Analysing the contribution of ATM/ATR pathway activation in establishing the premature senescence of E2F1/E2F2-/- bone-marrow-derived macrophages

E2F1 and E2F2 transcription factors have an important role during the regulation of cell cycle. In experiments done with E2F1/E2F2 knockout mice, it has been described that bone-marrow-derived macrophages (BMDM) undergo an early rapid proliferation event related to DNA hyper-replication. As a consequence, DNA damage response (DDR) pathway is triggered and E2F1/E2F2 knockout macrophages enter premature senescence related to G2/M phase arrest. The exact mechanism trough which DNA hyper-replication leads to DDR in absence of E2F1 and E2F2 remains undiscovered. To determine whether the ATR/ATM pathway, the master regulator of G2/M checkpoint, might be the surveillance mechanism in order to regulate uncontrolled proliferation in the DKO model, we monitored and analysis biochemical properties of BMDM cultures in the presence of caffeine, a potent inhibitor of ATM/ATR activity. Our results show that the addition of caffeine abolishes premature senescence in DKO BMDM, stimulates γ-H2AX accumulation and decreases Mcm2 expression

Unraveling the Role of Angiogenesis in Cancer Ecosystems

Activation of the tumor and stromal cell-driven angiogenic program is one of the first requirements in the tumor ecosystem for growth and dissemination. The understanding of the dynamic angiogenic tumor ecosystem has rapidly evolved over the last decades. Beginning with the canonical sprouting angiogenesis, followed by vasculogenesis and intussusception, and finishing with vasculogenic mimicry, the need for different neovascularization mechanisms is further explored. In addition, an overview of the orchestration of angiogenesis within the tumor ecosystem cellular and molecular components is provided. Clinical evidence has demonstrated the effectiveness of traditional vessel-directed antiangiogenics, stressing on the important role of angiogenesis in tumor establishment, dissemination, and growth. Particular focus is placed on the interaction between tumor cells and their surrounding ecosystem, which is now regarded as a promising target for the development of new antiangiogenics

Anti-tumor effects of anti-Semaphorin 4D antibody unravel a novel pro-invasive mechanism of vascular targeting agents

One of the main consequences of inhibition of neovessel growth and vessel pruning produced by angiogenesis inhibitors is increased intratumor hypoxia. Growing evidence indicates that tumor cells escape from this hypoxic environment to better nourished locations, presenting hypoxia as a positive stimulus for invasion. In particular, anti-VEGF/R therapies produce hypoxia-induced invasion and metastasis in a spontaneous mouse model of pancreatic neuroendocrine cancer (PanNET), RIP1-Tag2. Here, a novel vascular-targeting agent targeting semaphorin 4D (Sema4D) demonstrated impaired tumor growth and extended survival in the RIP1-Tag2 model. Surprisingly, although there was no induction of intratumor hypoxia by anti-Sema4D therapy, the increase in local invasion and distant metastases was comparable with the one produced by VEGFR inhibition. Mechanistically, the antitumor effect was due to an alteration in vascular function by modification of pericyte coverage involving platelet-derived growth factor B. On the other hand, the aggressive phenotype involved a macrophage-derived Sema4D signaling engagement, which induced their recruitment to the tumor invasive fronts and secretion of stromal cell–derived factor 1 (SDF1) that triggered tumor cell invasive behavior via CXCR4. A comprehensive clinical validation of the targets in different stages of PanNETs demonstrated the implication of both Sema4D and CXCR4 in tumor progression. Taken together, we demonstrate beneficial antitumor and prosurvival effects of anti-Sema4D antibody but also unravel a novel mechanism of tumor aggressivity. This mechanism implicates recruitment of Sema4D-positive macrophages to invasive fronts and their secretion of proinvasive molecules that ultimately induce local tumor invasion and distant metastasis in PanNETs.This work is supported by research grants from ERC (ERC-StG-281830) EU-FP7, MinECO (SAF2016-79347-R), ISCIII Spain (AES, DTS17/00194) and AGAUR-Generalitat de Catalunya (2017SGR771). Some of these include European Development Regional Funds (ERDF “a way to achieve Europe”). Vaccinex Inc. provided reagents and research money (<20.000 Eur) to support this work

