OXPHOS inhibitors, metabolism and targeted therapies in cancer.

More and more studies highlight the complex metabolic characteristics and plasticity of cancer cells. To address these specificities and explore the associated vulnerabilities, new metabolism-targeting therapeutic strategies are being developed. It is more and more accepted that cancer cells do not produce their energy only from aerobic glycolysis, as some subtypes strongly rely on mitochondrial respiration (OXPHOS). This review focuses on classical and promising OXPHOS inhibitors (OXPHOSi), unravelling their interest and modes of actions in cancer, particularly in combination with other strategies. Indeed, in monotherapy, OXPHOSi display limited efficiency as they mostly trigger cell death in cancer cell subtypes that strongly depend on mitochondrial respiration and are not able to shift to other metabolic pathways to produce energy. Nevertheless, they remain very interesting in combination with conventional therapeutic strategies such as chemotherapy and radiotherapy, increasing their anti-tumoral actions. In addition, OXPHOSi can be included in even more innovative strategies such as combinations with other metabolic drugs or immunotherapies.

Discovery of Mitochondrial Complex I Inhibitors as Anticancer and Radiosensitizer Drugs Based on Compensatory Stimulation of Lactate Release.

Cancer cells may stimulate glycolytic flux when O2 becomes insufficient. Increase in L-lactate release therefore appears as an escape mechanism to drugs targeting mitochondrial respiration but also represents a response that may be exploited to screen for compounds blocking either mitochondrial carriers of oxidizable substrates or the electron transport chain. Here, we developed a screening procedure based on the capacity of cancer cells to release L-lactate to gain insights on the development of mitochondrial complex I inhibitors. For this purpose, we synthesized derivatives of carboxyamidotriazole, a compound previously described as a potential OXPHOS inhibitor. Two series of derivatives were generated by cycloaddition between benzylazide and either cyanoacetamides or alkynes. A primary assay measuring L-lactate release as a compensatory mechanism upon OXPHOS inhibition led us to identify 15 hits among 28 derivatives. A secondary assay measuring O2 consumption in permeabilized cancer cells confirmed that 12 compounds among the hits exhibited reversible complex I inhibitory activity. Anticancer effects of a short list of 5 compounds identified to induce more L-lactate release than reference compound were then evaluated on cancer cells and tumor-mimicking 3D spheroids. Human and mouse cancer cell monolayers exhibiting high level of respiration in basal conditions were up to 3-fold more sensitive than less oxidative cancer cells. 3D tumor spheroids further revealed potency differences between selected compounds in terms of cytotoxicity but also radiosensitizing activity resulting from local reoxygenation. In conclusion, this study documents the feasibility to efficiently screen in 96-well plate format for mitochondrial complex I inhibitors based on the capacity of drug candidates to induce L-lactate release.

Transcriptional and Metabolic Investigation in 5'-Nucleotidase Deficient Cancer Cell Lines.

Enzymes of nucleoside and nucleotide metabolism regulate important cellular processes with potential impacts on nucleotide-unrelated parameters. We have used a set of CRISPR/Cas9-modified cell models expressing both, one, or none of the 5'-nucleotidases cN-II and CD73, together with RNA sequencing and targeted metabolomics, to decipher new regulatory roles of these proteins. We observed important transcriptional modifications between models as well as upon exposure to adenosine. Metabolite content varied differently between cell models in response to adenosine exposure but was rather similar in control conditions. Our original cell models allowed us to identify a new unobvious link between proteins in the nucleotide metabolism and other cellular pathways. Further analyses of our models, including additional experiments, could help us to better understand some of the roles played by these enzymes.

CD73 and cN-II regulate the cellular response to chemotherapeutic and hypoxic stress in lung adenocarcinoma cells.

