Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors
2021; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.15252/embr.202051412
ISSN1469-3178
AutoresDaria Sicari, Federica Grazia Centonze, Raphaël Pineau, Pierre‐Jean Le Reste, Luc Négroni, Sophie Chat, M. Aiman Mohtar, Daniel Thomas, Reynald Gillet, Ted R. Hupp, Éric Chevet, Aeid Igbaria,
Tópico(s)Redox biology and oxidative stress
ResumoArticle12 March 2021Open Access Transparent process Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors Daria Sicari Daria Sicari orcid.org/0000-0003-3847-129X Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Federica G Centonze Federica G Centonze Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Raphael Pineau Raphael Pineau Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Pierre-Jean Le Reste Pierre-Jean Le Reste Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Neurosurgery Department, University Hospital of Rennes, Rennes, France Search for more papers by this author Luc Negroni Luc Negroni orcid.org/0000-0002-6204-3917 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France UMR7104, Centre National de la Recherche Scientifique, Illkirch, France U1258, Institut National de la Santé et de la Recherche Médicale, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Sophie Chat Sophie Chat orcid.org/0000-0003-0671-5229 CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France Search for more papers by this author M Aiman Mohtar M Aiman Mohtar orcid.org/0000-0002-9015-9802 Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edinburgh, UK Search for more papers by this author Daniel Thomas Daniel Thomas orcid.org/0000-0002-9650-0434 CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France Search for more papers by this author Reynald Gillet Reynald Gillet orcid.org/0000-0001-9458-9503 CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France Search for more papers by this author Ted Hupp Ted Hupp Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edinburgh, UK International Centre for Cancer Vaccine Science, Gdansk, Poland Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet [email protected] orcid.org/0000-0001-5855-4522 Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Aeid Igbaria Corresponding Author Aeid Igbaria [email protected] orcid.org/0000-0003-3940-1824 Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel Search for more papers by this author Daria Sicari Daria Sicari orcid.org/0000-0003-3847-129X Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Federica G Centonze Federica G Centonze Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Raphael Pineau Raphael Pineau Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Pierre-Jean Le Reste Pierre-Jean Le Reste Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Neurosurgery Department, University Hospital of Rennes, Rennes, France Search for more papers by this author Luc Negroni Luc Negroni orcid.org/0000-0002-6204-3917 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France UMR7104, Centre National de la Recherche Scientifique, Illkirch, France U1258, Institut National de la Santé et de la Recherche Médicale, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Sophie Chat Sophie Chat orcid.org/0000-0003-0671-5229 CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France Search for more papers by this author M Aiman Mohtar M Aiman Mohtar orcid.org/0000-0002-9015-9802 Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edinburgh, UK Search for more papers by this author Daniel Thomas Daniel Thomas orcid.org/0000-0002-9650-0434 CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France Search for more papers by this author Reynald Gillet Reynald Gillet orcid.org/0000-0001-9458-9503 CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France Search for more papers by this author Ted Hupp Ted Hupp Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edinburgh, UK International Centre for Cancer Vaccine Science, Gdansk, Poland Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet [email protected] orcid.org/0000-0001-5855-4522 Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Search for more papers by this author Aeid Igbaria Corresponding Author Aeid Igbaria [email protected] orcid.org/0000-0003-3940-1824 Inserm U1242, University of Rennes, Rennes, France Centre de lutte contre le cancer Eugène Marquis, Rennes, France Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel