Cancer, stem cells and cancer stem cells: old ideas, new developments

It has been suggested that, at least in some forms of cancer, a sub-population of slow-cycling, therapy-resistant cancer stem cells exists that has the ability to reconstitute the tumor in its entirety. If true, this model implies that conventional therapies based on targeting highly cycling cells within the tumor will leave the slow-cycling stem cell population intact, giving them the opportunity to reinitiate the tumor at a later date. This review discusses the evidence for this model and the likely implications for cancer treatment

Foxo3 is required for the regulation of oxidative stress in erythropoiesis

Erythroid cells accumulate hemoglobin as they mature and as a result are highly prone to oxidative damage. However, mechanisms of transcriptional control of antioxidant defense in erythroid cells have thus far been poorly characterized. We observed that animals deficient in the forkhead box O3 (Foxo3) transcription factor died rapidly when exposed to erythroid oxidative stress-induced conditions, while wild-type mice showed no decreased viability. In view of this striking finding, we investigated the potential role of Foxo3 in the regulation of ROS in erythropoiesis. Foxo3 expression, nuclear localization, and transcriptional activity were all enhanced during normal erythroid cell maturation. Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of P21(CIP1/WAF1/Sdi1) (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role

Aging-like Phenotype and Defective Lineage Specification in SIRT1-Deleted Hematopoietic Stem and Progenitor Cells

Summary Aging hematopoietic stem cells (HSCs) exhibit defective lineage specification that is thought to be central to increased incidence of myeloid malignancies and compromised immune competence in the elderly. Mechanisms underlying these age-related defects remain largely unknown. We show that the deacetylase Sirtuin (SIRT)1 is required for homeostatic HSC maintenance. Differentiation of young SIRT1-deleted HSCs is skewed toward myeloid lineage associated with a significant decline in the lymphoid compartment, anemia, and altered expression of associated genes. Combined with HSC accumulation of damaged DNA and expression patterns of age-linked molecules, these have striking overlaps with aged HSCs. We further show that SIRT1 controls HSC homeostasis via the longevity transcription factor FOXO3. These findings suggest that SIRT1 is essential for HSC homeostasis and lineage specification. They also indicate that SIRT1 might contribute to delaying HSC aging

Macrophages support pathological erythropoiesis in Polycythemia Vera and Beta-Thalassemia

Regulation of erythropoiesis is achieved by integration of distinct signals. Among these, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages for physiological and pathological conditions of enhanced erythropoiesis. We utilized mouse models of induced anemia, Polycythemia vera and β-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively for recovery from induced anemia as well as the pathological progression of Polycythemia vera and β-thalassemia by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a critical direct impact of macrophages on proliferation and enucleation of erythroblasts from healthy individuals and Polycythemia vera or β-thalassemic patients. In summary, we identify a new mechanism that we named “Stress Erythropoiesis Macrophage-supporting Activity” (SEMA) that contributes to the pathophysiology of these disorders and will have critical scientific and therapeutic implications in the near future

Targeting the BRD4/FOXO3a/CDK6 Axis Sensitizes AKT Inhibition in Luminal Breast Cancer

BRD4 assembles transcriptional machinery at gene super-enhancer regions and governs the expression of genes that are critical for cancer progression. However, it remains unclear whether BRD4-mediated gene transcription is required for tumor cells to develop drug resistance. Our data show that prolonged treatment of luminal breast cancer cells with AKT inhibitors induces FOXO3a dephosphorylation, nuclear translocation, and disrupts its association with SirT6, eventually leading to FOXO3a acetylation as well as BRD4 recognition. Acetylated FOXO3a recognizes the BD2 domain of BRD4, recruits the BRD4/RNAPII complex to the CDK6 gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or CDK6 significantly overcomes the resistance of luminal breast cancer cells to AKT inhibitors in vitro and in vivo. Our study reports the involvement of BRD4/FOXO3a/CDK6 axis in AKTi resistance and provides potential therapeutic strategies for treating resistant breast cancer

Resveratrol Induces Erythroid Maturation by Activating FOXO3 and Improves in Vivo Erythropoiesis in Normal and Beta -Thalassemic Mice

