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Supplementary Video S1: PF-228's effect on NCI-446 SCLC cell line migration. NCI-H446 cell lines were grown to confluence in 12-well plates. Cell monolayers were wounded using a micropipette tip and floating cells were washed off with PBS. After overnight incubation, PF-228 at a concentration of 3µM or DMSO was added to culture medium. Cell movements within wounded area were monitored for 12h starting from the time drug was added using a Zeiss Axiovert 200M microscope (Zeiss, Thornwood, NY) at x200 magnification. Images were captured every 15-minutes from five different fields randomly selected in each well. They show that PF-228 decreased migration velocity in NCI-H446 cell lines.

PDF file - 276KB, Supplementary Figure 1. Chemical structures of 7ACC, 3BP and AR-C155858. Supplementary Figure 2. Effects of 7ACC on FaDu cell viability. Supplementary Figure 3. Pimonidazole labelling of tumor sections from control and 7ACC2-treated mice. Supplementary Figure 4. Effects of 3BP on lactate uptake and titration of the effects of 7ACC by extracellular lactate.

Supplementary Figure S1: Western blot densitometric quantifications. Supplementary Figure S2: FAK Inhibitor 14 (Inh14) and PF-562,271 (PF-271)'s effects on FAK expression/activity, cell proliferation, and apoptosis in NCI-H446 SCLC cell lines. Supplementary Figure S3: PF-573,228 (PF-228)'s effects on FAK expression/activity, cell proliferation, and apoptosis in NCI-H82 SCLC cell lines transduced with FAK shRNA. Supplementary Figure S4: phospho-Paxillin expression in NCI-H446 SCLC cell line where FAK has been inhibited with pharmacological and genetic methods.

High lactate concentration in tumors is associated with bad prognosis. Lactate is released by glycolytic cells in tumors and recaptured by oxidative cancer cells to feed the tricarboxylic acid (TCA) cycle after conversion into pyruvate. Monocarboxylate transporters (MCT) mediate these fluxes of proton-linked lactate and represent attractive targets to interrupt lactate shuttle and to inhibit tumor growth. Here, we investigated the properties of 7-aminocarboxycoumarins (7ACC) developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment. The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models. Contrary to the reference MCT1 inhibitor AR-C155858, 7ACC unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters. 7ACC delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors. MCT target engagement was confirmed by the lack of activity of 7ACC on bladder UM-UC-3 carcinoma that does not express functional MCT. 7ACC also inhibited SiHa tumor relapse after treatment with cisplatin. Finally, we found that contrary to AR-C155858, 7ACC did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment. In conclusion, our results indicate that 7ACC selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects. Mol Cancer Ther; 13(6); 1410–8. ©2014 AACR.

Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non–receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1–5 μmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2–M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKβ1−/− mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKβ1−/− mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.

Supplementary Data from Identification of Cyclooxygenase-2 as a Major Actor of the Transcriptomic Adaptation of Endothelial and Tumor Cells to Cyclic Hypoxia: Effect on Angiogenesis and Metastases

This file contains all supplementary figures with their legends, supplementary tables and supplementary methods

Caspases are well known for their role in apoptosis. Recently, nonapoptotic roles of caspases have been identified, however, these noncanonical roles are not well documented and the mechanisms involved are not fully understood. Here, we studied the role of cleaved caspase-3 using human- and mouse-proficient caspase-3 cancer cell lines and human-deficient caspase-3 cancer cells. Cleaved caspase-3 functioned as a transcription factor and directly bound to DNA. A DNA-binding domain was identified in the small subunit of caspase-3 and an active conformation was essential for caspase-3 transcriptional activity. Caspase-3 DNA binding enhanced angiogenesis by upregulating the expression of proangiogenic genes and by activating pathways that promoted endothelial cell activation. Some proapoptotic genes were downregulated in caspase-3–proficient cells. Inhibiting caspase-3 increased the efficacy of chemotherapy and decreased spontaneous tumor development. These data highlight a novel nonapoptotic role of caspase-3 and suggest that cleaved caspase-3 could be a new therapeutic target in cancer.Significance:These findings report a noncanonical function of caspase-3 by demonstrating its ability to transcriptionally regulate the VEGFR pathway.

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Supplementary Figure 1 from Lactate Influx through the Endothelial Cell Monocarboxylate Transporter MCT1 Supports an NF-κB/IL-8 Pathway that Drives Tumor Angiogenesis

Supplementary Methods from Lactate Influx through the Endothelial Cell Monocarboxylate Transporter MCT1 Supports an NF-κB/IL-8 Pathway that Drives Tumor Angiogenesis

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Supplementary Methods, Tables 1-2 from Preconditioned Endothelial Progenitor Cells Reduce Formation of Melanoma Metastases through SPARC-Driven Cell–Cell Interactions and Endocytosis

ROS in cancer cells play a key role in pathways regulating cell death, stemness maintenance, and metabolic reprogramming, all of which have been implicated in resistance to chemo/ immunotherapy. Adjusting ROS levels to reverse the resistance of cancer cells without impairing normal cell functions is a new therapeutic avenue. In this paper, we describe new inhibitors of NADPH oxidase (NOX), a key enzyme in many cells of the tumor microenvironment. The first inhibitor, called Nanoshutter-1, NS1, decreased the level of tumor-promoting “M2” macrophages differentiated from human blood monocytes. NS1 disrupted the active NADPH oxidase-2 (NOX2) complex at the membrane and in the mitochondria of the macrophages, as shown by confocal microscopy. As one of the characteristics of tumor invasion is hypoxia, we tested whether NS1 would affect vascular reactivity by reducing ROS or NO levels in wire and pressure myograph experiments on isolated blood vessels. The results show that NS1 vasodilated blood vessels and would likely reduce hypoxia. Finally, as both NOX2 and NOX4 are key proteins in tumors and their microenvironment, we investigated whether NS1 would probe these proteins differently. Models of NOX2 and NOX4 were generated by homology modeling, showing structural differences at their C-terminal NADPH site, in particular in their last Phe. Thus, the NADPH site presents an unexploited chemical space for addressing ligand specificity, which we exploited to design a novel NOX2-specific inhibitor targeting variable NOX2 residues. With the proper smart vehicle to target specific cells of the microenvironment as TAMs, NOX2-specific inhibitors could open the way to new precision therapies., French National Research Agency (ANR) PCVI-08-006 TRIGNOSTUMOR

STUDY QUESTIONIs it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation?SUMMARY ANSWEROur photodynamic therapy (PDT) approach has been shown to efficiently destroy leukemia cells from tumor-infiltration mimicking models (TIMs), indicating the feasibility of this technique to purge OT samples.WHAT IS KNOWN ALREADYAutotransplantation of cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women who require immediate cancer treatment. Up until now, more than 200 live births have already been reported after OT cryopreservation and transplantation. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age and in 2020, the estimated number of new leukemia cases was higher than 33 000 in girls between 0 and 19 years old. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient leading to leukemia recurrence.STUDY DESIGN, SIZE, DURATIONTo safely transplant the OT from leukemia patients and restore their fertility, our goal was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging of acute myelogenous leukemia cells from OT fragments (n = 4). Moreover, to ensure that such treatments are not harmful to follicle survival and development so they can be deemed a potential fertility restoration alternative, the effect of the ORN-based PDT purging procedure on follicles was assessed after xenografting the photodynamic-treated OT in SCID mice (n = 5). The work was carried out between September 2020 and April 2022 at the Catholic University of Louvain.PARTICIPANTS/MATERIALS, SETTING, METHODSAfter establishing the best ORN formulation, our PDT approach was used to eradicate HL60 cells from ex vivo TIMs prepared by microinjection of a cancer cell suspension into OT fragments. The purging efficiency was analyzed by droplet digital polymerase chain reaction and immunohistochemical analyses. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival and development, and tissue quality in terms of fibrotic areas and vascularization after 7-day xenotransplantation to immunodeficient mice.MAIN RESULTS AND THE ROLE OF CHANCEThe ex vivo purging of TIMs demonstrated that our PDT strategy could selectively eradicate the malignant cells from tissue fragments without affecting OT normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and PDT-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, our findings showed that the control and PDT-treated OT could be equally vascularized (7.65 ± 1.45% and 9.89 ± 2.21%, respectively). Similarly, the proportions of fibrotic area did not differ between the control (15.96 ± 5.94%) and PDT-treated groups (13.32 ± 3.05%).LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis study did not use OT fragments from leukemia patients, but TIMs created after injection of HL60 cells into OT from healthy patients. Therefore, while the results are promising, whether our PDT approach will be equally successful in eliminating malignant cells from leukemia patients remains to be assessed.WIDER IMPLICATIONS OF THE FINDINGSOur results showed that the purging procedure causes no significant impairment effect on follicle development and tissue quality, suggesting that our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of OT, allowing safe transplantation in cancer survivors.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A.); Fondation Louvain (awarded to C.A.A.; a Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and a Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer); and Foundation Against Cancer (grant number 2018-042 awarded to A.C.). The authors declare no competing interests.

The palladium-catalyzed uncaging of alloc-protected amines in living cells has been extensively stud- ied mostly in the form of nanostructures or mesostructures. However, the scarcity of kinetic and mechanistic studies of discrete palladium complexes for deallylation reactions has hindered progress. Herein, we report the development of a series of discrete palladium complexes bearing acetanilide lig- ands, which exhibit a good balance between reactivity and stability in living cells. We investigated the catalytic activity and cytotoxicity of these complexes in SiHa cells and found that acetanilide[tri(2- furyl)phosphine]palladium(II) triflate showed promising results. Under physiological conditions, this pal- ladium complex exhibited a second-order reaction rate of 30 M −1 s −1 , and liquid chromatography-mass spectrometry (LC-MS) studies suggested the possibility of a tandem Heck/Tsuji-Trost mechanism. Our re- sults demonstrate the potential of using these discrete palladium complexes for bioorthogonal chemistry studies in living cells.

Cycling hypoxia (cyH), neo-angiogenesis, and tumor-associated macrophages are key features of the tumor microenvironment. In this study, we demonstrate that cyH potentiates the induction by unpolarized and M1-like macrophages of endothelial inflammatory phenotype and adhesiveness for monocytes and cancer cells. This process triggers a positive feedback loop sustaining tumor inflammation. This work opens the door for innovative therapeutic strategies to treat tumor inflammation and metastasis.In cancers, the interaction between macrophages and endothelial cells (ECs) regulates tumor inflammation and metastasis. These cells are both affected by cycling hypoxia (cyH), also called intermittent hypoxia, a feature of the tumor microenvironment. cyH is also known to favor tumor inflammation and metastasis. Nonetheless, the potential impact of cyH on the dialog between macrophages and ECs is still unknown. In this work, the effects of unpolarized, M1-like, and M2-like macrophages exposed to normoxia, chronic hypoxia (chH), and cyH on endothelial adhesion molecule expression, pro-inflammatory gene expression, and EC adhesiveness for monocytes and cancer cells were investigated. cyH increased the ability of unpolarized and M1-like macrophages to induce EC inflammation and to increase the expression of the EC endothelial adhesion molecule ICAM1, respectively. Unpolarized, M1-like, and M2-like macrophages were all able to promote EC adhesive properties toward cancer cells. Furthermore, the ability of macrophages (mostly M1-like) to shift EC phenotype toward one allowing cancer cell and monocyte adhesion onto ECs was potentiated by cyH. These effects were specific to cyH because they were not observed with chH. Together, these results show that cyH amplifies the effects of macrophages on ECs, which may promote tumor inflammation and metastasis.

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.

In 2020, the estimated number of new leukemia cases was higher than 30,000 in girls between 0 and 19 years old. Due to cancer treatment, some of these patients may lose both endocrine and reproductive functions. Transplantation of cryopreserved ovarian tissue is not advised after cancer remission because it has a high risk of reintroducing malignant cells in the patient, potentially leading to leukemia recurrence. To safely transplant the ovarian tissue from these patients and restore their fertility, our goal was to develop a photodynamic therapy (PDT) strategy to eliminate leukemia ex vivo. To this end, we designed, optimized, and characterized OR141-loaded niosomes (ORN) to develop the most effective formulation for ex vivo purging ovarian fragments from chronic myelogenous leukemia cells. After establishing the best ORN formulation, the PDT efficiency of optimized ORN was determined for human ovarian stromal cells and acute myeloid leukemia cell line (HL60). Blank niosomes treatment on ovarian stromal cells causes no significant toxicity, showing that the composition of the nanoparticle is not toxic. On the other hand, the in vitro studies showed that while ovarian stromal cells were still viable (82.04 ± 2.79%) after the treatment by 0.5 µM ORN, the same treatment yielded 95.43 ± 3.89% toxicity and cell death in the cancer cells. In conclusion, our results showed that our novel PDT procedure could be a promising strategy to destroy leukemia cells in ovarian tissue fragments allowing safe transplantation in cancer survivors.

Epidemiological studies have shown that obese subjects have an increased risk of developing triple‐negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high‐fat diet in TNBC tumour‐bearing mice significantly increased tumour growth. By showing no effect of high‐fat diet in obesity‐resistant mice, we demonstrated the necessity to develop obesity‐related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity‐related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.

Immunogenic cell death (ICD) is a rapidly growing research area representing one of the emerging therapeutic strategies of cancer immunotherapy. ICD is an umbrella term covering several cell death modalities including apoptosis, necroptosis, ferroptosis and pyroptosis, and is the product of a balanced combination of adjuvanticity (damage-associated molecular patterns and chemokines/cytokines) and antigenicity (tumor associated antigens). Only a limited number of anti-cancer therapies are available to induce ICD in experimental cancer therapies and even much less is available for clinical use. To overcome this limitation, nanomaterials can be used to increase the immunogenicity of cancer cells killed by anti-cancer therapy, which in themselves are not necessarily immunogenic. In this review, we outline the current state of knowledge of ICD modalities and discuss achievements in using nanomaterials to increase the immunogenicity of dying cancer cells. The emerging trends in modulating the immunogenicity of dying cancer cells in experimental and translational cancer therapies and the challenges facing them are described. In conclusion, nanomaterials are expected to drive further progress in their use to increase efficacy of anti-cancer therapy based on ICD induction and in the future, it is necessary to validate these strategies in clinical settings, which will be a challenging research area.

(1) Background: The acidosis of the tumor micro-environment may have profound impact on cancer progression and on the efficacy of treatments. In the present study, we evaluated the impact of a treatment with UK-5099, a mitochondrial pyruvate carrier (MPC) inhibitor on tumor extracellular pH (pHe), (2) Methods: glucose consumption, lactate secretion and extracellular acidification rate (ECAR) were measured in vitro after exposure of cervix cancer SiHa cells and breast cancer 4T1 cells to UK-5099 (10 µM). Mice bearing the 4T1 tumor model were treated daily during four days with UK-5099 (3 mg/kg). The pHe was evaluated in vivo using either chemical exchange saturation transfer (CEST)-MRI with iopamidol as pHe reporter probe or 31P-NMR spectroscopy with 3-aminopropylphosphonate (3-APP). MR protocols were applied before and after 4 days of treatment, (3) Results: glucose consumption, lactate release and ECAR were increased in both cell lines after UK-5099 exposure. CEST-MRI showed a significant decrease in tumor pHe of 0.22 units in UK-5099-treated mice while there was no change over time for mice treated with the vehicle. Parametric images showed a large heterogeneity in response with 16% of voxels shifting to pHe values under 7.0. In contrast, 31P-NMR spectroscopy was unable to detect any significant variation in pHe, (4) Conclusions: MPC inhibition led to a moderate acidification of the extracellular medium in vivo. CEST-MRI provided high resolution parametric images (0.44 µL/voxel) of pHe highlighting the heterogeneity of response within the tumor when exposed to UK-5099.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze–thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKβ1−/− mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

Caspases are well known for their role in apoptosis. Recently, nonapoptotic roles of caspases have been identified, however, these noncanonical roles are not well documented and the mechanisms involved are not fully understood. Here, we studied the role of cleaved caspase-3 using human- and mouse-proficient caspase-3 cancer cell lines and human-deficient caspase-3 cancer cells. Cleaved caspase-3 functioned as a transcription factor and directly bound to DNA. A DNA-binding domain was identified in the small subunit of caspase-3 and an active conformation was essential for caspase-3 transcriptional activity. Caspase-3 DNA binding enhanced angiogenesis by upregulating the expression of proangiogenic genes and by activating pathways that promoted endothelial cell activation. Some proapoptotic genes were downregulated in caspase-3–proficient cells. Inhibiting caspase-3 increased the efficacy of chemotherapy and decreased spontaneous tumor development. These data highlight a novel nonapoptotic role of caspase-3 and suggest that cleaved caspase-3 could be a new therapeutic target in cancer. Significance: These findings report a noncanonical function of caspase-3 by demonstrating its ability to transcriptionally regulate the VEGFR pathway.

Although saturated fatty acid (FA) (SFA) and monounsaturated FA (MUFA) are synthesized in cancer cells from acetyl-CoA, polyunsaturated FAs (PUFAs) are necessarily obtained from diet. Depending on concentrations and metabolism, these different FAs may support tumor proliferation but also exert growth inhibitory effects. The mutual interplay between them also requires to integrate the FA oxidation component that may be concomitant with FA synthesis is cancer cells. New molecular mechanisms driving FA synthesis, lipotoxicity and anti-inflammatory activity of eicosanoids in mouse and human cancers were recently elicited. To block or take advantage of the above represent attractive perspectives of treatments to fight cancer progression. The various enzymatic reactions leading to SFA synthesis represent as many targets to prevent tumor growth. Ironically excess SFAs are per-se toxic for cancer cells and the introduction of a double bound to form MUFA is actually limiting lipotoxicity in cancer cells. Blocking stearoyl-CoA desaturase therefore represents another attractive modality. By contrast, dietary PUFAs may exert direct cytotoxic effects by promoting apoptosis or by generating anti-inflammatory eicosanoids. Altogether, these data point out the intricate relationship between SFA, MUFA and PUFA at the heart of the metabolism of proliferating cancer cells.

Background: Energy metabolism is described to be deregulated in cancer, and the Warburg effect is considered to be a major hallmark. Recently, cellular heterogeneity in tumors and the tumor microenvironment has been recognized to play an important role in several metabolic pathways in cancer. However, its contribution to papillary thyroid cancer (PTC) development and metabolism is still poorly understood. Methods: A proteomic analysis of five PTC was performed, and the cellular distribution of several upregulated metabolic proteins was investigated in the cancerous and stromal cells of these tumors. Results: Tandem mass spectrometry analysis revealed the upregulation of many metabolism-related proteins, among them pyruvate carboxylase (PC). PC knockdown in thyroid cell lines alters their proliferative and motility capacities, and measurements of oxygen consumption rates show that this enzyme is involved in the replenishment of the tricarboxylic acid cycle. Immunostainings of several upregulated metabolic proteins show that thyroid cancer cells have an increased mitochondrial oxidative metabolism compared to stromal cells. Conclusions: PTC has a very active tricarboxylic acid cycle, continuously replenished by a PC-mediated anaplerosis. This is specifically observed in the tumor cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Due to rising costs and the difficulty to identify new targets, drug repurposing appears as a viable strategy for the development of new anti-cancer treatments. Although the interest of disulfiram (DSF), an anti-alcohol drug, to treat cancer was reported for many years, it is only very recently that one anticancer mechanism-of-action was highlighted. This would involve the inhibition of the p97 segregase adaptor NPL4, which is essential for the turnover of proteins involved in multiple regulatory and stress-response intracellular pathways. However, recently DSF was also reported as one of the first phosphoglycerate dehydrogenase (PHGDH) inhibitors, a tetrameric enzyme catalyzing the initial step of the serine synthetic pathway that is highly expressed in numerous cancer types. Here, we investigated the structure-activity relationships (SAR) of PHGDH inhibition by disulfiram analogues as well as the mechanism of action of DSF on PHGDH via enzymatic and cell-based evaluation, mass spectrometric and mutagenesis experiments.

AIM: This study aimed at developing antibody-functionalized gold nanoparticles (AuNPs) to selectively target cancer cells and probing their potential radiosensitizing effects under proton irradiation.MATERIALS & METHODS: AuNPs were conjugated with cetuximab (Ctxb-AuNPs). Ctxb-AuNP uptake was evaluated by transmission electron microscopy and atomic absorption spectroscopy. Radioenhancing effect was assessed using conventional clonogenic assay.RESULTS & CONCLUSION: Ctxb-AuNPs specifically bound to and accumulated in EGFR-overexpressing A431 cells, compared with EGFR-negative MDA-MB-453 cells. Ctxb-AuNPs enhanced the effect of proton irradiation in A431 cells but not in MDA-MB-453 cells. These data indicate, for the first time, that combining enhanced uptake by specific targeting and radioenhancing effect, using conjugated AuNPs, is a promising strategy to increase cell killing by protontherapy.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies. ispartof: SCIENCE ADVANCES vol:7 issue:19 ispartof: location:United States status: published

The multidisciplinary field of photodynamic therapy (PDT) is a combination of photochemistry and photophysics sciences, which has shown tremendous potential for cancer therapy application. PDT employs a photosensitizing agent (PS) and light to form cytotoxic reactive oxygen species and subsequently oxidize light-exposed tissue. Despite numerous advantages of PDT and enormous progress in this field, common PSs are still far from ideal treatment because of their poor permeability, non-specific phototoxicity, side effects, hydrophobicity, weak bioavailability, and tendency to self-aggregation. To circumvent these limitations, PS can be encapsulated in liposomes, an advanced drug delivery system that has demonstrated the ability to enhance drug permeability into biological membranes and loading both hydrophobic and lipophilic agents. Moreover, liposomes can also be coated by targeting agents to improve delivery efficiency. The present review aims to summarize the principles of PDT, various PS generations, PS-loaded nanoparticles, liposomes, and their impact on PDT, then discuss recent photodynamic cancer therapy strategies using liposomes as PS-loaded vectors, and highlight future possibilities and perspectives.

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.

Specific diets for cancer patients have the potential to offer an adjuvant modality to conventional anticancer therapy. If the concept of starving cancer cells from nutrients to inhibit tumor growth is quite simple, the translation into the clinics is not straightforward. Several diets have been described including the Calorie-restricted diet based on a reduction in carbohydrate intake and the Ketogenic diet wherein the low carbohydrate content is compensated by a high fat intake. As for other diets that deviate from normal composition only by one or two amino acids, these diets most often revealed a reduction in tumor growth in mice, in particular when associated with chemo- or radiotherapy. By contrast, in cancer patients, the interest of these diets is almost exclusively supported by case reports precluding any conclusions on their real capacity to influence disease outcome. In parallel, the field of tumor lipid metabolism has emerged in the last decade offering a better understanding of how fatty acids are captured, synthesized or stored as lipid droplets in cancers. Fatty acids participate to cancer cell survival in the hypoxic and acidic tumor microenvironment and also support proliferation and invasiveness. Interestingly, while such addiction for fatty acids may account for cancer progression associated with high fat diet, it could also represent an Achilles heel for tumors. In particular n-3 polyunsaturated fatty acids represent a class of lipids that can exert potent cytotoxic effects in tumors and therefore represent an attractive diet supplementation to improve cancer patient outcomes.

The fundamental unit of chromatin is the nucleosome which is composed of a histone octamer and the DNA that wraps around it. Histones are globular proteins subject to various reversible covalent modifications that primarily occur on their flexible N-terminal ends (the so-called tail). Histone acetylation is one of these major alterations that may influence the chromatin conformation and consecutively influence gene expression (1). Histone acetylation is usually associated with an increase in transcriptional activity. Indeed, since acetylation occurs on positively charged lysines residues, the addition of an acetyl group on these residues changes the overall charge of the histone tail thereby leading to weaker binding of the nucleosomal components (2). As a direct consequence of histone acetylation, DNA becomes more accessible to transcription factors. Enzymes named histone acetyltransferases (HATs) catalyzed this mode of post-translational modifications (3). Other enzymes termed histone deacetylases (HDACs) are involved in the reverse process of histone deacetylation that restores the ionic interactions between positively charged histones and negatively charged DNA, thereby yielding a more compact chromatin structure (making it harder for transcription factors to bind to the DNA) (2,3).

Targeting endothelial cell (EC) metabolism should impair angiogenesis, regardless of how many angiogenic signals are present. The dependency of proliferating ECs on glucose and glutamine for energy and biomass production opens new opportunities for anti-angiogenic therapy in cancer. The aim of the present study was to investigate the role of pyruvate dehydrogenase kinase (PDK) inhibition with dichloroacetate (DCA), alone or in combination with the glutaminase-1 (GLS-1) inhibitor, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), on Human umbilical vein endothelial cells (HUVECs) metabolism, proliferation, apoptosis, migration, and vessel formation. We demonstrated that both drugs normalize HUVECs metabolism by decreasing glycolysis for DCA and by reducing glutamate production for BPTES. DCA and BPTES reduced HUVECs proliferation and migration but have no impact on tube formation. While DCA increased HUVECs respiration, BPTES decreased it. Using both drugs in combination further reduced HUVECs proliferation while normalizing respiration and apoptosis induction. Overall, we demonstrated that DCA, a metabolic drug under study to target cancer cells metabolism, also affects tumor angiogenesis. Combining DCA and BPTES may reduce adverse effect of each drug alone and favor tumor angiogenesis normalization.

The potential of dendritic cell (DC)-based immunotherapy to treat cancer is, nowadays, well documented. Still, the clinical success of immune checkpoint inhibitors has dampened the interest in anticancer DC vaccination. For highly life-threatening tumors that are regarded as nonimmunogenic, such as mesothelioma, however, T helper 1 immunity-biased DC-based immunotherapy could still represent an attractive strategy. In this study, we took advantage of photodynamic therapy (PDT) to induce immunogenic cell death to generate mesothelioma cell lysates for DC priming and evaluated such a vaccine to treat peritoneal mesothelioma. We found that the white light in vitro activation of the photosensitizer OR141 led to mesothelioma cell death, together with the release of bona fide danger signals that promote DC maturation. The administration of a PDT-based DC vaccine to mice bearing peritoneal mesothelioma led to highly significant survival when compared with sham or control animals treated with anti-CTLA4 antibodies. This was further supported by a strong CD8+ and CD4+ T cell response, characterized by an increased proliferation, cytotoxic activities and the expression of activation markers, including interferon gamma (IFN). Moreover, the PDT-based DC vaccine led to a significant increase in IFN+ T cells infiltered within mesothelioma, as determined by flow cytometry and immunohistochemistry. Finally, in vivo tracking of intraperitoneally administered DCs led us to document rapid chemotaxis towards tumor-occupied lymphatics (vs. lipopolysaccharide (LPS)-treated DC). DCs pulsed with PDT-killed mesothelioma cells also exhibited a significant increase in CCR7 receptors, together with an intrinsic capacity to migrate towards the lymph nodes. Altogether, these results indicate that PDT-based DC vaccination is particularly suited to induce a potent immune response against peritoneal mesothelioma.

The pandemic's impacts on European electricity markets have been enormous, especially in countries with abundant near-zero marginal cost of production like France. This article provides an in-depth quantitative study of the impacts of the crisis on the French electricity sector. During the lockdown episode, France has experienced unparalleled demand reductions (-11.5%) and energy price falls (-40%) resulting in revenue losses of 1.2 billion € (-45%) for market participants. This paper argues that the observed market outcomes during the crisis are somehow indicative of outcomes in a future with abundant renewable power, where prices will fall in a more sustainable way.

Acidosis, a common characteristic of the tumor microenvironment, is associated with alterations in metabolic preferences of cancer cells and progression of the disease. Here we identify the TGF-β2 isoform at the interface between these observations. We document that acidic pH promotes autocrine TGF-β2 signaling, which in turn favors the formation of lipid droplets (LD) that represent energy stores readily available to support anoikis resistance and cancer cell invasiveness. We find that, in cancer cells of various origins, acidosis-induced TGF-β2 activation promotes both partial epithelial-to-mesenchymal transition (EMT) and fatty acid metabolism, the latter supporting Smad2 acetylation. We show that upon TGF-β2 stimulation, PKC-zeta-mediated translocation of CD36 facilitates the uptake of fatty acids that are either stored as triglycerides in LD through DGAT1 or oxidized to generate ATP to fulfill immediate cellular needs. We also address how, by preventing fatty acid mobilization from LD, distant metastatic spreading may be inhibited., The tumour microenvironment is known to have an acidic pH but how this influences cancer cell phenotype is unclear. Here, the authors show that tumour cells upregulate TGF-β2 under acidosis, which leads to the increased formation of lipid droplets allowing for invasiveness and metastases.

Purpose: Optimal head and neck squamous cell carcinoma (HNSCC) patient selection for anti–EGFR-based therapy remains an unmet need since only a minority of patients derive long-term benefit from cetuximab treatment. We assessed the ability of state-of-the-art noninvasive in vivo metabolic imaging to probe metabolic shift in cetuximab-sensitive and -resistant HNSCC patient-derived tumor xenografts (PDTXs). Experimental Design: Three models selected based on their known sensitivity to cetuximab in patients (cetuximab-sensitive or acquired-resistant HNC007 PDTXs, cetuximab-naïve UCLHN4 PDTXs, and cetuximab-resistant HNC010 PDTXs) were inoculated in athymic nude mice. Results: Cetuximab induced tumor size stabilization in mice for 4 weeks in cetuximab-sensitive and -naïve models treated with weekly injections (30 mg/kg) of cetuximab. Hyperpolarized 13C-pyruvate–13C-lactate exchange was significantly decreased in vivo in cetuximab-sensitive xenograft models 8 days after treatment initiation, whereas it was not modified in cetuximab-resistant xenografts. Ex vivo analysis of sensitive tumors resected at day 8 after treatment highlighted specific metabolic changes, likely to participate in the decrease in the lactate to pyruvate ratio in vivo. Diffusion MRI showed a decrease in tumor cellularity in the HNC007-sensitive tumors, but failed to show sensitivity to cetuximab in the UCLHN4 model. Conclusions: This study constitutes the first in vivo demonstration of cetuximab-induced metabolic changes in cetuximab-sensitive HNSCC PDTXs that were not present in resistant tumors. Using metabolic imaging, we were able to identify hyperpolarized 13C-pyruvate as a potential marker for response and resistance to the EGFR inhibitor in HNSCC.

The management of locally advanced rectal cancer has passed a long way of developments, where total mesorectal excision and preoperative radiotherapy are crucial to secure clinical outcome. These and other aspects of multidisciplinary strategies are in-depth summarized in the literature, while our mini-review pursues a different goal. From an ethical and medical standpoint, we witness a delayed implementation of novel therapies given the cost/time consuming process of organizing randomized trials that would bridge an already excellent local control in cT3-4 node-positive disease with long-term survival. This unfortunate separation of clinical research and medical care provides a strong motivation to repurpose known pharmaceuticals that suit for treatment intensification with a focus on distant control. In the framework of on-going phase II-III IG/IMRT-SIB trials, we came across an intriguing translational observation that the ratio of circulating (protumor) myeloid-derived suppressor cells to (antitumor) central memory CD8+ T cells is drastically increased, a possible mechanism of tumor immuno-escape and spread. This finding prompts that restoring the CD45RO memory T-cell pool could be a part of integrated adjuvant interventions. Therefore, the immunocorrective potentials of modified IL-2 and the anti-diabetic drug metformin are thoroughly discussed in the context of tumor immunobiology, mTOR pathways and revised Warburg effect.

