ABCB1-dependent and independent mechanism of chemoresistance in human osteosarcoma: identification of new chemosensitizing strategies

ABC transporter B1/P-glycoprotein (ABCB1/Pgp) is a known factor of chemoresistance in osteosarcoma, because it effluxes several first-line drugs including doxorubicin. ABCB1 alone, however, does not fully explain chemoresistance. Finding new molecular mechanisms of chemoresistance and new chemosensitizing approaches are challenges still open. In the first part of this thesis, we analyzed 2D- and 3D-cultured doxorubicin-sensitive human osteosarcoma U2OS cells and the sublines with increasing resistance to doxorubicin (U-2OS/DX30, U-2OS/DX100, U-2OS/DX580). We found that in DX-sublines and in 3D-cultures ABCB1 is up-regulated by Ras/ERK1/2/HIF-1α axis. By contrast, ABC transporter A1 (ABCA1), which effluxes isopentenyl pyrophosphate, a lipidic activator of the anti-tumor population Vγ9Vδ2 T-cells, is negatively regulated by Ras/Akt/mTOR axis and is decreased when ABCB1 is high. Nanoparticles carrying the aminobisphosphonate zoledronic acid inhibit Ras-dependent pathways, decreasing ABCB1 and increasing ABCA1. This approach restores doxorubicin efficacy and immune-sensitivity in resistant osteosarcoma cells. In the second part, we performed a whole-genome expression profile of doxorubicin-sensitive and resistant osteosarcoma cells, to identify new molecular circuitries determining chemoresistance. Surprisingly, a huge number of small nucleolar RNAs (snoRNAs) are significantly up-regulated in resistant cells. SNORD3A, SNORA13 and SNORA28 induce doxorubicin resistance in an ABCB1-independent way, when over-expressed in sensitive cells. These snoRNAs up-regulate GADD45A and c-MYC, down-regulate TOP2A: these changes protect from doxorubicin-induced cytotoxicity. Indeed, GADD45A and c-MYC silencing, or TOP2A over-expression restore sensitivity to doxorubicin in resistant cells. This thesis identifies one ABCB1-dependent and one ABCB1-independent mechanism of resistance to doxorubicin in osteosarcoma and proposes new pharmacological or gene-targeting approaches to achieve chemosensitization.