Resveratrol is a polyphenolic stilbene with anti-oxidant, anti-inflammatory and anti-tumoral bioactivities . High concentrations of resveratrol (50 \u3bcM) have been reported to induce HbF synthesis in an in vitro model of normal and beta-thalassemic erythropoiesis (Fibach E. Int J Mol Med 2012; Rodrigue CM. BJH 2001) and to improve erythropoiesis in a mouse model for Fanconi Anemia (Zhang Q. Blood 2010). Beta thalassemia (b-thal) is characterized by ineffective erythropoiesis and increased cellular oxidative stress. We studied the effects of resveratrol (5 \ub5M) on erythropoiesis in vitro from peripheral CD34+ cells of healthy and b-thal subjects. Erythroid maturation was evaluated at 7, 9, 11 and 14 days of culture by cytofluorimetric analysis using the CD71-GPA-CD36 strategy that allows to separate CFU-E, Pro-E, Int-E and Late-Erythroblasts (Merryweather-Clarke AT. Blood 2011). Resveratrol reduced cell growth in both cell types, with a reduction of CFU-E, increased Int-E at day 7 and 9, and increased Int-E and Late-E at 11 and 14 days. The early maturation of erythroid progenitors was confirmed by morphological analysis of the cells. We sorted CFU-E cells (at 7 days) from resveratrol treated and untreated cells and analyzed the cell cycle, cyclinD1 and p21 expression. In both cell types resveratrol induced increased frequency of S-G2/M cells compared to untreated cells with increased p21 levels, suggesting decreased cycling of CFU-E with increased maturation of erythroblasts. No changes of gamma chain mRNA levels were present in cells treated with resveratrol (5 \ub5M). Since FOXO3 is a key regulator of erythroid redox required for normal erythroid maturation (Marinkovic D. JCI 2007), FOXO3 expression and activity was assessed in sorted CFU (7day) and Int-E (11 day) with and without resveratrol. FOXO3a mRNA levels were increased in resveratrol treated cells in both sorted cell populations. We used nuclear localization as a surrogate assay for FOXO3a activity and found resveratrol increased the overall expression of FOXO3 protein in the nucleus without impacting significantly the nuclear/cytoplasmic ratio. Interestingly, resveratrol did not appear to modify FOXO1 expression or subcellular localization. These results suggest that resveratrol enhances specifically expression of FOXO3 in human erythroblasts. Dietary resveratrol supplementation (2.4 mg/Kg) was studied in wild-type and Hbb3th+/- mice (2 months of age) for 6 months. In resveratrol Hbb3th+/- treated mice increased Hb levels (8.3\ub10.6 vs 10.3\ub10.5 g/dL, n=12; P<0.05) and decreased reticulocyte count (33.9\ub10.8 vs 23.7\ub1 8.2 %, n=12; P<0.05) were observed. Significant increased MCV (34.6\ub10.6 vs 41.6\ub1 5.4 fL, n=12; P<0.05) and MCH ( 9.7\ub1 0.6 vs 12.8 \ub1 2.1 pg, n=12; P<0.05) were also noted. Flow cytometric evidence of decreased ineffective erythropoiesis and reduced spleen/ body weight ratio were also observed. These data indicate that resveratrol affects erythroid maturation both in vitro and in vivo, and that these effects have possible therapeutic relevance for the treatment of thalassemias

The role of CD44 in the regulation of hematopoiesis

The daily production of the large number of circulating blood cells is the result of a controlled process called hematopoiesis. In humans, adult hematopoiesis takes place in the bone marrow microenvironment where the regulated production and presentation of both positive and negative-acting factors control hematopoietic cell proliferation, differentiation and survival. Many cell surface molecules, including growth factor receptors and adhesion molecules are involved in these processes, although their relative importance remains to be understood. In the present thesis, I have focused on an examination of the potential role of the CD44 family of adhesion molecules in the regulation of primitive hematopoietic cell proliferation and differentiation. CD44 is a widely expressed multifunctional cell surface glycoprotein which has been implicated in many different adhesion-dependent processes. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. The expression of CD44 isoforms was examined on primitive normal and myeloid leukemic hematopoietic progenitor cells. These studies have shown that the differentiation-associated changes of CD44 isoform expression is altered during leukemogenesis. In addition, monoclonal antibodies to distinct CD44 epitopes were found to have either inhibitory, enhancing, or no effect on normal, but not on CML, stromal-dependent hematopoiesis. These results provide evidence of early differentiation-associated changes in CD44 expression during normal hematopoiesis which may be deregulated in chronic phase CML as well as in a variety of other myeloid leukemias. They also identify CD44 as a putative regulatory component in the interactions of hematopoietic cells with stroma, and suggest that this function is absent in primitive CML hematopoietic cells. Future investigations of molecular mechanisms involved in the regulation of CD44 ligand-binding and its downstream effects should provide insights into the biological observations described at the cellular level in this thesis.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Application of Gene Expression Programming Model to Present a New Model for Bond Strength of Fiber Reinforced Polymer and Concrete

In this paper, the gene expression-programming model was applied to present a novel model for the bond strength of concrete and fiber-reinforced polymer estimation. In order to do this, collected data were divided into the trained and tested ones by gene expression programming (GEP) means. The input parameters are the width of fiber-reinforced polymer, the width of concrete, thickness of fiber-reinforced polymer, the elastic modulus of fiber-reinforced polymer (FRP), concrete cylinder compressive strength, and bond length. The output parameters are the bond strength of concrete and FRP. Finally, a novel relationship was derived using the GEP to predict the bond strength of FRP-to-concrete composite joints. Results showed that the presented relationship was more convenient than the other models and that it was a powerful tool to predict the bond strength values of the FRP-to-concrete composite. For example, R-square (R2) of the present work is 0.92 compared to that (< 0.82) reported for other models. Among themodels presented by other researchers, that of Dai et al. is more accurate than the other ones, and the model offered by Khalifa et al. has the lowest accuracy.Peer reviewe