Search for more papers by this author Author Information Daria Sicari1,2,12, Federica G Centonze1,2,13, Raphael Pineau1,2,13, Pierre-Jean Le Reste1,2,3, Luc Negroni4,5,6,7, Sophie Chat8, M Aiman Mohtar9,13, Daniel Thomas8, Reynald Gillet8, Ted Hupp9,10, Eric Chevet *,1,2 and Aeid Igbaria *,1,2,11 1Inserm U1242, University of Rennes, Rennes, France 2Centre de lutte contre le cancer Eugène Marquis, Rennes, France 3Neurosurgery Department, University Hospital of Rennes, Rennes, France 4Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France 5UMR7104, Centre National de la Recherche Scientifique, Illkirch, France 6U1258, Institut National de la Santé et de la Recherche Médicale, Illkirch, France 7Université de Strasbourg, Illkirch, France 8CNRS, Institut de Génétique et Développement de Rennes (IGDR), UMR6290, Univ. Rennes, Rennes, France 9Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edinburgh, UK 10International Centre for Cancer Vaccine Science, Gdansk, Poland 11Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel 12Present address: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy 13Present address: UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia *Corresponding author. Tel: +33 2 23237258; E-mail: [email protected] *Corresponding author. Tel: +972 8 6428411; E-mail: [email protected] EMBO Reports (2021)22:e51412https://doi.org/10.15252/embr.202051412 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions—a phenomenon we name ER to Cytosol Signaling (ERCYS). Synopsis Endoplasmic Reticulum (ER) stress in cancer cells causes a subset of ER proteins to escape to the cytosol where they bind and inhibit key signaling pathways to increase cancer cell fitness. ER stress mediated protein reflux is a conserved ER surveillance mechanism from yeast to mammals that plays a physiological role to relieve the ER from its contents upon ER stress. ER refluxed proteins gain new functions once they are in the cytosol. The refluxed PDI-like protein AGR2 inhibits p53 signaling by binding and inhibiting p53 protein Introduction The endoplasmic reticulum (ER) is the gateway to the secretory pathway thus maintaining the communication between the cell's intracellular space and extracellular environment. In addition, the ER is a sensing organelle that coordinates many stress signaling pathways (Higa & Chevet, 2012; Alexia et al,2013; Hetz et al,2015). Secretory and transmembrane proteins translocate into the ER through different translocation/membrane insertion molecular machines including the Sec61 channel. The ER is crowded with molecular chaperones and foldases that ensure these proteins' productive folding followed by their export en-route to their final destination (Rapoport, 2007). Diverse perturbations compromise the folding and maturation of secretory proteins in the ER thereby causing ER stress. To ensure productive folding, cells have also evolved various ER quality control (ERQC) systems allowing for further folding rounds (Adams et al,2019) and their degradation in the cytosol by a process termed ER-associated degradation (ERAD) (Travers et al,2000; Rutkowski et al,2006; Vembar & Brodsky, 2008). In addition to ERQC and ERAD, a pre-emptive quality control (pre-QC) mechanism was also described that averts protein entry into the secretory pathway under protein-folding stress resulting in their proteasomal degradation in the cytosol (Kang et al,2006). If these quality control systems are overwhelmed, ER stress activates a signaling pathway called the unfolded protein response (UPR) that aims at restoring ER homeostasis. However, if the UPR adaptive function fails, cell death programs are activated (Almanza et al,2019). The UPR is transduced by three transmembrane proteins (PERK, ATF6α and IRE1α) that sense and monitor the protein-folding status of the ER through their luminal domains and transmit signals to the rest of the cell through their cytosolic domain (Almanza et al,2019). In the past three decades, a subset of ER-resident proteins was reported to accumulate in the cytosol. This was observed in several human diseases including cancer and degenerative diseases (Turano et al,2002; Afshar et al,2005; Tarr et al,2010; Galligan & Petersen, 2012; Kanekura et al,2015; Wiersma et al,2015; Shim et al,2018). The localization of proteins identified as ER-resident to other cellular compartments has been extensively reported for instance for members of the protein disulfide