Strategies to Overcome Resistance to Immune-Based Therapies in Osteosarcoma.

Improving the prognosis and cure rate of HGOSs (high-grade osteosarcomas) is an absolute need. Immune-based treatment approaches have been increasingly taken into consideration, in particular for metastatic, relapsed and refractory HGOS patients, to ameliorate the clinical results currently achieved. This review is intended to give an overview on the immunotherapeutic treatments targeting, counteracting or exploiting the different immune cell compartments that are present in the HGOS tumor microenvironment. The principle at the basis of these strategies and the possible mechanisms that HGOS cells may use to escape these treatments are presented and discussed. Finally, a list of the currently ongoing immune-based trials in HGOS is provided, together with the results that have been obtained in recently completed clinical studies. The different strategies that are presently under investigation, which are generally aimed at abrogating the immune evasion of HGOS cells, will hopefully help to indicate new treatment protocols, leading to an improvement in the prognosis of patients with this tumor.

Electronic Coupling in 1,2,3-Triazole Bridged Ferrocenes and Its Impact on Reactive Oxygen Species Generation and Deleterious Activity in Cancer Cells.

Mixed-valence (MV) binuclear ferrocenyl compounds have long been studied as models for testing theories of electron transfer and in attempts to design molecular-scale electronic devices (e.g., molecular wires). In contrary to that, far less attention has been paid to MV binuclear ferrocenes as anticancer agents. Herein, we discuss the synthesis of six 1,2,3-triazole ferrocenyl compounds for combined (spectro)electrochemical, electron paramagnetic resonance (EPR), computational, and anticancer activity studies. Our synthetic approach was based on the copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction and enabled us to obtain in one step compounds bearing either one, two, or three ferrocenyl entities linked to the common 1,2,3-triazole core. Thus, two series of complexes were obtained, which pertain to derivatives of 3'-azido-3'-deoxythymidine (AZT) and 3-azidopropionylferrocene, respectively. Based on the experimental and theoretical data, the two mono-oxidized species corresponding to binuclear AZT and trinuclear 3-azidopropionylferrocene complexes have been categorized as class II mixed-valence according to the classification proposed by Robin and Day. Of importance is the observation that these two compounds are more active against human A549 and H1975 non-small-cell lung cancer cells than their congeners, which do not show MV characteristics. Moreover, the anticancer activity of MV species competes or surpasses, dependent on the cell line, the activity of reference anticancer drugs such as cisplatin, tamoxifen, and 5-fluorouracil. The most active from the entire series of compounds was the binuclear thymidine derivative with the lowest IC50 value of 5 ± 2 µM against lung H1975 cancer cells. The major mechanism of antiproliferative activity for the investigated MV compounds is based on reactive oxygen species generation in cancer cells. This hypothesis was substantiated by EPR spin-trapping experiments and the observation of decreased anticancer activity in the presence of N-acetyl cysteine (NAC) free-radical scavenger.

Click ferrocenyl-erlotinib conjugates active against erlotinib-resistant non-small cell lung cancer cells in vitro.

Thanks to development of erlotinib and other target therapy drugs the lung cancer treatment have improved a lot in recent years. However, erlotinib-resistant lung cancer remains an unsolved clinical problem which demands for new therapeutics to be developed. Herein we report the synthesis of a library of 1,4- and 1,5-triazole ferrocenyl derivatives of erlotinib together with their anticancer activity studies against erlotinib-sensitive A549 and H1395 as well as erlotinib-resistant H1650 and H1975 cells. Studies showed that extend of anticancer activity is mainly related to the length of the spacer between the triazole and the ferrocenyl entity. Among the series of investigated compounds two isomers commonly bearing C(O)CH2CH2 spacer have shown superior to erlotinib activity against erlotinib-resistant H1650 and H1975 cells whereas compound with short methylene spacer devoid of any activity. In-depth biological studies for the most active compound showed differences in its mechanism of action in compare to erlotinib. The latter is known EGFR inhibitor whereas their ferrocenyl congener exerts anticancer activity mainly as ROS-inducer which activates mitochondrial pathway of apoptosis in cancer cells. However, docking studies suggested that the most active compound can also binds to the active site of EGFR TK in a similar way as erlotinib.

Small Nucleolar RNAs Determine Resistance to Doxorubicin in Human Osteosarcoma.

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.

Mitochondrial ROS drive resistance to chemotherapy and immune-killing in hypoxic non-small cell lung cancer.

BACKGROUND: Solid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 T-lymphocytes. The molecular circuitries determining this double resistance are not known. METHODS: We analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-ß (C/EBP-ß) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34+NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy. RESULTS: By up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced O2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-ß mRNA mediated by HIF-1α. HIF-1α/C/EBP-ß mRNA binding increases the splicing of C/EBP-ß toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-ß LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34+NSG mice recapitulated the patients' chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1α/LAP cascade. CONCLUSIONS: The impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-ß LAP as a new predictive and prognostic factor in NSCLC patients.

Targeting Mitochondrial Oncometabolites: A New Approach to Overcome Drug Resistance in Cancer.

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.

Cholesterol metabolism: At the cross road between cancer cells and immune environment.

Mevalonate pathway is a highly conserved pathway that produces isoprenoids and cholesterol, and it is often increased in cancer cells. Cholesterol, upstream metabolites including isoprenoids and cholesterol derivatives such as oxysterols modulate cell proliferation, motility, stemness and drug resistance. Moreover, when produced by cancer cells or immune infiltrating cells, they modulate the activity of immune populations of the tumor microenvironment. In this review, we will focus on the recent findings demonstrating that cholesterol derivatives may regulate tumor immune recognition or immune escape, playing a critical role in the immune surveillance. Since the mevalonate pathway is druggable, a deeper knowledge of the metabolic cross talks existing between the mevalonate pathway of cancer cells and immune cells may help to identify novel agents targeting cholesterol metabolism, able to boost the anti-tumor activity of the immune populations.

Insights into P-Glycoprotein Inhibitors: New Inducers of Immunogenic Cell Death.

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.

ABCA1/ABCB1 Ratio Determines Chemo- and Immune-Sensitivity in Human Osteosarcoma.

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 ABCB1highABCA1low phenotype is indicative of chemo-immune-resistance. We propose aminobisphosphonates as new chemo-immune-sensitizing tools against drug-resistant osteosarcomas.