5171 parental cells and spheroids by RT-PCR. The CA5171 spheroids expressed CHI3L1 3 folds higher than CA5171 parental cells. (C) The representative images of spheroids from OVCAR3 parental cells treated with recombinant human CHI3L1 protein. OVCAR3 parental cells treated with CHI3L1 (1000 ng/mL) generated larger spheroid sizes than treated with PBS or 100 ng/mL of CHI3L1. (D) Percentages of ALDH+ OVCAR3 tumor cells treated with different concentrations of CHI3L1, as detected by Aldefluor assays and flow cytometric analysis. OVCAR3 parental cells under stem cell conditions treated with CHI3L1 increased the ratio of ALDH+ OVCAR3 cancer cells. (E) Immunoblotting analysis of expression of stemness-related molecules in OVCAR3 tumor cells treated with different concentrations of CHI3L1. Cultured OVCAR3 parental cells under stem cell conditions with CHI3L1 regulated expression of β-catenin and SOX2 with no change in Nanog or OCT4. (F) Percentages of apoptotic cells of OVCAR3 tumor cells treated with different concentrations of CHI3L1, as detected in apoptotic assays and by flow cytometric analysis. CHI3L1 enhanced the viability of OVCAR3 parental cells under stem cell conditions.

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    Expression of CHI3L1 in ovarian cancer cells altered the quantity of spheroids and stemness properties and stimulated both phospho-Akt and phospho-Erk pathways under stem cell conditions. (A) CHI3L1 RNA expression levels of OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants, as detected by RT-PCR. (B) CHI3L1 protein concentrations in culture media of OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants as detected by ELISA. CHI3L1 mRNA and protein expression levels were both high in OVCAR3 CHI3L1 transfectants, whereas they were low in CA5171 shCHI3L1 transfectants, as detected by RT-PCR and ELISA. (C) Representative images of spheroid formation of OVCAR3 parental cells and their CHI3L1 transfectants, and CA5171 parental cells and their shCHI3L1 transfectants. (D) Bar graphs of the numbers of spheroids in OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants. Numbers and sizes of spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions were both increased compared with controls (mock), but those of CA5171 shCHI3L1 transfectants were both decreased compared with controls. (E) Representative images of the percentages of ALDH+ OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants, measured by flow cytometry. (F) Bar graphs of the percentages of ALDH+ OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants (right) by Aldefluor assays. The ratio of ALDH+ cells in spheroids of OVCAR3 CHI3L1 transfectants was increased compared with controls (mock), whereas that of ALDH+ cells in spheroids of CA5171 shCHI3L1 transfectants was decreased. (G) Expression levels of various stemness-related genes in OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants, by immunoblotting analysis. Expression of β-catenin and SOX2 was increased in spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions but decreased in spheroids of CA5171 shCHI3L1 transfectants, and there was no change in Nanog or OCT4. (H) Phosphorylation of various signaling molecules of OVCAR3 tumor cells treated with different concentrations of CHI3L1 detected by immunoblotting. CHI3L1 stimulated phosphorylation of Akt and Erk in spheroids of OVCAR3 parental cells under stem cell conditions. (I) Phosphorylation of various signaling molecules of OVCAR3 parental cells and their CHI3L1 transfectants and of CA5171 parental cells and their shCHI3L1 transfectants, as detected by immunoblotting. Under stem cell conditions, phosphorylation of Akt and Erk increased in spheroids of OVCAR3 CHI3L1 transfectants and decreased in spheroids of CA5171 shCHI3L1 transfectants.

