Limits...
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 and AGR2 thioredoxin mutant (AGR2-AXXA) purification and activity on organoids formation.(A) Coomassie stained SDS-PAGE gel showing GST induction with IPTG (left panels) and purified GST control proteins (right panel). Asterisk (*) indicate GST product. (B) Coomassie stained SDS-PAGE gel showing GST-AGR2 induction with IPTG (left panels) and purified AGR2-GST proteins (right panel). Asterisk (**) indicate AGR2 fused to GST product. (C) Coomassie stained SDS-PAGE gel showing GST-AGR2 cleaved with thrombin (left panels) and purified AGR2 was confirmed using an anti-AGR2 or anti-GST antibody. (D) The concentration of the purified AGR2 was obtained from a Bovine serum albumin (BSA) standard curve. (E) Representative images of the organoids formed following different dose of AGR2 (+) as compared to untreated cells (-). (F) The bar graph shows the mean of organoids per well (mean ± SEM) after 10 days of culture from three independent experiments. (G) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST induction with IPTG (left panels) and AGR2-AXXA-GST cleaved with thrombin (right panel). Asterisks (**) indicate AGR2-AXXA-GST product. (H) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST cleaved with thrombin (left panels) and purified AGR2-AXXA was confirmed using an anti-AGR2 or anti-GST antibody. (I) The concentration of the purified AGR2-AXXA was obtained using a Bovine serum albumin (BSA) standard curve. (J) The bar graphs show the number of organoids per well in absence (-eAGR2) of eAGR2 as compared to organoids in presence either of AGR2-AXXA mutant (+eAGR2-AXXA) or AGR2 wt (+eAGR2) in H1838 Sh-AGR2 depleted cells. Data are mean ± SEM. *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.13887.011
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940162&req=5

fig6s1: AGR2 and AGR2 thioredoxin mutant (AGR2-AXXA) purification and activity on organoids formation.(A) Coomassie stained SDS-PAGE gel showing GST induction with IPTG (left panels) and purified GST control proteins (right panel). Asterisk (*) indicate GST product. (B) Coomassie stained SDS-PAGE gel showing GST-AGR2 induction with IPTG (left panels) and purified AGR2-GST proteins (right panel). Asterisk (**) indicate AGR2 fused to GST product. (C) Coomassie stained SDS-PAGE gel showing GST-AGR2 cleaved with thrombin (left panels) and purified AGR2 was confirmed using an anti-AGR2 or anti-GST antibody. (D) The concentration of the purified AGR2 was obtained from a Bovine serum albumin (BSA) standard curve. (E) Representative images of the organoids formed following different dose of AGR2 (+) as compared to untreated cells (-). (F) The bar graph shows the mean of organoids per well (mean ± SEM) after 10 days of culture from three independent experiments. (G) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST induction with IPTG (left panels) and AGR2-AXXA-GST cleaved with thrombin (right panel). Asterisks (**) indicate AGR2-AXXA-GST product. (H) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST cleaved with thrombin (left panels) and purified AGR2-AXXA was confirmed using an anti-AGR2 or anti-GST antibody. (I) The concentration of the purified AGR2-AXXA was obtained using a Bovine serum albumin (BSA) standard curve. (J) The bar graphs show the number of organoids per well in absence (-eAGR2) of eAGR2 as compared to organoids in presence either of AGR2-AXXA mutant (+eAGR2-AXXA) or AGR2 wt (+eAGR2) in H1838 Sh-AGR2 depleted cells. Data are mean ± SEM. *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.13887.011

Mentions: Given that eAGR2 is secreted by tumor organoids (Figure 5A) and that AGR2 has been reported in the serum of cancer patients with an average level of eAGR2 ranging from 0.5 to 20 ng/ml (Chen et al., 2010), we assessed whether eAGR2 might exhibit specific extracellular functions. To this end, we used different adenocarcinoma cell lines depleted for AGR2 (A549-Sh-AGR2, H23-Sh-AGR2 and H1838-Sh-AGR2). Remarkably, the addition of recombinant human AGR2 (40 ng/ml) (Figure 6—figure supplement 1A–F) to the medium of AGR2-depleted tumor cell lines reversed the cell growth inhibition induced by AGR2 depletion (Figure 6A). Moreover, the addition of eAGR2 to the ECM of AGR2-depleted organoids restored the formation of tumor organoids (Figure 5B–C).10.7554/eLife.13887.010Figure 6.Extracellular AGR2 boosts the organoid-initiating frequency.


Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
AGR2 and AGR2 thioredoxin mutant (AGR2-AXXA) purification and activity on organoids formation.(A) Coomassie stained SDS-PAGE gel showing GST induction with IPTG (left panels) and purified GST control proteins (right panel). Asterisk (*) indicate GST product. (B) Coomassie stained SDS-PAGE gel showing GST-AGR2 induction with IPTG (left panels) and purified AGR2-GST proteins (right panel). Asterisk (**) indicate AGR2 fused to GST product. (C) Coomassie stained SDS-PAGE gel showing GST-AGR2 cleaved with thrombin (left panels) and purified AGR2 was confirmed using an anti-AGR2 or anti-GST antibody. (D) The concentration of the purified AGR2 was obtained from a Bovine serum albumin (BSA) standard curve. (E) Representative images of the organoids formed following different dose of AGR2 (+) as compared to untreated cells (-). (F) The bar graph shows the mean of organoids per well (mean ± SEM) after 10 days of culture from three independent experiments. (G) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST induction with IPTG (left panels) and AGR2-AXXA-GST cleaved with thrombin (right panel). Asterisks (**) indicate AGR2-AXXA-GST product. (H) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST cleaved with thrombin (left panels) and purified AGR2-AXXA was confirmed using an anti-AGR2 or anti-GST antibody. (I) The concentration of the purified AGR2-AXXA was obtained using a Bovine serum albumin (BSA) standard curve. (J) The bar graphs show the number of organoids per well in absence (-eAGR2) of eAGR2 as compared to organoids in presence either of AGR2-AXXA mutant (+eAGR2-AXXA) or AGR2 wt (+eAGR2) in H1838 Sh-AGR2 depleted cells. Data are mean ± SEM. *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.13887.011
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940162&req=5

fig6s1: AGR2 and AGR2 thioredoxin mutant (AGR2-AXXA) purification and activity on organoids formation.(A) Coomassie stained SDS-PAGE gel showing GST induction with IPTG (left panels) and purified GST control proteins (right panel). Asterisk (*) indicate GST product. (B) Coomassie stained SDS-PAGE gel showing GST-AGR2 induction with IPTG (left panels) and purified AGR2-GST proteins (right panel). Asterisk (**) indicate AGR2 fused to GST product. (C) Coomassie stained SDS-PAGE gel showing GST-AGR2 cleaved with thrombin (left panels) and purified AGR2 was confirmed using an anti-AGR2 or anti-GST antibody. (D) The concentration of the purified AGR2 was obtained from a Bovine serum albumin (BSA) standard curve. (E) Representative images of the organoids formed following different dose of AGR2 (+) as compared to untreated cells (-). (F) The bar graph shows the mean of organoids per well (mean ± SEM) after 10 days of culture from three independent experiments. (G) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST induction with IPTG (left panels) and AGR2-AXXA-GST cleaved with thrombin (right panel). Asterisks (**) indicate AGR2-AXXA-GST product. (H) Coomassie stained SDS-PAGE gel showing AGR2-AXXA-GST cleaved with thrombin (left panels) and purified AGR2-AXXA was confirmed using an anti-AGR2 or anti-GST antibody. (I) The concentration of the purified AGR2-AXXA was obtained using a Bovine serum albumin (BSA) standard curve. (J) The bar graphs show the number of organoids per well in absence (-eAGR2) of eAGR2 as compared to organoids in presence either of AGR2-AXXA mutant (+eAGR2-AXXA) or AGR2 wt (+eAGR2) in H1838 Sh-AGR2 depleted cells. Data are mean ± SEM. *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.13887.011
Mentions: Given that eAGR2 is secreted by tumor organoids (Figure 5A) and that AGR2 has been reported in the serum of cancer patients with an average level of eAGR2 ranging from 0.5 to 20 ng/ml (Chen et al., 2010), we assessed whether eAGR2 might exhibit specific extracellular functions. To this end, we used different adenocarcinoma cell lines depleted for AGR2 (A549-Sh-AGR2, H23-Sh-AGR2 and H1838-Sh-AGR2). Remarkably, the addition of recombinant human AGR2 (40 ng/ml) (Figure 6—figure supplement 1A–F) to the medium of AGR2-depleted tumor cell lines reversed the cell growth inhibition induced by AGR2 depletion (Figure 6A). Moreover, the addition of eAGR2 to the ECM of AGR2-depleted organoids restored the formation of tumor organoids (Figure 5B–C).10.7554/eLife.13887.010Figure 6.Extracellular AGR2 boosts the organoid-initiating frequency.

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