Abstract 3774: Regulation of the pro-invasive properties of neuroblastoma-stem cells by membrane type-1 matrix metalloproteinase.
Authors: Belounis Assila , Nyalendo Carine , Cournoyer Sonia , Hadj-Mimoune Sarah , Benoit Alexandre , Lasalle Elliot , Girard Jonathan , Beaunoyer Mona , Teira Pierre , Sartelet Hervé 
Published: August 2014

Neuroblastoma (NB) is an aggressive childhood cancer that represents the leading cause of cancer deaths in children. Despite aggressive therapy, more than half of the children with advanced NB usually die because of uncontrolled metastatic disease. In order to develop new therapeutic strategies to limit NB's metastatic potential, it is crucial to identify key molecular targets governing the invasive process. The study of Cancer stem cells (CSCs) may be of interest in this regard because after chemotherapy, CSCs persist in tumors and cause relapse and metastasis. We had recently demonstrated that CD133 allowed to detect CSCs in NB. Membrane type-1 matrix metalloproteinase (MT1-MMP) is important in the metastatic process and its expression was correlated with unfavourable outcome in NBs. Our preliminary data had indicated that MT1-MMP was highly expressed in CD133high NB.

The purpose of this study is to characterize the interaction between CD133 and MT1-MMP in NB and determinate the role of MT1-MMP in pro-invasive properties of CSCs. We constructed paraffin-embedded blocks of tissue microarrays (TMA) from 235 patients. In vitro experiments were performed on four established NB cell lines (SK-N-DZ, SK-N-FI and SK-N-SH and SJNB-10). We performed immunohistochemical studies on paraffin-embedded TMA sections with two antibodies (CD133, MT1-MMP). To verify correlation of expression of MT1-MMP and CD133, we realized western blot and immunofluorescence (IF) of both proteins in non treated versus treated NB cell lines. CD133high NB cells were isolated by flow cytometry. CD133high and CD133low NB cells were grown within a 3D collagen matrix and the cell migration assay was tested in collagen-coated transwells. All experiments were performed with or without an anti-MT1-MMP neutralizing antibody or GM6001, a broad-spectrum MMP inhibitor.

To assess the interaction between CD133 and MT1-MMP, lysates were subjected to immunoprecipitation (IP) using an anti-CD133 antibody, followed by immunodetection with an anti-MT1-MMP antibody. Finally, we determined which domain of MT1-MMP is involved in its interaction with CD133. Different dominant negative mutants of MT1-MMP (catalytically inactive E240A, cytoplasmic domain-deleted CΔ20 and non-phosphorylatable Y573F) were transfected into NB cells followed by an IP.

There is a correlation between MT1-MMP and CD133 expressions in tumors of patients and in cell lines. Cells selected after chemotherapy express both CD133 and MT1-MMP. CD133high cells presented higher migration and invasion properties than CD133low which were MT1-MMP dependent. IF and IP showed a colocalization and interaction between CD133 and MT1-MMP. The cytoplasmic domain of MT1-MMP seems to be responsible for the interaction with CD133.

These results contribute to a better understanding CSCs properties in NB and may be of great interest to improve new therapeutic strategies.

Citation Format: Assila Belounis, Carine Nyalendo, Sonia Cournoyer, Sarah Hadj-Mimoune, Alexandre Benoit, Elliot Lasalle, Jonathan Girard, Mona Beaunoyer, Pierre Teira, Hervé Sartelet. Regulation of the pro-invasive properties of neuroblastoma-stem cells by membrane type-1 matrix metalloproteinase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2013-3774