Raphael Pineau

IRE1 Signaling Is Essential for Ischemia-Induced Vascular Endothelial Growth Factor-A Expression and Contributes to Angiogenesis and Tumor Growth In vivo

In solid tumors, cancer cells subjected to ischemic conditions trigger distinct signaling pathways contributing to angiogenic stimulation and tumor development. Characteristic features of tumor ischemia include hypoxia and glucose deprivation, leading to the activation of hypoxia-inducible factor-1-dependent signaling pathways and to complex signaling events known as the unfolded protein response. Here, we show that the activation of the endoplasmic reticulum stress sensor IRE1 is a common determinant linking hypoxia- and hypoglycemia-dependent responses to the up-regulation of vascular endothelial growth factor-A (VEGF-A). Tumor cells expressing a dominant-negative IRE1 transgene as well as Ire1alpha-null mouse embryonic fibroblasts were unable to trigger VEGF-A up-regulation upon either oxygen or glucose deprivation. These data correlated with a reduction of tumor angiogenesis and growth in vivo. Our results therefore suggest an essential role for IRE1-dependent signaling pathways in response to ischemia and identify this protein as a potential therapeutic target to control both the angiogenic switch and tumor development.

Inositol-requiring enzyme 1α is a key regulator of angiogenesis and invasion in malignant glioma

Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1α correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1β, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.

Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα

Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development.

High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor

BackgroundEpidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma.MethodsExpression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown.ResultsEREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α.ConclusionEREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

Detection of collagens in brain tumors based on FTIR imaging and chemometrics

Fourier transform infrared (FTIR) imaging has been used as a molecular histopathology tool on brain tissue sections after intracranial implantation and development of glioma tumors. Healthy brain tissue (contralateral lobe) as well as solid and diffuse tumor tissues were compared for their collagen contents. IR spectra were extracted from IR images for determining the secondary structure of protein contents and compared to pure product spectra of collagens (types I, III, IV, V, and VI). Multivariate statistical analyses of variance and correspondence factorial analysis were performed to differentiate healthy and tumor brain tissues as well as their classification according to their secondary structure profiles. Secondary structure profiles revealed that no collagen was present in healthy tissues; they are also significantly different from solid and diffuse tumors (p < 0.05). Solid and diffuse tumors could be discriminated with respect to the secondary structure profile of fibrillar and non-fibrillar collagens, respectively. We can thus propose to develop FTIR imaging for histopathology examination of tumors on the basis of collagen contents.

Inhibition of FGF receptor activity in glioma implanted into the mouse brain using the tetracyclin-regulated expression system.

We have investigated growth and vascularization of malignant glioma in mice upon conditional inhibition of fibroblast growth factor (FGF) receptor activity. C6 rat glioma cells were transfected with a dominant-negative fibroblast growth factor receptor-2 (FGFR2-DN) cDNA under the control of a tetracycline-regulated expression promoter (tet off) and implanted in the brain of immunodeficient mice. Magnetic resonance imaging analysis showed a significant decrease in tumor growth 14 days after implantation when FGFR2-DN was expressed compared to control. This size difference disappeared after 20 days. However, after 20 days, tumor and endothelial cells apoptosis were higher in the FGFR2-DN group and consequently angiogenesis was decreased whereas tumor cells were similarly associated with blood vessels at the tumor periphery. Pericyte coverage was not different between the two groups but a higher amount of pericytes not associated with vessels was found in the FGFR2-DN expressing group. This demonstrates, that conditional expression of inhibitor of FGF receptor activity in gliomas implanted in the brain of immunodeficient mice can be achieved efficiently, and that FGFs are major players in glioma development and in glioma angiogenesis.

Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity

IRE1α is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. The protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes. In an orthotopic xenograft model of human glioma, invalidation of IRE1α RNase or/and kinase activities generated tumors with remarkably distinct phenotypes. Contrasting with the extensive angiogenesis observed in tumors derived from control cells, the double kinase/RNase invalidation reprogrammed mesenchymal differentiation of cancer cells and produced avascular and infiltrative glioblastomas with blood vessel co-option. In comparison, selective invalidation of IRE1α RNase did not compromise tumor angiogenesis but still elicited invasive features and vessel co-option. In vitro, IRE1α RNase deficient cells were also endowed with a higher ability to migrate. Constitutive activation of both enzymes led to wild-type-like lesions. The presence of IRE1α, but not its RNase activity, is therefore required for glioblastoma neovascularization, whereas invasion results only from RNase inhibition. In this model, two key mechanisms of tumor progression and cancer cell survival are functionally linked to IRE1α.

Dual roles for CXCL4 chemokines and CXCR3 in angiogenesis and invasion of pancreatic cancer

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties.

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

Computational Modelling of Metastasis Development in Renal Cell Carcinoma.

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.