Plant-derived conjugated linolenic acids (CLnA) have been widely studied for their preventive and therapeutic properties against diverse diseases such as cancer. In particular, punicic acid (PunA), a conjugated linolenic acid isomer (C18:3 c9t11c13) present at up to 83% in pomegranate seed oil, has been shown to exert anti-cancer effects, although the mechanism behind its cytotoxicity remains unclear. Ferroptosis, a cell death triggered by an overwhelming accumulation of lipid peroxides, has recently arisen as a potential mechanism underlying CLnA cytotoxicity. In the present study, we show that PunA is highly cytotoxic to HCT-116 colorectal and FaDu hypopharyngeal carcinoma cells grown either in monolayers or as three-dimensional spheroids. Moreover, our data indicate that PunA triggers ferroptosis in carcinoma cells. It induces significant lipid peroxidation and its effects are prevented by the addition of ferroptosis inhibitors. A combination with docosahexaenoic acid (DHA), a known polyunsaturated fatty acid with anticancer properties, synergistically increases PunA cytotoxicity. Our findings highlight the potential of using PunA as a ferroptosis-sensitizing phytochemical for the prevention and treatment of cancer.

Tumor acidosis promotes disease progression through a stimulation of fatty acid (FA) metabolism in cancer cells. Instead of blocking the use of FAs by acidic cancer cells, we examined whether excess uptake of specific FAs could lead to antitumor effects. We found that n-3 but also remarkably n-6 polyunsaturated FA (PUFA) selectively induced ferroptosis in cancer cells under ambient acidosis. Upon exceeding buffering capacity of triglyceride storage into lipid droplets, n-3 and n-6 PUFA peroxidation led to cytotoxic effects in proportion to the number of double bonds and even more so in the presence of diacylglycerol acyltransferase inhibitors (DGATi). Finally, an n-3 long-chain PUFA-rich diet significantly delayed mouse tumor growth when compared with a monounsaturated FA-rich diet, an effect further accentuated by administration of DGATi or ferroptosis inducers. These data point out dietary PUFA as a selective adjuvant antitumor modality that may efficiently complement pharmacological approaches.

This Review by Corbet and Feron summarizes recent data showing that tumour acidosis influences cancer metabolism and contributes to cancer progression; it also highlights advances in therapeutic modalities aimed at either inhibiting or exploiting tumour acidification. The high metabolic demand of cancer cells leads to an accumulation of H+ ions in the tumour microenvironment. The disorganized tumour vasculature prevents an efficient wash-out of H+ ions released into the extracellular medium but also favours the development of tumour hypoxic regions associated with a shift towards glycolytic metabolism. Under hypoxia, the final balance of glycolysis, including breakdown of generated ATP, is the production of lactate and a stoichiometric amount of H+ ions. Another major source of H+ ions results from hydration of CO2 produced in the more oxidative tumour areas. All of these events occur at high rates in tumours to fulfil bioenergetic and biosynthetic needs. This Review summarizes the current understanding of how H+-generating metabolic processes segregate within tumours according to the distance from blood vessels and inversely how ambient acidosis influences tumour metabolism, reducing glycolysis while promoting mitochondrial activity. The Review also presents novel insights supporting the participation of acidosis in cancer progression via stimulation of autophagy and immunosuppression. Finally, recent advances in the different therapeutic modalities aiming to either block pH-regulatory systems or exploit acidosis will be discussed.

BACKGROUND: Activation of free fatty acid receptor 2 (FFAR2) by microbiota-derived metabolites (e.g., propionate) reduces leukaemic cell proliferation in vitro. This study aims to test whether Ffar2 expression per se also influences leukaemia cell growth in vivo. METHODS: Bcr-Abl-expressing BaF cells were used as a leukaemia model and the role of Ffar2 was evaluated in Balb/c mice after lentiviral shRNA transduction. RESULTS: Our data formally establish that reduced leukaemic cell proliferation is associated with increased Ffar2 expression in vivo and in vitro. Going beyond association, we point out that decreasing Ffar2 expression fosters cancer cell growth in vitro and in vivo. CONCLUSIONS: Our data demonstrate the role of Ffar2 in the control of leukaemic cell proliferation in vivo and indicate that a modulation of Ffar2 expression through nutritional tools or pharmacological agents may constitute an attractive therapeutic approach to tackle leukaemia progression in humans.