Recurrence of glioblastoma after radio-chemotherapy is associated with an angiogenic switch to the CXCL12-CXCR4 pathway Recurrence of glioblastoma after radio-chemotherapy is associated with an angiogenic switch to the CXCL12-CXCR4 pathway

Glioblastomas (GBMs) are the most aggressive primary brain tumors in adult and remain a therapeutic challenge. Targeting key apoptosis regulators with the ultimate aim to restore apoptosis in tumor cells could be an interesting therapeutic strategy. The inhibitors of apoptosis proteins (IAPs) are regulators of cell death and represent attractive targets, especially because they can be antagonized by SMAC mimetics. In this study, we first investigated the expression of cIAP1, cIAP2, XIAP and ML-IAP in human GBM samples and in four different cell lines. We showed that all GBM samples and GBM cell lines expressed all these IAPs, although the expression of each IAP varied from one case to another. We then showed that high level of ML-IAP predicted worse progression-free survival and overall survival in both univariate and multivariate analyses in two independent cohorts of 58 and 43 primary human GBMs. We then used GDC-0152, a SMAC mimetic that antagonizes these IAPs and confirmed that GDC-0152 treatment in vitro decreased IAPs in all the cell lines studied. It affected cell line viability and triggered apoptosis, although the effect was higher in U87MG and GL261 than in GBM6 and GBM9 cell lines. In vivo, GDC-0152 effect on U87MG orthotopic xenografts was dose dependent; it postponed tumor formation and slowed down tumor growth, significantly improving survival of GBM-bearing mice. This study revealed for the first time that ML-IAP protein expression correlates with GBM patient survival and that its antagonist GDC-0152 improves outcome in xenografted mouse.

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo

Glioblastoma (GBM) is characterized by highly aggressive growth and invasive behavior. Due to the highly lethal nature of GBM, new therapies are urgently needed and repositioning of existing drugs is a promising approach. We have previously shown the activity of Proscillaridin A (ProA), a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase (NKA) pump, against proliferation and migration of GBM cell lines. ProA inhibited tumor growth in vivo and increased mice survival after orthotopic grafting of GBM cells. This study aims to decipher the mechanism of action of ProA in GBM tumor and stem-like cells. ProA displayed cytotoxic activity on tumor and stem-like cells grown in 2D and 3D culture, but not on healthy cells as astrocytes or oligodendrocytes. Even at sub-cytotoxic concentration, ProA impaired cell migration and disturbed EB1 accumulation at microtubule (MT) plus-ends and MT dynamics instability. ProA activates GSK3β downstream of NKA inhibition, leading to EB1 phosphorylation on S155 and T166, EB1 comet length shortening and MT dynamics alteration, and finally inhibition of cell migration and cytotoxicity. Similar results were observed with digoxin. Therefore, we disclosed here a novel pathway by which ProA and digoxin modulate MT-governed functions in GBM tumor and stem-like cells. Altogether, our results support ProA and digoxin as potent candidates for drug repositioning in GBM.

A2B5+ cells isolated from human glioblastomas exhibit cancer stem cell properties. The A2B5 epitope belongs to the sialoganglioside family and is synthetized by the ST8 alpha-N-acetyl-neuraminidase α-2,8-sialyltransferase 3 (ST8SIA3) enzyme. Glycolipids represent attractive targets for solid tumors; therefore, the aim of this study was to decipher A2B5 function in glioblastomas. To this end, we developed cell lines expressing various levels of A2B5 either by genetically manipulating ST8SIA3 or by using neuraminidase. The overexpression of ST8SIA3 in low-A2B5-expressing cells resulted in a dramatic increase of A2B5 immunoreactivity. ST8SIA3 overexpression increased cell proliferation, migration, and clonogenicity in vitro and tumor growth when cells were intracranially grafted. Conversely, lentiviral ST8SIA3 inactivation in low-A2B5-expressing cells resulted in reduced proliferation, migration, and clonogenicity in vitro and extended mouse survival. Furthermore, in the shST8SIA3 cells, we found an active apoptotic phenotype. In high-A2B5-expressing cancer stem cells, lentiviral delivery of shST8SIA3 stopped cell growth. Neuraminidase treatment, which modifies the A2B5 epitope, impaired cell survival, proliferation, self-renewal, and migration. Our findings prove the crucial role of the A2B5 epitope in the promotion of proliferation, migration, clonogenicity, and tumorigenesis, pointing at A2B5 as an attractive therapeutic target for glioblastomas.