Josep Roma

uPA deficiency exacerbates muscular dystrophy in MDX mice.

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.

Notch Pathway Inhibition Significantly Reduces Rhabdomyosarcoma Invasiveness and Mobility In Vitro

Purpose: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal and alveolar RMS. Patients with metastatic disease continue to have very poor prognosis although aggressive therapies and recurrences are common in advanced localized disease. The oncogenic potential of the Notch pathway has been established in some cancers of the adult and in some pediatric malignancies. Experimental Design: A real-time PCR assay was used to ascertain the expression of several Notch pathway components in a wide panel of RMS and cell lines. Four γ-secretase inhibitors (GSIs) were tested for pathway inhibition and the degree of inhibition was assessed by analysis of Hes1 and Hey1 expression. The putative effects of Notch pathway inhibition were evaluated by wound-healing, matrigel/transwell invasion, cell-cycle, and apoptosis assays. Results: The Notch pathway was widely expressed and activated in RMS and underwent substantial inhibition when treated with GSIs or transfected with a dominant negative form of MAML1. RMS cells showed a significant decrease in its mobility and invasiveness when the Notch pathway was properly inhibited; conversely, its inhibition had no noticeable effect on cell cycle or apoptosis. Conclusion: Pharmacological or genetic blockage of the pathway significantly reduced invasiveness of RMS cell lines, thereby suggesting a possible role of the Notch pathway in the regulation of the metastatic process in RMS. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

Notch, Wnt, and Hedgehog Pathways in Rhabdomyosarcoma: From Single Pathways to an Integrated Network

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Regarding histopathological criteria, RMS can be divided into 2 main subtypes: embryonal and alveolar. These subtypes differ considerably in their clinical phenotype and molecular features. Abnormal regulation or mutation of signalling pathways that regulate normal embryonic development such as Notch, Hedgehog, and Wnt is a recurrent feature in tumorigenesis. Herein, the general features of each of the three pathways, their implication in cancer and particularly in RMS are reviewed. Finally, the cross-talking among these three pathways and the possibility of better understanding of the horizontal communication among them, leading to the development of more potent therapeutic approaches, are discussed.

BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30–40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

Detection of bone marrow micrometastasis and microcirculating disease in rhabdomyosarcoma by a real-time RT-PCR assay

Purpose: To assess if molecular detection of minimal disseminated disease by real-time reverse transcription and polymerase chain reaction (RT-PCR) could contribute to a better treatment stratification in patients with rhabdomyosarcoma (RMS). Methods: Relative quantification of the tumor-mRNA present in serial samples of bone marrow (BM) and peripheral blood (PB) from 16 patients with RMS (7 alveolar and 9 embryonal) was performed by a real-time RT-PCR assay. Expression of MyoD1 and acetylcholine receptor (AChR) was analyzed in all samples, along with PAX3/7-FKHR in samples from alveolar tumors. Results: A good correlation was found between the expression of PAX3/7-FKHR and AChR, while MyoD1 was more sensitive but less specific. In this study, patients with positive PB at the end of treatment showed a poorer prognosis than patients with negative PB. Moreover, in this patient cohort, metastatic relapses were preceded by the detection of microcirculating disease in all cases. Conclusion: The detection of minimal circulating and micrometastatic disease by real-time RT-PCR, based on the expression of multiple genes, yields highly reproducible results. Patients with positive PB after treatment show poorer survival than patients without microcirculating disease.

MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma

BackgroundRhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells.ResultsHerein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells.ConclusionsAltogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.

Notch-mediated induction of N-cadherin and α9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells

Background:Rhabdomyosarcoma (RMS) is the commonest type of soft-tissue sarcoma in children. Patients with metastatic RMS continue to have very poor prognosis. Recently, several works have demonstrated a connection between Notch pathway activation and the regulation of cell motility and invasiveness. However, the molecular mechanisms of this possible relationship remain unclear.Methods:The Notch pathway was manipulated pharmacologically and genetically. The mRNA changes were analysed by quantitative PCR and protein variations by western blot and immunofluorescence. Finally, the capabilities of RMS cells to adhere, heal a wound and invade were assessed in the presence of neuronal cadherin (N-cadherin)- and α9-integrin-blocking antibodies.Results:Cells treated with γ-secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti-α9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness.Conclusion:Neuronal cadherin and α9-integrin are postulated as leading actors in the association between the Notch pathway and promotion of cell adhesion, motility and invasion, pointing to these proteins and the Notch pathway itself as interesting putative targets for new molecular therapies against metastases in RMS.

MicroRNA-497 impairs the growth of chemoresistant neuroblastoma cells by targeting cell cycle, survival and vascular permeability genes.

Despite multimodal therapies, a high percentage of high-risk neuroblastoma (NB) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR). MicroRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor. We found several genes implicated in the MDR to be overexpressed in high-risk NB which could be targeted by microRNAs simultaneously. Our functional screening identified several of those microRNAs that reduced proliferation of chemoresistant NB cell lines, the best of which was miR-497. Low expression of miR-497 correlated with poor patient outcome. The overexpression of miR-497 reduced the proliferation of multiple chemoresistant NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy.

Distribution and effects of a single intramuscular injection of India ink in mice.

Our goal has been to study the distribution and effects following a single intramuscular injection of a substance using India ink as a tracer. We injected 30 microl India ink in the gastrocnemius muscle group of C57Bl10 mice. Hematoxylin-Eosin, Trichrome stains and polyclonal anti-laminin, anti-collagen-IV and anti-dystrophin were used. The liquid spreads in all directions mainly following the perimysial and epimysial septae. The collagen bundles act as physical barriers preventing passage of the ink particles. In the area of the injection site, necrosis of the fibres is associated with disruption of the basement membrane. In the zones adjacent and distal to the injection site, the liquid progresses by pushing the muscle fibres apart with preservation of the basement membrane. Research based on intramuscular injection of substances should take the following into consideration: a) anatomy of the muscle group injected, b) routes of distribution of the substance, c) types of lesions produced with relation to the site of injection of the liquid, and d) size of the particles of the injected substance.

Characterisation of novel splicing variants of the tyrosine hydroxylase C-terminal domain in human neuroblastic tumours.

Abstract Alternative splicing of human tyrosine hydroxylase (hTH) transcripts appears to occur mainly in the N-terminal domain, giving rise to at least eight different isoforms. We recently reported the existence of hTH transcript variants resulting from splicing of exons 8 and 9, within a region previously thought to be constant. The mRNA distribution of these novel hTH isoforms in neuroblastic tumours and in foetal adrenal glands was analysed by conventional and real-time RT-PCR. The presence of the target protein was determined by Western blotting, immunoprecipitation and protein analysis. Transcripts lacking exons 8 and 9 were widely distributed in the tissues analysed. Characterisation of full-length mRNA revealed that splicing of exons 8 and 9 was always associated with splicing of exons 2 (hTH-Δ2,8,9) or 1b and 2 (hTH-Δ1b,2,8,9). In addition, one variant detected on Western blots in several tumours fits the predicted size (58 kDa) of the isoforms lacking exons 8 and 9. In conclusion, the two novel isoforms reported here (hTH-Δ2,8,9 and hTH-Δ1b,2,8,9) represent the first full-length isoforms with alternative splicing in the hTH C-terminal domain. These results demonstrate for the first time the existence of hTH isoforms Δ2,8,9 and Δ1b,2,8,9. Their general distribution in neuroblastoma and adrenal glands and translation into protein suggest a significant functional role for these novel hTH isoforms, which merit further study.