isomerase (PDI) family, for GRP78/BiP or for calreticulin (Turano et al,2002; Afshar et al,2005; Tarr et al,2010; Galligan & Petersen, 2012; Wiersma et al,2015; Shim et al,2018). Despite this recurring observation, the mechanisms by which ER-resident proteins relocate in the cytosol and the potential functions of those proteins in this compartment remain unclear. Recently, we showed that protein-folding stress causes ER-resident proteins to be refluxed to the cytosol in the yeast Saccharomyces Cerevisiae (Igbaria et al,2019). This mechanism requires ER and cytosolic chaperones and co-chaperones but is independent of ERAD and of protein degradation (Igbaria et al,2019). Here, we found that ER stress-mediated protein reflux is conserved in mammalian cells and in cancer cells isolated from human and murine tumors in which it aims at debulking the ER upon stress. Moreover, we found that this process to be constitutively active in tumor cells and lead to cytosolic gain-of-functions of the refluxed protein as inhibitor of tumor suppressors, thereby exhibiting pro-oncogenic features. Results ER-resident proteins are refluxed from the ER lumen to the cytosol in cancer cells isolated from human and murine GBM tumors To study the role of ER protein reflux in tumors, we initially focused on Glioblastoma multiforme (GBM) in which the unfolded protein response (UPR) sustains tumor aggressiveness (Obacz et al,2017). Mouse GBM cells (GL261) were grafted orthotopically in the brain of immunocompetent C57BL/6 mice and 30 days post-injection, tumors were resected, dissociated, and isolated tumor cells subjected to subcellular fractionation using previously validated fractionation protocols (Holden & Horton, 2009). Immunoblot analysis of the digitonin fraction (enriched in cytosolic proteins) from freshly isolated tumor cells was compared with that of dissociated control tissue from the opposite hemisphere of the brain (non-tumor). It revealed higher levels of select ER-resident proteins in tumor than in the non-tumor cells' cytosolic fractions (Fig 1A-1E and Appendix Fig S1A and B). Several ER-resident proteins (ERp29/PDIA9 and ERp57/PDIA3) were enriched up to ∼ 70% in the cytosolic fraction compared with only ∼ 10% enrichment in non-tumor controls. These results indicate that tumor cells are more prone to exhibit reflux of ER proteins to the cytosol than non-tumoral cells. To rule out the possibility that ER stress in tumor cells may change the composition of the ER membrane thereby sensitizing it to digitonin treatment (detergent), we consequently used a digitonin-free subcellular protein fractionation protocol (Lodish, 2000). Cells were disrupted using a 26-gauge needle, and then, differential centrifugation was applied as shown in Appendix Fig S1C. After analyzing the cytosolic fractions, we observed results comparable to those obtained with the digitonin-based protocol (Fig 1A and Appendix Fig S1D). We confirmed these findings in another GBM model, the human U87 cells orthotopically implanted in the brain of NSG mice (Appendix Fig S1E). These two different protocols further strengthen the notion that ER proteins do exit the ER to reach the cytosol more actively in tumor cells. Figure 1. ER proteins are rerouted to the cytosol in human and mouse Glioblastoma (GBM) tumors A. Representative Western blots after subcellular protein fractionation experiment in isolated murine-derived non-tumor (NT) and GBM tumor (T) tissues. B. Total levels of the ER-luminal proteins tested from the cell lysate derived from tissues used in (A). C–E. Quantification of the protein levels of ER luminal proteins in the cytosolic fraction as shown in (A). n = 5 biological replicates and the horizontal line represent the sample mean. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. P-values < 0.05 were considered significant). F, G. Human-derived GBM tumors were processed as in (A). Representative Western blot was performed (F), and the percentage of ER-luminal protein cytosolic localization (G) were quantified. Data are the average from six different tumors. N = 6 Bars and error bars indicate mean Download figure Download PowerPoint We next asked whether this phenomenon is also observed in human GBM samples freshly isolated from patients at surgery (Table S1). We tested the subcellular localization of ER-resident proteins in freshly isolated human tumor cells. Tumor tissues were dissociated, and digitonin