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    Inhibition of phospho-Akt and phosphor-Erk pathways impaired spheroid formation. (A) LY249002 and PD98059 inhibited phospho-Akt and phosphor-Erk pathways stimulated by CHI3L1 under stem cell conditions. Under these conditions, CHI3L1 stimulated phosphorylation of Akt and Erk in spheroids of OVCAR3 parental cells, and LY294002 and PD98059 inhibited phosphorylation of Akt and Erk, respectively, stimulated by CHI3L1 in spheroids of OVCAR3 parental cells. (B) Representative image of OVCAR3 parental cells and their CHI3L1 transfectants cultured in stem cell media with LY294002 or PD98059 and photographed after 48 h. (C) Representative image of CA5171 parental cells and their shCHI3L1 transfectants cultured in stem cell media with LY294002 or PD98059 and photographed after 48 h. LY294002 significantly decreased numbers and sizes of spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions and PD98059 decreased them partially; they both had the same respective effect on spheroids of the CA5171/mock group. (D) Percentages of ALDH+ OVCAR3 tumor cells treated with CHI3L1 and LY294002 or PD98059, detected by Aldefluor assays and flow cytometric analysis. LY294002 decreased the ratio of ALDH+ cells in spheroids of OVCAR3 parental cells cultured with CHI3L1 under stem cell conditions, and PD98059 decreased it partially. (E) Percentages of ALDH+ OVCAR3 parental cells and their CHI3L1 transfectants treated with CHI3L1 and LY294002 or PD98059, detected by Aldefluor assays and flow cytometric analysis. LY294002 significantly decreased the ratio of ALDH+ cells in spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions, and PD98059 decreased it partially. (F) Percentages of apoptotic cells of OVCAR3 spheroids in the presence of paclitaxel and CHI3L1 treated with LY294002 or PD98059, detected by apoptotic and flow cytometric analyses. LY294002 considerably suppressed cell viability conferred by CHI3L1 in spheroids of OVCAR3 parental cells treated with paclitaxel under stem cell conditions, and PD98059 suppressed it partially. (G) Percentages of apoptotic OVCAR3 parental cells and their CHI3L1 transfectants treated with paclitaxel and LY294002 or PD98059, detected by apoptotic assays and flow cytometric analysis. LY294002 significantly suppressed cell viability in spheroids of OVCAR3 CHI3L1 transfectants treated with paclitaxel under stem cell conditions, and PD98059 suppressed it partially. (H) Expression levels of various stemness-related genes in OVCAR3 parental cells and their CHI3L1 transfectants after treatment with LY294002 or PD98059, detected by immunoblotting. LY294002 significantly suppressed expression of SOX2 and β-catenin in spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions, but PD98059 suppressed only SOX2 partially. Protein levels of Nanog and OCT-4 did not change.

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    Knockdown of SOX2 or β-catenin impaired the formation of spheroids stimulated by CHI3L1. (A) The expression levels of SOX2 and β-catenin in OVCAR3 CHI3L1 transfectants after transfection with siRNA SOX2 or siRNA β-catenin, as detected by immunoblotting. Protein suppression of β-catenin by siRNA in spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions alone prevented SOX2 protein expression, but protein suppression of SOX2 by siRNA did not affect the protein expression of β-catenin. (B) OVCAR3 parental cells and their CHI3L1 transfectants after transfection with siRNA SOX2 or siRNA β-catenin for 48 h were harvested and cultured to spheroids under stem cell conditions and photographed (40×) after 48 h. The formation of spheroids of OVCAR3 CHI3L1 transfectants decreased after the suppression of SOX2 or β-catenin protein expression by siRNA. (C) Representative images of the percentages of ALDH+ OVCAR3 parental cells and their CHI3L1 transfectants after transfection with siRNA SOX2 or siRNA β-catenin and culture to spheroids, measured by flow cytometry. (D) Bar graphs of the percentages of ALDH+ OVCAR3 parental cells and their CHI3L1 transfectants after transfection with siRNA SOX2 or siRNA β-catenin and culture to spheroids, measured by Aldefluor assays after 48 h. The ratio of ALDH+ cells in spheroids of OVCAR3 CHI3L1 transfectants under stem cell conditions was also decreased after the suppression of SOX2 or β-catenin protein expression by siRNA. (E) Percentages of apoptotic cells of OVCAR3 CHI3L1 transfectants transfected with siRNA SOX2 or siRNA β-catenin and cultured to spheroids with paclitaxel, as measured by apoptotic assays and flow cytometric analysis after 48 hours. siRNA SOX2 or siRNA β-catenin decreased the viability of OVCAR3/CHI3L1 transfectants under stem cell conditions and paclitaxel treatment. (F) In vivo limiting dilution assays for spheroids of OVCAR3 CHI3L1 transfectants. (G) In vivo limiting dilution assays for spheroids of CA5171 shCHI3L1 transfectants. The tumorigenicity of spheroids of OVCAR3 CHI3L1 transfectants increased faster than in OVCAR3 parental cells, as did the tumorigenicity of spheroids of CA5171 parental cells compared to CA5171 shCHI3L1 transfectants.

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    Schematic of a model of CHI3L1 promotion of tumor spheroid formation and the characteristics of cancer stem-like cells. When ovarian tumors initiate and grow, chronic inflammation arises in the tumor microenvironment. Cancer cells and/or inflammatory cells secrete abundant CHI3L1 in ascites or extracellular matrix. CHI3L1 regulates cancer cells to form tumor spheroids by enhancing expression of stemness-related genes through activation of Akt and Erk signaling pathways. These tumor spheroids then increase chemoresistance and survival of tumor cells, finally migrating to new sites to generate metastasis. A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0300.

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