Clara Guerzoni

BCR-ABL suppresses C/EBPα expression through inhibitory action of hnRNP E2

The arrest of differentiation is a feature of both chronic myelogenous leukemia cells in myeloid blast crisis and myeloid precursors that ectopically express the p210BCR-ABL oncoprotein; however, its underlying mechanisms remain poorly understood. Here we show that expression of BCR-ABL in myeloid precursor cells leads to transcriptional suppression of the granulocyte colony–stimulating factor receptor G-CSF-R (encoded by CSF3R), possibly through down-modulation of C/EBPα—the principal regulator of granulocytic differentiation. Expression of C/EBPα protein is barely detectable in primary marrow cells taken from individuals affected with chronic myeloid leukemia in blast crisis. In contrast, CEBPA RNA is clearly present. Ectopic expression of C/EBPα induces granulocytic differentiation of myeloid precursor cells expressing BCR-ABL. Expression of C/EBPα is suppressed at the translational level by interaction of the poly(rC)-binding protein hnRNP E2 with CEBPA mRNA, and ectopic expression of hnRNP E2 in myeloid precursor cells down-regulates both C/EBPα and G-CSF-R and leads to rapid cell death on treatment with G-CSF (encoded by CSF3). Our results indicate that BCR-ABL regulates the expression of C/EBPα by inducing hnRNP E2—which inhibits the translation of CEBPA mRNA.

NVP-BEZ235 as a New Therapeutic Option for Sarcomas

Purpose: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide 3-kinase–mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors (osteosarcoma, Ewings sarcoma, and rhabdomyosarcoma). Experimental Design: Antiproliferative activity as well as the effects on migration and metastasis were evaluated in a panel of osteosarcoma, Ewings sarcoma, as well as rhabdomyosarcoma cell lines. Moreover, simultaneous and sequential treatments were done in association with two of the most important conventional drugs in the treatment of sarcoma, doxorubicin and vincristine. Results: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the experimental conditions tested, the compound induced disease stasis, by arresting cells in G1 phase of cell cycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitation of its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxic agents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phosphoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellular signal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signaling toward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235 with other small signaling inhibitors. Here, we showed synergistic effects when the compound was associated with a anti–insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 also inhibited cell migration and metastasis. Combination with vincristine further potentiated the antimetastatic effects. Conclusions: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advanced cancer patients. Clin Cancer Res; 16(2); 530–40

hnRNP A1 Nucleocytoplasmic Shuttling Activity Is Required for Normal Myelopoiesis and BCR/ABL Leukemogenesis

ABSTRACT hnRNP A1 is a nucleocytoplasmic shuttling heterogeneous nuclear ribonucleoprotein that accompanies eukaryotic mRNAs from the active site of transcription to that of translation. Although the importance of hnRNP A1 as a regulator of nuclear pre-mRNA and mRNA processing and export is well established, it is unknown whether this is relevant for the control of proliferation, survival, and differentiation of normal and transformed cells. We show here that hnRNP A1 levels are increased in myeloid progenitor cells expressing the p210BCR/ABL oncoprotein, in mononuclear cells from chronic myelogenous leukemia (CML) blast crisis patients, and during disease progression. In addition, in myeloid progenitor 32Dcl3 cells, BCR/ABL stabilizes hnRNP A1 by preventing its ubiquitin/proteasome-dependent degradation. To assess the potential role of hnRNP A1 nucleocytoplasmic shuttling activity in normal and leukemic myelopoiesis, a mutant defective in nuclear export was ectopically expressed in parental and BCR/ABL-transformed myeloid precursor 32Dcl3 cells, in normal murine marrow cells, and in mononuclear cells from a CML patient in accelerated phase. In normal cells, expression of this mutant enhanced the susceptibility to apoptosis induced by interleukin-3 deprivation, suppressed granulocytic differentiation, and induced massive cell death of granulocyte colony-stimulating factor-treated cultures. In BCR/ABL-transformed cells, its expression was associated with suppression of colony formation and reduced tumorigenic potential in vivo. Moreover, interference with hnRNP A1 shuttling activity resulted in downmodulation of C/EBPα, the major regulator of granulocytic differentiation, and Bcl-XL, an important survival factor for hematopoietic cells. Together, these results suggest that the shuttling activity of hnRNP A1 is important for the nucleocytoplasmic trafficking of mRNAs that encode proteins influencing the phenotype of normal and BCR/ABL-transformed myeloid progenitors.