fractions tested for the presence of ER luminal proteins using immunoblotting. In the majority of tumors (80% of the tested tumors), ∼ 50% of the ER proteins evaluated were detected in the digitonin fraction including ERDJ3/DNAJB11 in its N-glycosylated state (Fig 1F-G and Appendix Fig S1F). Moreover, individual tumors exhibited heterogenous refluxed protein patterns, which might reflect inter-tumor heterogeneity (Fig 1F and G). In both GBM tumors (human- or murine-derived, N-linked-glycoproteins (such as ERDJ3/DNAJB11) were found in the digitonin fraction thus indicating that the refluxed proteins had been translocated into the ER and modified by N-Linked glycosylation, before being refluxed to the cytosol (Appendix Fig S1G-H). These data indicate that in GBM, ER protein reflux might be selectively regulated by different factors such as tumor heterogeneity, genetic background, or activation status of UPR. These findings will certainly stimulate others to replicate and extend these data in other tumor models. ER stress mediates ER-resident proteins reflux from the ER to the cytosol We next sought to identify factors regulating ER-to-cytosol protein reflux. Recently, we reported that ER luminal proteins were refluxed to the cytosol upon ER stress in the yeast S. cerevisiae (Igbaria et al,2019; Lajoie & Snapp, 2020) in a chaperone-mediated process (Igbaria et al,2019). We tested whether ER stress/UPR activation—two factors that showed a correlation with protein reflux in S. cerevisiae—would also cause ER protein reflux in mammalian cells. As such we monitored the localization of an engineered ER-targeted super-folder GFP (ER-sfGFP) using confocal microscopy, to follow the fate of ER-sfGFP in living cells. The cells were also transfected with the cytosolically localized mCherry used as a cytosolic marker. Notably, cells treated with Tunicamycin (Tm), which perturbs protein folding by inhibiting N-linked glycosylation, Thapsigargin (Tg) which inhibits the sarco-endoplasmic reticulum Ca2 + ATPase or Brefeldin A (BFA) that prevents protein transport from the ER to the Golgi apparatus, showed enhanced colocalization of ER-sfGFP with the cytosolic mCherry. The cytosolic colocalization of ER-sfGFP/mCherry reached a maximum after 24hrs of treatment (Fig 2A and Appendix Fig S2A). To confirm that the ER-resident proteins found in the cytosol after ER stress originated from the ER lumen, we engineered an ER-targeted photoactivatable fluorescent protein (FP) called mEOS3.2 by adding an ER signal peptide and a KDEL ER-retention sequence that discriminates newly synthesized proteins from the pre-existing pool. Indeed, UV exposure shifts the excitation maxima of the mEOS3.2 from 488nm to 573nm, allowing detection of proteins synthesized before a UV pulse exposure (Appendix Fig S2B). Notably, after a UV pulse and Tm or DMSO treatments for 24 h, the mEOS3.2573 pool was mainly localized in the ER of the DMSO treated cells, but cells treated with Tm or BFA showed a significant fraction of mEOS3.2573 localized in the cytosol (Appendix Fig S2B). These results indicated that during stress pre-existing and ER localized proteins are refluxed to the cytosol where they might exist in a folded, functional state. Figure 2. Pre-existing, ER-targeted mEOS3.2 and ER endogenous proteins reflux to the cytosol during ER stress A. Left: Representative images of cells were transfected with super-folder GFP (sfGFP) treated with 250 ng/ml Tunicamycin (Tm), 50 nM Thapsigargin (Tg), and 250 ng/ml Brefeldin-A (BFA) for 24 h. Right: Quantification of the microscopy images of cells expressing ER-targeted sfGFP and the cytosolically localized mCherry. Data values are the mean ± SD of technical replicates (n = 10) from three independent experiments (****P < 0.0001). One-way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software. Scale bar 15 μm. B, C. Subcellular protein fractionation of several ER-resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 h using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) (B) or differential centrifugation (C) protocols, representative Western blots are showed. D, E. Quantification of the subcellular protein fractionation of several ER endogenous proteins in HEK293T cells treated with different concentrations of Tm and Tg for 16 h from panels (B and C), respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad). F. Mass spec analysis of soluble ER-targeted glycoproteins in HEK293T cells treated with Tg and analyzed as described in materials and methods. Biological triplicates and data analyses were carried using Cytoscape v3.8.0 for network representation (PMID: 14597658) with the Cytoscape Stringapp for enrichments (PMID: 30450911). Statistics were done using the default settings of the Cytoscape app. G. Mass spectrometry analysis of cytosolically located soluble ER glycoproteins in HEK293T cells treated with Tg compared with that found in human GBM tumor cells-derived cytosols. Download figure Download PowerPoint We next tested whether endogenous ER-resident proteins were also refluxed to the cytosol in cultured cells that were exposed to various ER stress inducers. Subcellular protein fractionation using minimal concentration of digitonin that results in proper separation of the different subcellular fractions was carried out in cells subjected to ER stress induced by Tm or Tg. This was followed by an analysis of the localization of different endogenous ER-resident proteins including the soluble ERp29, PRDX4, PDIA3, and the integral protein calnexin (CANX). We found that soluble ER luminal proteins were enriched in the digitonin fraction up to 50-55% (Fig 2B and D) but not calnexin, thus indicating that ER reflux could be exclusive for soluble proteins. We then compared those results to those obtained from the detergent-free protocol (Lodish, 2000) using differential centrifugation, to rule out the possibility that ER stress inducers may alter the ER membrane properties toward digitonin. As shown in Fig 2C and E, we observed results similar to those obtained with the digitonin-based protocol (Fig 2B and D). We further investigated this by examining the integrity of the ER membrane in those cells. We obtained pellets post-digitonin fraction and after the 100,000xg centrifugation and treated them with proteinase K in the absence or presence of TritonX-100. We reasoned that if the ER membrane is damaged/ruptured due to digitonin or differential centrifugation protocols, ER luminal proteins should be sensitive to proteinase-K-mediated proteolysis. As shown in Appendix Fig S2C and D, while proteinase K was active toward the cytosolic portion of Calnexin, the ER luminal proteins were protected from proteinase-K in the absence of TritonX-100 in post-digitonin pellet. Similar results were also observed in the 100,000 g pellet (Appendix Fig S2C and D). Moreover, if the ER membrane is damaged or ruptured due to ER stress, we could expect that it should be permeable to small metabolite such as Glutathione. As such using a version of the redox sensitive eroGFP that is attached to the ER membrane with the eroGFP facing the luminal side, we found that the redox state of eroGFP remained oxidized even during ER stress (Appendix Fig S2E). Those data indicate that during ER protein reflux, the ER membrane is not significantly damaged neither after digitonin treatment nor following differential centrifugation protocols used in our study. Next, we sought to systematically characterize the spectrum of proteins from the secretory pathway refluxed from the ER. To this end, we enriched N-glycosylated proteins from the digitonin fraction extracted from HEK293T cells treated with Tg (Appendix Fig S2F). The purified material was subjected to mass spectrometry analysis. We focused on soluble glycoproteins that were enriched in the cytosolic fraction after Tg treatment compared with control. We identified 26 different soluble secretory N-glycoproteins present in the cytosol (Table S2). Gene Ontology-based analysis showed that these proteins mostly emanated from both ER and lysosomal compartments. Moreover, 23 out of these 26 proteins were part of a unique functional network (Fig 2F and Appendix Table S2), thus suggesting functional implications to this observation. We also performed a similar analysis on digitonin fractions from GBM tumor cells (isolated from patients) and compared them with our previous analysis of Tg treated HEK293T fractions. Interestingly, about 60% of hits were enriched in both fractionation approaches (Fig 2G and Appendix Table S2). This observation led us to hypothesize that during ER stress the reflux of ER proteins to the cytosol may play an important role to decrease the protein load within the ER in order to regain homeostasis. ER protein reflux was also observed in other cancer cell lines such as GL261, U87, and A549 (lung adenocarcinoma) (Fig 