Induction of ER stress-mediated apoptosis through SOD1 upregulation by deficiency of CHI3L1 inhibits lung metastasis

Abstract

Chitinase-3-like protein 1 (CHI3L1), which is secreted by immune and inflammatory cells, is associated with several inflammatory diseases. However, the basic cellular pathophysiological functions of CHI3L1 are not well characterized. To investigate the novel pathophysiological function of CHI3L1, we performed LC-MS/MS analysis of cells transfected with Myc-vector and Myc-CHI3L1. We analyzed the changes in the protein distribution in Myc-CHI3L1 transfected-cells, and identified 451 differentially expressed proteins (DEPs) compared with Myc-vector-transfected-cells. The biological function of the 451 DEPs was analyzed and it was found that the proteins with endoplasmic reticulum (ER)-associated function were much more highly expressed in CHI3L1-overexpressing cells. We then compared and analyzed the effect of CHI3L1 on the ER chaperon levels in normal lung cells and cancer cells. We identified that CHI3L1 is localized in the ER. In normal cells, the depletion of CHI3L1 did not induce ER stress. However, the depletion of CHI3L1 induces ER stress and eventually activates the unfolded protein response, especially the activation of Protein kinase R-like endoplasmic reticulum kinase (PERK), which regulates protein synthesis in cancer cells. CHI3L1 may not affect ER stress owing to the lack of misfolded proteins in normal cells, but instead activate ER stress as a defense mechanism only in cancer cells. Under ER stress conditions induced by the application of thapsigargin, the depletion of CHI3L1 induces ER stress through the upregulation of PERK and PERK downstream factors (eIF2α and ATF4) in both normal and cancer cells. However, these signaling activations occur more often in cancer cells than in normal cells. The expression of Grp78 and PERK in the tissues of patients with lung cancer was higher compared with healthy tissues. It is well known that ER stress-mediated PERK-eIF2α-ATF4 signaling activation causes apoptotic cell death. ER stress-mediated apoptosis induced by the depletion of CHI3L1 occurs in cancer cells, but rarely occurs in normal cells. Consistent with results from the in vitro model, ER stress-mediated apoptosis was greatly increased during tumor growth and in the lung metastatic tissue of CHI3L1-knockout (KO) mice. The analysis of "big data" identified superoxide dismutase-1 (SOD1) as a novel target of CHI3L1 and interacted with CHI3L1. The depletion of CHI3L1 increased SOD1 expression, resulting in ER stress. Furthermore, the depletion of SOD1 reduced the expression of ER chaperones and ER-mediated apoptotic marker proteins, as well as apoptotic cell death induced by the depletion of CHI3L1 in in vivo and in vitro models. These results suggest that the depletion of CHI3L1 increases ER stress-mediated apoptotic cell death through SOD1 expression, and subsequently inhibits lung metastasis.

Keywords: Chitinas-3-like 1; ER stress; metastasis; superoxide dismutase-1.

Figure 1

ER associated proteins are the most enriched in the downregulated DEPs in CHI3L1 expressing cells. (A) Venn diagram of identified differential expressed proteins (DEPs) from LC-MS/MS. 1027 DEPs are shared between both groups (1053 for the Myc-vector and 1120 for the Myc-CHI3L1 transfected cells). (B) Heatmap of expression profiles for differential proteins. The color scale represents average log signal intensity values. Red indicated upregulated protein, and blue indicated the downregulated protein in comparison groups. (C) Volcano plot of DEPs between Myc-vector and Myc-CHI3L1 transfected cells. Red dots indicate upregulated DEPs (log₂FC > 1), blue dots indicate downregulated DEPs (log₂FC < -1), and gray dots indicate no significant difference between Myc-vector and Myc-CHI3L1 transfected cells. FDR < 0.05; p < 0.05. In total 451 DEPs, including 209 upregulated and 242 downregulated DEPs, were identified. (D) The gene ontology categories of DEPs in biological process. Red bar graph indicates upregulated DEPs in Myc-CHI3L1 transfected cells compared with control. The top 10 significant Biological Processes terms of upregulated DEPs in Myc-CHI3L1 transfected cells. The enrichment p value in biological processes is indicated.

Figure 2

CHI3L1 is localized in ER and associated with ER chaperone Grp78. (A) The 4% paraformaldehyde fixed MRC5 (normal lung cell) and A549 (lung cancer cell) cells were co-stained with anti-CHI3L1 and anit-Grp78 (endoplasmic reticulum marker). Scale bar, 10 μm. (B) MRC5 and A549 cel0ls were fractioned into three distinct fractions, including cytosolic, membrane bound organelle, and nuclear fractions. Isolated proteins were subjected to immunoblot analysis with indicated antibodies. (C, D) MRC5 and A549 cell lysates were immunoprecipitated using anti-CHI3L1 antibody, and then subjected to immunoblot analysis with the indicated antibodies.

Figure 3

Depletion of CHI3L1 activates ER stress and induces PERK and eIF2α in A549 cells. (A) MRC5 (normal lung cell) and A549 (lung cancer cell) cells were transfected with control siRNA (si-cont.) or CHI3L1 siRNA (si-CHI3L1). The cell lysates were subjected to immunoblot analysis with the indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. ***, P < 0.001; n.s., not significant. (B) MRC5 cells were transfected with si-control or si-CHI3L1, then treated with 1 uM thapsigargin (ER stress inducer) for 18 h. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. ***, P < 0.001; n.s., not significant. (C) A549 cells were transfected with si-control or si-CHI3L1, then treated with 1 uM thapsigargin for 18 h. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. ***, P < 0.001. (D) MRC5 (normal lung cell) and A549 (lung cancer cell) cells were transfected with control siRNA (si-cont.) or CHI3L1 siRNA (si-CHI3L1). The cell lysates were subjected to immunoblot analysis with the indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. n.s., not significant. (E) MRC5 cells were transfected with si-control or si-CHI3L1, then treated with 1 uM thapsigargin (ER stress inducer) for 18 h. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (F) A549 cells were transfected with si-control or si-CHI3L1, then treated with 1 uM thapsigargin for 18 h. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. ***, P < 0.001.