Impact of a Single Nucleotide Polymorphism in the MDM2 Gene on Neuroblastoma Development and Aggressiveness: Results of a Pilot Study on 239 Patients

Purpose: MDM2 is a key negative regulator of p53 activity, and a single nucleotide polymorphism (SNP309, T>G change; rs 2279744) in its promoter increases the affinity for the transcription factor SP1, enhancing MDM2 expression. We carried out a pilot study to investigate the effect of this polymorphism on development and behavior of neuroblastoma, an extracranial pediatric tumor with unfrequent genetic inactivation of p53. Experimental Design: We genotyped the MDM2-SNP309 alleles of tumor DNA from 239 neuroblastoma patients and peripheral blood DNA from 237 controls. In 40 of 239 neuroblastomas, the MDM2-SNP309 alleles were also genotyped in peripheral blood DNA. Data were analyzed by two-sided Fishers exact test, log-rank test, and Kaplan-Meier statistics. Where appropriate, data are reported with 95% confidence intervals (CI). Results: The frequency of both the T/G and G/G genotypes or the G/G or T/G genotype only was higher in neuroblastoma DNA samples than in controls: 60.3% (95% CI, 54.1-66.5) versus 47.3% (95% CI, 40.9-53.6), 30.4% (95% CI, 22.4-37.8) versus 15.0% (95% CI, 9.2-20.7), and 52.0% (95% CI, 45.0-59.9) versus 41.9% (95% CI, 35.3-48.5), respectively; Two-Sided Fishers Exact Test P values were 0.006, 0.003, and 0.048, respectively; Odds ratios were 1.69 (95% CI, 1.18-2.43), 2.45 (95% CI, 1.37-4.39) and 1.51 (95% CI, 1.02-2.22), respectively. A significant association (P = 0.016) between heterozygous (T/G)/homozygous (G/G) genotypes at SNP309 and advanced clinical stages was also shown. Homozygous/heterozygous SNP309 variant carriers had a shorter 5-year overall survival than patients with the wild-type allele (P = 0.046; log-rank test). A shorter overall survival in patients with heterozygous/homozygous SNP309 was also observed in the subgroups with age at diagnosis >1 year and adrenal primary tumor (P = 0.024 and P = 0.014, respectively). Conclusions: Data from this pilot study suggest that the MDM2 G/G and T/G-SNP309 alleles are markers of increased predisposition to tumor development and disease aggressiveness in neuroblastoma. However, additional studies with larger patient cohorts are required for a definitive assessment of the clinical relevance of these data.

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility

BackgroundAkt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells.ResultsWe confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1α. EF1α contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1α expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1α siRNAs with specific pAkt inhibitors whereas EF1α downregulation slightly attenuated the decreased invasion induced by Akt inhibitors.ConclusionWe show here that EF1α is a pAkt-interacting protein which regulates pAkt levels. Since EF1α is often overexpressed in breast cancer, the consequences of EF1α increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells

ABSTRACT Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell–cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled–coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

CD99 Drives Terminal Differentiation of Osteosarcoma Cells by Acting as a Spatial Regulator of ERK 1/2

Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99‐transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane‐bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21WAF1/CIP1, leading to G0/G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate.

CD99 Triggering in Ewing Sarcoma Delivers a Lethal Signal through p53 Pathway Reactivation and Cooperates with Doxorubicin

Purpose: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. Experimental Design: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. Results: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS. Conclusions: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death. Clin Cancer Res; 21(1); 146–56. ©2014 AACR.

CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling

CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.