3A–H and Appendix Fig S3A–F) using the two aforementioned subcellular protein fractionation protocols. Next, analysis of immunogold-labeled electron microscopy images unveiled that PDIA3 distributed to non-ER locations in cells treated with ER stressors compared with DMSO treated cells (Fig 4A–C). It is worth to note that in cancer cells the amount of ER proteins refluxed to the cytosol was higher than in non-cancer cells (Appendix Fig S3G). Figure 3. ER protein reflux is constitutive in cancer cells A–C. Subcellular protein fractionation of several ER-resident proteins in (A) GL261, (B) U87 and (C) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin. (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) D–F. Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261, U87, and A549 cells as in (A–C), respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad). G. Subcellular protein fractionation of several ER-resident proteins in A549 cells treated with the indicated concentrations of Tm or Tg using differential centrifugation protocol. H. Quantification of the subcellular protein fractionation of several ER endogenous proteins in A549 as in (G). Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad). Download figure Download PowerPoint Figure 4. PDI proteins are redistributed to the cytosol during ER stress A, B. Representative transmission electron microscopy images of the gold particles distribution (after immunogold labeling with PDIA3 antibodies) in A549 cells treated with DMSO or Tm. In the inserts, gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles. (Scale bar 500 nm). n represents the nucleus, and m represents the mitochondria. C. Violin plots of the gold particles distribution from the electron microscopy experiment (immunogold labeling of PDIA3) as shown in (B, C) *P-value = 0.0486. n = 8 for DMSO and n = 8 for Tm. Thick horizontal lines represent mean ± SD -lighter dashed lines. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad). Download figure Download PowerPoint These data indicate that upon ER stress, ER luminal proteins (and ER-targeted sfGFP/mEOS3.2) are refluxed to the cytosol. Fluorescence microscopy with ER-sfGFP and ER-mEOS3.2 in HEK293T cells (Fig 2A, Appendix Fig S2A and Fig 4) confirmed the results obtained using cell fractionation and serve as alternative detergent-free methods to monitor reflux from the ER. Moreover, the ER-mEOS3.2 experiment showed that ER protein reflux occurred for proteins that already resided in the ER rather than as a result of the pre-emptive quality control mechanism (Kang et al,2006). ER stress-mediated reflux as an ER to CYtosol Signaling (ERCYS) pathway to inhibit tumor suppressors Thus far, we have shown that ER protein reflux is constitutive in some cancer cells as is the activation of the UPR and we thus hypothesized that, as the UPR, it may play an adaptive and pro-oncogenic function contributing to cancer cells increased fitness. To investigate possible adaptive mechanisms of the reflux process, we evaluated the nature of refluxed proteins in A549 cells subjected to ER stress (Fig 3, Appendix Fig S3A–F and Fig 5A). We focused on Anterior GRadient 2 (AGR2, PDIA17) that was highly enriched in the digitonin fractions upon ER stress (Fig 5A and Appendix Fig S3A–C). AGR2 is a PDI family member thought to catalyze protein folding through thiol-disulfide based reactions (Chevet et al,2013). In many studies, it has been shown to exert pro-oncogenic functions through yet ill-defined mechanisms. For instance, AGR2 was shown to interact with and to inhibit the activity of the p53 tumor suppressor (Pohler et al,2004). Here, we propose a model in which ER stress in cancer cells may cause constitutive AGR2 reflux to the cytosol, where AGR2 might in turn gain new functions to interact and inhibit p53. To test this hypothesis, co-immunoprecipitation experiments showed that upon stress AGR2 was translocated to the cytosol and interacted with wild-type (wt) p53 in A549 cells treated with Tm, Tg, or BFA (Fig 5B). This was shown by measuring wt p53 transcriptional and p21 protein expression levels (a downstream target of p53 signaling). Tm, Tg, or BFA treatment reduced p21 protein levels, as well as wt p53 phosphorylation and transcriptional activity as shown in cells transfected with a luciferase reporter under the p53-DNA-binding site (Fig 5C). Moreover, AGR2
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