Altérations métaboliques cellulaires (la voie de biosynthèse des acides gras monoinsaturés comme cible thérapeutique)

La stéaroyl Co-A désaturase (SCD) est l enzyme clé du métabolisme des acides gras mono-insaturés (AGMI). Son activité 9 désaturase introduit une double liaison cis en position 9 des acides gras saturés (AGS), formant des AGMI. Une altération de la voie de biosynthèse des AGMI est impliquée dans de nombreuses pathologies, telles que le cancer et les maladies cardiovasculaires. Les cellules cancéreuses présentent une synthèse de novo en acide gras accrue avec une accumulation d'AGMI. Ce changement dans le métabolisme des acides gras est associé à la surexpression de la SCD1. Plusieurs études ont démontré que l'inhibition de SCD1 conduit au blocage de la prolifération et l'induction de l'apoptose dans les cellules cancéreuses. Néanmoins, les mécanismes d'activation mort cellulaire restent à être mieux compris. Dans cette étude, nous avons démontré que l extinction de SCD1 par siRNA, inhibiteur synthétique ou naturel induit l abolition de la synthèse de novo AGMI dans les cellules cancéreuses ou non. L activation de la mort cellulaire par apoptose lors de l inhibition de SCD1 n est observée que dans les cellules cancéreuses. En outre, la déplétion en SCD1 induite un stress du réticulum endoplasmique, ces caractéristiques étant l épissage de l'ARNm XBP1, la phosphorylation de eIF2a et augmentation de l'expression CHOP. Toutefois, l'activation du stress du RE lors de l abolition de SCD1 est particuliers puisque nous ne mettons pas en évidence de modification de l expression de la protéine chaperonne GRP78, une autre caractéristique du stress du RE. Enfin, nous avons montré que l'induction de CHOP participe à l activation de la mort cellulaire lors de l extinction de SCD1. En effet, la surexpression de constructions dominants négatifs et anti-CHOP restaure partiellement la viabilité des cellules cancéreuses déplétées en SCD1. Pour conclure, ces résultats suggèrent que l inhibition de la synthèse de novo en AGMI via l extinction de SCD1 pourrait être une cible thérapeutique prometteuse contre le cancer en induisant la mort cellulaire par l activation de la voie du stress du réticulum endoplasmique et du facteur de transcription CHOP. Nous nous sommes également intéressés à la régulation de SCD par différents AGMI dans un modèle cellulaire en lien avec la pathologie athéromateuse. De nombreux facteurs de risque participent au développement de cette pathologie, parmi lesquels les acides gras trans (AGT). En effet, des études épidémiologiques ont mis en corrélation la consommation d AGT d origine industrielle et le risque de maladie cardiovasculaire. Les AGT pourraient jouer leurs effets athérogènes par l altération du métabolisme lipidiques des cellules vasculaires. L'accumulation de lipides dans les cellules musculaires lisses vasculaires (CML) est une caractéristique de l'athérosclérose et une conséquence de la lipogenèse accrue. L expression de la SCD est associée à l'induction de la lipogenèse et développement de l'athérosclérose. Nous nous sommes intéressés à la régulation de l'activité SCD1 dans les CML exposés à des isomères d AGMI en C18 [l acide cis-9oléique(OL), l'acide trans-11 vaccénique (TVA) ou l acide trans-9 élaïdique (ELA)]. Nous avons montré que la SCD, présente dans les CML était régulée différemment selon l isomère en C18 :1. En effet, nous observons une augmentation de l expression et de l activité de SCD1 sous l effet d un traitement par ELA et une diminution importante pour le traitement par OL. L effet du TVA sur l expression et l activité dans les CML reste modeste mais une diminution est néanmoins trouvée. Nous avons corrélé l'activité de SCD avec son niveau d'expression protéique. En effet, celle-ci est augmentée par l ELA et diminuée par l'OL. Cette régulation n est pas post-traductionnelle et l expression de SCD1 lors des traitements par l OL et l ELA est moduler au niveau transcriptionnel.Pour conclure, nous avons démontré une modulation de l'activité SCD par des AGMI (C18: 1) de configuration cis et [...]Stearoyl Co-A desaturase (SCD) is the key enzyme of the metabolism of mono-unsaturated fatty acids (MUFA). Its activity 9 desaturase introduces a double bond cis in position 9 of saturated fatty acids (SFA), induced formation of MUFA. Impaired biosynthesis of MUFA is involved in many diseases such as cancer and cardiovascular disease. Cancer cells have a de novo synthesis of fatty acids increased with an accumulation MUFA. This change in the metabolism of fatty acids is associated with overexpression SCD1 which catalyzes the conversion of saturated fatty acids, monounsaturated fat. Several reports have shown that inhibition of SCD1 leads to blockage of proliferation and induction of apoptosis in cancer cells. However, the mechanism of cell death activation remains to be understood. In this study, we demonstrated that the extinction of SCD1 by siRNA, synthetic or natural inhibitor induces the abolition of de novo MUFA synthesis in cancer cells or not. SCD1 inhibition activates cell death by apoptosis only in cancer cells. In addition, depletion of SCD1 induced endoplasmic reticulum stress, these features being XBP1 mRNA splicing, phosphorylation of eIF2a and increased expression of CHOP. However, activation of ER stress in the abolition of SCD1 is special because we do not show changes in expression of the chaperone protein GRP78, another characteristic of ER stress. Finally, we showed that induction of CHOP is involved in activation of cell death during shutdown of SCD1. Indeed, overexpression of dominant negative constructs and anti-CHOP partially restores the viability of cancer cells depleted of SCD1. In conclusion, these results suggest that inhibition of de novo synthesis of MUFA through the extinction of SCD1 could be a promising therapeutic target against cancer by inducing cell death through the activation of the stress and endoplasmic reticulum transcription factor CHOP. We are also interested in the regulation of SCD by different MUFA in a cellular model linked with atherosclerotic disease. Many risk factors contribute to the development of this disease, including trans fatty acid (TFA). Indeed, epidemiological studies have correlated the consumption of TFA from industrial sources and the risk of cardiovascular disease. TFA could play their atherogenic effects by altering the lipid metabolism of vascular cells. The accumulation of lipids in vascular smooth muscle cells (SMC) is a feature of atherosclerosis and a consequence of increased lipogenesis. The expression of SCD is associated with the induction of lipogenesis and development of atherosclerosis. We are interested in the regulation of SCD1 activity in SMCs exposed to isomers of MUFA C18 [cis-9 oleic (OL), trans-11 vaccenic acid (TVA) and acid trans -9 elaidic (ELA)]. We showed that SCD which is present in SMC was regulated differently depending on the isomer C18: 1. Indeed, we observed an increase in the expression and activity of SCD1 as a result of treatment with ELA and a significant decrease for treatment with OL. The effect of the TVA on the expression and activity in SMCs remains modest decrease is nevertheless found. We correlated the activity of SCD with its level of protein expression. Indeed, it is increased by the ELA and decreased by OL. This regulation is posttranslational and expression of SCD1 during treatment with the OL and the ELA is modulated at the transcriptional level. To conclude, we demonstrated a modulation of SCD activity by MUFA (C18: 1) cis and trans-mediated regulation of SCD1 gene transcription.DIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

