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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

Extracellular AGR2 disrupts apico-basal polarity and lumen formation.(A, C, E) HBEC cultured organoids in the presence (+ eAGR2) or absence (-eAGR2) of AGR2 in the ECM, stained for GM130 (A), ZO-1 (B) and F-Actin (C). Scale bars, 50 μm. Note the repositioning or fragmentation of Golgi following the presence of eAGR2. (B, D, F) Fluorescence intensity profile along the white line across the organoid in the presence (+ eAGR2, right panel) or absence (-eAGR2, left panel) of AGR2 in the ECM, stained with DAPI and GM130 (B), ZO-1 (D) and F-Actin (F). (G) Quantification of organoids with lumens; n>100 for each condition, (n=3).DOI:http://dx.doi.org/10.7554/eLife.13887.013
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fig7: Extracellular AGR2 disrupts apico-basal polarity and lumen formation.(A, C, E) HBEC cultured organoids in the presence (+ eAGR2) or absence (-eAGR2) of AGR2 in the ECM, stained for GM130 (A), ZO-1 (B) and F-Actin (C). Scale bars, 50 μm. Note the repositioning or fragmentation of Golgi following the presence of eAGR2. (B, D, F) Fluorescence intensity profile along the white line across the organoid in the presence (+ eAGR2, right panel) or absence (-eAGR2, left panel) of AGR2 in the ECM, stained with DAPI and GM130 (B), ZO-1 (D) and F-Actin (F). (G) Quantification of organoids with lumens; n>100 for each condition, (n=3).DOI:http://dx.doi.org/10.7554/eLife.13887.013

Mentions: Confocal microscopy analysis of non-tumorigenic HBEC organoids was then used to further evaluate the impact of eAGR2 on organoid architecture. We showed that eAGR2-induced re-distribution of polarization markers using confocal microscopy in HBEC organoids (Figure 7A–D). As shown in Figure 7A and B (bottom and right panels), the apical marker GM130 was randomly distributed throughout the HBEC organoids in presence of eAGR2 in the ECM, in contrast to its restriction to the apical localization in HBEC organoids in the absence of eAGR2 (Figure 7A and B (top and left panels). Fluorescence intensity cross-section profile (Figure 7B) revealed that the intensity of GM130 (green) had two side peaks that referred to the control HBEC organoid shell (- eAGR2). In presence of eAGR2, GM130 distributed randomly within the HBEC organoid (Figure 7B). Similarly, control HBEC organoids exhibited a lumen lined by the tight junction marker ZO-1 (Figure 7C and D; top and left panels), whereas ZO-1 was no longer restricted to the lumen compartment upon eAGR2 exposure (Figure 7C and D; bottom and right panels). Thus, eAGR2 in the ECM, resulted in a disruption of apicobasal polarity, of non-tumorigenic organoids. Therefore, we hypothesize that eAGR2 determines the orientation of epithelial polarity and thereby lumen formation. Hence, we next investigated the development of lumen in HBEC organoids, in the absence (- eAGR2) or in the presence (+ eAGR2) in the ECM of eAGR2 (Figure 7E–G) using apical F-actin (Figure 7E,F). The presence of eAGR2, at the time of plating, gave rise to organoids unable to form a lumen (+ eAGR2) (Figure 7E–G) thereby showing that eAGR2 interferes with the formation of hollow organoid. Collectively, these data provide further evidence that eAGR2, in the ECM, induced loss of cell polarity and lumen formation.10.7554/eLife.13887.013Figure 7.Extracellular AGR2 disrupts apico-basal polarity and lumen formation.


Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
Extracellular AGR2 disrupts apico-basal polarity and lumen formation.(A, C, E) HBEC cultured organoids in the presence (+ eAGR2) or absence (-eAGR2) of AGR2 in the ECM, stained for GM130 (A), ZO-1 (B) and F-Actin (C). Scale bars, 50 μm. Note the repositioning or fragmentation of Golgi following the presence of eAGR2. (B, D, F) Fluorescence intensity profile along the white line across the organoid in the presence (+ eAGR2, right panel) or absence (-eAGR2, left panel) of AGR2 in the ECM, stained with DAPI and GM130 (B), ZO-1 (D) and F-Actin (F). (G) Quantification of organoids with lumens; n>100 for each condition, (n=3).DOI:http://dx.doi.org/10.7554/eLife.13887.013
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fig7: Extracellular AGR2 disrupts apico-basal polarity and lumen formation.(A, C, E) HBEC cultured organoids in the presence (+ eAGR2) or absence (-eAGR2) of AGR2 in the ECM, stained for GM130 (A), ZO-1 (B) and F-Actin (C). Scale bars, 50 μm. Note the repositioning or fragmentation of Golgi following the presence of eAGR2. (B, D, F) Fluorescence intensity profile along the white line across the organoid in the presence (+ eAGR2, right panel) or absence (-eAGR2, left panel) of AGR2 in the ECM, stained with DAPI and GM130 (B), ZO-1 (D) and F-Actin (F). (G) Quantification of organoids with lumens; n>100 for each condition, (n=3).DOI:http://dx.doi.org/10.7554/eLife.13887.013
Mentions: Confocal microscopy analysis of non-tumorigenic HBEC organoids was then used to further evaluate the impact of eAGR2 on organoid architecture. We showed that eAGR2-induced re-distribution of polarization markers using confocal microscopy in HBEC organoids (Figure 7A–D). As shown in Figure 7A and B (bottom and right panels), the apical marker GM130 was randomly distributed throughout the HBEC organoids in presence of eAGR2 in the ECM, in contrast to its restriction to the apical localization in HBEC organoids in the absence of eAGR2 (Figure 7A and B (top and left panels). Fluorescence intensity cross-section profile (Figure 7B) revealed that the intensity of GM130 (green) had two side peaks that referred to the control HBEC organoid shell (- eAGR2). In presence of eAGR2, GM130 distributed randomly within the HBEC organoid (Figure 7B). Similarly, control HBEC organoids exhibited a lumen lined by the tight junction marker ZO-1 (Figure 7C and D; top and left panels), whereas ZO-1 was no longer restricted to the lumen compartment upon eAGR2 exposure (Figure 7C and D; bottom and right panels). Thus, eAGR2 in the ECM, resulted in a disruption of apicobasal polarity, of non-tumorigenic organoids. Therefore, we hypothesize that eAGR2 determines the orientation of epithelial polarity and thereby lumen formation. Hence, we next investigated the development of lumen in HBEC organoids, in the absence (- eAGR2) or in the presence (+ eAGR2) in the ECM of eAGR2 (Figure 7E–G) using apical F-actin (Figure 7E,F). The presence of eAGR2, at the time of plating, gave rise to organoids unable to form a lumen (+ eAGR2) (Figure 7E–G) thereby showing that eAGR2 interferes with the formation of hollow organoid. Collectively, these data provide further evidence that eAGR2, in the ECM, induced loss of cell polarity and lumen formation.10.7554/eLife.13887.013Figure 7.Extracellular AGR2 disrupts apico-basal polarity and lumen formation.

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