Late endosomal secretion is an unconventional secretion mechanism that depends on the SNARE protein VAMP7. We previously showed that VAMP7 mediates the secretion of the ER protein Reticulon3. However, the functional relevance and molecular mechanism of this secretory pathway remain unclear. Here, we show that VAMP7 knockout cells exhibit impaired secretion of ER- and mitochondrial-derived proteins and signs of ER and mitochondrial stress. In addition, pharmacological induction of organellar stress enhances the VAMP7-dependent secretion. We assess the pathophysiological significance of this mechanism using a preclinical glioblastoma model. VAMP7 knockout glioblastoma cells implanted in male rat brain develop into more necrotic tumors with reduced macrophage infiltration compared to controls, suggesting that VAMP7-dependent late endosomal secretion contributes to the tumor microenvironment and affects macrophage infiltration. Together, our results support a model in which late endosomal secretion functions as an organelle quality-control and stress-communication mechanism, with particular relevance to cancer.

Anterior Gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein that belongs to the protein disulphide isomerase (PDI) family, and whose expression and secretion are induced by stress. Extracellular (secreted) AGR2 has been proposed as a marker of ER stress-related proteostasis alterations. Cancer cells frequently overexpress intracellular AGR2 (iAGR2) and secrete extracellular AGR2 (eAGR2). These features are associated with tumour progression and may serve as potential biomarkers in epithelial ovarian cancer (EOC). To investigate the roles of both iAGR2 and eAGR2 in EOC, we first generated EOC cells overexpressing iAGR2 and secreting eAGR2. Antibodies blocking eAGR2 reduced the proliferation and migration of these overexpressing cells. Concurrently, supplementation of parental cells with recombinant eAGR2 partially rescued these properties, further supporting a functional extracellular role for AGR2 in EOC. Quantitative proteomics, complemented by analysis of the TCGA database, revealed that eAGR2 modulated the expression of proteins involved in autophagy. This suggests that eAGR2-induced signalling may enhance catabolic activity under stress conditions, thereby increasing nutrient availability and, in turn, facilitating protein synthesis. This was reflected in the increased translational activity observed in AGR2-overexpressing and eAGR2-stimulated cells. Our results highlight two distinct, compartmentalised roles for AGR2. Specifically, iAGR2 acts as an ER-resident PDI, enhancing protein folding and ER quality control. In a complementary manner, eAGR2 functions as a metabolic regulator that may relieve constraints on tumour cell aggressiveness by maintaining autophagic flux and promoting protein synthesis. Overall, these findings support a dual-compartment model in which iAGR2 couples ER proteostasis with the metabolic and translational stimulation mediated by eAGR2.