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

Scd1 depletion did not affect viability of non cancer cells.

<p>Normal human dermal fibroblasts (NHDF) were transfected with siRNA control (scr) and siRNA targeting Scd1 (Scd1.A and Scd1.B). NHDF cells were harvested after 72 h siRNA treatment and harvested for further analyses. <b>A</b>) Total proteins were prepared for Scd1 expression analysis by western-blotting. <b>B</b>) NHDF treated 72 h with siRNA were incubated for further 6 h with [¹⁴C] stearic acid and total lipid extraction was carried out. Conversion of [¹⁴C] stearic acid into oleic acid was performed by HPLC. Scd activity was expressed as the % ratio of [¹⁴C] oleic acid to [¹⁴C] oleic and stearic acids. Values represent the mean ± SEM for at least two separate experiments. <b>C</b>) Proliferation status of siRNA-treated NHDF was determined at indicated time by the CyQuant® proliferation assay. Each value is the mean of relative fluorescence units ± SEM of three independent experiments. <b>D</b>) NHDF were collected 72 h post-transfection and total cell death analysis was performed by flow cytometry after staining with propidium iodide. Results were the mean ± SEM of PI positive cells (%) of one representative from two experiments performed in triplicate. *, p<0.05 vs. siRNA scr-treated cells by Anova analysis followed by Tuckey test.</p

Scd1 knockdown promoted apoptosis-cell death.