Figure 4

Depletion of CHI3L1 induces ER stress mediated apoptosis via PERK- eIF2α-CHOP pathway in A549 cells. (A) A549 cells were transfected with si-control or si-CHI3L1, then treated with 1 uM thapsigargin (ER stress inducer) for 18 h. The cell viability was measured using the MTT assay. The data are presented as the mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001. (B) Representative fluorescence microscopic images showing DAPI (blue) and TUNEL (green) staining in A549 cells transfected with si-control or si-CHI3L1 in the absence or presence of the thapsigargin (1 μM). Scale bar, 10 μm. The number of positively stained cells was counted in three different fields and averaged. The data are presented as the mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001. (C) A549 cells were transfected with si-control or si-CHI3L1, then treated with 1 uM thapsigargin for 18 h. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (D) si-control or si-CHI3L1 transfected A549 cells were seeded in the upper chamber and incubated at 37 °C for 18 h. The migrated cells on the bottom chamber were stained with 0.1% crystal violet. Data are presented as mean ±SD from three independent experiments. Scale bar, 100 μm. The number of migrated cells was counted in three different fields and averaged. The data are presented as the mean ± SD of three independent experiments. ***, P < 0.05.

Figure 5

The metastatic lung tissues of CHI3L1 KO mice induces ER stress mediated apoptosis. (A) The lung tissue extracts of NSCLC patients were subjected to immunoblot analysis with indicated antibodies. (B) The serums of NSCLC patients were subjected to ELISA analysis with indicated proteins. (C) The metastatic lung tissue extracts of mice were subjected to immunoblot analysis with indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. ***, P < 0.001. (D) Representative immunohistochemical images of metastatic lung tissues in mice using anti-CHI3L1, anti-Grp78, and anti-p-PERK antibodies in each group. Scale bar, 100 μm. (E) The metastatic lung tissues extracts of mice were subjected to immunoblot analysis with indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± SD from two independent experiments. ***, P < 0.001. (F) Representative immunohistochemical images of metastatic lung tissues in mice using anti-CHOP and anti-cleaved caspase12 antibodies in each group. Scale bar, 100 μm.

Figure 6

SOD1 is involved in the depletion of CHI3L1-induced ER stress. (A) A549 cells were transfected with either Myc-vector or Myc tagged Chi3L1 plasmids. The cell lysates were immunoprecipitated using the anti-Myc antibody, and then subjected to immunoblot analysis with the indicated antibodies. (B) A549 cells were transfected with either Myc-vector or Myc tagged Chi3L1 plasmids. The cell lysates were immunoprecipitated using the anti-Myc antibody, and then subjected to immunoblot analysis with the indicated antibodies. (C) A549 cells were transfected with control siRNA or si-CHI3L1.The cell lysates were subjected to immunoblot analysis with the indicated antibodies (D) The metastatic lung tissues extracts were subjected to immunoblot analysis with anti-SOD1 antibodies (upper). Representative immunohistochemical images of metastatic lung tissues using anti-SOD1 antibodies in each group (lower). Scale bar, 100 μm. (E) A549 cells were transfected with si-control, si-CHI3L1, or si-SOD1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (F) A549 cells were transfected with si-control, si-CHI3L1, or si-SOD1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (G) A549 cells were transfected with si-control, si-CHI3L1, or si-SOD1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (H) Representative fluorescence microscopic images showing DAPI (blue) and TUNEL (green) staining in A549 cells transfected with si--control, si-CHI3L1, or si-SOD1. Scale bar, 20 μm. The number of positively stained cells was counted in three different fields and averaged. The data are presented as the mean ± SD of three independent experiments. ***, P < 0.001.

Figure 7

CHI3L1 KO mice suppress lung metastasis through SOD1 expression. (A-G) A549 cells (1X10⁷ cells) were injected intravenously scramble si-RNA or si-SOD1 were injected intravenously thrice a week for seven weeks. n=6 per group. (A) The number of metastatic nodules on the lung surface was counted and quantified (n=6). (B) The tumor surface areas on the lung tissue were measured from H&E staining images and quantified as a percentage of the total lung surface area (n=6). (C) Representative H&E staining images of metastatic lung tissues excised from each group. H&E staining were repeated from three independent experiments. (D) The lung tissue extracts were subjected to immunoblot analysis with indicated antibodies. The positive cells in immunohistochemistry staining was measured and calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. (E) Representative immunohistochemical images of lung tissues using indicated antibodies in each group. Immunohistochemical staining were repeated from three independent experiments. The positive cells in immunohistochemistry staining was measured and calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. (F) The lung tissue extracts were subjected to immunoblot analysis with indicated antibodies. The intensity of each band was measured and the ratio of the amount of each protein to β-actin was calculated. Data are presented as mean ± standard deviation (SD) from two independent experiments. (G) Representative immunohistochemical images of lung tissues using indicated antibodies in each group. Immunohistochemical staining were repeated from three independent experiments.