The ATP Binding Cassette transporter B1 (ABCB1) induces chemoresistance in osteosarcoma, because it effluxes doxorubicin, reducing the intracellular accumulation, toxicity, and immunogenic cell death induced by the drug. The ATP Binding Cassette transporter A1 (ABCA1) effluxes isopentenyl pyrophosphate (IPP), a strong activator of anti-tumor Vγ9Vδ2 T-cells. Recruiting this population may represent an alternative strategy to rescue doxorubicin efficacy in ABCB1-expressing osteosarcoma. In this work, we analyzed how ABCA1 and ABCB1 are regulated in osteosarcoma, and if increasing the ABCA1-dependent activation of Vγ9Vδ2 T-cells could be an effective strategy against ABCB1-expressing osteosarcoma. We used 2D-cultured doxorubicin-sensitive human U-2OS and Saos-2 cells, their doxorubicin-resistant sublines (U-2OS/DX580 and Saos-2/DX580), and 3D cultures of U-2OS and Saos-2 cells. DX580-sublines and 3D cultures had higher levels of ABCB1 and higher resistance to doxorubicin than parental cells. Surprisingly, they had reduced ABCA1 levels, IPP efflux, and Vγ9Vδ2 T-cell-induced killing. In these chemo-immune-resistant cells, the Ras/Akt/mTOR axis inhibits the ABCA1-transcription induced by Liver X Receptor α (LXRα); Ras/ERK1/2/HIF-1α axis up-regulates ABCB1. Targeting the farnesylation of Ras with self-assembling nanoparticles encapsulating zoledronic acid (NZ) simultaneously inhibited both axes. In humanized mice, NZ reduced the growth of chemo-immune-resistant osteosarcomas, increased intratumor necro-apoptosis, and ABCA1/ABCB1 ratio and Vγ9Vδ2 T-cell infiltration. We suggest that the ABCB1^highABCA1^low phenotype is indicative of chemo-immune-resistance. We propose aminobisphosphonates as new chemo-immune-sensitizing tools against drug-resistant osteosarcomas.

Doxorubicin is a strong inducer of immunogenic cell death (ICD), but it is ineffective in P-glycoprotein (Pgp)-expressing cells. Indeed, Pgp effluxes doxorubicin and impairs the immunesensitizing functions of calreticulin (CRT), an “eat-me” signal mediating ICD. It is unknown if classical Pgp inhibitors, designed to reverse chemoresistance, may restore ICD. We addressed this question by using Pgp-expressing cancer cells, treated with Tariquidar, a clinically approved Pgp inhibitor, and R-3 compound, a N,N-bis(alkanol)amine aryl ester derivative with the same potency of Tariquidar as Pgp inhibitor. In Pgp-expressing/doxorubicin-resistant cells, Tariquidar and R-3 increased doxorubicin accumulation and toxicity, reduced Pgp activity, and increased CRT translocation and ATP and HMGB1 release. Unexpectedly, only R-3 promoted phagocytosis by dendritic cells and activation of antitumor CD8⁺T-lymphocytes. Although Tariquidar did not alter the amount of Pgp present on cell surface, R-3 promoted Pgp internalization and ubiquitination, disrupting its interaction with CRT. Pgp knock-out restores doxorubicin-induced ICD in MDA-MB-231/DX cells that recapitulated the phenotype of R-3-treated cells. Our work demonstrates that plasma membrane-associated Pgp prevents a complete ICD notwithstanding the release of ATP and HMGB1, and the exposure of CRT. Pharmacological compounds reducing Pgp activity and amount may act as promising chemo- and immunesensitizing agents.

Doxorubicin (Dox) is one of the most important first-line drugs used in osteosarcoma therapy. Multiple and not fully clarified mechanisms, however, determine resistance to Dox. With the aim of identifying new markers associated with Dox-resistance, we found a global up-regulation of small nucleolar RNAs (snoRNAs) in human Dox-resistant osteosarcoma cells. We investigated if and how snoRNAs are linked to resistance. After RT-PCR validation of snoRNAs up-regulated in osteosarcoma cells with different degrees of resistance to Dox, we overexpressed them in Dox-sensitive cells. We then evaluated Dox cytotoxicity and changes in genes relevant for osteosarcoma pathogenesis by PCR arrays. SNORD3A, SNORA13 and SNORA28 reduced Dox-cytotoxicity when over-expressed in Dox-sensitive cells. In these cells, GADD45A and MYC were up-regulated, TOP2A was down-regulated. The same profile was detected in cells with acquired resistance to Dox. GADD45A/MYC-silencing and TOP2A-over-expression counteracted the resistance to Dox induced by snoRNAs. We reported for the first time that snoRNAs induce resistance to Dox in human osteosarcoma, by modulating the expression of genes involved in DNA damaging sensing, DNA repair, ribosome biogenesis, and proliferation. Targeting snoRNAs or down-stream genes may open new treatment perspectives in chemoresistant osteosarcomas.

Drug resistance is the main obstacle for a successful cancer therapy. There are many mechanisms by which cancers avoid drug-mediated death, including alterations in cellular metabolism and apoptotic programs. Mitochondria represent the cell’s powerhouse and the connection between carbohydrate, lipid and proteins metabolism, as well as crucial controllers of apoptosis, playing an important role not only in tumor growth and progression, but also in drug response. Alterations in tricarboxylic acid cycle (TCA) caused by mutations in three TCA enzymes—isocitrate dehydrogenase, succinate dehydrogenase and fumarate hydratase—lead to the accumulation of 2-hydroxyglutarate, succinate and fumarate respectively, collectively known as oncometabolites. Oncometabolites have pleiotropic effects on cancer biology. For instance, they generate a pseudohypoxic phenotype and induce epigenetic changes, two factors that may promote cancer drug resistance leading to disease progression and poor therapy outcome. This review sums up the most recent findings about the role of TCA-derived oncometabolites in cancer aggressiveness and drug resistance, highlighting possible pharmacological strategies targeting oncometabolites production in order to improve the efficacy of cancer treatment.