Osteopontin/SPP1 is overexpressed in glioblastoma and through distinct domains participates in glioma stem cell renewal and glioma-microenvironment interactions
K. Stepniak, M. Kocyk, M. Maleszewska, A. Gieryng, P. Przanowski, B. Wojtas, P. Nauman2, K. Kotulska, W. Grajkowska, B. Kaminska
Osteopontin/SPP1 (a secreted phosphoprotein1) is involved in immune responses, bone/tissue remodeling and biomineralization. SPP1 is overexpressed in many cancers, including glioblastoma (GBM) and contributes to tumor progression by regulating migration, invasion and cancer stem cell self-renewal. There are five SPP1 splice variants generated by alternative splicing and posttranslational protein modifications. We found that the expression of specific SPP1 isoforms is upregulated in resected GBMs and GBM cell lines, correlates with poor prognosis in GBM patients. Tumor-derived SPP1 interacts with cell surface receptors of microglia, myeloid cells residing in the central nervous system that infiltrate and support glioma invasion. Glioma-derived SPP1 induces a pro-tumoral, M2 activation. Knockdown of Spp1 in C6 glioma cells with lentivirally delivered shRNA reduced the number of tumor spheres and diminished expression of M2 markers in primary microglial cultures. To assess the role of functional domains of Spp1 in glioma stem cell self-renewal and glioma-microglia interactions, shRNA resistant constructs coding for a wild type and mutated Spp1 were created. A rescue experiment with Spp1 variants lacking C-terminal, CD44 binding domain or with point mutations in specific functional domains was performed. Our results show that the CD44 binding domain of Spp1 is necessary for a sphere forming activity, while mutations in a thrombin cleavage site and a integrin binding site disturb interactions with microglia. Altogether, we demonstrate that tumor-derived Spp1 supports glioma stem cell self-renewal and shapes glioma microenvironment.
Studies supported by a 2012/04/A/NZ3/00630 grant from the National Science Center.