Role of the mechanotransductor PIEZO1 in megakaryocyte differentiation.

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34+ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 µM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.

HDAC6 regulates human erythroid differentiation through modulation of JAK2 signalling.

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34+ -cells-derived erythroid progenitors. ACY-1215 exposure on CD34+ -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. This was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. In erythroid cells, ACY-1215 and shRNA-mediated HDAC6 knockdown inhibited the EPO-dependent JAK2 phosphorylation. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure, which decreased the 14-3-3ζ/LNK interaction while increased LNK ability to interact with JAK2. Thus, in addition to its previously described role in the enucleation of mouse fetal liver erythroblasts, we identified here a new mechanism of HDAC6-dependent control of erythropoiesis through 14-3-3ζ acetylation level, LNK availability and finally JAK2 activation in response to EPO, which is crucial downstream of EPO-R activation for human erythroid cell survival, proliferation and differentiation.

Mechanisms of chronic alcohol exposure-induced aggressiveness in cellular model of HCC and recovery after alcohol withdrawal.

Alcohol-related liver disease is the most prevalent chronic liver disease worldwide, accounting for 30% of hepatocellular carcinoma (HCC) cases and HCC-specific deaths. However, the knowledge on mechanisms by which alcohol consumption leads to cancer progression and its aggressiveness is limited. Better understanding of the clinical features and the mechanisms of alcohol-induced HCC are of critical importance for prevention and the development of novel treatments. Early stage Huh-7 and advanced SNU449 liver cancer cell lines were subjected to chronic alcohol exposure (CAE), at different doses for 6 months followed by 1-month alcohol withdrawal period. ADH activity and ALDH expression were much lower in SNU449 compared with Huh-7 cells and at the 270 mM dose, CAE decreased cell viability by about 50% and 80%, respectively, in Huh-7 and SNU449 cells but induced mortality only in Huh-7 cells. Thus, Huh-7 may be more vulnerable to ethanol toxicity because of the higher levels of acetaldehyde. CAE induced a dose-dependent increase in cell migration and invasion and also in the expression of cancer stem cells markers (CD133, CD44, CD90). CAE in Huh-7 cells selectively activated ERK1/2 and inhibited GSK3ß signaling pathways. Most of the changes induced by CAE were reversed after alcohol withdrawal. Interestingly, we confirmed the increase in CD133 mRNA levels in the tumoral tissue of patients with ethanol-related HCC compared to other HCC etiologies. Our results may explain the benefits observed in epidemiological studies showing a significant increase of overall survival in abstinent compared with non-abstinent patients.

Histone Deacetylases Function in the Control of Early Hematopoiesis and Erythropoiesis.

Numerous studies have highlighted the role of post-translational modifications in the regulation of cell proliferation, differentiation and death. Among these modifications, acetylation modifies the physicochemical properties of proteins and modulates their activity, stability, localization and affinity for partner proteins. Through the deacetylation of a wide variety of functional and structural, nuclear and cytoplasmic proteins, histone deacetylases (HDACs) modulate important cellular processes, including hematopoiesis, during which different HDACs, by controlling gene expression or by regulating non-histone protein functions, act sequentially to provide a fine regulation of the differentiation process both in early hematopoietic stem cells and in more mature progenitors. Considering that HDAC inhibitors represent promising targets in cancer treatment, it is necessary to decipher the role of HDACs during hematopoiesis which could be impacted by these therapies. This review will highlight the main mechanisms by which HDACs control the hematopoietic stem cell fate, particularly in the erythroid lineage.

Optical genome mapping, a promising alternative to gold standard cytogenetic approaches in a series of acute lymphoblastic leukemias.

Acute lymphoblastic leukemias (ALL) are characterized by a large number of cytogenetic abnormalities of clinical interest that require the use of several complementary techniques. Optical genome mapping (OGM) is based on analysis of ultra-high molecular weight DNA molecules that provides a high-resolution genome-wide analysis highlighting copy number and structural anomalies, including balanced translocations. We compared OGM to standard techniques (karyotyping, fluorescent in situ hybridization, single nucleotide polymorphism-array and reverse transcription multiplex ligation-dependent probe amplification) in 10 selected B or T-ALL. Eighty abnormalities were found using standard techniques of which 72 (90%) were correctly detected using OGM. Eight discrepancies were identified, while 12 additional anomalies were found by OGM. Among the discrepancies, four were detected in raw data but not retained because of filtering issues. However, four were truly missed, either because of a low variant allele frequency or because of a low coverage of some regions. Of the additional anomalies revealed by OGM, seven were confirmed by another technique, some of which are recurrent in ALL such as LMO2-TRA and MYC-TRB fusions. Despite false positive anomalies due to background noise and a case of inter-sample contamination secondarily identified, the OGM technology was relatively simple to use with little practice. Thus, OGM represents a promising alternative to cytogenetic techniques currently performed for ALL characterization. It enables a time and cost effective analysis allowing identification of complex cytogenetic events, including those currently inaccessible to standard techniques.

