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Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

View Article: PubMed Central - PubMed

ABSTRACT

The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.

Doi:: http://dx.doi.org/10.7554/eLife.13887.001

No MeSH data available.


Related in: MedlinePlus

AGR2 is overexpressed in lung cancer cell lines and tumor tissues.(A) Analysis by immunofluorescence of AGR2 expression in normal human bronchial epithelial cells (HBEC) and three lung cancer cell lines (A549, H23 and H1838) grown in 2D culture. Scale bars, 50 μm. (B) Quantification of AGR2 protein expression in cell lines according to immunofluorescence. The stacked bars show the percent contribution of high and low AGR2-positive cells relative to the total number of cells per field. (C) Expression of AGR2 protein detected by Western blot in a panel of human lung epithelial cell lines. Values correspond to three independent experiments. Data are mean ± SEM. (D) Confocal cross-sections of organoids stained with AGR2 antibody (red) and DAPI (blue) for nucleus, in normal HBECs and three lung cancer cell lines (A549, H23 and H1838). Scale bars, 50 μm. (E) AGR2 expression determined by immunohistochemistry in sections of formalin-fixed paraffin-embedded normal human lung samples and in the different lung adenocarcinoma subtypes. II = squamous cell carcinoma, IV = adenocarcinoma, VI = large cell carcinoma as compared to normal tissues (I,III,V) (x200). (F) Pulmonary lung carcinoma showing brown, nuclear immunostaining for TTF-1 expression and cytoplasmic immunostaining for AGR2 expression (dual color Multiplex TTF-1 + AGR2 immunostain; x200). (G) Kaplan-Meier survival curves of lung cancer patients. The cumulative survival was related to different levels of AGR2 expression: Group 1, low to moderately positive stains (n=17); and Group 2, strongly positive AGR2 stains (n=16), as defined in Materials and methods and Supplementary file 1A.DOI:http://dx.doi.org/10.7554/eLife.13887.003
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fig1: AGR2 is overexpressed in lung cancer cell lines and tumor tissues.(A) Analysis by immunofluorescence of AGR2 expression in normal human bronchial epithelial cells (HBEC) and three lung cancer cell lines (A549, H23 and H1838) grown in 2D culture. Scale bars, 50 μm. (B) Quantification of AGR2 protein expression in cell lines according to immunofluorescence. The stacked bars show the percent contribution of high and low AGR2-positive cells relative to the total number of cells per field. (C) Expression of AGR2 protein detected by Western blot in a panel of human lung epithelial cell lines. Values correspond to three independent experiments. Data are mean ± SEM. (D) Confocal cross-sections of organoids stained with AGR2 antibody (red) and DAPI (blue) for nucleus, in normal HBECs and three lung cancer cell lines (A549, H23 and H1838). Scale bars, 50 μm. (E) AGR2 expression determined by immunohistochemistry in sections of formalin-fixed paraffin-embedded normal human lung samples and in the different lung adenocarcinoma subtypes. II = squamous cell carcinoma, IV = adenocarcinoma, VI = large cell carcinoma as compared to normal tissues (I,III,V) (x200). (F) Pulmonary lung carcinoma showing brown, nuclear immunostaining for TTF-1 expression and cytoplasmic immunostaining for AGR2 expression (dual color Multiplex TTF-1 + AGR2 immunostain; x200). (G) Kaplan-Meier survival curves of lung cancer patients. The cumulative survival was related to different levels of AGR2 expression: Group 1, low to moderately positive stains (n=17); and Group 2, strongly positive AGR2 stains (n=16), as defined in Materials and methods and Supplementary file 1A.DOI:http://dx.doi.org/10.7554/eLife.13887.003

Mentions: To evaluate the correlation between AGR2 expression levels and lung cancer, we monitored AGR2 endogenous expression in a panel of human lung bronchial epithelial cell lines. High AGR2 expression was only observed in lung tumor cell lines (A549, H23, H1838) compared to a non-tumorigenic human bronchial epithelial cell (HBEC) (Figure 1A–C). Moreover, the expression pattern of AGR2 in tumor and non-tumor bronchial organoids (Figure 1D) was similar to that observed in 2D culture (Figure 1A). Immunohistochemistry of AGR2 in a cohort of 34 non-small cell lung cancer (NSCLC) patients (Supplementary file 1A) revealed that AGR2 was overexpressed in tumors compared to adjacent non-tumor tissue (Figure 1E). Consequently, AGR2 expression was increased in NSCLC tissues (Figure 1E), and was essentially restricted to type II pneumocytes (Figure 1F). We then used a log-rank test with Kaplan–Meier estimates to analyze the cohort in order to stratify patient samples as having high, low/intermediate AGR2 expression status (Supplementary file 1A). High AGR2 expression correlated with low survival rate and the low/intermediate AGR2 expression with high survival rate in NSCLCs patients (Figure 1G). Hence NSCLC patients can be sorted into poor and good prognosis groups as a function of high or low/intermediate AGR2 expression levels, respectively. Taken together, these results demonstrate in vitro and in vivo correlations between AGR2 expression levels and lung cancer, suggesting a function for this protein in tumor development, progression and aggressiveness.10.7554/eLife.13887.003Figure 1.AGR2 is overexpressed in lung cancer cell lines and tumor tissues.


Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
AGR2 is overexpressed in lung cancer cell lines and tumor tissues.(A) Analysis by immunofluorescence of AGR2 expression in normal human bronchial epithelial cells (HBEC) and three lung cancer cell lines (A549, H23 and H1838) grown in 2D culture. Scale bars, 50 μm. (B) Quantification of AGR2 protein expression in cell lines according to immunofluorescence. The stacked bars show the percent contribution of high and low AGR2-positive cells relative to the total number of cells per field. (C) Expression of AGR2 protein detected by Western blot in a panel of human lung epithelial cell lines. Values correspond to three independent experiments. Data are mean ± SEM. (D) Confocal cross-sections of organoids stained with AGR2 antibody (red) and DAPI (blue) for nucleus, in normal HBECs and three lung cancer cell lines (A549, H23 and H1838). Scale bars, 50 μm. (E) AGR2 expression determined by immunohistochemistry in sections of formalin-fixed paraffin-embedded normal human lung samples and in the different lung adenocarcinoma subtypes. II = squamous cell carcinoma, IV = adenocarcinoma, VI = large cell carcinoma as compared to normal tissues (I,III,V) (x200). (F) Pulmonary lung carcinoma showing brown, nuclear immunostaining for TTF-1 expression and cytoplasmic immunostaining for AGR2 expression (dual color Multiplex TTF-1 + AGR2 immunostain; x200). (G) Kaplan-Meier survival curves of lung cancer patients. The cumulative survival was related to different levels of AGR2 expression: Group 1, low to moderately positive stains (n=17); and Group 2, strongly positive AGR2 stains (n=16), as defined in Materials and methods and Supplementary file 1A.DOI:http://dx.doi.org/10.7554/eLife.13887.003
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fig1: AGR2 is overexpressed in lung cancer cell lines and tumor tissues.(A) Analysis by immunofluorescence of AGR2 expression in normal human bronchial epithelial cells (HBEC) and three lung cancer cell lines (A549, H23 and H1838) grown in 2D culture. Scale bars, 50 μm. (B) Quantification of AGR2 protein expression in cell lines according to immunofluorescence. The stacked bars show the percent contribution of high and low AGR2-positive cells relative to the total number of cells per field. (C) Expression of AGR2 protein detected by Western blot in a panel of human lung epithelial cell lines. Values correspond to three independent experiments. Data are mean ± SEM. (D) Confocal cross-sections of organoids stained with AGR2 antibody (red) and DAPI (blue) for nucleus, in normal HBECs and three lung cancer cell lines (A549, H23 and H1838). Scale bars, 50 μm. (E) AGR2 expression determined by immunohistochemistry in sections of formalin-fixed paraffin-embedded normal human lung samples and in the different lung adenocarcinoma subtypes. II = squamous cell carcinoma, IV = adenocarcinoma, VI = large cell carcinoma as compared to normal tissues (I,III,V) (x200). (F) Pulmonary lung carcinoma showing brown, nuclear immunostaining for TTF-1 expression and cytoplasmic immunostaining for AGR2 expression (dual color Multiplex TTF-1 + AGR2 immunostain; x200). (G) Kaplan-Meier survival curves of lung cancer patients. The cumulative survival was related to different levels of AGR2 expression: Group 1, low to moderately positive stains (n=17); and Group 2, strongly positive AGR2 stains (n=16), as defined in Materials and methods and Supplementary file 1A.DOI:http://dx.doi.org/10.7554/eLife.13887.003
Mentions: To evaluate the correlation between AGR2 expression levels and lung cancer, we monitored AGR2 endogenous expression in a panel of human lung bronchial epithelial cell lines. High AGR2 expression was only observed in lung tumor cell lines (A549, H23, H1838) compared to a non-tumorigenic human bronchial epithelial cell (HBEC) (Figure 1A–C). Moreover, the expression pattern of AGR2 in tumor and non-tumor bronchial organoids (Figure 1D) was similar to that observed in 2D culture (Figure 1A). Immunohistochemistry of AGR2 in a cohort of 34 non-small cell lung cancer (NSCLC) patients (Supplementary file 1A) revealed that AGR2 was overexpressed in tumors compared to adjacent non-tumor tissue (Figure 1E). Consequently, AGR2 expression was increased in NSCLC tissues (Figure 1E), and was essentially restricted to type II pneumocytes (Figure 1F). We then used a log-rank test with Kaplan–Meier estimates to analyze the cohort in order to stratify patient samples as having high, low/intermediate AGR2 expression status (Supplementary file 1A). High AGR2 expression correlated with low survival rate and the low/intermediate AGR2 expression with high survival rate in NSCLCs patients (Figure 1G). Hence NSCLC patients can be sorted into poor and good prognosis groups as a function of high or low/intermediate AGR2 expression levels, respectively. Taken together, these results demonstrate in vitro and in vivo correlations between AGR2 expression levels and lung cancer, suggesting a function for this protein in tumor development, progression and aggressiveness.10.7554/eLife.13887.003Figure 1.AGR2 is overexpressed in lung cancer cell lines and tumor tissues.

View Article: PubMed Central - PubMed

ABSTRACT

The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.

Doi:: http://dx.doi.org/10.7554/eLife.13887.001

No MeSH data available.


Related in: MedlinePlus