Abstract Background Anterior Gradient 2 (AGR2) is an endoplasmic reticulum (ER) resident protein involved in protein folding, predominantly expressed in mucin-secreting epithelial cells. Under stress, AGR2 is secreted into the extracellular milieu (eAGR2), where it disrupts epithelial integrity, attracts monocytes, and activates fibroblasts. We explored the relationship between AGR2 tissue expression in IBD patients and the efficacy of a neutralizing anti-eAGR2 antibody (TH-009) in murine models of colonic inflammation and fibrosis Methods AGR2 expression was analyzed by immunohistochemistry on ileal and colonic biopsies from IBD patients and controls. In mice, the efficacy of TH-009 was evaluated in models of colitis (3% DSS) and digestive fibrosis (3 cycles of 1.5% DSS) Results 132 ileal and colonic endoscopic biopsies from 66 patients with Crohn’s disease (CD), 50 patients with ulcerative colitis (UC), and 16 controls, and 96 surgical samples from 11 CD patients, 24 UC patients, and 11 controls were included. AGR2 expression correlated with mucosal inflammation (R² = 0.416, p<0.0001) and tissue damage (R² = 0.329, p<0.0001). All patients with mucosal inflammation showed AGR2 overexpression, while 38% of histologically healed patients had high AGR2, with a relapse rate at 18 months three times higher than those whose AGR2 levels were comparable to controls.In a murine acute colitis model (3% DSS), intravenous (IV) treatment with TH-009 at doses of 5 and 20 µg showed an improvement in weight (p<0.005 and p=0.0001, respectively), in the global colitis DAI score (p=0.002 and p=0.001), and in the histological colitis score (p=0.04 for 5 µg) compared to controls. Intraperitoneal and subcutaneous administration were also effective. There was a significant reduction in tissue infiltration by macrophages (p<0.01) and T lymphocytes (p<0.001), in myeloperoxidase activity, and in the tissue and serum expression of pro-inflammatory cytokines TNFα and IL-6. TH-009-treated mice restored intestinal permeability, measured by FITC-Dextran blood passage, to levels comparable to unexposed DSS mice, unlike DSS-only exposed mice (p<0.01). In a fibrosis model (3 cycles of 1.5% DSS), preventive and curative treatment with TH-009 reduced digestive fibrosis by 49% (p<0.0001), and reduced submucosal fibrosis thickness by 59% (p<0.0001). There was a significant decrease in the colonic expression of fibrosis-related genes (Collagen A1, αSMA, fibrinogen, and TGFβ). Conclusion AGR2 is a novel therapeutic target in IBD, correlating with inflammation and complications. Its expression is not affected by major therapies and is overexpressed in 40% of patients with deep histological healing. TH-009 improves inflammation, reverses fibrosis, and restores epithelial barrier functions. References 1.Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. Fessart D, Domblides C, Avril T, et al. Elife. 2016 May 30;5:e13887. 2.Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation. Maurel M, Obacz J, Avril T, et al. EMBO Mol Med. 2019 Jun;11(6):e10120. 3.Potential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homologue in Crohn's Disease Fibrosis. Vieujean S, Hu S, Bequet E, et al. J Crohns Colitis. 2021 Oct 7;15(10):1737-1750.

While locating in different microenvironments, glioblastoma stem-like cells (GSCs) receive maintenance signals and information to exploit neurovascular tracts. Although the cell adhesion mechanisms to blood vessels have been explored, the mediators guiding GSC interaction with the endothelial cells and their matrix remain incompletely resolved. Here, we identify junctional adhesion molecule C (JAMC) as a key regulator of heterophilic and homophilic interactions of GSC to endothelial surfaces. Using decellularized matrices, co-cultures, and organotypic brain slices, we demonstrate that JAMC restrains GSC spreading. JAMC-/- GSCs exhibit extended spreading on endothelial-borne supports, with exacerbated invasive, migratory, and mesenchymal-like behaviors, further eroding mice survival. Spatial transcriptomics of human samples confirmed the association between invasion and JAMC expression pattern. Quantitative proteomics unveiled that JAMC deletion elicits integrin upregulation, concurrent with a downregulation of the integrin negative regulator, SHARPIN. The landscape of adhesion molecules anchoring GSCs to vascular surfaces may coordinate cell migration in glioblastoma territories.

In cancer research, murine models play a crucial role as highly valuable preclinical tools. Here, we present a protocol to generate a murine model of glioblastoma through the direct intracranial injection of tumor cells. We describe steps for cell culture, intracranial implantation, and standard-of-care treatments. We then detail procedures for monitoring tumor growth using bioluminescent imaging.For complete details on the use and execution of this protocol, please refer to Pelizzari-Raymundo et al.1

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

Cholangiocarcinoma is a devastating liver cancer characterized by high aggressiveness and therapy resistance, resulting in poor prognosis. Long non-coding RNAs and signals imposed by oncogenic pathways, such as transforming growth factor β (TGFβ), frequently contribute to cholangiocarcinogenesis. Here, we explore novel effectors of TGFβ signalling in cholangiocarcinoma. LINC00313 is identified as a novel TGFβ target gene. Gene expression and genome-wide chromatin accessibility profiling reveal that nuclear LINC00313 transcriptionally regulates genes involved in Wnt signalling, such as the transcriptional activator TCF7. LINC00313 gain-of-function enhances TCF/LEF-dependent transcription, promotes colony formation in vitro and accelerates tumour growth in vivo. Genes affected by LINC00313 over-expression in CCA tumours are associated with KRAS and TP53 mutations and reduce overall patient survival. Mechanistically, ACTL6A and BRG1, subunits of the SWI/SNF chromatin remodelling complex, interact with LINC00313 and affect TCF7 and SULF2 transcription. We propose a model whereby TGFβ induces LINC00313 in order to regulate the expression of hallmark Wnt pathway genes, in co-operation with SWI/SNF. By modulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and promotes cholangiocarcinogenesis.

