Notch-mediated induction of N-cadherin and α 9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RD06/0020/1021 and PI11/00740), Fundació la Marató de TV3, Asociación Española Contra el Cáncer, Fundació SMALL and Fundació A. BOSCH.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. 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 α 9-integrin-blocking antibodies. Cells treated with γ -secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α 9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α 9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti- α 9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness. Neuronal cadherin and α 9-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

Caracterización del mecanismo de activación de la vía Hedgehog en el rabdomiosarcoma : el papel oncogénico de los ligandos /

El rabdomiosarcoma (RMS) es el sarcoma de partes blandas más común en la infancia y se considera que se origina en células embrionarias destinadas a la formación de músculo esquelético. Histológicamente, el RMS se divide en 2 grupos principales: embrionario (RMSe) y alveolar (RMSa), los cuales difieren tanto en su presentación clínica, respuesta a terapia y pronóstico, siendo el subtipo alveolar el que presenta un pronóstico más desfavorable. La vía de señalización Hedgehog (HH) tiene un papel clave en el desarrollo y la progresión de muchos tipos de cáncer, incluyendo el RMS. Aunque esté ampliamente aceptada la activación de esta vía de señalización así como su papel oncogénico en el RMS, se desconoce el mecanismo molecular que impulsa su activación. En esta tesis se describe por primera vez en el RMS la contribución de los ligandos HH (Sonic, Indian y Desert) en la activación de la vía HH así como su implicación en la oncogénesis, apuntando a la existencia de una activación autocrina de esta vía de señalización. En este trabajo también se muestra el efecto de la inhibición farmacológica de la vía HH con inhibidores de Smoothened (Sonidegib y Vismodegib) y de los ligandos HH (MEDI-5304). De manera interesante, se describe por primera vez el efecto deletéreo de Vismodegib, provocando la inducción indeseada de la invasividad celular mediante la activación off-target de la vía Unfolded protein response (UPR). En conjunto, estos resultados establecen el mecanismo de activación de la vía HH y esclarecen el papel oncogénico de esta vía de señalización y, en particular, el de los ligandos HH. Asimismo, un número creciente de publicaciones describen el papel clave de la vía HH en el mantenimiento de las células madre tumorales en un amplia variedad de neoplasias, una pequeña subpoblación celular considerada la responsable de la iniciación, progresión y propagación de los tumores. En el caso del RMS, ha sido reportada la existencia de células madre tumorales en el subtipo embrionario pero no en el alveolar, el subtipo más agresivo de RMS. Debido a la falta de marcadores adecuados para el aislamiento de esta subpoblación celular, en este trabajo se realizaron ensayos basados en la formación de estructuras celulares (holoclones y esferas) enriquecidas en células madre tumorales para su selección y posterior estudio. La capacidad de las células de RMS para formar estas estructuras nos ha permitido demostrar la existencia de un subgrupo de células con capacidad de auto-renovación, por primera vez también en el subtipo alveolar. Además, en este trabajo se muestra la implicación de la vía HH en el mantenimiento de las células iniciadoras de RMS. Tanto los holoclones como las esferas mostraron una sobre-activación de esta vía de señalización; su inhibición farmacológica mediante el tratamiento con Sonidegib y MEDI-5304 resultó en una reducción en la formación de ambas estructuras celulares, mostrando por primera vez el papel clave de los ligandos HH para el mantenimiento de esta subpoblación celular. Por lo tanto, nuestros resultados apuntan a que la vía HH tiene un papel clave en el mantenimiento de las células iniciadoras de RMS y que su inhibición tendría un gran potencial terapéutico para prevenir recidivas locales y metástasis. En resumen, en esta tesis se describe la implicación de los ligandos HH en la oncogenicidad del RMS así como su papel en el mantenimiento de las células iniciadoras de RMS, posicionándolos como nuevas dianas terapéuticas para el desarrollo de futuras terapias contra el RMS.Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and is thought to derive from embryonic cells committed to developing into skeletal muscle. Histologically, RMS is divided into 2 major subtypes: embryonal (eRMS) and alveolar (aRMS), which differ in their clinical presentation, response to therapy and prognostic, being the alveolar subtype the most aggressive. Hedgehog (HH) pathway is considered to play an important role in the development and progression of several types of cancer, including RMS. Although a consistent activation of the HH pathway is well established in RMS, the molecular mechanism that drives its activation remains unknown. This work describes for first time the contribution of HH ligands (Sonic, Indian and Desert) in the HH pathway activation as well as their implication in oncogenesis, pointing to an autocrine ligand-dependent activation of the pathway in this neoplasia. A comparison of pharmacologic Smoothened inhibition (Sonidegib and Vismodegib) and HH ligand blocking (MEDI-5304) is provided. Interestingly, a first description of pernicious induction of cell invasiveness through the off-target activation of Unfolded protein response (UPR) after Vismodegib treatment is also reported. Taken together, these results establish the molecular mechanism of HH pathway activation and clarify the oncogenic role of this signalling pathway and, in particular, of the HH ligands. Likewise, a growing body of evidence describes the key role of HH pathway in cancer stem cells (CSC) maintenance in some neoplasias, a small subpopulation of cells considered to be the responsible of the initiation, progression and propagation of tumors. Although the existence of CSC has been reported in eRMS, their existence in aRMS, the most malignant subtype, has not been demonstrated to date. Given the lack of suitable markers to identify this subpopulation in aRMS, we used cancer stem cell-enriched supracellular structures (spheres and holoclones) to study this subpopulation. This strategy allowed us to demonstrate the capacity of both aRMS and eRMS cells to form these structures and retain self-renewal capacity. Furthermore, cells contained in spheres and holoclones showed a significant HH pathway induction, the pharmacologic inhibition of which impairs the formation of both holoclones and spheres, showing for first time the key role of HH ligands in the maintenance of RMS-initiating cells. Our findings point to a crucial role of this pathway in the maintenance of these structures and suggest that HHpathway targeting in CSC may have great potential in preventing local relapses and metastases. In summary, our findings describe the implication of HH ligands in the RMS oncogenesis as well as their role in the maintenance of RMS-initiating cells, pointing them as new therapeutic targets for the development of futures therapies against RMS