Recent advances in the pathophysiology of PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases; PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation, as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets.

PIEZO1 activation delays erythroid differentiation of normal and hereditary xerocytosis-derived human progenitor cells.

Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and decreased α/ß-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STAT5 and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 - both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis.

Characterization of a new V gene replacement in the absence of activation-induced cytidine deaminase and its contribution to human B-cell receptor diversity.

In B cells, B-cell receptor (BCR) immunoglobulin revision is a common route for modifying unwanted antibody specificities via a mechanism called VH replacement. This in vivo process, mostly affecting heavy-chain rearrangement, involves the replacement of all or part of a previously rearranged IGHV gene with another germline IGHV gene located upstream. Two different mechanisms of IGHV replacement have been reported: type 1, involving the recombination activating genes complex and requiring a framework region 3 internal recombination signal; and type 2, involving an unidentified mechanism different from that of type 1. In the case of light-chain loci, BCR immunoglobulin editing ensures that a second V-J rearrangement occurs. This helps to maintain tolerance, by generating a novel BCR with a new antigenic specificity. We report that human B cells can, surprisingly, undergo type 2 replacement associated with κ light-chain rearrangements. The de novo IGKV-IGKJ products result from the partial replacement of a previously rearranged IGKV gene by a new germline IGKV gene, in-frame and without deletion or addition of nucleotides. There are wrcy/rgyw motifs at the 'IGKV donor-IGKV recipient chimera junction' as described for type 2 IGHV replacement, but activation-induced cytidine deaminase (AID) expression was not detected. This unusual mechanism of homologous recombination seems to be a variant of gene conversion-like recombination, which does not require AID. The recombination phenomenon described here provides new insight into immunoglobulin locus recombination and BCR immunoglobulin repertoire diversity.

MR1B, a natural spliced isoform of the MHC-related 1 protein, is expressed as homodimers at the cell surface and activates MAIT cells.

The MHC-related 1 (MR1) protein is a monomorphic, evolutionarily conserved MHC class I-like molecule, which is necessary for the development and functions of mucosal-associated invariant T (MAIT) cells, a new subset of innate-like lymphocytes. Multiple isoforms of the MR1 gene are naturally transcribed, but only the full-length MR1A has been analyzed so far. Using transfected cell lines expressing an alternative spliced transcript, MR1B, characterized by the absence of the α3 extracellular domain, we show that MR1B is transcribed and glycosylated but remains in an immature (endoglycosidase H-sensitive) state. MR1B mostly accumulates in the ER, without interacting with proteins of the peptide-loading complex such as tapasin. Interestingly, it is nevertheless found expressed at the cell surface, independently of ß2-microglobulin, in a homodimeric form. MR1B is functional as its overexpression induces MAIT cell activation in vitro in the presence of bacteria. Altogether, these data show that MR1B displays several remarkable features, and probably plays a physiological role complementary to MR1A with respect to MAIT cell development and/or function.

Phorbol myristate acetate, but not CD40L, induces the differentiation of CLL B cells into Ab-secreting cells.

In this study, we investigated the capacity of chronic lymphocytic leukemia (CLL) B cells to undergo terminal differentiation into Ig-secreting plasma cells in T cell-independent and T cell-dependent responses. We used a two-step model involving stimulation with phorbol myristate acetate (PMA) and CD40L, together with cytokines (PMA/c and CD40L/c), for 7 days. We describe immunophenotypic modifications, changes in the levels of mRNA and protein for transcription factors and morphological and functional events occurring during the differentiation of CLL B cells into antibody-secreting cells (ASCs). The induction of differentiation differed significantly between the CD40L/c and PMA/c culture systems. The PMA/c culture system allowed CLL B cells to differentiate into IgM-secreting cells with an immunophenotype and molecular profile resembling those of preplasmablasts. By contrast, CD40L/c-stimulated cells had a phenotype and morphology similar to those of activated B cells and resembling those of the CLL B cells residing in the lymph node and bone marrow. These data suggest that the CLL B cells are not frozen permanently at a stage of differentiation and are able to differentiate into ASCs as appropriate stimulation are provided. The data presented here raise questions about the molecular processes and stimulation required for CLL B-cell differentiation and about the inability of CD40 ligand to induce differentiation of the CLL B cells.