BACKGROUND:Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils. METHODS:We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings. RESULTS:We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors. CONCLUSIONS:Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.

We read with great interest the manuscript by Tian et al published in Gut. Tian et al report that in colorectal cancer (CRC) at stages III/IV, the presence of extracellular AGR2 in the tumour environment is due to its secretion by tumourassociated neutrophils (TANs). This, in turn, promotes tumour cells migration and invasion. Although these findings are original and exciting, it is essential to put them in the context of the biology of anterior gradient (AGR) proteins. AGR2 is the most studied member of the AGR family, which contains three proteins and belongs to the superfamily of protein disulfide isomerases. AGR13 exhibit all the features of endoplasmic reticulum (ER) resident proteins by containing a signal peptide and an ER retention motif. AGR2 and AGR3 can be secreted in the extracellular milieu to trigger cell migration, and for AGR2 only, epithelialtomesenchymal transition, chemoattraction of monocytes and myofibroblast activation. 6 Moreover, AGR2 is strongly expressed in endodermderived organs (lung stomach, colon prostate, intestine) and as such, is almost exclusively expressed in mucosal epithelial cells. Its overexpression has been associated with tumour aggressiveness in various cancers. Tian et al claim that in CRC, AGR2 is only produced by tumourinfiltrating neutrophils (TANs) which impacts on the migration of CRC cells through the activation of signalling pathways depending on CD98hccCT and Rho GTPases. Although this result is interesting, it is surprising that no expression of AGR2 is detected in tumour epithelial cells. We aimed at further documenting the expression levels of AGR2 protein in TANinfiltrated CRC. To do so, 21 CRC samples from our European institutions and covering 3 aetiologies including microsatellite stable (MSS) and mismatch repair and instable (MSI) adenocarcinomas (ADK) and CRC from patients with inflammatory bowel disease (IBD) associated with primary sclerosing cholangitis (IBD+PSC) or not (IBDCRC) at stages III/IV. These samples were processed for immunohistochemistry with antiAGR2 (antiAGR2 (M03) 1C3, Abnova, 1/800) and antiMPO antibody as a neutrophil marker (antiMPO (MAB3174), R&D System, 1/1000) and the images quantified (figure 1A,B). We also show data from the Human Protein Atlas (figure 1C,D). Mutually exclusive staining of AGR2 and MPO in CRC stages III/IV from various aetiologies were found thereby indicating that if TAN do express AGR2, it is marginal (figure 1A). Riener et al showed that AGR2 expression is lost or decreased in the majority of leftsided CRC, and is significantly associated with reduced overall patient survival suggesting that AGR2 might be a tumour suppressor. However, these results neither did correspond to the Tian et al article nor to their previous report in which results in discrepancies were speculated to come from variabilities in subjects or experimentalists. Hence one should rely on large international cohorts of patients with CRC to link AGR2 expression to specific aetiologies. Recent results from our laboratories did not confirm AGR2 loss of expression Letter

Figure S1: CD90 mRNA expression on NCI and glioma cell lines, glioma and GBM specimens; Figure S2: CD90 mRNA and protein are expressed on all GBM cells; Figure S3: Expression of adhesion/migration genes in CD90low and CD90high RNS cell lines and EMT associated genes in CD90low and CD90high GBM patients; Figure S4: CD90 affects cell-cell/matrix adhesion of U251 and U87 GBM cell lines; Figure S5: CD90 affects migration of U251, U87 and primary GBM cell lines; Figure S6: CD90 signaling involves SRC and FAK kinases; Figure S7: CD90-dependent migration involves MEK1, Rac1 and JNK signaling molecules; TABLE S1: Patients demographic and clinical characteristics; TABLE S2: Top10 genes up-regulated in CD90low and CD90high GBM patients1.

