ECTS Abstracts (2015) 1 P134

Calpain-6 expression identifies a stem cell population in osteosarcoma

Caroline Andrique2, Laetitia Morardet2, Claire-Sophie Devignes2, Sylvain Provot1 & Dominique Modrowski1

1INSERM U1132, Paris, France; 2Paris 7 University, Paris, France.

Identification of cancer stem cells in carcinomas has proved to be useful to understand cancer progression and for prognostic purpose. However, the properties of osteosarcoma stem cells remain challenging and controversial, mainly due to the lack of functional markers to study such cells in vivo. Previously, we identified calpain-6 as a protective factor involved in chemoresistance process of osteosarcoma. To investigate the mechanisms controlling its expression we characterised 7285 bases of the regulatory sequence in calpain-6 gene. This sequence comprises an active promoter and multiple functional binding sites for embryonic stem cell factors such as Oct4, Nanog and Sox2 as shown by rapid cDNA end amplification and chromatin precipitation. Silencing Oct4, Nanog or Sox2 was sufficient to reduce basal and hypoxia dependent up-regulation of calpain-6 expression and the activity of the regulatory sequence cloned upstream the luciferase gene reporter. This indicates that calpain-6 is controlled by the stem cell transcription factors. To further document a possible relationship between Calpain-6 and a stem cell phenotype, we used GFP as gene reporter, to identify the cells in which the calpain-6 promoter was activated. Culturing osteosarcoma cell lines on non-adherent plastic and in minimal medium allowed obtaining spheroids that were previously shown to be enriched in tumourigenic stem-like cells. Calpain-6 protein was up regulated in spheres obtained from human 143B cells as compared with adherent cultures. Moreover, GFP positive cells sorted from adherent cultures have higher capacities to form spheroids than GFP negative cells. These GFP positive cells also expressed higher RNA levels of the embryonic stem cell markers, c-MYC and ABCB1. Five weeks after injection into the tibia of BALB/c mice, GFP-positive K7M2 cells formed tumours that produced a high luminescent signal as compared with tumours formed from GFP-negative cells that are largely necrotic. In in vitro scratch tests, migrating cells were found to express high levels of calpain-6 and GFP-positive cells displayed higher capacities for migration than negative ones, whereas, calpain-6 shRNA reduced these capacities. Finally, intra bone injection of GFP-positive cells resulted in more metastatic lesions in lungs than negative cells indicating that calpain-6 is involved in metastatic process. Altogether, our data show that calpain-6 expression is regulated by transcription factors that control multipotency and renewal of embryonic stem cells. Calpain-6 identifies an osteosarcoma cell population that express stem markers and with higher chemoresistance, migration capacities and tumourigenicity. The reporter system driven by calpain-6 regulatory sequence may therefore represent a powerful tool to further study stem cells in osteosarcoma.

Disclosure: The authors declared no competing interests. This work was supported by Société française du cancer de l’enfant.

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