Notch-mediated induction of N-Cadherin and a9-Integrin confers higher invasive phenotype on rhabdomyosarcoma cells

Br J Cancer. 2012 Oct 9;107(8):1374-83.

Masià A1, Almazán-Moga A1, Velasco P2, Reventós J1, Torán N3,Sánchez de Toledo J2, Roma J1, Gallego S1,2.

1Research Unit in Biomedicine and Translational and Pediatric Oncology, Vall d’Hebron Institut de Recerca (VHIR); 2Pediatric Oncology and Hematology Unit and 3Department of Pathology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.

PMID: 22976797

 

Abstract

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 analyzed 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 N-Cadherin- and a9-Integrin-blocking antibodies. Cells treated with g-secretase inhibitor showed lower adhesion capability and down-regulation of N-Cadherin and a9-Integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-Cadherin and a9-Integrin. Treatment with anti-N-Cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti-a9-Integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness. N-Cadherin and a9-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.

Keywords: Rhabdomyosarcoma, Notch, NCAD, ITGA9, invasion, soft tissue sarcomas.

 

Supplement:

RMS patients with metastatic disease continue to have poor prognosis and the major cause of death in these patients is the formation of distant metastases. The identification of molecular components and mechanisms that control metastasis in RMS may be useful for developing targeted therapies focused on reducing metastasis in this neoplasia.

Although the mechanism by which increased N-Cadherin expression promotes malignancy is not fully understood, it is thought to promote adhesion of malignant cells to N-Cadherin-expressing stromal or endothelial cells, thereby facilitating invasion and migration of tumor cells to distant sites. Although several works have demonstrated that N-Cadherin expression suffices to promote metastasis in some cancers and an aberrant N-Cadherin expression has been described in RMS, no previous publications have shown the importance of N-Cadherin for RMS invasiveness. Herein, a central role of N-Cadherin in RMS cell adhesion and motility is demonstrated; furthermore, a new mechanism of N-Cadherin induction, mediated by Notch pathway activation, is also proposed. Likewise, a9-Integrin – forming a heterodimer with b1-Integrin – has previously been implicated in processes, such as cell migration, involving dynamic interaction among cells and their microenvironment. However, its possible role in RMS remained unknown. Therefore, this work represents the first description of a pro-invasive role of a9-Integrin in RMS.

The use of pharmacologic Notch pathway inhibitors, such as g-secretase inhibitors (GSIs), has been proposed as a therapeutic alternative in several cancers, focused essentially on reducing cell proliferation. Several GSIs have been actively studied as potential inhibitors of the generation of the b-amyloid peptide associated with Alzheimer’s disease (Selkoe D et al, 2003). More recently, some GSIs have begun to be studied in phase I-II trials for patients with advanced breast cancer and acute T-cell leukemias (Krop IE et al, 2006; Deangelo DJ et al, 2006). Beyond the use of GSIs as anti-proliferation agents, the results of our experiments point to GSIs as compounds able to reduce the invasive features of tumor cells and, therefore, suitable for use in the fight against metastasis.

Taken together, the results of this work reveal that the Notch pathway plays a significant role in the regulation of N-Cadherin and a9-Integrin expression in RMS and, therefore, the Notch pathway should be considered a key regulator of the mechanisms, such as cell motility and invasiveness, which lead to tumor progression. Hence, Notch-inhibiting molecules can be considered as possible therapeutic agents against metastasis. Furthermore, the importance of N-Cadherin and a9-Integrin as players directly involved in cell attachment, migration and invasiveness in RMS is also demonstrated, consequently pointing to these proteins as new candidates for target-specific therapies focused on reducing metastasis in this neoplasia.

 Josep Roma Castanyer-1

Acknowledgments: 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.

 

Contact: Soledad Gallego, Pediatric Oncology and Hematology Unit, Hospital Vall d’Hebron, Passeig Vall d’Hebron 119, 08035 Barcelona, Spain; Phone: +34934893090; Fax +34934893089, E-mail: sgallego@vhebron.net; or Josep Roma, Laboratory of Translational Research in Pediatric Cancer, Vall d’Hebron Research Institute (VHIR), Spain; Phone: +34934893000 ext 4936; Fax +34932746708, E-mail: josep.roma@vhir.org

 Josep Roma Castanyer-3

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