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Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser 665 and Tyr 685 . It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr 416 and Ser 144 , respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Glioblastomas represent approximatively half of all gliomas and are the most deadly and aggressive form. Their therapeutic resistance and tumor relapse rely on a subpopulation of cells that are called Glioma Stem Cells (GSCs). Here, we investigated the role of the long non-coding RNA HOXA-AS2 in GSC biology using descriptive and functional analyses of glioma samples classified according to their isocitrate dehydrogenase ( IDH ) gene mutation status, and of GSC lines. We found that HOXA-AS2 is overexpressed only in aggressive ( IDHwt ) glioma and GSC lines. ShRNA-based depletion of HOXA-AS2 in GSCs decreased cell proliferation and altered the expression of several hundreds of genes. Integrative analysis revealed that these expression changes were not associated with changes in DNA methylation or chromatin signatures at the promoter of the majority of genes deregulated following HOXA-AS2 silencing in GSCs, suggesting a post-transcriptional regulation. In addition, transcription factor binding motif enrichment and correlation analyses indicated that HOXA-AS2 affects, directly or indirectly, the expression of key transcription factors implicated in GCS biology, including E2F8, E2F1, STAT1, and ATF3, thus contributing to GCS aggressiveness by promoting their proliferation and modulating the inflammation pathway.

Deficient mismatch repair system (dMMR)/microsatellite instability (MSI) is found in about 5% of metastatic colorectal cancers (mCRCs) with a major therapeutic impact for immune checkpoint inhibitor (ICI) use. We conducted a multicentre study including all consecutive patients with a dMMR/MSI mCRC. MSI status was determined using the Pentaplex panel and expression of the four MMR proteins was evaluated by immunohistochemistry (IHC). The primary endpoint was the rate of discordance of dMMR/MSI status between primary tumours and paired metastases. We included 99 patients with a dMMR/MSI primary CRC and 117 paired metastases. Only four discrepancies (3.4%) with a dMMR/MSI primary CRC and a pMMR/MSS metastasis were initially identified and reviewed by expert pathologists and molecular biologists. Two cases were false discrepancies due to human or technical errors. One discordant case could not be confirmed due to the low level of tumour cells. The last case had a confirmed discrepancy with a dMMR/MSI primary CRC and a pMMR/MSS peritoneal metastasis. Our study demonstrated a high concordance rate of dMMR/MSI status between primary CRCs and their metastases. The analysis of one sample, either from the primary tumour or metastasis, with consistent dMMR and MSI status seems to be sufficient prior to treatment with ICI.

The high expression of MEOX2 transcription factor is closely associated with poor overall survival in glioma. MEOX2 has recently been described as an interesting prognostic biomarker, especially for lower grade glioma. MEOX2 has never been studied in glioma stem-like cells (GSC), responsible for glioma recurrence. The aim of our study was to investigate the role of MEOX2 in GSC. Loss of function approach using siRNA was used to assess the impact of MEOX2 on GSC viability and stemness phenotype. MEOX2 was localized in the nucleus and its expression was heterogeneous between GSCs. MEOX2 expression depends on the methylation state of its promoter and is strongly associated with IDH mutations. MEOX2 is involved in cell proliferation and viability regulation through ERK/MAPK and PI3K/AKT pathways. MEOX2 loss of function correlated with GSC differentiation and acquisition of neuronal lineage characteristics. Besides, inhibition of MEOX2 is correlated with increased expression of CDH10 and decreased pFAK. In this study, we unraveled, for the first time, MEOX2 contribution to cell viability and proliferation through AKT/ERK pathway and its potential involvement in phenotype and adhesion properties of GSC.

The Hippo signaling pathway is a highly conserved pathway involved in tissue development and regeneration that controls organ size through the regulation of cell proliferation and apoptosis. The core Hippo pathway is composed of a block of kinases, MST1/2 (Mammalian STE20-like protein kinase 1/2) and LATS1/2 (Large tumor suppressor 1/2), which inhibits nuclear translocation of YAP/TAZ (Yes-Associated Protein 1/Transcriptional co-activator with PDZ-binding motif) and its downstream association with the TEAD (TEA domain) family of transcription factors. This pathway was recently shown to be involved in tumorigenesis and metastasis in several cancers such as lung, breast, or colorectal cancers but is still poorly investigated in brain tumors. Gliomas are the most common and the most lethal primary brain tumors representing about 80% of malignant central nervous system neoplasms. Despite intensive clinical protocol, the prognosis for patients remains very poor due to systematic relapse and treatment failure. Growing evidence demonstrating the role of Hippo signaling in cancer biology and the lack of efficient treatments for malignant gliomas support the idea that this pathway could represent a potential target paving the way for alternative therapeutics. Based on recent advances in the Hippo pathway deciphering, the main goal of this review is to highlight the role of this pathway in gliomas by a state-of-the-art synthesis.

Background: In most countries, participation in colorectal cancer (CRC) screening programs with the immunological fecal occult blood test (iFOBT) is low. Mutations of RAS and BRAF occur early in colorectal carcinogenesis and "liquid biopsy" allows detection of mutated circulating tumor DNA (ctDNA). This prospective study aims to evaluate the performance of RAS and BRAF-mutated ctDNA in detecting CRC and advanced adenomas (AA)., Methods: One hundred and thirty patients who underwent colonoscopy for suspicion of colorectal lesion were included and divided into four groups: 20 CRC, 39 AA, 31 non-advanced adenoma and/or hyperplastic polyp(s) (NAA) and 40 with no lesion. Mutated ctDNA was analyzed by droplet digital PCR., Results: ctDNA was detected in 45.0% of CRC, in 2.6% of AA and none of the NAA and "no-lesion" groups. All patients with stage II to IV mutated CRC had detectable ctDNA ( n = 8/8). Among the mutated AA, only one patient had detectable ctDNA (4.3%), maybe due to limited technical sensitivity or to a low rate of ctDNA or even the absence ctDNA in plasma. Specificity and sensitivity of KRAS- and BRAF-mutated ctDNA for the detection of all CRC and AA were 100% and 16.9%, respectively., Conclusions: ctDNA had high sensitivity in detection of advanced mutated CRC but was unable to sensitively detect AA. ctDNA analysis was easy to perform and readily accepted by the population but requires combination with other circulating biomarkers before replacing iFOBT.

Tumor DNA mismatch repair (MMR) deficiency testing is important to the identification of Lynch syndrome and decision making regarding adjuvant chemotherapy in stage II colorectal cancer (CRC) and has become an indispensable test in metastatic tumors due to the high efficacy of immune checkpoint inhibitor (ICI) in deficient MMR (dMMR) tumors. CRCs greatly benefit from this testing as approximately 15% of them are dMMR but only 3% to 5% are at a metastatic stage. MMR status can be determined by two different methods, microsatellite instability (MSI) testing on tumor DNA, and immunohistochemistry of the MMR proteins on tumor tissue. Recent studies have reported a rate of 3% to 10% of discordance between these two tests. Moreover, some reports suggest possible intra- and inter-tumoral heterogeneity of MMR and MSI status. These issues are important to know and to clarify in order to define therapeutic strategy in CRC. This review aims to detail the standard techniques used for the determination of MMR and MSI status, along with their advantages and limits. We review the discordances that may arise between these two tests, tumor heterogeneity of MMR and MSI status, and possible explanations. We also discuss the strategies designed to distinguish sporadic versus germline dMMR/MSI CRC. Finally, we present new and accurate methods aimed at determining MMR/MSI status ., Competing Interests: Abbreviations:

Background: Colorectal cancers (CRC) with brain metastases (BM) are scarcely described. The main objective of this study was to determine the molecular profile of CRC with BM. Methods: We included 82 CRC patients with BM. KRAS , NRAS , BRAF and mismatch repair (MMR) status were investigated on primary tumors ( n = 82) and BM ( n = 38). ALK, ROS1, cMET, HER-2, PD-1, PD-L1, CD3 and CD8 status were evaluated by immunohistochemistry, and when recommended, by fluorescence in situ hybridization. Results: In primary tumors, KRAS , NRAS and BRAF mutations were observed in 56%, 6%, and 6% of cases, respectively. No ROS1 , ALK and cMET rearrangement was detected. Only one tumor presented HER-2 amplification. Molecular profiles were mostly concordant between BM and paired primary tumors, except for 9% of discordances for RAS mutation. CD3, CD8, PD-1 and PD-L1 expressions presented some discordance between primary tumors and BM. In multivariate analysis, multiple BM, lung metastases and PD-L1+ tumor were predictive of poor overall survival. Conclusions: CRCs with BM are associated with high frequency of RAS mutations and significant discordance for RAS mutational status between BM and paired primary tumors. Multiple BM, lung metastases and PD-L1+ have been identified as prognostic factors and can guide therapeutic decisions for CRC patients with BM., Competing Interests: The authors declare no conflict of interest.

"Glioma Stem Cells" (GSCs) are known to play a role in glioblastoma (GBM) recurrence. Homologous recombination (HR) defects and cell cycle checkpoint abnormalities can contribute concurrently to the radioresistance of GSCs. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for HR and its inhibition has been shown to sensitize GSCs to irradiation. The aim of this study was to examine the consequences of ionizing radiation (IR) for cell cycle progression in GSCs. In addition, we intended to assess the potential effect of RAD51 inhibition on cell cycle progression. Five radiosensitive GSC lines and five GSC lines that were previously characterized as radioresistant were exposed to 4Gy IR, and cell cycle analysis was done by fluorescence-activated cell sorting (FACS) at 24, 48, 72, and 96 h with or without RAD51 inhibitor. Following 4Gy IR, all GSC lines presented a significant increase in G2 phase at 24 h, which was maintained over 72 h. In the presence of RAD51 inhibitor, radioresistant GSCs showed delayed G2 arrest post-irradiation for up to 48 h. This study demonstrates that all GSCs can promote G2 arrest in response to radiation-induced DNA damage. However, following RAD51 inhibition, the cell cycle checkpoint response differed. This study contributes to the characterization of the radioresistance mechanisms of GSCs, thereby supporting the rationale of targeting RAD51-dependent repair pathways in view of radiosensitizing GSCs.

Approximately 30% of patients with wild type RAS metastatic colorectal cancer are non-responders to anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR mAbs), possibly due to undetected tumoral subclones harboring RAS mutations. The aim of this study was to analyze the distribution of RAS mutations in different areas of the primary tumor, metastatic lymph nodes and distant metastasis. A retrospective cohort of 18 patients with a colorectal cancer (CRC) was included in the study. Multiregion analysis was performed in 60 spatially separated tumor areas according to the pathological tumor node metastasis (pTNM) staging and KRAS , NRAS and BRAF mutations were tested using pyrosequencing. In primary tumors, intra-tumoral heterogeneity for RAS mutation was found in 33% of cases. Inter-tumoral heterogeneity for RAS mutation between primary tumors and metastatic lymph nodes or distant metastasis was found in 36% of cases. Moreover, 28% of tumors had multiple RAS mutated subclones in the same tumor. A high proportion of CRCs presented intra- and/or inter-tumoral heterogeneity, which has relevant clinical implications for anti-EGFR mAbs prescription. These results suggest the need for multiple RAS testing in different parts of the same tumor and/or more sensitive techniques., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Glioblastoma (GBM) is the most malignant type of primary brain tumor with a very poor prognosis. The actual standard protocol of treatment for GBM patients consists of radiotherapy and concomitant temozolomide (TMZ). However, the therapeutic efficacy of this treatment is limited due to tumor recurrence and TMZ resistance. Recently isolated, glioma stem-like cells (GSCs) are thought to represent the population of tumorigenic cells responsible for GBM resistance and recurrence following surgery and chemotherapy. In addition, MGMT (O6-methylguanine-methyltransferase) methylation is considered as one of the principal mechanisms contributing to TMZ sensitivity of GBM. In this study we have isolated GSCs from 10 adult GBM patients and investigated the relationship between MGMT methylation status and Temozolomide (TMZ) sensitivity of these lines grown either in stem-like or differentiation promoting conditions. Sensitivity to TMZ was significantly associated with MGMT methylation status in cells committed to differentiation but not in stem-like cells. In addition, patients harboring highly methylated MGMT promoters had a longer overall survival. These results reveal the importance of the differentiation process when considering the predictive value of MGMT status in GSCs for clinical response to TMZ.