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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.


Structure model showing the CXXS (81–84) sequence.(A) The CXXS residues (ball-and-stick model in bottom left and top right) in the AGR2 crystal structure to relate to the position of these vs the KTEL motif. (B) Zoom in of CXXS motif along with residues 78–80 (stick model). S84 forms hydrogen bonded interaction with the carboxylic acid of D79 side chain and backbone carbonyl of L78. (C) Y124 (stick model) from an adjacent loop interacts with H83, which in turn forms a hydrogen bond to C81; providing a good interaction network. (D) AXXA mutant. In silico mutation of C81 and S84 into Alanine illustrates the loss of interactions as compared to that seen in (B) between S84 and L78/D79, and in (C) between Y127-H83-C81.DOI:http://dx.doi.org/10.7554/eLife.13887.012
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fig6s2: Structure model showing the CXXS (81–84) sequence.(A) The CXXS residues (ball-and-stick model in bottom left and top right) in the AGR2 crystal structure to relate to the position of these vs the KTEL motif. (B) Zoom in of CXXS motif along with residues 78–80 (stick model). S84 forms hydrogen bonded interaction with the carboxylic acid of D79 side chain and backbone carbonyl of L78. (C) Y124 (stick model) from an adjacent loop interacts with H83, which in turn forms a hydrogen bond to C81; providing a good interaction network. (D) AXXA mutant. In silico mutation of C81 and S84 into Alanine illustrates the loss of interactions as compared to that seen in (B) between S84 and L78/D79, and in (C) between Y127-H83-C81.DOI:http://dx.doi.org/10.7554/eLife.13887.012

Mentions: Next, we investigated whether eAGR2 could play a direct extracellular role through its thioredoxin-like domain (CXXS motif) (Figure 6E). To this end, we purified recombinant human AGR2-AXXA, the inactive form of AGR2 thioredoxin-like domain (Figure 6—figure supplement 1G–J). AGR2-AXXA mutant, in the ECM of AGR2-depleted organoids, did not restore the formation of tumor organoids (Figure 6F). However, the AGR2-AXXA mutant might be improperly folded as displayed in Figure 6—figure supplement 2. The sequence C81-S84 is a loop connecting a β-sheet strand with an α-helix. The mutation to A could affect the structure, since we thereby loose the key interactions stabilizing the 'folding'/closure of that loop (Figure 6—figure supplement 2). S84 forms strong hydrogen bonds to both the carboxylic acid of D79 and the backbone carbonyl of L78 (Figure 6—figure supplement 2). Thus, we used another inactive form of AGR2 thioredoxin-like domain, the AGR2-SXXS mutant. In contrast to the AGR2-AXXA mutant, AGR2-SXXS mutant restored the formation of AGR2-depleted tumor organoids (Figure 6G). These results show that the single cysteine residue in the AGR2 thioredoxin-like domain is not essential for the formation of tumor organoids. To further assess the redox function of eAGR2 in such process, we treated tumor organoids depleted in AGR2 (H23-Sh-AGR2) with dithiothreitol (DTT) at a concentration of 5µM and no cell death was detected (Figure 6H). The addition of DTT to the medium of AGR2-depleted H23 cells neither reversed the cell growth inhibition induced by AGR2 depletion (Figure 6I) nor restored the formation of tumor organoids (Figure 6J). Thus, these results demonstrate that treating cells with reducing agent cannot mimic the effects of eAGR2.


Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
Structure model showing the CXXS (81–84) sequence.(A) The CXXS residues (ball-and-stick model in bottom left and top right) in the AGR2 crystal structure to relate to the position of these vs the KTEL motif. (B) Zoom in of CXXS motif along with residues 78–80 (stick model). S84 forms hydrogen bonded interaction with the carboxylic acid of D79 side chain and backbone carbonyl of L78. (C) Y124 (stick model) from an adjacent loop interacts with H83, which in turn forms a hydrogen bond to C81; providing a good interaction network. (D) AXXA mutant. In silico mutation of C81 and S84 into Alanine illustrates the loss of interactions as compared to that seen in (B) between S84 and L78/D79, and in (C) between Y127-H83-C81.DOI:http://dx.doi.org/10.7554/eLife.13887.012
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fig6s2: Structure model showing the CXXS (81–84) sequence.(A) The CXXS residues (ball-and-stick model in bottom left and top right) in the AGR2 crystal structure to relate to the position of these vs the KTEL motif. (B) Zoom in of CXXS motif along with residues 78–80 (stick model). S84 forms hydrogen bonded interaction with the carboxylic acid of D79 side chain and backbone carbonyl of L78. (C) Y124 (stick model) from an adjacent loop interacts with H83, which in turn forms a hydrogen bond to C81; providing a good interaction network. (D) AXXA mutant. In silico mutation of C81 and S84 into Alanine illustrates the loss of interactions as compared to that seen in (B) between S84 and L78/D79, and in (C) between Y127-H83-C81.DOI:http://dx.doi.org/10.7554/eLife.13887.012
Mentions: Next, we investigated whether eAGR2 could play a direct extracellular role through its thioredoxin-like domain (CXXS motif) (Figure 6E). To this end, we purified recombinant human AGR2-AXXA, the inactive form of AGR2 thioredoxin-like domain (Figure 6—figure supplement 1G–J). AGR2-AXXA mutant, in the ECM of AGR2-depleted organoids, did not restore the formation of tumor organoids (Figure 6F). However, the AGR2-AXXA mutant might be improperly folded as displayed in Figure 6—figure supplement 2. The sequence C81-S84 is a loop connecting a β-sheet strand with an α-helix. The mutation to A could affect the structure, since we thereby loose the key interactions stabilizing the 'folding'/closure of that loop (Figure 6—figure supplement 2). S84 forms strong hydrogen bonds to both the carboxylic acid of D79 and the backbone carbonyl of L78 (Figure 6—figure supplement 2). Thus, we used another inactive form of AGR2 thioredoxin-like domain, the AGR2-SXXS mutant. In contrast to the AGR2-AXXA mutant, AGR2-SXXS mutant restored the formation of AGR2-depleted tumor organoids (Figure 6G). These results show that the single cysteine residue in the AGR2 thioredoxin-like domain is not essential for the formation of tumor organoids. To further assess the redox function of eAGR2 in such process, we treated tumor organoids depleted in AGR2 (H23-Sh-AGR2) with dithiothreitol (DTT) at a concentration of 5µM and no cell death was detected (Figure 6H). The addition of DTT to the medium of AGR2-depleted H23 cells neither reversed the cell growth inhibition induced by AGR2 depletion (Figure 6I) nor restored the formation of tumor organoids (Figure 6J). Thus, these results demonstrate that treating cells with reducing agent cannot mimic the effects of eAGR2.

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.