Temozolomide-Acquired Resistance Is Associated with Modulation of the Integrin Repertoire in Glioblastoma, Impact of α5β1 Integrin

Despite extensive treatment, glioblastoma inevitably recurs, leading to an overall survival of around 16 months. Understanding why and how tumours resist to radio/chemotherapies is crucial to overcome this unmet oncological challenge. Primary and acquired resistance to Temozolomide (TMZ), the standard-of-care chemotherapeutic drug, have been the subjects of several studies. This work aimed to evaluate molecular and phenotypic changes occurring during and after TMZ treatment in a glioblastoma cell model, the U87MG. These initially TMZ-sensitive cells acquire long-lasting resistance even after removal of the drug. Transcriptomic analysis revealed that profound changes occurred between parental and resistant cells, particularly at the level of the integrin repertoire. Focusing on α5β1 integrin, which we proposed earlier as a glioblastoma therapeutic target, we demonstrated that its expression was decreased in the presence of TMZ but restored after removal of the drug. In this glioblastoma model of recurrence, α5β1 integrin plays an important role in the proliferation and migration of tumoral cells. We also demonstrated that reactivating p53 by MDM2 inhibitors concomitantly with the inhibition of this integrin in recurrent cells may overcome the TMZ resistance. Our results may explain some integrin-based targeted therapy failure as integrin expressions are highly switchable during the time of treatment. We also propose an alternative way to alter the viability of recurrent glioblastoma cells expressing a high level of α5β1 integrin.

Molecular Characterization and Functional Analysis of the Nattectin-like Toxin from the Venomous Fish Thalassophryne maculosa

TmC4-47.2 is a toxin with myotoxic activity found in the venom of Thalassophryne maculosa, a venomous fish commonly found in Latin America whose envenomation produces an injury characterized by delayed neutrophil migration, production of major pro-inflammatory cytokines, and necrosis at the wound site, as well as a specific systemic immune response. However, there are few studies on the protein structure and functions associated with it. Here, the toxin was identified from the crude venom by chromatography and protein purification systems. TmC4-47.2 shows high homology with the Nattectin from Thalassophryne nattereri venom, with 6 cysteines and QPD domain for binding to galactose. We confirm its hemagglutinating and microbicide abilities independent of carbohydrate binding, supporting its classification as a nattectin-like lectin. After performing the characterization of TmC4-47.2, we verified its ability to induce an increase in the rolling and adherence of leukocytes in cremaster post-capillary venules dependent on the α5β1 integrin. Finally, we could observe the inflammatory activity of TmC4-47.2 through the production of IL-6 and eotaxin in the peritoneal cavity with sustained recruitment of eosinophils and neutrophils up to 24 h. Together, our study characterized a nattectin-like protein from T. maculosa, pointing to its role as a molecule involved in the carbohydrate-independent agglutination response and modulation of eosinophilic and neutrophilic inflammation.

NetrinG1+ cancer-associated fibroblasts generate unique extracellular vesicles that support the survival of pancreatic cancer cells under nutritional stress

It is projected that, in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). This fibrous stroma, known as desmoplasia, causes the collapse of local blood vessels rendering a nutrient-deprived milieu. Hence, PDAC cells are nurtured by local CAF-secreted products, which include, among others, CAF-generated small extracellular vesicles (sEVs). It is well-accepted that upon culturing functionally tumor-promoting CAFs under pathophysiological-relevant conditions (e.g., within self-produced ECM), these cells express NetrinG1 (NetG1) and sustain endosomal pools rich in active α5β1-integrin, traits indicative of poor patient survival. We herein report that NetG1+ CAFs generate sEVs that rescue PDAC cells from nutrient-deprived induced apoptosis. Two unique sEVs, NetG1+ and α5β1-integrin+, were uncovered. The former constitutes cargo of CAF-generated exomeres, and the latter is detected in classic exosomes. Proteomic and metabolomic analyses showed that the sEV-dependent PDAC survival is, at least in part, dictated by the cargo packaged within sEVs in a NetG1-dependent manner. Indeed, despite producing a similar number of vesicles, selected key proteins and metabolites (e.g., glutamine) were incorporated within the unique sEVs. Finally, we found that NetG1 and α5β1-integrin were detected in sEVs collected from plasma of PDAC patients, while their concomitant levels were significantly lower in plasma of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF sEVs opens a new investigative avenue in tumor-stroma interactions and stroma staging detection.

SURG-05. Α5Β1 INTEGRIN TARGETING LIPOSOME DELIVERY OF MIR-603 SUPPRESSED GLIOBLASTOMA STEM CELL STATE AND ENHANCED RADIATION SENSITIVITY

INTRODUCTION Despite the potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a surgery mediated polyethylenimine (PEI)/liposome-based strategy for the delivery of miR-603, a master regulatory miRNA that suppresses glioblastoma stem cell state by simultaneous down-regulation of insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). METHODS miR-603 was complexed with PEI, a cationic polymer designed to facilitate miRNA from the endolysosomal compartment. The miR-603/PEI complex was encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5β1 integrin that is overexpressed in glioblastoma. RESULTS Patient-derived glioblastoma cells internalized PR_b coated liposomes but not the non-coated liposomes. The internalization of PR_b liposomes encapsulating miR-603/PEI was associated with orders of magnitude increase in intra-cellular miR-603 levels and decreased IGF1 and IGF1R mRNA/protein levels. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI showed altered morphology and decreased expression of stem cell marker, suggesting the treated cells have exited the cancer stem cell state. Finally, treatment of the PR_b liposomes encapsulating miR-603/PEI sensitized glioblastoma cells to ionizing radiation (IR) in patient-derived glioblastoma cells. These results were not observed in liposomes missing the PR_b peptide, PEI, or miR-603. CONCLUSION These results suggest that intra-tumoral injection of PR_b functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a strategy for glioblastomas therapy. A first-in-human trial is currently underway to test this strategy.

Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.

Trop2 Forms a Stable Dimer with Significant Structural Differences within the Membrane-Distal Region as Compared to EpCAM

Trop2 is a cell-surface transmembrane glycoprotein involved in the maintenance of epithelial tissue integrity and is an important carcinoma marker. It shares similar claudin-interaction capacity with its paralogue EpCAM, and both are implicated in signaling triggered by proteolytic cleavage within the ectodomain. However, the cell proliferation-regulating interactions with IGF-1, neuregulin-1, and α5β1 integrin appear to be Trop2-specific. To illuminate the structural differences between Trop2 and EpCAM, we report the first crystal structure of a Trop2 ectodomain dimer and compare it to the analogous part of EpCAM. While the overall fold of the two proteins is similar, the dimers differ. In Trop2, the inter-subunit contacts are more extensive than in EpCAM, and there are two major differences in the membrane-distal regions. The immunogenic N-terminal domain is in Trop2 almost colinear with the dimer interface plain and consequently more laterally exposed, and the cleft of yet unknown functionality between the two subunits is almost absent. Furthermore, the site of initial signaling-associated proteolytic cleavage in Trop2 is accessible in the dimeric state, while in EpCAM dimer destabilization is required. The structural differences highlight the divergent evolutionary path of the two proteins and pave the way for their structure-based utilization in therapy.

P04.18 Heterogeneity and plasticity of integrin α5β1 expression in glioblastoma stem cells

BACKGROUND Glioblastoma (GBM) is the most frequent and deadliest type of central nervous system tumors. Despite the treatment by the Stupp protocol, almost all patients relapse and new therapeutic protocols have been unsuccessful for ameliorating patient survival. Molecular heterogeneity of GBM and existence of glioma stem cells (GSC) may be linked to therapy resistance and recurrence. We demonstrated earlier that α5β1 integrin is a GBM therapeutic target which participate to therapy resistance; a high expression in patient tumors is linked to a worse prognosis. Expression of α5β1 integrin is heterogeneous inter- and intra-tumorally. We particularly addressed the role of glioma stem cell plasticity in the modulation of the integrin expression. Stem cells reside in specific niches (perivascular or hypoxic niches) in the tumor and are at the origin of the more differentiated tumor cell bulk. Metabolism is known to change between the different GSC states and may be affected by or may affect the integrin expression. The aim of our work is therefore to consider the expression of the integrin α5β1 in relationship with GSC differentiation or in hypoxic environment and with cell metabolism. MATERIAL AND METHODS Ten different patient-derived glioma stem cell lines were investigated. Cell culture in stem cell medium (neurospheres) or differentiation medium (adherent cell monolayer) was made in normoxia (21% O2) or hypoxia (1%O2). Alternatively, chemically-induced hypoxia (cobalt chloride/desferoxiamine) was used. Integrin expression was kinetically checked at the mRNA (RT-qPCR) or protein (Western blot) levels. Cell metabolism was investigated with the Seahorse Xfp technology and by HRMAS-NMR. RESULTS No GSC lines (neurospheres) expressed the α5β1 integrin. Interestingly, only half of them did after differentiation suggesting a first level of heterogeneity. A second level of heterogeneity was observed in hypoxic conditions provoking induction of integrin α5β1 expression in only some non-differentiated GSC. Three categories of GSC were thus characterized: one able to express the integrin in hypoxia and after differentiation, one never expressing it and the third one only after differentiation. Cell metabolism differed between GSC before and after differentiation and in presence of integrin α5β1 antagonists. Specific glioma regulator network analysis revealed new targets to be inhibited concomitantly with the integrin. CONCLUSION Data suggest that α5β1 integrin expression may be induced by different signaling pathways. Molecular switches may occur either when stem cells differentiate to tumor cells but also directly in stem cells in hypoxic niches. Characterization of α5β1 integrin expression drivers may help to find new therapeutic targets but also to delineate subpopulation of patients who would benefit from an anti-integrin strategy.

Targeted Liposomes Encapsulating miR-603 Complexes Enhance Radiation Sensitivity of Patient-Derived Glioblastoma Stem-Like Cells

Despite potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a strategy for miRNA delivery in the treatment of glioblastoma, the most common form of adult brain cancer, that involves complexation of miRNA with polyethylenimine (PEI) and encapsulation in targeted liposomes. miRNA 603 (miR-603) is a master regulatory miRNA that suppresses glioblastoma radiation resistance through down-regulation of insulin-like growth factor 1 (IGF1) signaling. miR-603 was complexed with PEI, a cationic polymer, and encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5β1 integrin that is overexpressed in glioblastomas. Cultured patient-derived glioblastoma cells internalized PR_b-functionalized liposomes but not the non-targeted liposomes. The integrin targeting and complexation of the miRNA with PEI were associated with a 22-fold increase in intracellular miR-603 levels, and corresponding decreases in IGF1 and IGF1 receptor (IGF1R) mRNA expression. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI sensitized the cells to ionizing radiation (IR), a standard of care treatment for glioblastomas. These results suggest that PR_b-functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a therapeutic platform for glioblastomas.