Complement as a biological tool to control tumor growth

Deposits of complement components have been documented in several human tumors suggesting a potential involvement of the complement system in tumor immune surveillance. In vitro and in vivo studies have revealed a double role played by this system in tumor progression. Complement activation in the cancer microenvironment has been shown to promote cancer growth through the release of the chemotactic peptide C5a recruiting myeloid suppressor cells. There is also evidence that tumor progression can be controlled by complement activated on the surface of cancer cells through one of the three pathways of complement activation. The aim of this review is to discuss the protective role of complement in cancer with special focus on the beneficial effect of complement-fixing antibodies that are efficient activators of the classical pathway and contribute to inhibit tumor expansion as a result of MAC-mediated cancer cell killing and complement-mediated inflammatory process. Cancer cells are heterogeneous in their susceptibility to complement-induced killing that generally depends on stable and relatively high expression of the antigen and the ability of therapeutic antibodies to activate complement. A new generation of monoclonal antibodies are being developed with structural modification leading to hexamer formation and enhanced complement activation. An important progress in cancer immunotherapy has been made with the generation of bispecific antibodies targeting tumor antigens and able to neutralize complement regulators overexpressed on cancer cells. A great effort is being devoted to implementing combined therapy of traditional approaches based on surgery, chemotherapy and radiotherapy and complement-fixing therapeutic antibodies. An effective control of tumor growth by complement is likely to be obtained on residual cancer cells following conventional therapy to reduce the tumor mass, prevent recurrences and avoid disabilities

Dual effect of Semaphorin 4D blockade in neuroendocrine tumor malignization: from vessels to macrophages

[eng] Tumor progression is a complex conundrum of events that involve not only tumor cells, but also their surrounding microenvironment. Accounting to the natural dependency of tumors on angiogenesis, its therapeutic targeting remains a valid stroma-directed strategy in the fight against cancer. However, standard antiangiogenics fail to produce enduring beneficial effects due to the appearance of resistance, often as a consequence of intratumor hypoxia triggered by vessel trimming. In the case of neuroendocrine tumors (NETs), which are characterized by their low aggressiveness, high heterogeneity and vessel content, new therapeutic approaches are being explored to overcome such hurdles, where conventional therapies stumble. In this context, blockade of semaphorin 4D (Sema4D), a proangiogenic molecule with homeostatic roles in the immune system, by a monoclonal antibody (anti-Sema4D) has proved beneficial antitumor effects in the RIP1-Tag2 preclinical mouse model of pancreatic NET (PanNET). Unfortunately, the decrease in tumor burden and increase in survival of anti-Sema4D treated mice followed an increase in local invasiveness and metastasis. Contrary to the thoroughly described mechanisms governing malignization after antiangiogenic therapies, no intratumoral hypoxia was detected after Sema4D blockade. In this doctoral thesis, aiming to decipher this novel form of resistance by which anti- Sema4D treatment acts as a double-edged sword in PanNETs, the two systems involved in Sema4D signaling were studied: the vascular and the immune system. We first described a beneficial antiangiogenic effect, characterized by structural changes in tumor vessels mediated by a pericyte-endothelial cell crosstalk. Incidentally, we found that the aggressive phenotype involved the recruitment of Sema4D-positive tumor-associated macrophages (TAMs) to the tumor ecosystem, which, after becoming activated by anti- Sema4D treatment, triggered tumor cell migration and invasion. Mechanistically, functional characterization of Sema4D-positive TAMs’ secretome revealed cytokine CXCL12 to be one of the molecules involved in the proinvasive program, suggesting the implication of CXCL12/CXCR4 signaling. Comprehensive clinical validation further shed light on the implication of both macrophage-derived Sema4D and CXCR4 in the malignization steps of tumor development in PanNET patients, which undoubtedly unleashes a new range of approaches merging the immunotherapy and the antiangiogenic fields in their shared fight against cancer.[spa] Considerando la dependencia natural de los tumores por la angiogénesis, su explotación como diana dirigida contra el microambiente en el tratamiento del cáncer, supone una válida estrategia terapéutica. No obstante, los antiangiogénicos estándar fracasan a la hora de producir efectos duraderos debido a la aparición de resistencia, habitualmente como consecuencia de la hipoxia intratumoral. En el caso de los tumores neuroendocrinos (NETs), caracterizados por su alta heterogeneidad y alto contenido vascular, donde la terapia convencional falla, están siendo explorados nuevos abordajes terapéuticos. En este contexto, el bloqueo de la semaforina 4D (Sema4D), una molécula proangiogénica con un papel homeostático en el sistema inmune, utilizando un anticuerpo monoclonal (anti- Sema4D) ha demostrado efectos antitumorales beneficiosos, en un modelo murino de cáncer de páncreas neuroendocrino (PanNET). Lamentablemente, al descenso en el volumen tumoral y al aumento en la supervivencia de los ratones tratados con anti- Sema4D les siguen un aumento en la invasión local y la metástasis. Al contrario de lo esperado, no se detectó hipoxia intratumoral tras el bloqueo de la Sema4D. Con el objetivo de descifrar esta nueva forma de resistencia, en la cual, el tratamiento anti-Sema4D actúa como un arma de doble filo en PanNETs, estudiamos los dos sistemas implicados en la señalización vía Sema4D: el sistema vascular y el inmune. Primeramente, describimos un efecto antiangiogénico beneficioso, caracterizado por un cambio estructural de los vasos tumorales, y mediado por una comunicación cruzada entre células endoteliales y pericitos. A continuación, encontramos que el fenotipo agresivo involucra el reclutamiento de macrófagos positivos para Sema4D al ecosistema tumoral, los cuales, tras activarse por el tratamiento anti-Sema4D, potencian la migración e invasión de las células tumorales. La caracterización funcional de los macrófagos desveló la contribución de la citoquina CXCL12 al programa proinvasivo, sugiriendo una implicación de la señalización vía CXCL12/CXCR4. Finalmente, una validación clínica integral en pacientes de PanNETs arrojó luz sobre la participación de Sema4D derivada de los macrófagos y CXCR4 durante el desarrollo tumoral. En conjunto, nuestros datos reconducen el abanico de estrategias terapéuticas existentes hacia un nuevo enfoque que combina la inmunoterapia y la antiangiogénesis en la lucha común contra el cáncer

Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity

Anti-tumor effects of anti-Semaphorin 4D antibody unravel a novel pro-invasive mechanism of vascular targeting agents

One of the main consequences of inhibition of neovessel growth and vessel pruning produced by angiogenesis inhibitors is increased intratumor hypoxia. Growing evidence indicates that tumor cells escape from this hypoxic environment to better nourished locations, presenting hypoxia as a positive stimulus for invasion. In particular, anti-VEGF/R therapies produce hypoxia-induced invasion and metastasis in a spontaneous mouse model of pancreatic neuroendocrine cancer (PanNET), RIP1-Tag2. Here, a novel vascular-targeting agent targeting semaphorin 4D (Sema4D) demonstrated impaired tumor growth and extended survival in the RIP1-Tag2 model. Surprisingly, although there was no induction of intratumor hypoxia by anti-Sema4D therapy, the increase in local invasion and distant metastases was comparable with the one produced by VEGFR inhibition. Mechanistically, the antitumor effect was due to an alteration in vascular function by modification of pericyte coverage involving platelet-derived growth factor B. On the other hand, the aggressive phenotype involved a macrophage-derived Sema4D signaling engagement, which induced their recruitment to the tumor invasive fronts and secretion of stromal cell–derived factor 1 (SDF1) that triggered tumor cell invasive behavior via CXCR4. A comprehensive clinical validation of the targets in different stages of PanNETs demonstrated the implication of both Sema4D and CXCR4 in tumor progression. Taken together, we demonstrate beneficial antitumor and prosurvival effects of anti-Sema4D antibody but also unravel a novel mechanism of tumor aggressivity. This mechanism implicates recruitment of Sema4D-positive macrophages to invasive fronts and their secretion of proinvasive molecules that ultimately induce local tumor invasion and distant metastasis in PanNETs.This work is supported by research grants from ERC (ERC-StG-281830) EU-FP7, MinECO (SAF2016-79347-R), ISCIII Spain (AES, DTS17/00194) and AGAUR-Generalitat de Catalunya (2017SGR771). Some of these include European Development Regional Funds (ERDF “a way to achieve Europe”). Vaccinex Inc. provided reagents and research money (<20.000 Eur) to support this work

Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α.

Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs-degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR