Farnesyl diphosphate synthase is important for the maintenance of glioblastoma stemness.

Glioblastoma is a highly malignant tumor that easily acquires resistance to treatment. The stem-cell-like character (stemness) has been thought to be closely associated with the treatment resistance of glioblastoma cells. In this study, we determined that farnesyl diphosphate synthase (FDPS), a key enzyme in isoprenoid biosynthesis, plays an important role in maintaining glioblastoma stemness. A comparison of the mRNA expression in patient-derived glioblastoma sphere cells, which maintain stemness, and their differentiated counterparts, which lose stemness, via RNA sequencing showed that most of the altered genes were networked in the cholesterol biosynthesis pathway. We screened Federal Drug Administration (FDA)-approved drugs targeting specific enzymes in the cholesterol biosynthesis pathway for their ability to inhibit glioblastoma sphere formation. Inhibitors of FDPS, such as alendronate and zoledronate, significantly reduced the formation of glioblastoma spheres, and alendronate was effective at a lower molar concentration than zoledronate. Knockdown of FDPS using short hairpin RNA also completely inhibited the formation of secondary spheres. FDPS mRNA in patients with glioblastoma was associated with malignancy in three independent microarray data sets. RNA sequencing showed that alendronate treatment reduced the embryonic stem cell signature and activated development- and necrosis-related pathways in glioblastoma spheres. These results suggest that FDPS is important for the maintenance of glioblastoma stemness and that alendronate, a drug widely used to treat osteoporosis, can be repositioned to treat glioblastoma.ope

Identification of the molecular pathways mediating the anti-AML activity of statins

La leucémie myéloïde aiguë (LMA) est fréquente chez l’adulte. Malgré un taux de survie très faible des patients après 5 ans (environ 27%), le traitement de la LMA a peu changé au cours des quarante dernières années. Par le biais d’un criblage de viabilité à haut débit réalisé dans le but d’identifier de meilleures molécules anti-LMA, nous avons mis en évidence la capacité des statines (inhibiteurs de l’hydroxy-methyl-glutary-CoA reductase (HMGCR)) à cibler de manière différentielle les différents sous-types de LMA. De manière intéressante, les échantillons de LMA appartenant à des patients classés «bon prognostic», sont 10 fois plus sensibles aux statines que les spécimens provenant de patients présentant un pronostic défavorable. Cet effet discriminatoire est perdu avec l’analogue de statine A405, synthétisé par notre équipe et qui n’a pas d’activité anti-HMGCR, suggérant que l’inhibition d’HMGCR est essentielle pour l’effet discriminatoire des statines. Les statines sont des médicaments hypocholestérolémiants et sont largement connues pour leur action inhibitrice sur l’HMGCR, une enzyme limitante de la voie de synthèse du mévalonate. En plus d’être d’excellents inhibiteurs de la production de cholestérol, les statines sont également connues pour leurs effets pléiotropiques sur d’autres branches de la voie du mévalonate. Ces branches jouent des rôles importants dans diverses fonctions cellulaires telles que la transduction du signal, la synthèse des protéines et la régulation du cytosquelette. Dans cette étude, nous avons émis l’hypothèse selon laquelle l’effet anti-LMA différentiel des statines est modulé par une ou plusieurs sous-branche(s) de la voie du mévalonate. Comme preuve de principe, nous avons confirmé que la réduction de l’activité d’HMGCR, médiée par shARN dans la lignée cellulaire OCI-AML5, conduit à une augmentation significative de la sensibilité aux statines mais pas à l’analogue A405. Nous avons également démontré que la perte complète du récepteur HMGCR, obtenue par CRISPR, est létale et que la supplémentation en mévalonate rétablit à la fois la viabilité cellulaire et la sensibilité aux statines. Pour disséquer davantage l’activité anti-LMA des statines, nous avons utilisé la technique d’ARN interférence pour cibler chacune des sous-branches en aval de la production de mévalonate et étudier la conséquence de ceci sur la viabilité cellulaire ainsi que sur la sensibilité aux statines. Notre étude a montré que la suppression individuelle des enzymes des sous-branches n’affecte pas significativement la sensibilité des cellules OCI-AML-5 aux statines. Fait intéressant, nous avons observé que la réduction de l’enzyme au point de ramification de la voie, la farnésyl diphosphate synthase (FDPS), est létale dans les cellules OCI-AML-5. De manière surprenante, nous avons également observé que la réduction de FDPS provoque une réponse biphasique aux statines. Alors qu’une concentration élevée d’atorvastatine aggrave le phénotype causé par la réduction du FDPS, une faible concentration conduit à un sauvetage presque complet de ce phénotype. Ces résultats ont permis de mettre en évidence un nouveau mécanisme de résistance aux statines dans la LMA. Dans l’ensemble, cette étude souligne l’importance de comprendre le contexte métabolique de la LMA avant traitement, afin de prévenir la résistance au médicaments.Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. The overall 5-year survival is as low as ~27%; however treatment for AML has only recently evolved with marginal impact on this low survival. In a large viability screen performed to identify better anti-AML molecules, we identified statins (3-Hydroxy-3-Methyl-glutary-CoA reductase (HMGCR) inhibitors) for their ability to differentially target AML specimens. Interestingly, AML specimens belonging to good outcome AML patients were 10 times more sensitive to statins compared to specimens belonging to adverse outcome patients. This discriminatory effect is lost with statin analog A405, synthesized by our team, which lacks anti-HMGCR activity, suggesting that HMGCR inhibition is essential for statin’s discriminatory effect. Statins are cholesterol- lowering drugs and are widely known for their inhibitory action on HMGCR, a rate-limiting enzyme in the mevalonate pathway. In addition to being excellent inhibitors of cholesterol production, statins are also known for their independent pleiotropic effects attributed by other products of the mevalonate pathway. These branches play important roles in various cellular functions such as signal transduction, protein synthesis and cytoskeletal regulation. In this study, we hypothesized that statin’s anti-AML differential effect is modulated by sub-branch(es) of the mevalonate pathway. As proof of principle, we confirmed that shRNA-mediated reduction of HMGCR activity in OCI-AML-5 cell line led to a significant increase of sensitivity to statins but not to its analog A405. We further demonstrated that complete loss of HMGCR using CRISPR technology was lethal and that mevalonate supplementation rescued both cell viability and sensitivity to statins. To further dissect the anti-AML activity of statins, we used RNAi technology to target each of the downstream sub-branches of the mevalonate pathway and investigated the consequences on cell viability and statin sensitivity. Our study showed that knocking down enzymes of single sub-branches did not affect significantly OCI-AML-5 cells sensitivity to statins. Interestingly, we observed that reduction of branch point enzyme, farnesyl diphosphate synthase (FDPS) was lethal in OCI-AML-5 cells. Unexpectedly, we also observed that FDPS knockdown caused a biphasic response to statin. While high concentrations of atorvastatin aggravates the phenotype caused by FDPS reduction, low concentrations led to a near complete rescue of this phenotype. These results uncovered a novel unsuspected statin resistance mechanism in AML. Overall, this study highlights the importance of understanding the metabolic background of AML prior to drug treatment, in order to prevent drug resistance

Novel Insights into the Biological Effects of the Isoprenoid Derivative N6-Isopentenyladenosine: Involvement of the Metabolic Sensor Ampk in Angiogenesis Inhibition

2012 - 2013N6-isopentenyladenosine (iPA) is a modified adenosine characterized by an isopentenyl chain derived by dimethylallyl pyrophosphate (DMAPP), an intermediate of the metabolic pathway of mevalonate, that is known to be deregulated in cancer. iPA is an endogenous isoprenoid-derived product present in mammalian cells as a free nucleoside in the cytoplasm, or in a tRNA-bound form, displaying well established pleiotropic biological effects, including a direct anti-tumor activity against several cancers. However, the precise mechanism of action of iPA in inhibiting cancer cell proliferation remains to be clarified. In this work, we investigated whether iPA could directly interfere with the angiogenic process, fundamental to cancer growth and progression, and if the growth and proliferation of human melanoma cells, known for their highly angiogenic phenotype, could be affected by the treatment with iPA. Finally, we investigated if iPA could have an immunomodulatory role targeting directly human natural killer (NK) cells, components of innate immunity that participate in immunity against neoplastic cells, in order to provide a cooperative and multifactorial mode of action of iPA to arrest cancer growth. To evaluate the potential involvement of iPA in angiogenesis, we employed human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, by evaluating the viability, proliferation, migration, invasion, tube formation, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 μM. We demonstrated for the first time that iPA was monophosphorylated into iPA 5'-monophosphate (iPAMP) by adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pre-treatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. Thus, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology... [edited by author]XII n.s

Statins in Cancer Prevention and Therapy

Statins, a class of HMG-CoA reductase inhibitors best known for their cholesterol-reducing and cardiovascular protective activity, have also demonstrated promise in cancer prevention and treatment. This review focuses on their potential applications in head and neck cancer (HNC), a common malignancy for which established treatment often fails despite incurring debilitating adverse effects. Preclinical and clinical studies have suggested that statins may enhance HNC sensitivity to radiation and other conventional therapies while protecting normal tissue, but the underlying mechanisms remain poorly defined, likely involving both cholesterol-dependent and -independent effects on diverse cancer-related pathways. This review brings together recent discoveries concerning the anticancer activity of statins relevant to HNC, highlighting their anti-inflammatory activity and impacts on DNA-damage response. We also explore molecular targets and mechanisms and discuss the potential to integrate statins into conventional HNC treatment regimens to improve patient outcomes

Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors

Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies

The development of a novel therapeutic strategy for the treatment of prostate cancer by targeting metabolic signalling

Prostate cancer is one of the most prevalent cancers worldwide. The early stages of prostate cancer (PCa) are highly dependent on the androgen receptor (AR) pathway and hence therapies target this signalling axis. This approach is successful initially but invariably fails and the tumours progress to castration resistant prostate cancer (CRPC), for which few therapeutic options exist. Therefore, there is a great need to identify and characterize novel therapeutic targets for this stage of the disease. Cancer cells undergo alterations that allow them to survive and proliferate, and metabolic reprogramming is one of the most important manifestations in cancer progression. Therefore, targeting tumour metabolism is an attractive approach to treat cancer. Screening for novel metabolic targets was performed using an siRNA library. 26 metabolic factors were identified to affect proliferation and/or migration, and these were found to be involved in essential pathways including lipogenesis, heme-biosynthesis, redox homeostasis, and glycolysis. The lead targets were validated in a range of cell lines and additional assays performed to investigate the effect upon cell cycle and cell death. UROS, the fourth step of heme synthesis, was further investigated and depletion of this enzyme significantly inhibited prostate cancer proliferation and migration, promoted cell cycle arrest and induced cell death. Further, inhibition of heme synthesis using the inhibitor succinylacetone was found to significantly induce caspase-independent cell death and to sensitise cells to ROS. Importantly, the inhibitory activity of succinylacetone in combination with ROS showed specificity for cancer cell lines. Targeting heme synthesis therefore represents a novel targeted treatment option for prostate cancer and further work is needed to develop this into a therapeutic strategy

Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

Chronic Lymphocytic Leukemia (CLL) is an incurable disease, characterized by the accumulation of malignant B-lymphocytes in the blood stream (quiescent state) and homing tissues (where they can proliferate). In CLL, the targeting of B-cell receptor signaling through a Burton's tyrosine kinase inhibitor (ibrutinib) has rendered outstanding clinical results. However, complete remission is not guaranteed due to drug resistance or relapse, revealing the need for novel approaches for CLL treatment. The characterization of metabolic rewiring in proliferative cancer cells is already being applied for diagnostic and therapeutic purposes, but our knowledge of quiescent cell metabolism—relevant for CLL cells—is still fragmentary. Recently, we reported that glutamine metabolism in primary CLL cells bearing the del11q deletion is different from their del11q negative counterparts, making del11q cells especially sensitive to glutaminase and glycolysis inhibitors. In this work, we used our primary CLL lymphocyte bank and compounds interfering with central carbon metabolism to define metabolic traits associated with ibrutinib resistance. We observe a differential basal metabolite uptake linked to ibrutinib resistance, favoring glutamine uptake and catabolism. Upon ibrutinib treatment, the redox balance in ibrutinib resistant cells is shifted toward NADPH accumulation, without an increase in glutamine uptake, suggesting alternative metabolic rewiring such as the activation of fatty acid oxidation. In accordance to this idea, the curtailing of fatty acid oxidation by CPT1 inhibition (etomoxir) re-sensitized resistant cells to ibrutinib. Our results suggest that fatty acid oxidation could be explored as a target to overcome ibrutinib resistance

The Potential of Isoprenoids in Adjuvant Cancer Therapy to Reduce Adverse Effects of Statins

The mevalonate pathway provides sterols for membrane structure and nonsterol intermediates for the post-translational modification and membrane anchorage of growth-related proteins, including the Ras, Rac, and Rho GTPase family. Mevalonate-derived products are also essential for the Hedgehog pathway, steroid hormone signaling, and the nuclear localization of Yes-associated protein and transcriptional co-activator with PDZ-binding motif, all of which playing roles in tumorigenesis and cancer stem cell function. The phosphatidylinositol-4,5-bisphosphate 3-kinase-AKT-mammalian target of rapamycin complex 1 pathway, p53 with gain-of-function mutation, and oncoprotein MYC upregulate the mevalonate pathway, whereas adenosine monophosphate-activated protein kinase and tumor suppressor protein RB are the downregulators. The rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is under a multivalent regulation. Sterol regulatory element binding protein 2 mediates the sterol-controlled transcriptional downregulation of HMGCR. UbiA prenyltransferase domain-containing protein-1 regulates the ubiquitination and proteasome-mediated degradation of HMGCR, which is accelerated by 24, 25-dihydrolanosterol and the diterpene geranylgeraniol. Statins, competitive inhibitors of HMGCR, deplete cells of mevalonate-derived intermediates and consequently inhibit cell proliferation and induce apoptosis. Clinical application of statins is marred by dose-limiting toxicities and mixed outcomes on cancer risk, survival and mortality, partially resulting from the statin-mediated compensatory upregulation of HMGCR and indiscriminate inhibition of HMGCR in normal and tumor cells. Tumor HMGCR is resistant to the sterol-mediated transcriptional control; consequently, HMGCR is upregulated in cancers derived from adrenal gland, blood and lymph, brain, breast, colon, connective tissue, embryo, esophagus, liver, lung, ovary, pancreas, prostate, skin, and stomach. Nevertheless, tumor HMGCR remains sensitive to isoprenoid-mediated degradation. Isoprenoids including monoterpenes (carvacrol, L-carvone, geraniol, perillyl alcohol), sesquiterpenes (cacalol, farnesol, β-ionone), diterpene (geranylgeranyl acetone), “mixed” isoprenoids (tocotrienols), and their derivatives suppress the growth of tumor cells with little impact on non-malignant cells. In cancer cells derived from breast, colon, liver, mesothelium, prostate, pancreas, and skin, statins and isoprenoids, including tocotrienols, geraniol, limonene, β-ionone and perillyl alcohol, synergistically suppress cell proliferation and associated signaling pathways. A blend of dietary lovastatin and δ-tocotrienol, each at no-effect doses, suppress the growth of implanted murine B16 melanomas in C57BL6 mice. Isoprenoids have potential as adjuvant agents to reduce the toxicities of statins in cancer prevention or therapy

Tumor Promoting Functions of Lipid Storage in Clear Cell Renal Cell Carcinoma

Two hallmarks of clear cell renal cell carcinoma (ccRCC) are constitutive hypoxia inducible factor (HIF) signaling and abundant intracellular lipid droplets (LDs). However, regulation of lipid storage and its role in ccRCC are incompletely understood. In this study, we explored the function of the LD coat protein perilipin 2 (PLIN2) within the context of ccRCC. Transcriptional profiling of primary ccRCC samples revealed that expression of PLIN2 was elevated in tumors and correlated with HIF-2α, but not HIF-1α, activation. HIF-2α dependent PLIN2 expression promoted lipid storage, proliferation, and viability in xenograft tumors. Mechanistically, lipid storage maintained integrity of the endoplasmic reticulum (ER), which is functionally and physically associated with LDs. Specifically, PLIN2 dependent lipid storage suppressed cytotoxic ER stress responses that otherwise result from elevated protein synthetic activity characteristic of ccRCC cells. Thus, in addition to promoting ccRCC proliferation and anabolic metabolism, HIF-2α modulates lipid storage to sustain ER homeostasis, particularly under conditions of nutrient and oxygen limitation, thereby promoting tumor cell survival. We also examined the mechanisms underlying the tumor promoting functions of PLIN2-dependent lipid storage in ccRCC. Most phospholipid and triacylglycerol (TAG) biosynthetic enzymes reside in the ER, and lipids are exchanged between the ER and LD. The phospholipid composition of the ER membrane is tightly regulated to support biosynthetic functions and protein homeostasis within the ER lumen. In addition, perturbation of the phospholipid membrane is sufficient to induce ER stress. We tested the hypothesis that (TAG) synthesis and storage prevents ER stress by guarding against perturbations in ER phospholipid composition. Phospholipid and TAG biosynthesis share a common pathway involving the synthesis of diacylglyerol. We found that ablation of the acyl-CoA:diacylglycerol (DGAT) enzymes necessary for TAG biosynthesis enhances sensitivity of ccRCC cells to conditions that perturb phospholipid homeostasis and trigger ER stress. These include exposure to the saturated fatty acid palmitate, hypoxia, and importantly, growth as a sub-cutaneous xenograft tumor. Collectively, our results reveal a novel function for the well-documented “clear cell” phenotype and identifying ER stress as a targetable vulnerability created by HIF-2α/PLIN2 suppression in this common renal malignancy