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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

The Gluthation peroxidase (GPX) enzymes are part of the protective system against lipid peroxydation that includes prevention of oxydation and reduction of already oxidized lipid through enzymatic reactions catalyzed by GSH. GPX4 is one of the five GPX able to incorporate selenium. It is also the only GPX able to directly reduce in the membrane the oxidized fatty acids and cholesterol. Recent reports identified GPX4 as the central inhibitor of ferroptosis, a process during which iron-induced peroxydation of membrane lipids causes a specific cell death that can be reverted by lipophilic antioxydants or by iron chelators. GPX4 has recently been involved during mice erythropoiesis: GPX4-/-mice present a hemolytic anemia and a high apoptotic rate in spleen erythroid progenitors. Although transcriptomics and proteomics found it expressed in human erythroid precursors, its role during human erythropoiesis has not been described. Using an in-vitroerythroid differentiation protocol from CD34+cells obtained from apheresis, we confirmed that GPX4 expression was induced at RNA and protein level during differentiation. RSL3, a specific GPX4 inhibitor, didn't affect early steps of erythropoiesis (i.eclonogenic potential and progenitor amplification) nor the early maturation of erythroid precursors (assessed by sequential CD49d/CD235/CD71 staining) but led to a significant decrease in the enucleation rate as assessed by Hoechst staining using flow cytometry (74%±9 DMSO versus 35%±6 RSL3, p Using Western Blot, we observed that RSL3 exposure induced a strong GPX4 depletion in erythroid progenitors while it didn't affect GPX1, another member of the GPX family expressed in erythroid cells. In order to confirm that enucleation defect was related to GPX4 knockdown, we used an Sh-RNA strategy that allowed a 62%±6 GPX4 decrease at RNA level and a 46%±5 at protein level. We observed a significant defect in terminal enucleation in the cells transduced with shGPX4-lentiviruses in comparison with sh-Scramble (59%±5 Sh-Scr versus 39%±6 Sh-Gpx4, p We investigated then whether GPX4-knock-down affected quantitatively or qualitatively the membrane lipid content, which was shown to be involved in the enucleation process. Addition of Cholesterol to RSL3 in the medium partially restored the enucleation rate. However, lipidomics failed to show any significant difference in the total membrane lipid content (and particularly in the cholesterol content) after RSL3 exposure in comparison to DMSO. Since cholesterol is particularly abundant in the lipid rafts, we investigated whether the lipid distribution was qualitatively altered within the cell membrane. We observed a disruption of membrane lipid rafts when cells were exposed to RSL3, as shown by a 60%±12 decrease in the mean cholera toxin fluorescence intensity. GPX4 presence in lipid rafts was confirmed using immunofluorescence showing their co-localization at cell surface in human primary erythroblasts. Since lipid rafts play a role in the contractile ring that separates pyrenocyte from reticulocyte, we evaluated the myosin-light chain phosphorylation using flow cytometry and Western Blot and found it drastically decreased in GPX4-knockdown conditions. In summary, we identified GPX4 as a new actor of human terminal erythroid differentiation, independently to its function in ferroptosis control. We described its interaction at cell surface with lipid rafts that are required for the assembly of the contractile ring and cytokinesis leading to the nucleus extrusion. Disclosures No relevant conflicts of interest to declare.

International audience; 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/GATAI ratio and decreased alpha/beta-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 STATS 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.

Background and Aims Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of renal function due to high serum levels of uremic toxins. Anemia is a major trouble in CKD patients that contributes to a faster deterioration of renal failure, leading to cardiovascular disease and increasing morbimortality. Erythropoietin (EPO) is known to contribute to CKD-associated anemia. Thus, accumulation of uremic toxins in blood impairs EPO synthesis, leading to a subsequent impairment of erythropoiesis in the bone marrow. Very few molecular clues explain why erythropoiesis is affected in CKD or explain why erythropoiesis-stimulating agents (ESA) are not efficient in some patients with CKD. The current study aims to characterize the impact of one of the most representative uremic toxins, Indoxyl Sulfate (IS), in CKD-related anemia. IS is a protein-bound uremic toxin derived from the tryptophan dietary metabolism which is difficult to remove by dialysis. Our study demonstrates the molecular effects of IS on the growth and the differentiation of red blood cells in an erythroid cell line and in primary cell cultures CD34+. Method Firstly, we examined in vitro the time-courses of IS under clinically relevant concentrations of IS (250 µM -1 mM) in a human leukemic cell line in which proliferation is induced by EPO, the UT7/EPO cell line. Cell apoptosis, proliferation, differentiation and cell cycle analysis were assessed by the MACSQuant flow cytometry. Erythroid gene expression analysis was assessed by RT-qPCR (Quantstudio 7 flex). The ratio A260/280 assessed the quality of nucleic acids. Western blotting experiments were performed to study protein expression. Human primary CD34+ cells were obtained from mobilized peripheral blood mononuclear cells (MNC) of healthy subjects and were isolated by magnetic microbeads separation on MACS columns. Results IS at 250 µM and 1 mM increased apoptosis of UT7/EPO cell line at 48h compared to control condition. On the other hand, we found no significant effect of IS on the phenotype of UT7/EPO, when using CD235a (Glycophorin A), as a marker for the detection of the erythroid cell lineage. Ki67 cellular levels, a cell proliferation marker, was not altered between control and IS experiments. This indicated that IS did not affect proliferation in UT7/EPO. At 48h, at the clinically relevant concentration of IS (250 µM), we observed an increase of the cell phase cycle Sub-G1. The analysis of erythropoiesis related genes shows that HIF2α was deregulated with IS (250 µM). Finally, in the Epo-EpoR signalling pathway, we studied the activation of the Jak2/Stat5 proteins. Results in human primary CD34+ cells confirmed the apoptotic effect of IS observed in UT7/EPO. Conclusion Our findings suggest that IS, a representative protein-bound uremic toxin, could affect cell viability, apoptosis and the cell cycle. This study suggests clues to develop new therapies for CKD-associated anemia.

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.

The precursor for nerve growth factor (proNGF) is expressed in some cancers but its clinicopathological significance is unclear. The present study aimed to define the clinicopathological significance of proNGF in thyroid cancer. ProNGF expression was analysed by immunohistochemistry in two cohorts of cancer versus benign tumors (adenoma) and normal thyroid tissues. In the first cohort (40 thyroid cancers, 40 thyroid adenomas and 80 normal thyroid tissues), proNGF was found overexpressed in cancers compared to adenomas and normal samples (p

Starvation is a strong physiological stimulus of macroautophagy/autophagy. In this study, we addressed the question as to whether it would be possible to measure autophagy in blood cells after nutrient deprivation. Fasting of mice for 48 h (which causes ∼20% weight loss) or starvation of human volunteers for up to 4 d (which causes

One of the mechanisms of cancer-associated genomic instability involves a transient phase of polyploidization, in most cases tetraploidization, followed by asymmetric divisions and chromosome loss. Increases in ploidy are consistently accompanied by the activation of an endoplasmic reticulum (ER) stress response, resulting in the translocation of calreticulin to the outer surface of the plasma membrane where it stimulates anticancer immune responses. Conversely, immunoselection leads to a coordinated reduction in ploidy, ER stress, and calreticulin exposure. To simultaneously investigate the ER stress and ploidy, we developed an image cytofluorometric method that allows to measure DNA content, ER stress-associated phosphorylation of eIF2α, and calreticulin exposure at the cell surface. Here, we specify this methodology, which is useful for investigating the correlation between ploidy and ER stress at the single cell level.

One of the mechanisms of cancer-associated genomic instability involves a transient phase of polyploidization, in most cases tetraploidization, followed by asymmetric divisions and chromosome loss. Increases in ploidy are consistently accompanied by the activation of an endoplasmic reticulum (ER) stress response, resulting in the translocation of calreticulin to the outer surface of the plasma membrane where it stimulates anticancer immune responses. Conversely, immunoselection leads to a coordinated reduction in ploidy, ER stress, and calreticulin exposure. To simultaneously investigate the ER stress and ploidy, we developed an image cytofluorometric method that allows to measure DNA content, ER stress-associated phosphorylation of eIF2α, and calreticulin exposure at the cell surface. Here, we specify this methodology, which is useful for investigating the correlation between ploidy and ER stress at the single cell level.

The Trk family is composed of three principal members (A, B and C), also termed neurotrophin tyrosine kinase receptors (NTRK1, NTRK2, NTRK3), with several additional splice variants, which in humans are located on three different chromosomes (1, 9, 15). The Trk proteins are receptors for the neurotrophin family of growth factors (NGF, BDNF, NT-3, and NT4/5); they also bind and respond more weakly to the proforms of these neurotrophins. Both the neurotrophins and pro-neurotrophins utilize other receptors (p75NTR, sortilin) that can interact with the Trks and affect their activity. The Trks are predominantly expressed in nervous tissues, where they are essential for the growth, development, and maintenance of select types of both peripheral and central neurons and are involved in neurodegenerative diseases. The Trks are also found in some nonneuronal tissues and in a wide variety of tumors, where they can play an active role in tumor progression and metastasis. The activated Trk receptors primarily bind and signal through Shc, FRS2, and PLCγ, which in turn activate PI3K, the Erks, and the hydrolysis of inositol phospholipids, among other events, leading to the modulation of gene transcription.

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, τB = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of ≥8 compared with a Gleason score of 7 and 6 (P 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves.

The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.

The membrane protein sortilin is involved in intracellular trafficking and has emerged as a key player in the regulation of neuronal viability and function. Few studies have suggested that sortilin may also be implicated in cancer, but its expression in human tumors and potential value as a therapeutic target is unknown. In this study, the level of sortilin was analyzed in a series of 318 clinically annotated breast cancers and 53 normal adjacent tissues by immunohistochemistry. Sortilin was specifically localized in epithelial cells and was detected in 65% of cancers compared to 46% of normal tissues (p=0.0088). Sortilin was detected in 79% of invasive ductal carcinomas compared to only 54% of invasive lobular carcinomas (p Citation Format: Jay Pundavela, Severine Roselli, Yohann Demont, Sam Faulkner, John Attia, Sheridan Keene, Marjorie M Walker, Hubert Hondermarck. The neuronal protein sortilin is expressed in aggressive breast cancers and participates in tumor cell growth and invasion [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-11.

International audience; The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA ( ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappa B and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

The neuronal membrane protein sortilin has been reported in a few cancer cell lines, but its expression and impact in human tumors is unclear. In this study, sortilin was analyzed by immunohistochemistry in a series of 318 clinically annotated breast cancers and 53 normal breast tissues. Sortilin was detected in epithelial cells, with increased levels in cancers, as compared to normal tissues (p = 0.0088). It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001). There was an association between sortilin expression and lymph node involvement (p = 0.0093), suggesting a relationship with metastatic potential. In cell culture, sortilin levels were higher in cancer cell lines compared to non-tumorigenic breast epithelial cells and siRNA knockdown of sortilin inhibited cancer cell adhesion, while proliferation and apoptosis were not affected. Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation. In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.