PIEZO1 is essential for the survival and proliferation of acute myeloid leukemia cells.

INTRODUCTION: Leukemogenesis is a complex process that interconnects tumoral cells with their microenvironment, but the effect of mechanosensing in acute myeloid leukemia (AML) blasts is poorly known. PIEZO1 perceives and transmits the constraints of the environment to human cells by acting as a non-selective calcium channel, but very little is known about its role in leukemogenesis. RESULTS: For the first time, we show that PIEZO1 is preferentially expressed in healthy hematopoietic stem and progenitor cells in human hematopoiesis, and globally overexpressed in AML cells. In AML subtypes, PIEZO1 expression associates with favorable outcomes as better overall (OS) and disease-free survival (DFS). If PIEZO1 is expressed and functional in THP1 leukemic myeloid cell line, its chemical activation doesn't impact the proliferation, differentiation, nor survival of cells. However, the downregulation of PIEZO1 expression dramatically reduces the proliferation and the survival of THP1 cells. We show that PIEZO1 knock-down blocks the cell cycle in G0/G1 phases of AML cells, impairs the DNA damage response pathways, and critically increases cell death by triggering extrinsic apoptosis pathways. CONCLUSIONS: Altogether, our results reveal a new role for PIEZO1 mechanosensing in the survival and proliferation of leukemic blasts, which could pave the way for new therapeutic strategies to target AML cells.

A proteomic study of the downregulation of TRIM37 on chondrocytes: Implications for the MULIBREY syndrome.

MULIBREY nanism which results from autosomal recessive mutations in TRIM37 impacts skeletal development, leading to growth delay with complications in multiple organs. In this study, we employed a combined proteomics and qPCR screening approach to investigate the molecular alterations in the CHON-002 cell line by comparing CHON-002 wild-type (WT) cells to CHON-002 TRIM37 knockdown (KD) cells. Our proteomic analysis demonstrated that TRIM37 depletion predominantly affects the expression of extracellular matrix proteins (ECM). Specifically, nanoLC-MS/MS experiments revealed an upregulation of SPARC, and collagen products (COL1A1, COL3A1, COL5A1) in response to TRIM37 KD. Concurrently, large-scale qPCR assays targeting osteogenesis-related genes corroborated these dysregulations of SPARC at the mRNA level. Gene ontology enrichment analysis highlighted the involvement of dysregulated proteins in ECM organization and TGF-ß signaling pathways, indicating a role for TRIM37 in maintaining ECM integrity and regulating chondrocyte proliferation. These findings suggest that TRIM37 deficiency in chondrocytes change ECM protein composition and could impairs long bone growth, contributing to the pathophysiology of MULIBREY nanism.

STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells.

Calcium is a ubiquitous messenger that regulates a wide range of cellular functions, but its involvement in the pathophysiology of acute myeloid leukemia (AML) is not widely investigated. Here, we identified, from an analysis of The Cancer Genome Atlas and genotype-tissue expression databases, stromal interaction molecule 2 (STIM2) as being highly expressed in AML with monocytic differentiation and negatively correlated with overall survival. This was confirmed on a validation cohort of 407 AML patients. We then investigated the role of STIM2 in cell proliferation, differentiation, and survival in two leukemic cell lines with monocytic potential and in normal hematopoietic stem cells. STIM2 expression increased at the RNA and protein levels upon monocyte differentiation. Phenotypically, STIM2 knockdown drastically inhibited cell proliferation and induced genomic stress with DNA double-strand breaks, as shown by increased levels of phosphorylate histone H2AXγ (p-H2AXγ), followed by activation of the cellular tumor antigen p53 pathway, decreased expression of cell cycle regulators such as cyclin-dependent kinase 1 (CDK1)-cyclin B1 and M-phase inducer phosphatase 3 (CDC25c), and a decreased apoptosis threshold with a low antiapoptotic/proapoptotic protein ratio. Our study reports STIM2 as a new actor regulating genomic stability and p53 response in terms of cell cycle and apoptosis of human normal and malignant monocytic cells.

Aortic valve calcification is promoted by interleukin-8 and restricted through antagonizing CXC motif chemokine receptor 2.

AIMS: Inflammatory cytokines play a critical role in the progression of calcific aortic valve disease (CAVD), for which there is currently no pharmacological treatment. The aim of this study was to test the hypothesis that interleukin-8 (IL-8), known to be involved in arterial calcification, also promotes aortic valve calcification (AVC) and to evaluate whether pharmacologically blocking the IL-8 receptor, CXC motif chemokine receptor 2 (CXCR2), could be effective in preventing AVC progression. METHODS AND RESULTS: A cohort of 195 patients (median age 73, 74% men) diagnosed with aortic valve stenosis (severe in 16.9% of cases) were prospectively followed by CT for a median time of 2.6 years. A Cox proportional hazards regression analysis indicated that baseline IL-8 serum concentrations were associated with rapid progression of AVC, defined as an annualized change in the calcification score by CT ≥ 110 AU/year, after adjustment for age, gender, bicuspid anatomy, and baseline disease severity. In vitro, exposure of primary human aortic valvular interstitial cells (hVICs) to 15 pg/mL IL-8 induced a two-fold increase in inorganic phosphate (Pi)-induced calcification. IL-8 promoted NFκB pathway activation, MMP-12 expression, and elastin degradation in hVICs exposed to Pi. These effects were prevented by SCH527123, an antagonist of CXCR2. The expression of CXCR2 was confirmed in hVICs and samples of aortic valves isolated from patients with CAVD, in which the receptor was mainly found in calcified areas, along with MMP-12 and a degraded form of elastin. Finally, in a rat model of chronic kidney disease-associated CAVD, SCH527123 treatment (1 mg/kg/day given orally for 11 weeks) limited the decrease in aortic cusp separation, the increase in maximal velocity of the transaortic jet, and the increase in aortic mean pressure gradient measured by echocardiography, effects that were associated with a reduction in hydroxyapatite deposition and MMP-12 expression in the aortic valves. CONCLUSION: Overall, these results highlight, for the first time, a significant role for IL-8 in the progression of CAVD by promoting calcification via a CXCR2- and MMP-12-dependent mechanism that leads to elastin degradation, and identify CXCR2 as a promising therapeutic target for the treatment of CAVD.

Indoxyl sulfate impairs erythropoiesis at BFU-E stage in chronic kidney disease.

Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of kidney function. It is associated with high serum levels of uremic toxins (UT), such as Indoxyl Sulfate (IS), which may participate in the genesis of several uremic complications. Anemia is one of the major complications in CKD patients that contribute to cardiovascular disease, increase morbi-mortality, and is associated with a deterioration of kidney failure in these patients. Our study aimed to characterize the impact of IS on CKD-related erythropoiesis. Using cellular and pre-clinical models, we studied cellular and molecular effects of IS on the growth and differentiation of erythroid cells. First, we examined the effect of clinically relevant concentrations of IS (up to 250 µM) in the UT7/EPO cell line. IS at 250 µM increased apoptosis of UT7/EPO cells at 48 h compared to the control condition. We confirmed this apoptotic effect of IS in erythropoiesis in human primary CD34+ cells during the later stages of erythropoiesis. Then, in IS-treated human primary CD34+ cells and in a (5/6 Nx) mice model, a blockage at the burst-forming unit-erythroid (BFU-E) stage of erythropoiesis was also observed. Finally, IS deregulates a number of erythropoietic related genes such as GATA-1, Erythropoietin-Receptor (EPO-R), and ß-globin. Our findings suggest that IS could affect cell viability and differentiation of erythroid progenitors by altering erythropoiesis and contributing to the development of anemia in CKD.

Rescuing SLAMF3 Expression Restores Sorafenib Response in Hepatocellular Carcinoma Cells through the Induction of Mesenchymal-to-Epithelial Transition.

BACKGROUND: Acquired resistance to sorafenib in hepatocellular carcinoma (HCC) patients results in poor prognosis. Epithelial-to-mesenchymal transition (EMT) is the major mechanism implicated in the resistance to sorafenib. We have reported the tumor suppressor role of SLAMF3 (signaling lymphocytic activation molecules family 3) in HCC progression and highlighted its implication in controlling the MRP-1 transporter activity. These data suggest the implication of SLAMF3 in sorafenib resistance mechanisms. METHODS: We evaluated the resistance to sorafenib in Huh-7 cells treated with progressive doses (Res cells). We investigated the link between acquired resistance to sorafenib and SLAMF3 expression by flow cytometry and Western blot methods. Furthermore, we analyzed the EMT and the stem cell potential of cells resistant to sorafenib. RESULTS: Sorafenib resistance was confirmed in Res cells by analyzing the cell viability in the presence of sorafenib. The mesenchymal transition, in Res cells, was confirmed by high migratory index and the expression of EMT antigens. Interestingly, we found that loss of SLAMF3 expression corresponded to sorafenib-resistant phenotypes. The overexpression of SLAMF3 reversed EMT, decreased metastatic potential and inhibited mTOR/ERK1/2 in Res cells. CONCLUSIONS: We propose that rescuing SLAMF3 expression in resistant cells could represent a potential therapeutic strategy to enhance sorafenib efficacy in HCC patients.

Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a dominant red cell membrane disorder characterized by an increased leak of potassium from the inside to outside the red blood cell membrane, associated with loss of water leading to red cell dehydration and chronic hemolysis. 90% of cases are related to heterozygous gain of function mutations in PIEZO1, encoding a mechanotransductor that translates a mechanical stimulus into a biological signaling. Data are still required to understand better PIEZO1-HX pathophysiology. Recent studies identified proteomics as an accurate and high-input tool to study erythroid progenitors and circulating red cell physiology. Here, we isolated red blood cells from 5 controls and 5 HX patients carrying an identified and pathogenic PIEZO1 mutation and performed a comparative deep proteomic analysis. A total of 603 proteins were identified among which 56 were differentially expressed (40 over expressed and 16 under expressed) between controls and HX with a homogenous expression profile within each group. We observed relevant modifications in the protein expression profile related to PIEZO1 mutations, identifying two main "knots". The first contained both proteins of the chaperonin containing TCP1 complex involved in the assembly of unfolded proteins, and proteins involved in translation. The second contained proteins involved in ubiquitination. Deregulation of proteins involved in protein biosynthesis was also observed in in vitro-produced reticulocytes after Yoda1 exposure. Thus, our work identifies significant changes in the protein content of PIEZO1-HX erythrocytes, revealing a "PIEZO1 signature" and identifying potentially targetable pathways in this disease characterized by a heterogeneous clinical expression and contra-indication of splenectomy.

Orai3-Mediates Cisplatin-Resistance in Non-Small Cell Lung Cancer Cells by Enriching Cancer Stem Cell Population through PI3K/AKT Pathway.

The development of the resistance to platinum salts is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Among the reasons underlying this resistance is the enrichment of cancer stem cells (CSCs) populations. Several studies have reported the involvement of calcium channels in chemoresistance. The Orai3 channel is overexpressed and constitutes a predictive marker of metastasis in NSCLC tumors. Here, we investigated its role in CSCs populations induced by Cisplatin (CDDP) in two NSCLC cell lines. We found that CDDP treatment increased Orai3 expression, but not Orai1 or STIM1 expression, as well as an enhancement of CSCs markers. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to CDDP and led to a reduction in the expression of Nanog and SOX-2. Orai3 contributed to SOCE (Store-operated Calcium entry) in both CDDP-treated and CD133+ subpopulation cells that overexpress Nanog and SOX-2. Interestingly, the ectopic overexpression of Orai3, in the two NSCLC cell lines, lead to an increase of SOCE and expression of CSCs markers. Furthermore, CD133+ cells were unable to overexpress neither Nanog nor SOX-2 when incubated with PI3K inhibitor. Finally, Orai3 silencing reduced Akt phosphorylation. Our work reveals a link between Orai3, CSCs and resistance to CDDP in NSCLC cells.

TRIM37 is highly expressed during mitosis in CHON-002 chondrocytes cell line and is regulated by miR-223.

Multiple molecular disorders can affect mechanisms regulating proliferation and differentiation of growth plate chondrocytes. Mutations in the TRIM37 gene cause the Mulibrey nanism, a heritable growth disorder. Since chondrocytes are instrumental in long bone growth that is deficient in nanism, we hypothesized that TRIM37 defect could contribute to dysregulation of the chondrocyte cell cycle. Western blotting, confocal microscopy and imaging flow cytometry determined TRIM37 expression in CHON-002 cell lineage. We showed that TRIM37 is expressed during mitosis of chondrocytes and directly impacted their proliferation. During the chondrocyte cell cycle, TRIM37 was present in both nucleus and cytoplasm. During M phase we observed an increase of the TRIM37-Tubulin co-localization in comparison with G1, S and G2 phases. TRIM37 knock down inhibited proliferation, together with cell cycle anomalies and increased autophagy, while overexpression accordingly enhanced cell proliferation. We demonstrated that microRNA-223 directly targets TRIM37, and suggest that miR-223 regulates TRIM37 gene expression during the cell cycle. In summary, our results give clues to explain why TRIM37 deficiency in chondrocytes impacts bone growth. Modulating TRIM37 using miR-223 could be an approach to increase chondrogenesis.