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Abstract Anti-PD1 immunotherapy, as a single agent or in combination with standard chemotherapies, has significantly improved the outcome of many patients with cancers. However, resistance to anti-PD1 antibodies often decreases the long-term therapeutic benefits. Despite this observation in clinical practice, the molecular mechanisms associated with resistance to anti-PD1 antibody therapy have not yet been elucidated. To identify the mechanisms of resistance associated with anti-PD1 antibody therapy, we developed cellular models including purified T cells and different cancer cell lines from glioblastoma, lung adenocarcinoma, breast cancer and ovarian carcinoma. A murine model of lung cancer was also used. Longitudinal blood samples of patients treated with anti-PD1 therapy were also used to perform a proof-of-concept study of our findings. We found that anti-PD1 exposure of T-cell promotes an enrichment of exosomal miRNA-4315. We also noted that exosomal miRNA-4315 induced a phenomenon of apopto-resistance to conventional chemotherapies in cancer cells receiving exosomal miRNA-4315. At molecular level, we discern that the apopto-resistance phenomenon was associated with the miRNA-4315-mediated downregulation of Bim, a proapoptotic protein. In cellular and mice models, we observed that the BH3 mimetic agent ABT263 circumvented this resistance. A longitudinal study using patient blood showed that miRNA-4315 and cytochrome c can be used to define the time period during which the addition of ABT263 therapy may effectively increase cancer cell death and bypass anti-PD1 resistance.This work provides a blood biomarker (exosomal miRNA-4315) for patient stratification developing a phenomenon of resistance to anti-PD1 antibody therapy and also identifies a therapeutic alternative (the use of a BH3 mimetic drug) to limit this resistance phenomenon.

MicroRNAs (miRNAs or miRs) play crucial roles in biological and pathological processes. Some miRNAs also appear as promising biomarkers and therapeutic tools. However, the epitranscriptomic regulation of miRNAs is not yet fully elucidated in all of their fields of application. We report that adenosine methylation of miR-200b-3p inhibits its repressive function toward its mRNA targets such as XIAP by blocking the formation of the miRNA/3′ UTRmRNA duplex. Our data indicate that the adenosine methylation of miR-200b-3p is associated with the survival of glioblastoma patients. Collectively, our data support the idea that the adenosine methylation of miR-200b-3p can be used as a prodrug having a selective cytotoxicity against cancer cells (while being harmless to peripheral blood mononuclear cells [PBMCs], astrocytes, neurons, and hepatocytes).

Abstract Background Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). Methods RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3’UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. Results Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. Conclusion Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively. Graphical abstract

In the last decade, microRNAs (miRs) have been described as biomarkers and therapeutic agents. Based on this finding, our aim here is to know if (1) miRNA-370-3p can be used as a biomarker associated with a favorable survival and if (2) miRNA-370-3p can be used as a therapeutic tool that increases the efficiency of standard anti-GBM treatment. A first approach using the data available on the “Prognostic miRNA Database” indicated that the expression level of miRNA-370-3p in GBM (T-miR-370-3p) is not associated with a prognosis value for survival. A second approach quantifying the expression level of cell-free circulating miRNA-370-3p (cfc-miR-370-3p) also indicated that cfc-miR-370-3p is not associated with a prognosis value for survival. To investigate whether miR-370-3p can be used in vivo to increase the anti-GBM effect of TMZ, we then used the model of LN18-induced GBMs in mice. Our data indicated that the miRNA-370-3p/TMZ treatment was two times more efficient than the TMZ treatment for decreasing the tumor volume. In addition, our study correlated the decrease of tumor volume induced by the miRNA-370-3p/TMZ treatment with the decrease in FOXM1 and MGMT (i.e., two targets of miR-370-3p).Our data thus support the idea that miR-370-3p could be used as therapeutic tool for anti-glioblastoma therapy, but not as a biomarker. Keywords: GBM, miRNA, temozolomide

Glioblastoma (GBM) is the most aggressive brain tumor, and despite initial response to chemo- and radio-therapy, the persistence of glioblastoma stem cells (GSCs) unfortunately always results in tumor recurrence. It is now largely admitted that tumor cells recruit normal cells, including mesenchymal stem cells (MSCs), and components of their environment, to participate in tumor progression, building up what is called the tumor microenvironment (TME). While growth factors and cytokines constitute essential messengers to pass on signals between tumor and TME, recent uncovering of extracellular vesicles (EVs), composed of microvesicles (MVs) and exosomes, opened new perspectives to define the modalities of this communication. In the GBM context particularly, we investigated what could be the nature of the EV exchange between GSCs and MSCs. We show that GSCs MVs can activate MSCs into cancer-associated fibroblasts (CAFs)-like cells, that subsequently increase their secretion of exosomes. Moreover, a significant decrease in anti-tumoral miR-100-5p, miR-9-5p and let-7d-5p was observed in these exosomes. This clearly suggests a miRNA-mediated GBM tumor promotion by MSCs exosomes, after their activation by GBM MVs.

The acknowledgment that pollutants might influence the epigenome raises serious concerns regarding their long-term impact on the development of chronic diseases. The herbicide glyphosate has been scrutinized for an impact on cancer incidence, but reports demonstrate the difficulty of linking estimates of exposure and response analysis. An approach to better apprehend a potential risk impact for cancer is to follow a synergistic approach, as cancer rarely occurs in response to one risk factor. The known influence of glyphosate on estrogen-regulated pathway makes it a logical target of investigation in breast cancer research. We have used nonneoplastic MCF10A cells in a repeated glyphosate exposure pattern over 21 days. Glyphosate triggered a significant reduction in DNA methylation, as shown by the level of 5-methylcytosine DNA; however, in contrast to strong demethylating agent and cancer promoter UP peptide, glyphosate-treated cells did not lead to tumor development. Whereas UP acts through a DNMT1/PCNA/UHRF1 pathway, glyphosate triggered increased activity of ten-eleven translocation (TET)3. Combining glyphosate with enhanced expression of microRNA (miR) 182-5p associated with breast cancer induced tumor development in 50% of mice. Culture of primary cells from resected tumors revealed a luminal B (ER+/PR-/HER2-) phenotype in response to glyphosate-miR182-5p exposure with sensitivity to tamoxifen and invasive and migratory potentials. Tumor development could be prevented either by specifically inhibiting miR 182-5p or by treating glyphosate-miR 182-5p-cells with dimethyloxallyl glycine, an inhibitor of TET pathway. Looking for potential epigenetic marks of TET-mediated gene regulation under glyphosate exposure, we identified MTRNR2L2 and DUX4 genes, the hypomethylation of which was sustained even after stopping glyphosate exposure for 6 weeks. Our findings reveal that low pressure but sustained DNA hypomethylation occurring via the TET pathway primes cells for oncogenic response in the presence of another potential risk factor. These results warrant further investigation of glyphosate-mediated breast cancer risk.

Abstract Extracellular vesicles (EVs) have been shown as key mediators of extracellular small RNA transport. However, carriers of cell-free messenger RNA (cf-mRNA) in human biofluids and their association with cancer remain poorly understood. Here, we performed a transcriptomic analysis of size-fractionated plasma from lung cancer, liver cancer, multiple myeloma, and healthy donors. Morphology and size distribution analysis showed the successful separation of large and medium particles from other soluble plasma protein fractions. We developed a strategy to purify and sequence ultra-low amounts of cf-mRNA from particle and protein enriched subpopulations with the implementation of RNA spike-ins to control for technical variability and to normalize for intrinsic drastic differences in cf-mRNA amount carried in each plasma fraction. We found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. We observed specific enrichment patterns of cancer-associated cf-mRNA in each particle and protein enriched subpopulation. The EV-enriched differentiating genes were associated with specific biological pathways, such as immune systems, liver function, and toxic substance regulation in lung cancer, liver cancer, and multiple myeloma, respectively. Our results suggest that dissecting the complexity of EV subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.