Thrombopoietin dose-response curves reflect the number of doublings undergone by human megakaryocyte progenitors

audience: researcherTo assess the variation of thrombopoietin (TPO) responsiveness associated with MK progenitor amplification, TPO dose-response curves were obtained for MK clones derived from normal human CD34+CD41+ cells which had been single-cell plated in serum-deprived medium with TPO concentrations of 0-1000 pg/mL per well. On day 5, the number of MK cells per well was determined in situ and expressed as number of doublings (NbD). Dose-response curves of the means of the frequency of clones of each size vs logTPO concentration showed highly significant differences in the TPO concentration needed for half-maximum generation of clones of different sizes (TPO50). Based on 3 independent experiments, TPO50, expressed in pg/mL, was 1.89 ±SD 0.87 for 1 cell MK clone; 7.75 ±1.40 for 2-3 cell MK clone; 38.5 ±8.73 for 4-7 cell MK clone and 93.6 ±27.7 for 8-15 cell MK clone (p<0.001). Compared to the CD42- fraction of CD34+CD41+ progenitors, the corresponding CD42+ fraction had a lower TPO50 (p<0.001), underwent lesser NbD (p<0.001) and expressed a 10-fold greater mean mpl receptor density. In order to interpret the conclusion that the most responsive progenitors were those which proliferated less, we tested a prediction of the generation-age model, namely that the percentage of progenitors undergoing a given number of residual doublings in vitro (NbDr) depends on the number of previous doublings they had undergone in vivo (NbDp). It was demonstrated that logTPO50 had a positive correlation with NbDr and negative correlation with NbDp (p<0.001). The reciprocal relationship between NbDp and NbDr was evidence for a generation-age model in which TPO responsiveness increases by a factor of approximately 4 per doubling. This model suggests that progenitors which have completed their proliferative program have maximum factor responsiveness and are preferentially induced to terminal differentiation

Inhibition of the GTPase Rac1 Mediates the Antimigratory Effects of Metformin in Prostate Cancer Cells

International audienceCell migration is a critical step in the progression of prostate cancer to the metastatic state, the lethal form of the disease. The antidiabetic drug metformin has been shown to display antitumoral properties in prostate cancer cell and animal models; however, its role in the formation of metastases remains poorly documented. Here, we show that metformin reduces the formation of metastases to fewer solid organs in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin leads to a major inhibition of Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cAMP, and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1, or P-Rex, as well as the inhibition of the adenylate cyclase, was able to reverse the antimigratory effects of metformin. These results establish a novel mechanism of action for metformin and highlight its potential antimetastatic properties in prostate cancer

Targeting Cancer Cell Metabolism: The Combination of Metformin and 2-Deoxyglucose Induces p53-Dependent Apoptosis in Prostate Cancer Cells

International audienceTargeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, exerts antitumoral and antiproliferative action. In this study, the addition of metformin to 2-deoxyglucose (2DG) inhibited mitochondrial respiration and glycolysis in prostate cancer cells leading to a severe depletion in ATP. The combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone, leading to 96% inhibition of cell viability in LNCaP prostate cancer cells. In contrast, a moderate effect on cell viability was observed in normal prostate epithelial cells. At the cellular level, the combination of metformin and 2DG induced p53-dependent apoptosis via the energy sensor pathway AMP kinase, and the reexpression of a functional p53 in p53-deficient prostate cancer cells restored caspase-3 activity. In addition to apoptosis, the combination of metformin and 2DG arrested prostate cancer cells in G(2)-M. This G(2)-M arrest was independent of p53 and correlated with a stronger decrease in cell viability than obtained with either drug. Finally, metformin inhibited 2DG-induced autophagy, decreased beclin 1 expression, and triggered a switch from a survival process to cell death. Our study reinforces the growing interest of metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anticancerous treatment

Cancer cell-derived long pentraxin 3 (PTX3) promotes melanoma migration through a toll-like receptor 4 (TLR4)/NF-kappa B signaling pathway

Cutaneous melanoma is one of the most aggressive cancers characterized by a high plasticity, a propensity for metastasis, and drug resistance. Melanomas are composed of phenotypically diverse subpopulations of tumor cells with heterogeneous molecular profiles that reflect intrinsic invasive abilities. In an attempt to identify novel factors of the melanoma invasive cell state, we previously investigated the nature of the invasive secretome by using a comparative proteomic approach. Here, we have extended this analysis to show that PTX3, an acute phase inflammatory glycoprotein, is one such factor secreted by invasive melanoma to promote tumor cell invasiveness. Elevated PTX3 production was observed in the population of MITFlow invasive cells but not in the population of MITFhigh differentiated melanoma cells. Consistently, MITF knockdown increased PTX3 expression in MITFhigh proliferative and poorly invasive cells. High levels of PTX3 were found in tissues and blood of metastatic melanoma patients, and in BRAF inhibitor-resistant melanoma cells displaying a mesenchymal invasive MITFlow phenotype. Genetic silencing of PTX3 in invasive melanoma cells dramatically impaired migration and invasion in vitro and in experimental lung extravasation assay in xenografted mice. In contrast, addition of melanoma-derived or recombinant PTX3, or expression of PTX3 enhanced motility of low migratory cells. Mechanistically, autocrine production of PTX3 by melanoma cells triggered an IKIC/NF kappa B signaling pathway that promotes migration, invasion, and expression of the EMT factor TWIST1. Finally, we found that TLR4 and MYD88 knockdown inhibited PTX3-induced melanoma cell migration, suggesting that PTX3 functions through a TLR4-dependent pathway. Our work reveals that tumor-derived PTX3 contributes to melanoma cell invasion via targetable inflammation-related pathways. In addition to providing new insights into the biology of melanoma invasive behavior, this study underscores the notion that secreted PTX3 represents a potential biomarker and therapeutic target in a subpopulation of MITFlow invasive and/or refractory melanoma

Blockade of pro-fibrotic response mediated by the miR-143/-145 cluster prevents targeted therapy-induced phenotypic plasticity and resistance in melanoma

Abstract Lineage dedifferentiation towards a mesenchymal-like state is a common mechanism of adaptive response and resistance to targeted therapy in melanoma. Yet, the transcriptional network driving this phenotypic plasticity remains elusive. Remarkably, this cellular state displays myofibroblast and fibrotic features and escapes MAPK inhibitors (MAPKi) through extracellular matrix (ECM) remodeling activities. Here we show that the anti-fibrotic drug Nintedanib/BIBF1120 is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy and delay tumor relapse in a pre-clinical model of melanoma. We also uncovered the molecular networks that regulate the acquisition of this resistant phenotype and its reversion by Nintedanib, pointing the miR-143/-145 pro-fibrotic cluster as a driver of the therapy-resistant mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile of resistant cells. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p collaborated to mediate phenotypic transition towards a drug resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics as well as contributing to a fine-tuning of mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof-of-principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy