Downregulation of Elovl5 promotes breast cancer metastasis through a lipid-droplet accumulation-mediated induction of TGF-ß receptors.

Metastatic breast cancer cannot be cured, and alteration of fatty acid metabolism contributes to tumor progression and metastasis. Here, we were interested in the elongation of very long-chain fatty acids protein 5 (Elovl5) in breast cancer. We observed that breast cancer tumors had a lower expression of Elovl5 than normal breast tissues. Furthermore, low expression of Elovl5 is associated with a worse prognosis in ER+ breast cancer patients. In accordance with this finding, decrease of Elovl5 expression was more pronounced in ER+ breast tumors from patients with metastases in lymph nodes. Although downregulation of Elovl5 expression limited breast cancer cell proliferation and cancer progression, suppression of Elovl5 promoted EMT, cell invasion and lung metastases in murine breast cancer models. The loss of Elovl5 expression induced upregulation of TGF-ß receptors mediated by a lipid-droplet accumulation-dependent Smad2 acetylation. As expected, inhibition of TGF-ß receptors restored proliferation and dampened invasion in low Elovl5 expressing cancer cells. Interestingly, the abolition of lipid-droplet formation by inhibition of diacylglycerol acyltransferase activity reversed induction of TGF-ß receptors, cell invasion, and lung metastasis triggered by Elovl5 knockdown. Altogether, we showed that Elovl5 is involved in metastasis through lipid droplets-regulated TGF-ß receptor expression and is a predictive biomarker of metastatic ER+ breast cancer.

NLRP3 inflammasome-mediated cytokine production and pyroptosis cell death in breast cancer.

Breast cancer is the most diagnosed malignancy in women. Increasing evidence has highlighted the importance of chronic inflammation at the local and/or systemic level in breast cancer pathobiology, influencing its progression, metastatic potential and therapeutic outcome by altering the tumor immune microenvironment. These processes are mediated by a variety of cytokines, chemokines and growth factors that exert their biological functions either locally or distantly. Inflammasomes are protein signaling complexes that form in response to damage- and pathogen-associated molecular patterns (DAMPS and PAMPS), triggering the release of pro-inflammatory cytokines. The dysregulation of inflammasome activation can lead to the development of inflammatory diseases, neurodegeneration, and cancer. A crucial signaling pathway leading to acute and chronic inflammation occurs through the activation of NLRP3 inflammasome followed by caspase 1-dependent release of IL-1ß and IL-18 pro-inflammatory cytokines, as well as, by gasdermin D-mediated pyroptotic cell death. In this review we focus on the role of NLRP3 inflammasome and its components in breast cancer signaling, highlighting that a more detailed understanding of the clinical relevance of these pathways could significantly contribute to the development of novel therapeutic strategies for breast cancer.

Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis.

Mitochondria have emerged as key actors of innate and adaptive immunity. Mitophagy has a pivotal role in cell homeostasis, but its contribution to macrophage functions and host defense remains to be delineated. Here, we showed that lipopolysaccharide (LPS) in combination with IFN-γ inhibited PINK1-dependent mitophagy in macrophages through a STAT1-dependent activation of the inflammatory caspases 1 and 11. In addition, we demonstrated that the inhibition of mitophagy triggered classical macrophage activation in a mitochondrial ROS-dependent manner. In a murine model of polymicrobial infection (cecal ligature and puncture), adoptive transfer of Pink1-deficient bone marrow or pharmacological inhibition of mitophagy promoted macrophage activation, which favored bactericidal clearance and led to a better survival rate. Reciprocally, mitochondrial uncouplers that promote mitophagy reversed LPS/IFN-γ-mediated activation of macrophages and led to immunoparalysis with impaired bacterial clearance and lowered survival. In critically ill patients, we showed that mitophagy was inhibited in blood monocytes of patients with sepsis as compared with nonseptic patients. Overall, this work demonstrates that the inhibition of mitophagy is a physiological mechanism that contributes to the activation of myeloid cells and improves the outcome of sepsis.

Docosahexaenoic acid inhibits both NLRP3 inflammasome assembly and JNK-mediated mature IL-1ß secretion in 5-fluorouracil-treated MDSC: implication in cancer treatment.

Limitation of 5-fluorouracil (5-FU) anticancer efficacy is due to IL-1ß secretion by myeloid-derived suppressor cells (MDSC), according to a previous pre-clinical report. Release of mature IL-1ß is a consequence of 5-FU-mediated NLRP3 activation and subsequent caspase-1 activity in MDSC. IL-1ß sustains tumor growth recovery in 5-FU-treated mice. Docosahexaenoic acid (DHA) belongs to omega-3 fatty acid family and harbors both anticancer and anti-inflammatory properties, which could improve 5-FU chemotherapy. Here, we demonstrate that DHA inhibits 5-FU-induced IL-1ß secretion and caspase-1 activity in a MDSC cell line (MSC-2). Accordingly, we showed that DHA-enriched diet reduces circulating IL-1ß concentration and tumor recurrence in 5-FU-treated tumor-bearing mice. Treatment with 5-FU led to JNK activation through ROS production in MDSC. JNK inhibitor SP600125 as well as DHA-mediated JNK inactivation decreased IL-1ß secretion. The repression of 5-FU-induced caspase-1 activity by DHA supplementation is partially due to ß-arrestin-2-dependent inhibition of NLRP3 inflammasome activity but was independent of JNK pathway. Interestingly, we showed that DHA, through ß-arrestin-2-mediated inhibition of JNK pathway, reduces V5-tagged mature IL-1ß release induced by 5-FU, in MDSC stably overexpressing a V5-tagged mature IL-1ß form. Finally, we found a negative correlation between DHA content in plasma and the induction of caspase-1 activity in HLA-DR- CD33+ CD15+ MDSC of patients treated with 5-FU-based chemotherapy, strongly suggesting that our data are clinical relevant. Together, these data provide new insights on the regulation of IL-1ß secretion by DHA and on its potential benefit in 5-FU-based chemotherapy.

Nlrp3 Gene Expression in Circulating Leukocytes Declines During Healthy Aging.

Aging is often associated with elevated levels of low grade inflammation supposed to drive age-associated diseases. Here, we conducted a cross-sectional study on 58 healthy volunteers, aged from 19 to 81, to investigate the relationship between age and the expression of three inflammasome component genes (Nlrp3, Asc, Casp1), the up-stream transcription factor NFkB, and the pro-inflammatory cytokine Il-1ß in leukocytes. We also assessed C-reactive protein (CRP) and IL-1ß in plasma, as additional inflammatory markers. We did not find any support to the hypothesis that inflammasone activation increases with age. Expression of Asc, Casp1, NFkB, and Il-1ß did not vary with age, body mass index (BMI), and CRP levels. In addition, expression did not differ between males and females or between smokers and non-smokers. A notable exception was the expression of Nlrp3 which varied non-linearly with age. Specifically, Nlrp3 expression strongly declined during aging, in subjects who were between 50 and 81 years old. CRP was higher in women and increased as a function of age-corrected BMI, while only four subjects showed detectable amount of IL-1ß in plasma. Further work on larger cohorts with a longitudinal monitoring should be conducted to corroborate the finding that healthy aging is associated with a decrease in inflammasome activation.

Protective effects of polyphenol-rich infusions from carob (Ceratonia siliqua) leaves and cladodes of Opuntia ficus-indica against inflammation associated with diet-induced obesity and DSS-induced colitis in Swiss mice.

In the present study, we have investigated the effects of polyphenol-rich infusions from carob leaves and OFI-cladodes on inflammation associated with obesity and dextran sulfate sodium (DSS)-induced ulcerative colitis in Swiss mice. In vitro studies revealed that aqueous extracts of carob leaves and OFI-cladodes exhibited anti-inflammatory properties marked by the inhibition of IL-6, TNF-α and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells concomitant with NF-κß nucleus translocation inhibition. For in vivo investigations, Swiss male mice were subjected to control or high fat diet (HFD). At the 8th week after the start of study, animals received or not 1% infusion of either carob leaves or OFI-cladode for 4 weeks and were subjected to 2% DSS administration in drinking water over last 7 days. After sacrifice, pro-inflammatory cytokines levels in plasma and their mRNA expression in different organs were determined. Results showed that carob leaf and OFI-cladode infusions reduced inflammation severity associated with HFD-induced obesity and DSS-induced acute colitis indicated by decrease in pro-inflammatory cytokines expression (as such TNF-α, IL1b and IL-6) in colon, adipose tissue and spleen. In addition, plasma levels of IL-6 and TNF-α were also curtailed in response to infusions treatment. Thus, carob leaf and OFI-cladode infusions prevented intestinal permeability through the restoration of tight junction proteins (Zo1, occludins) and immune homeostasis. Hence, the anti-inflammatory effect of carob leaves and OFI-cladodes could be attributed to their polyphenols which might alleviate inflammation severity associated with obesity and colitis.

Correction: Phenolic extract from oleaster (Olea europaea var. Sylvestris) leaves reduces colon cancer growth and induces caspase-dependent apoptosis in colon cancer cells via the mitochondrial apoptotic pathway.

[This corrects the article DOI: 10.1371/journal.pone.0170823.].

Phenolic extract from oleaster (Olea europaea var. Sylvestris) leaves reduces colon cancer growth and induces caspase-dependent apoptosis in colon cancer cells via the mitochondrial apoptotic pathway.

Dietary polyphenols, derived from natural products, have received a great interest for their chemopreventive properties against cancer. In this study, we investigated the effects of phenolic extract of the oleaster leaves (PEOL) on tumor growth in mouse model and on cell death in colon cancer cell lines. We assessed the effect of oleaster leaf infusion on HCT116 (human colon cancer cell line) xenograft growth in athymic nude mice. We observed that oleaster leaf polyphenol-rich infusion limited HCT116 tumor growth in vivo. Investigations of PEOL on two human CRC cell lines showed that PEOL induced apoptosis in HCT116 and HCT8 cells. We demonstrated an activation of caspase-3, -7 and -9 by PEOL and that pre-treatment with the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), prevented PEOL-induced cell death. We observed an involvement of the mitochondrial pathway in PEOL-induced apoptosis evidenced by reactive oxygen species (ROS) production, a decrease of mitochondrial membrane potential, and cytochrome c release. Increase in intracellular Ca2+ concentration induced by PEOL represents the early event involved in mitochondrial dysfunction, ROS-induced endoplasmic reticulum (ER) stress and apoptosis induced by PEOL, as ruthenium red, an inhibitor of mitochondrial calcium uptake inhibited apoptotic effect of PEOL, BAPTA/AM inhibited PEOL-induced ROS generation and finally, N-acetyl-L-cysteine reversed ER stress and apoptotic effect of PEOL. These results demonstrate that polyphenols from oleaster leaves might have a strong potential as chemopreventive agent in colorectal cancer.

Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRß subcellular localization.

Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRß-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRß. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRß with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRß in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRß and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRß, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRß could be a promising target in cancer therapy.

Trans-10, cis-12 conjugated linoleic acid induced cell death in human colon cancer cells through reactive oxygen species-mediated ER stress.

Dietary conjugated linoleic acids (CLA) are fatty acid isomers with anticancer activities produced naturally in ruminants or from vegetable oil processing. The anticancer effects of CLA differ upon the cancer origin and the CLA isomers. In this study, we carried out to precise the effects of CLA isomers, c9,t11 and t10,c12 CLA, on mechanisms of cell death induction in colon cancer cells. We first showed that only t10,c12 CLA treatment (25 and 50µM) for 72h triggered apoptosis in colon cancer cells without affecting viability of normal-derived colon epithelial cells. Exposure of colon cancer cells to t10,c12 CLA activated ER stress characterized by induction of eIF2α phoshorylation, splicing of Xbp1 mRNA and CHOP expression. Furthermore, we evidenced that inhibition of CHOP expression and JNK signaling decreased t10,c12 CLA-mediated cancer cell death. Finally, we showed that CHOP induction by t10,c12 CLA was dependent on ROS production and that the anti-oxidant N-acetyl-cysteine reduced CHOP induction-dependent cell death. These results highlight that t10,c12 CLA exerts its cytotoxic effect through ROS generation and a subsequent ER stress-dependent apoptosis in colon cancer cells.

Recent advances on stearoyl-Coa desaturase regulation in fatty liver diseases.

Stearoyl-CoA desaturase 1 (SCD-1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of monounsaturated fatty acids. Indeed, SCD-1 is the critical control point regulating hepatic lipogenesis and lipid oxidation. Due to its central role in lipid metabolism in the liver, recent studies have focused on the involvement of SCD-1 in the development of fatty liver during obesity, diabetes mellitus, hypertension, excessive alcohol consumption, and in subjects with high triglyceride blood concentrations. The accumulation of fat in liver cells can be a sign that harmful conditions are developing, possibly associated with or leading to inflammation of the liver. This review evaluates the recent advances in our understanding of the regulation of SCD-1 expression and its role in the development of nonalcoholic and alcoholic hepatosteatosis. Animal models presenting a liver-specific loss or inhibition of SCD-1, as well as dietary interventions, have highlighted the important role of the enzyme in the accumulation of fat (fatty infiltration) in hepatocytes during both alcoholic and nonalcoholic liver diseases. The data summarized in this article support the notion that SCD-1 plays a direct role in the development of fatty liver diseases, and is not simply a marker of an unfavorable diet or hepatic disorder. Accordingly, SCD-1 represents a promising therapeutic target for the treatment of hepatic steatosis.

High pancreatic n-3 fatty acids prevent STZ-induced diabetes in fat-1 mice: inflammatory pathway inhibition.

OBJECTIVE: Because of confounding factors, the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on type 1 diabetes remain to be clarified. We therefore evaluated whether fat-1 transgenic mice, a well-controlled experimental model endogenously synthesizing n-3 PUFA, were protected against streptozotocin (STZ)-induced diabetes. We then aimed to elucidate the in vivo response at the pancreatic level. RESEARCH DESIGN AND METHODS: ß-Cell destruction was produced by multiple low-doses STZ (MLD-STZ). Blood glucose level, plasma insulin level, and plasma lipid analysis were then performed. Pancreatic mRNA expression of cytokines, the monocyte chemoattractant protein, and GLUT2 were evaluated as well as pancreas nuclear factor (NF)-κB p65 and inhibitor of κB (IκB) protein expression. Insulin and cleaved caspase-3 immunostaining and lipidomic analysis were performed in the pancreas. RESULTS: STZ-induced fat-1 mice did not develop hyperglycemia compared with wild-type mice, and ß-cell destruction was prevented as evidenced by lack of histological pancreatic damage or reduced insulin level. The prevention of ß-cell destruction was associated with no proinflammatory cytokine induction (tumor necrosis factor-α, interleukin-1ß, inducible nitric oxide synthase) in the pancreas, a decreased NF-κB, and increased IκB pancreatic protein expression. In the fat-1-treated mice, proinflammatory arachidonic-derived mediators as prostaglandin E2 and 12-hydroxyeicosatetraenoic acid were decreased and the anti-inflammatory lipoxin A4 was detected. Moreover, the 18-hydroxyeicosapentaenoic acid, precursor of the anti-inflammatory resolvin E1, was highly increased. CONCLUSIONS: Collectively, these findings indicate that fat-1 mice were protected against MLD-STZ-induced diabetes and pointed out for the first time in vivo the beneficial effects of n-3 PUFA at the pancreatic level, on each step of the development of the pathology-inflammation, ß-cell damage-through cytokine response and lipid mediator production.

The metabolic cascade leading to eicosanoid precursors--desaturases, elongases, and phospholipases A2--is altered in Zucker fatty rats.

Metabolic syndrome characterized by insulin resistance and obesity is accompanied by severe lipid metabolism perturbations and chronic low-grade inflammation. However, many unresolved questions remained regarding the regulation that underlie dyslipidemia, particularly the regulation of the metabolic cascade (synthesis and release) leading to eicosanoid precursors release. This study was undertaken to investigate the regulation of desaturases/elongases and phospholipases A(2) during the establishment of metabolic syndrome. Our results showed that delta-6 desaturase as well as elongase-6 expressions were upregulated in 3-month-old Zucker fatty rats as compared to lean littermates, independently of SREBP-1c activation. We also demonstrated for the first time an increase of liver group VII phospholipase A(2) gene expression in the obese animals together with a strong specific inhibition of type IVA and VIA phospholipases A(2). These results suggest that the regulation of unsaturated fatty acids biosynthesis and signalling cascade could contribute to the development of liver lipid dysregulation related to metabolic syndrome and may be considered as new potential targets in such pathological conditions.

Inhibition of stearoyl-CoA desaturase 1 expression induces CHOP-dependent cell death in human cancer cells.

BACKGROUND: Cancer cells present a sustained de novo fatty acid synthesis with an increase of saturated and monounsaturated fatty acid (MUFA) production. This change in fatty acid metabolism is associated with overexpression of stearoyl-CoA desaturase 1 (Scd1), which catalyses the transformation of saturated fatty acids into monounsaturated fatty acids (e.g., oleic acid). Several reports demonstrated that inhibition of Scd1 led to the blocking of proliferation and induction of apoptosis in cancer cells. Nevertheless, mechanisms of cell death activation remain to be better understood. PRINCIPAL FINDINGS: In this study, we demonstrated that Scd1 extinction by siRNA triggered abolition of de novo MUFA synthesis in cancer and non-cancer cells. Scd1 inhibition-activated cell death was only observed in cancer cells with induction of caspase 3 activity and PARP-cleavage. Exogenous supplementation with oleic acid did not reverse the Scd1 ablation-mediated cell death. In addition, Scd1 depletion induced unfolded protein response (UPR) hallmarks such as Xbp1 mRNA splicing, phosphorylation of eIF2α and increase of CHOP expression. However, the chaperone GRP78 expression, another UPR hallmark, was not affected by Scd1 knockdown in these cancer cells indicating a peculiar UPR activation. Finally, we showed that CHOP induction participated to cell death activation by Scd1 extinction. Indeed, overexpression of dominant negative CHOP construct and extinction of CHOP partially restored viability in Scd1-depleted cancer cells. CONCLUSION: These results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.

Revisiting delta-6 desaturase regulation by C18 unsaturated fatty acids, depending on the nutritional status.

Dietary polyunsaturated fatty acids (PUFA) play a key role in regulating delta-6 desaturase (D6D), the key enzyme for long-chain PUFA biosynthesis. Nevertheless, the extent of their effects on this enzyme remains controversial and difficult to assess. It has been generally admitted that C18 unsaturated fatty acids (UFAs) regulate negatively delta-6 desaturase (D6D). This inhibition has been evidenced in regard to a high glucose/fat free (HG/FF) diet used in reference. However, several nutritional investigations did not evidence any inhibition of desaturases when feeding fatty acids. Because the choice of the basal diet appeared to be of primary importance in such experiments, our goal was to reconsider the specific role of dietary UFAs on D6D regulation, depending on nutritional conditions. For that, sixteen adult Wistar rats were fed purified linoleic acid, alpha-linolenic acid or oleic acid, included in one of two diets at 4% by weight: an HG/FF or a high starch base (HS) where the pure UFAs replaced a mixed vegetable oil. Our results showed first that D6D specific activity was significantly greater when measured in presence of an HG/FF than with an HS/4% vegetable oil diet. Secondly, we found that linoleic and alpha-linolenic acids added to HG/FF reduced the specific activity of D6D. In contrast, when pure UFAs were added to an HS base, D6D specific activities remained unchanged or increased. Concordant results were obtained on D6D mRNA expression. Altogether, this study evidenced the importance of the nutritional status in D6D regulation by C18 UFAs: when used as control, HG/FF diet stimulates D6D compared with a standard control diet containing starch and 4% fats, leading to an overestimation of the D6D regulation by UFAs. Then, UFAs should be considered as repressors for unsaturated fatty acid biosynthesis only in very specific nutritional conditions.

A new subfamily of structurally related human F-box proteins.

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.

Essential role of human CDT1 in DNA replication and chromatin licensing.

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.