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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 knockdown reduces tumorigenicity and metastatic potential.(A–C) Representative brightfield pictures of tumor organoids grown in 3D from cells harboring Sh-ctl or Sh-AGR2 (three independent experiments). The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. The p values (determined by Student's t test) are relative to Sh-ctl cells. *p≤0.05. **p≤0.01 and ***p≤0.001. (D, F) Representative pictures of the lungs in mice injected with Sh-ctl cells or Sh-AGR2 cells allowed to developed tumors for 2 weeks. Note the extensive lesions (white areas) on the surface of lungs from Sh-ctl mice and the relatively normal appearance of the lungs from Sh-AGR2 mice. (E, G) Representative H&E-stained sections for lungs injected with Sh-ctl cells or Sh-AGR2 cells (montage of x5 magnification of lung sections). Scale bars, 1 mm. Box plot showing the mean number of tumors per mouse in Sh-ctl (n=6) vs Sh-AGR2 (n=6) groups. The p values (determined by Student's t test) are relative to Sh-ctl cells. **p≤0.01.DOI:http://dx.doi.org/10.7554/eLife.13887.006
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fig3: AGR2 knockdown reduces tumorigenicity and metastatic potential.(A–C) Representative brightfield pictures of tumor organoids grown in 3D from cells harboring Sh-ctl or Sh-AGR2 (three independent experiments). The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. The p values (determined by Student's t test) are relative to Sh-ctl cells. *p≤0.05. **p≤0.01 and ***p≤0.001. (D, F) Representative pictures of the lungs in mice injected with Sh-ctl cells or Sh-AGR2 cells allowed to developed tumors for 2 weeks. Note the extensive lesions (white areas) on the surface of lungs from Sh-ctl mice and the relatively normal appearance of the lungs from Sh-AGR2 mice. (E, G) Representative H&E-stained sections for lungs injected with Sh-ctl cells or Sh-AGR2 cells (montage of x5 magnification of lung sections). Scale bars, 1 mm. Box plot showing the mean number of tumors per mouse in Sh-ctl (n=6) vs Sh-AGR2 (n=6) groups. The p values (determined by Student's t test) are relative to Sh-ctl cells. **p≤0.01.DOI:http://dx.doi.org/10.7554/eLife.13887.006

Mentions: To test the relevance of AGR2 overexpression in tumorigenesis, we silenced its expression in lung adenocarcinoma cell lines (A549, H23 and H1838) using lentivirus-mediated infection with AGR2 shRNA (Sh-AGR2). First, we monitored the efficacy of AGR2 depletion using immunofluorescence (Figure 2A,D,G) and Western blotting (Figure 2B,E,H). Cell growth was strongly inhibited in Sh-AGR2 cells compared to control cells (Sh-ctl) (Figure 2C,F,I–J and Figure 2—figure supplement 1) with no cell death detected (Figure 2K). Interestingly, soft-agar colony formation (Figure 2L–N) (used as indicator of malignant transformation) significantly reduced upon AGR2 silencing in adenocarcinoma cell lines, thereby indicating that AGR2 may favor tumor progression by increasing anchorage-independent growth. To further characterize the role of AGR2 in cancer, we assessed the impact of AGR2 depletion using the 3D human bronchial organoids system which we previously established (Fessart et al., 2013). AGR2 silencing dramatically decreased the formation of tumor organoids (Figure 3A–C). To determine whether AGR2 is required for tumor progression in vivo, we then evaluated the in vivo tumorigenic potential of Sh-ctl and Sh-AGR2 cells using two different adenocarcinoma cell lines (A549 and H1838), following tail vein injection in immunodeficient mice. Sh-ctl mice presented large metastases that were easily detected at the surface of the lungs compared to mice injected with Sh-AGR2 cells (Figure 3D,F). Moreover, AGR2 depletion significantly reduced total metastasis number (by ~50%) in both A549-Sh-AGR2 and H1838-Sh-AGR2 injected mice (Figure 3E,G). Taken together, these results show that overexpression of AGR2 in cancer cells increases their tumorigenic and metastatic potential.10.7554/eLife.13887.004Figure 2.AGR2 knock down in lung cancer cells decreases cell proliferation and malignant transformation.


Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
AGR2 knockdown reduces tumorigenicity and metastatic potential.(A–C) Representative brightfield pictures of tumor organoids grown in 3D from cells harboring Sh-ctl or Sh-AGR2 (three independent experiments). The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. The p values (determined by Student's t test) are relative to Sh-ctl cells. *p≤0.05. **p≤0.01 and ***p≤0.001. (D, F) Representative pictures of the lungs in mice injected with Sh-ctl cells or Sh-AGR2 cells allowed to developed tumors for 2 weeks. Note the extensive lesions (white areas) on the surface of lungs from Sh-ctl mice and the relatively normal appearance of the lungs from Sh-AGR2 mice. (E, G) Representative H&E-stained sections for lungs injected with Sh-ctl cells or Sh-AGR2 cells (montage of x5 magnification of lung sections). Scale bars, 1 mm. Box plot showing the mean number of tumors per mouse in Sh-ctl (n=6) vs Sh-AGR2 (n=6) groups. The p values (determined by Student's t test) are relative to Sh-ctl cells. **p≤0.01.DOI:http://dx.doi.org/10.7554/eLife.13887.006
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fig3: AGR2 knockdown reduces tumorigenicity and metastatic potential.(A–C) Representative brightfield pictures of tumor organoids grown in 3D from cells harboring Sh-ctl or Sh-AGR2 (three independent experiments). The bar graph shows the mean of organoids per well (mean ± SEM.) after 10 days of culture from three independent experiments. The p values (determined by Student's t test) are relative to Sh-ctl cells. *p≤0.05. **p≤0.01 and ***p≤0.001. (D, F) Representative pictures of the lungs in mice injected with Sh-ctl cells or Sh-AGR2 cells allowed to developed tumors for 2 weeks. Note the extensive lesions (white areas) on the surface of lungs from Sh-ctl mice and the relatively normal appearance of the lungs from Sh-AGR2 mice. (E, G) Representative H&E-stained sections for lungs injected with Sh-ctl cells or Sh-AGR2 cells (montage of x5 magnification of lung sections). Scale bars, 1 mm. Box plot showing the mean number of tumors per mouse in Sh-ctl (n=6) vs Sh-AGR2 (n=6) groups. The p values (determined by Student's t test) are relative to Sh-ctl cells. **p≤0.01.DOI:http://dx.doi.org/10.7554/eLife.13887.006
Mentions: To test the relevance of AGR2 overexpression in tumorigenesis, we silenced its expression in lung adenocarcinoma cell lines (A549, H23 and H1838) using lentivirus-mediated infection with AGR2 shRNA (Sh-AGR2). First, we monitored the efficacy of AGR2 depletion using immunofluorescence (Figure 2A,D,G) and Western blotting (Figure 2B,E,H). Cell growth was strongly inhibited in Sh-AGR2 cells compared to control cells (Sh-ctl) (Figure 2C,F,I–J and Figure 2—figure supplement 1) with no cell death detected (Figure 2K). Interestingly, soft-agar colony formation (Figure 2L–N) (used as indicator of malignant transformation) significantly reduced upon AGR2 silencing in adenocarcinoma cell lines, thereby indicating that AGR2 may favor tumor progression by increasing anchorage-independent growth. To further characterize the role of AGR2 in cancer, we assessed the impact of AGR2 depletion using the 3D human bronchial organoids system which we previously established (Fessart et al., 2013). AGR2 silencing dramatically decreased the formation of tumor organoids (Figure 3A–C). To determine whether AGR2 is required for tumor progression in vivo, we then evaluated the in vivo tumorigenic potential of Sh-ctl and Sh-AGR2 cells using two different adenocarcinoma cell lines (A549 and H1838), following tail vein injection in immunodeficient mice. Sh-ctl mice presented large metastases that were easily detected at the surface of the lungs compared to mice injected with Sh-AGR2 cells (Figure 3D,F). Moreover, AGR2 depletion significantly reduced total metastasis number (by ~50%) in both A549-Sh-AGR2 and H1838-Sh-AGR2 injected mice (Figure 3E,G). Taken together, these results show that overexpression of AGR2 in cancer cells increases their tumorigenic and metastatic potential.10.7554/eLife.13887.004Figure 2.AGR2 knock down in lung cancer cells decreases cell proliferation and malignant transformation.

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