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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

Enhanced cell proliferation and transformation after AGR2 overexpression in HBECs.(A) Analysis by immunofluorescence of AGR2 overexpression in HBECs. Scale bars, 50 μm. (B) Analysis of AGR2 sub-cellular localization in infected HEBCs by immunofluorescence. Calnexin (CANX) and Bip (GRP78) are used as an ER localization control and GM130 is used as a Golgi localization control. Scale bars, 50 μm. (C) Up-regulation of AGR2 protein concentrations in control cells (HBEC-EV) and in cells infected with AGR2 (HBEC-AGR2), as analyzed by western blot. Calnexin (CANX) concentrations are shown as the loading control. One representative experiment (n = 3) is shown. (D) Growth of cells stably expressing AGR2 or empty vector (EV) (three independent experiments). Data are mean ± SEM. (E) Quantification of the percentage of BrdU positive cells. Data are presented as mean ± SEM of at least three independent experiments. **p<0.001. (F) Quantitation of cell death in AGR2 overexpression cells. Results are representative of three independent experiments. The cell death rate was determined by Trypan blue dye-exclusion assay. n.s: not significant. (G) Cell proliferation on HBEC overexpressing AGR2 cells. Representative images of control HBECs (HBEC-EV) and HBEC-AGR2 stained for BrdU (red) administrated 12 hr before, and with DAPI (blue). Images were subjected to high throughput imaging (bottom panels, analysis data). After acquisition of the dataset, images were segmented by a watershed transformation to identify individual cells (bottom right). Scale bars: 100 μm. (H) The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. Shown at the left are representative images of the organoids formed by each type of cell HBEC-AGR2 and HBEC-empty vector. The p values (determined by Student's t test) are relative to empty vector infected cells. **p≤0.01 and ***p≤0.001.DOI:http://dx.doi.org/10.7554/eLife.13887.007
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fig4: Enhanced cell proliferation and transformation after AGR2 overexpression in HBECs.(A) Analysis by immunofluorescence of AGR2 overexpression in HBECs. Scale bars, 50 μm. (B) Analysis of AGR2 sub-cellular localization in infected HEBCs by immunofluorescence. Calnexin (CANX) and Bip (GRP78) are used as an ER localization control and GM130 is used as a Golgi localization control. Scale bars, 50 μm. (C) Up-regulation of AGR2 protein concentrations in control cells (HBEC-EV) and in cells infected with AGR2 (HBEC-AGR2), as analyzed by western blot. Calnexin (CANX) concentrations are shown as the loading control. One representative experiment (n = 3) is shown. (D) Growth of cells stably expressing AGR2 or empty vector (EV) (three independent experiments). Data are mean ± SEM. (E) Quantification of the percentage of BrdU positive cells. Data are presented as mean ± SEM of at least three independent experiments. **p<0.001. (F) Quantitation of cell death in AGR2 overexpression cells. Results are representative of three independent experiments. The cell death rate was determined by Trypan blue dye-exclusion assay. n.s: not significant. (G) Cell proliferation on HBEC overexpressing AGR2 cells. Representative images of control HBECs (HBEC-EV) and HBEC-AGR2 stained for BrdU (red) administrated 12 hr before, and with DAPI (blue). Images were subjected to high throughput imaging (bottom panels, analysis data). After acquisition of the dataset, images were segmented by a watershed transformation to identify individual cells (bottom right). Scale bars: 100 μm. (H) The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. Shown at the left are representative images of the organoids formed by each type of cell HBEC-AGR2 and HBEC-empty vector. The p values (determined by Student's t test) are relative to empty vector infected cells. **p≤0.01 and ***p≤0.001.DOI:http://dx.doi.org/10.7554/eLife.13887.007

Mentions: To further confirm that AGR2 is essential for cancer tumorigenesis, we next investigated the effects of overexpressing AGR2 in non-tumorigenic HBECs using lentivirus-mediated infection with either empty vector (HBEC-EV) (Figure 4A, top panels) or AGR2 containing vector (HBEC-AGR2) (Figure 4A, bottom panels). In HBEC-AGR2 cells, AGR2 co-localized with calnexin (CANX), an ER resident type I integral membrane protein (Figure 4B, top panels) and BiP (GRP78), a soluble HSP70 molecular chaperone located in the lumen of the ER (Figure 4B, middle panels). In contrast and as expected, AGR2 did not co-localize with GM130, a cis-Golgi marker (Figure 4B, bottom panels). These results show that AGR2 localized to the ER in HBEC-AGR2 cells (Figure 4B). Increased AGR2 expression in these cells (HBEC-AGR2) was also confirmed using Western blotting (Figure 4C). Remarkably, AGR2 overexpression enhanced cell proliferation (Figure 4D–G) and increased the formation of organoids (Figure 4H), thereby indicating that the expression of AGR2 dictates organoid-initiating frequency.10.7554/eLife.13887.007Figure 4.Enhanced cell proliferation and transformation after AGR2 overexpression in HBECs.


Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
Enhanced cell proliferation and transformation after AGR2 overexpression in HBECs.(A) Analysis by immunofluorescence of AGR2 overexpression in HBECs. Scale bars, 50 μm. (B) Analysis of AGR2 sub-cellular localization in infected HEBCs by immunofluorescence. Calnexin (CANX) and Bip (GRP78) are used as an ER localization control and GM130 is used as a Golgi localization control. Scale bars, 50 μm. (C) Up-regulation of AGR2 protein concentrations in control cells (HBEC-EV) and in cells infected with AGR2 (HBEC-AGR2), as analyzed by western blot. Calnexin (CANX) concentrations are shown as the loading control. One representative experiment (n = 3) is shown. (D) Growth of cells stably expressing AGR2 or empty vector (EV) (three independent experiments). Data are mean ± SEM. (E) Quantification of the percentage of BrdU positive cells. Data are presented as mean ± SEM of at least three independent experiments. **p<0.001. (F) Quantitation of cell death in AGR2 overexpression cells. Results are representative of three independent experiments. The cell death rate was determined by Trypan blue dye-exclusion assay. n.s: not significant. (G) Cell proliferation on HBEC overexpressing AGR2 cells. Representative images of control HBECs (HBEC-EV) and HBEC-AGR2 stained for BrdU (red) administrated 12 hr before, and with DAPI (blue). Images were subjected to high throughput imaging (bottom panels, analysis data). After acquisition of the dataset, images were segmented by a watershed transformation to identify individual cells (bottom right). Scale bars: 100 μm. (H) The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. Shown at the left are representative images of the organoids formed by each type of cell HBEC-AGR2 and HBEC-empty vector. The p values (determined by Student's t test) are relative to empty vector infected cells. **p≤0.01 and ***p≤0.001.DOI:http://dx.doi.org/10.7554/eLife.13887.007
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fig4: Enhanced cell proliferation and transformation after AGR2 overexpression in HBECs.(A) Analysis by immunofluorescence of AGR2 overexpression in HBECs. Scale bars, 50 μm. (B) Analysis of AGR2 sub-cellular localization in infected HEBCs by immunofluorescence. Calnexin (CANX) and Bip (GRP78) are used as an ER localization control and GM130 is used as a Golgi localization control. Scale bars, 50 μm. (C) Up-regulation of AGR2 protein concentrations in control cells (HBEC-EV) and in cells infected with AGR2 (HBEC-AGR2), as analyzed by western blot. Calnexin (CANX) concentrations are shown as the loading control. One representative experiment (n = 3) is shown. (D) Growth of cells stably expressing AGR2 or empty vector (EV) (three independent experiments). Data are mean ± SEM. (E) Quantification of the percentage of BrdU positive cells. Data are presented as mean ± SEM of at least three independent experiments. **p<0.001. (F) Quantitation of cell death in AGR2 overexpression cells. Results are representative of three independent experiments. The cell death rate was determined by Trypan blue dye-exclusion assay. n.s: not significant. (G) Cell proliferation on HBEC overexpressing AGR2 cells. Representative images of control HBECs (HBEC-EV) and HBEC-AGR2 stained for BrdU (red) administrated 12 hr before, and with DAPI (blue). Images were subjected to high throughput imaging (bottom panels, analysis data). After acquisition of the dataset, images were segmented by a watershed transformation to identify individual cells (bottom right). Scale bars: 100 μm. (H) The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. Shown at the left are representative images of the organoids formed by each type of cell HBEC-AGR2 and HBEC-empty vector. The p values (determined by Student's t test) are relative to empty vector infected cells. **p≤0.01 and ***p≤0.001.DOI:http://dx.doi.org/10.7554/eLife.13887.007
Mentions: To further confirm that AGR2 is essential for cancer tumorigenesis, we next investigated the effects of overexpressing AGR2 in non-tumorigenic HBECs using lentivirus-mediated infection with either empty vector (HBEC-EV) (Figure 4A, top panels) or AGR2 containing vector (HBEC-AGR2) (Figure 4A, bottom panels). In HBEC-AGR2 cells, AGR2 co-localized with calnexin (CANX), an ER resident type I integral membrane protein (Figure 4B, top panels) and BiP (GRP78), a soluble HSP70 molecular chaperone located in the lumen of the ER (Figure 4B, middle panels). In contrast and as expected, AGR2 did not co-localize with GM130, a cis-Golgi marker (Figure 4B, bottom panels). These results show that AGR2 localized to the ER in HBEC-AGR2 cells (Figure 4B). Increased AGR2 expression in these cells (HBEC-AGR2) was also confirmed using Western blotting (Figure 4C). Remarkably, AGR2 overexpression enhanced cell proliferation (Figure 4D–G) and increased the formation of organoids (Figure 4H), thereby indicating that the expression of AGR2 dictates organoid-initiating frequency.10.7554/eLife.13887.007Figure 4.Enhanced cell proliferation and transformation after AGR2 overexpression in HBECs.

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