Supplementary Table 1, Figures 2-9 from IRE1 Signaling Is Essential for Ischemia-Induced Vascular Endothelial Growth Factor-A Expression and Contributes to Angiogenesis and Tumor Growth <i>In vivo</i>

Background & Aims: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer worldwide with an increasing incidence and limited therapeutic options. Therefore, there is an urgent need to open the field to new concepts for identifying clinically relevant therapeutic targets and biomarkers. Here, we explored the role and the clinical relevance of circular RNA (circRNA) circLTBP2 in iCCA.Methods: Transforming growth factor b (TGFb)-regulated circRNAs were identified by dedicated microarrays in human HuCC-T1 iCCA cell line, and their clinical relevance was evaluated in independent cohorts of patients. Gain and loss of function of circLTBP2 combined with functional tests was performed in vitro and in vivo in mice. RNA pulldown, microRNA sequencing, and RNA immunoprecipitation were performed to explore the sponging activity of circLTBP2. Results: CircLTBP2 (has_circ_0032603) was identified as a novel TGFb-induced circRNA in several cholangiocarcinoma cell lines. CircLTBP2 promotes tumour cell proliferation, migration, and resistance to gemcitabine-induced apoptosis in vitro and tumour growth in vivo. Mechanistically, circLTBP2 acts as a competitive RNA regulating notably the activity of the tumour suppressor microRNA miR-338-3p, leading to the overexpression of its pro-metastatic targets. The restoration of miR-338-3p levels in iCCA cells reversed the pro-tumourigenic effects driven by circLTBP2, including the resistance to gemcitabine-induced apoptosis. In addition, circLTBP2 expression predicted a reduced survival, as detected in not only tumour tissues but also serum extracellular vesicles isolated from patients with iCCA.Conclusions: CircLTBP2 is a novel effector of the pro-tumourigenic arm of TGFb and a clinically relevant biomarker easily detected from liquid biopsies in iCCA.Impact and implications: Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer with limited therapeutic options. Opening the field to new concepts is urgently needed to improve the survival of patients. Here, we evaluated the role and the clinical relevance of circular RNA. We report that TGFb-induced circLTBP2 contributes to CCA carcinogenesis and may constitute a clinically relevant prognostic biomarker detected in liquid biopsies. (c) 2023 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Inositol-requiring enzyme 1 (IRE1) is a major mediator of the unfolded protein response (UPR), which is activated upon endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues, a stress over-come by relying on IRE1 signaling as an adaptive mechanism. Herein, we report the discovery of structurally new IRE1 inhibitors identified through the structural exploration of its kinase domain. Characterization in in vitro and in cellular models showed that they inhibit IRE1 signaling and sensitize glioblastoma (GB) cells to the standard chemotherapeutic, temozolomide (TMZ). Finally, we demonstrate that one of these inhibitors, Z4P, permeates the blood-brain barrier (BBB), inhibits GB growth, and prevents relapse in vivo when administered together with TMZ. The hit compound disclosed herein satisfies an unmet need for targeted, non-toxic IRE1 inhibitors and our results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB.

PDF file - 809K, Table S1 List of genes deregulated in IRE1_DN cells. Table S2 Potential IRE1 cleavage sites on PER1 mRNA. Table S3 Characteristics of human glioma biopsies used in this study. Table S4 Primer Pairs used for PCR and qPCR studies. Table S5 siRNA sequences. Figure S1 PER1 mRNA expression in IRE1 signaling deficient cells. Figure S2 Validation of ski2, xrn1/2 siRNA efficacy. Figure S3 Potential IRE1 cleavage sites on PER1 mRNA. Figure S4 Expression of PER1 mRNA in PER1 silenced cells. Figure S5 XBP1s expression in glioma biopsies by immunohistochemistry. Figure S6: stress independent regulation of PER1 by IRE1α. Figure S7: (A) Two-dimensional modeling of potential Per1 mRNA cleavage sites using the M-Fold program. (B) In vitro RNA cleavage assay. Figure S8: Expression of Per1 mRNA in U87 cells upon lentiviral transduction. Figure S9: determination of protein (A-D) and mRNA (E) expression levels of potential PER1 targets in EV and IRE_DN cells transduced or not with shPER1. Figure S10: impact of CXCL3 re-expression on cell proliferation and neurosphere formation in IRE1_DN cells. Figure S11: (A-F) Immunohistochemical analysis of 3 typical glioblastoma paraffin sections using anti sXBP1 antibodies.