BACKGROUND: Cytosolic 5'-nucleotidase II (cN-II) and ecto-5'-nucleotidase (CD73) are enzymes involved in the nucleotide metabolism by dephosphorylating nucleoside monophosphates. Both enzymes are involved in cancer by modifying anticancer drug activity, cancer cell biology and immune modulation. METHODS: We have modified lung cancer cells (NCI-H292) to become deficient for either or both enzymes using the CRISPR/Cas9 technique, and studied the implication of the two enzymes in the cellular response to different stress condition i.e. chemotherapeutic agents, hypoxia and nucleotide stress. RESULTS: Our results show that there is no significant role of these enzymes in cell proliferation under hypoxic stress. Similarly, cN-II and CD73 are not involved in wound healing ability under CoCl2-mediated HIF-1α stabilization. Furthermore, our results show that CD73-deficiency is associated with increased apoptosis in response to 1600 µM adenosine, decreased sensitivity to mitomycin and enhanced sensitivity to vincristine. cN-II deficiency increased in vivo tumor growth and sensitivity to vincristine and mitomycin C. CONCLUSIONS: Our study gives new insights into the biological roles of cN-II and CD73 under stress conditions in this particular cancer cell line. Further experiments will help deciphering the molecular mechanisms underlying the observed differences.

Enhanced migration of breast and lung cancer cells deficient for cN-II and CD73 via COX-2/PGE2/AKT axis regulation.

PURPOSE: Purine metabolism involves various intracellular and extracellular enzymes, including cN-II and CD73 that dephosphorylate intracellular and extracellular nucleoside monophosphates into their corresponding nucleosides. We conducted a study to better understand the biological roles of these enzymes in breast and lung cancer cells. METHODS: We modified cN-II and/or CD73 expression in human breast cancer cells (MDA-MB-231), human lung cancer cells (NCI-H292) and murine breast cancer cells (4T1) using the CRISPR/Cas9 technique, and evaluated their impact on various cellular parameters such as proliferation, migration, invasion, intracellular nucleotide pools and nucleotide metabolism-related gene expression under extracellular nucleotide stress conditions. RESULTS: Intracellular nucleotide contents were found to be altered in the modified cancer cell models both at their basal levels and after exposure to adenosine or AMP. Altered cN-II and CD73 levels were also found to be associated with cell migration and invasion alterations, involving TIMP-2, MMP-2 and MMP-9 expression, as well as alterations in the COX-2/PGE2/AKT pathway. CONCLUSION: Our results highlight new cell-specific roles of cN-II and CD73 in cancer cell biology and provide insight into their interactions with different intracellular pathways.

Regulation of tumor infiltrated innate immune cells by adenosine.

Cancer has the ability to escape the immune system using different molecular actors. Adenosine is known to be involved in mechanisms which control inflammatory reactions and prevent excessive immune response. This purine nucleoside can be translocated from the cell or produced in the extracellular space by 5'-ectonucleotidases. Once bound to its receptors on the surface of immune effector cells, adenosine activates various molecular pathways, which lead to functional inhibition of the cell or its death. Some tumors are infiltrated by the different cells of immune system but are able to use adenosine as an immunosuppressive molecule and thus inhibit immune anticancer response. This mechanism is well described on adaptive cells, but much less on innate cells. This review outlines major effects of adenosine on innate immune cells, its consequences on cancer progression, and possible ways to block the adenosine-dependent immunosuppressive effect.

The cytosolic 5'-nucleotidase cN-II lowers the adaptability to glucose deprivation in human breast cancer cells.

The cytosolic 5'-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence.

Haploinsufficiency networks identify targetable patterns of allelic deficiency in low mutation ovarian cancer.

Identification of specific oncogenic gene changes has enabled the modern generation of targeted cancer therapeutics. In high-grade serous ovarian cancer (OV), the bulk of genetic changes is not somatic point mutations, but rather somatic copy-number alterations (SCNAs). The impact of SCNAs on tumour biology remains poorly understood. Here we build haploinsufficiency network analyses to identify which SCNA patterns are most disruptive in OV. Of all KEGG pathways (N=187), autophagy is the most significantly disrupted by coincident gene deletions. Compared with 20 other cancer types, OV is most severely disrupted in autophagy and in compensatory proteostasis pathways. Network analysis prioritizes MAP1LC3B (LC3) and BECN1 as most impactful. Knockdown of LC3 and BECN1 expression confers sensitivity to cells undergoing autophagic stress independent of platinum resistance status. The results support the use of pathway network tools to evaluate how the copy-number landscape of a tumour may guide therapy.