Hedgehog Pathway Inhibition Hampers Sphere and Holoclone Formation in Rhabdomyosarcoma

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RTICC-RD12/0036/0016 and RD12/0036/0027; PI11/00740 and PI14/00647), Fundació A. BOSCH, and ajuts predoctorals VHIR.Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal (eRMS) and alveolar (aRMS). Among the cellular heterogeneity of tumors, the existence of a small fraction of cells called cancer stem cells (CSC), thought to be responsible for the onset and propagation of cancer, has been demonstrated in some neoplasia. Although the existence of CSC has been reported for eRMS, their existence in aRMS, the most malignant subtype, has not been demonstrated to date. Given the lack of suitable markers to identify this subpopulation in aRMS, we used cancer stem cell-enriched supracellular structures (spheres and holoclones) to study this subpopulation. This strategy allowed us to demonstrate the capacity of both aRMS and eRMS cells to form these structures and retain self-renewal capacity. Furthermore, cells contained in spheres and holoclones showed significant Hedgehog pathway induction, the inhibition of which (pharmacologic or genetic) impairs the formation of both holoclones and spheres. Our findings point to a crucial role of this pathway in the maintenance of these structures and suggest that Hedgehog pathway targeting in CSC may have great potential in preventing local relapses and metastases

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

Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma : the oncogenic role of the ligands

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RTICC-RD12/0036/0016, /0020, /0035, /0057; and PI14/00647), Fundació A BOSCH, Fundació Amics Joan Petit, ajuts predoctorals del VHIR and RIS3CAT grants COMRDI15-1-0014 (ACCIÓ and FEDER).Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers

Notch-mediated induction of N-cadherin and α 9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RD06/0020/1021 and PI11/00740), Fundació la Marató de TV3, Asociación Española Contra el Cáncer, Fundació SMALL and Fundació A. BOSCH.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. 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 α 9-integrin-blocking antibodies. Cells treated with γ -secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α 9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α 9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti- α 9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness. Neuronal cadherin and α 9-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

Hedgehog Pathway Inhibition Hampers Sphere and Holoclone Formation in Rhabdomyosarcoma

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RTICC-RD12/0036/0016 and RD12/0036/0027; PI11/00740 and PI14/00647), Fundació A. BOSCH, and ajuts predoctorals VHIR.Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal (eRMS) and alveolar (aRMS). Among the cellular heterogeneity of tumors, the existence of a small fraction of cells called cancer stem cells (CSC), thought to be responsible for the onset and propagation of cancer, has been demonstrated in some neoplasia. Although the existence of CSC has been reported for eRMS, their existence in aRMS, the most malignant subtype, has not been demonstrated to date. Given the lack of suitable markers to identify this subpopulation in aRMS, we used cancer stem cell-enriched supracellular structures (spheres and holoclones) to study this subpopulation. This strategy allowed us to demonstrate the capacity of both aRMS and eRMS cells to form these structures and retain self-renewal capacity. Furthermore, cells contained in spheres and holoclones showed significant Hedgehog pathway induction, the inhibition of which (pharmacologic or genetic) impairs the formation of both holoclones and spheres. Our findings point to a crucial role of this pathway in the maintenance of these structures and suggest that Hedgehog pathway targeting in CSC may have great potential in preventing local relapses and metastases