<p><b>A</b>) U2OS cells were treated with siRNA scr (control) and targeting Scd1 (Scd1.A and Scd1.B), and collected 24 h, 48 h or 72 h post-transfection. Proliferation status was determined by the CyQuant® proliferation assay. Each value is the mean of relative fluorescence units ± SEM of triplicate and representative of three independent experiments. <b>B</b>) U2OS and SW480 cells were cultured for 72 h post-siRNA transfection and harvested for trypan blue dye exclusion assay. Values are the mean ± SEM of triplicate and representative of two others independent experiments. <b>C</b>) U2OS ad SW480 cells treated 72 h with siRNA were collected and total cell death was analysed by flow cytometry after staining with propidium iodide (PI). Data represent the mean ± SEM of three independent experiments. <b>D</b>) U2OS cells were treated for 48 h with Scd1 inhibitors at indicated concentrations and were harvested for propidium iodide staining. Data represent the mean ± SEM from three experiments. <b>E</b>) U2OS cells were collected 72 h post-transfection and prepared for caspase 3 activity measurement by flow cytometry as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014363#s4" target="_blank">materials and methods</a>. Data are shown as fold increase over the control (siRNA scr) and represent the mean ± SEM of two independent experiments. <b>F</b>) Whole-cell lysates were prepared 72 h post-transfection with siRNA and PARP cleavage (c-PARP) level was determined by Western-blot. <b>G</b>) U2OS cells were treated for 72 h with siRNA control (scr) and targeting Scd1 in absence (vehicle) or presence of 100 µM oleic acid bound to BSA. Cell number was quantified by CyQuant® proliferation assay as previously described. Data are shown as fold change over the vehicle siRNA scr-treated cells and represent the mean of relative fluorescence units ± SEM of triplicate. *, ** p<0.05 vs. siRNA scr-treated vehicle and oleic acid cells, respectively, by Anova analysis followed by Tuckey test.</p

Inhibition of Scd1 partially induced UPR markers.

<p>U2OS and SW480 cells were treated with siRNA control (scr) and siRNA against Scd1 for 72h. <b>A</b>) Samples were prepared for mRNA analyses of Xbp1 processing by semi-quantitative RT-PCR. The PCR products were run on a 3% agarose gel and the spliced Xbp1 (sXbp1), unspliced Xbp1 (uXbp1) and hybrid Xbp1 (hXbp1) mRNA species were observed in siRNA-treated cells (ctr) and thapsigargine-treated cells (Tg) as positive control of UPR activation. <b>B</b>) Total protein lysates were prepared from untreated and thapsigargin-treated cells (Tg, 0.2 µM, 16 h) and analysed for eiF2α phosphorylation and GRP78 up-regulation by western-blotting. <b>C and D</b>) Scd1-depleted U2OS and SW480 cells were prepared as in 3B) and analysed by western-blotting for Scd1, GRP78 and phospho-eiF2α expression. <b>E</b>) U2OS cells were treated with 5 and 10 µM of Scd1 inhibitors (MF-438 and CVT-11127) and were collected after 10 h and 24 h of treatment for phospho-eiF2α expression analysis by western-blotting. Blots are representative of at least two independent experiments.</p

Inhibition of CHOP activity reduced siRNA Scd1-mediated U2OS cell death.

<p><b>A</b>) U2OS and SW480 cells were treated with siRNA control (scr) and siRNA against Scd1 for 72h. Total RNA was isolated and CHOP mRNA expression normalized to β-actin mRNA expression was quantified by real time RT-PCR. Results were represented as mean fold induction ± SEM relative to siRNA scr-treated cells from at least three independent experiments. *, p<0.05 vs. siRNA scr-treated cells by Anova analysis followed by Tuckey test. <b>B</b>) Nuclear extracts from U2OS were prepared 72 h after siRNA addition. CHOP expression was analysed by western-blotting and lamin A/C was loading control of nuclear extract samples. <b>C</b>) U2OS cells were transiently transfected with empty (ctr) or dominant-negative CHOP (DN-CHOP) expression vector and selected for three days in G418. Resistant cells transfected by ctr or DN-CHOP construct were treated by siRNA control scr or against Scd1 (Scd1.A and Scd1.B) for 72 h and harvested for propidium iodide staining analysis by flow cytometry. Values were shown as fold increase over the controls (siRNA scr) and represent the mean ± SEM of two independent experiments. *, **, p<0.05 vs. siRNA scr-treated ctr and DN-CHOP cells, respectively, by Anova analysis followed by Tuckey test. #, p<0.05 by Anova analysis followed by Tuckey test. <b>D</b>) U2OS cells were prepared as C) and harvested for active caspase 3 analysis by flow cytometry. Values were shown as fold increase over the controls (siRNA scr) and represent the mean ± SEM of three independent experiments. *, **, p<0.05 vs. siRNA scr-treated ctr and DN-CHOP cells, respectively, by Anova analysis followed by Tuckey test. #, p<0.05 by Anova analysis followed by Tuckey test.</p

Efficient suppression of Scd1 expression and activity by siRNA targeting Scd1.

<p><b>A</b>) U2OS cells were transfected with siRNA control (scr) or with siRNA against Scd1 (Scd1.A and Scd1.B) and collected 24 and 48 h post-transfection for Scd1 mRNA expression by real time RT-PCR. Scd1 mRNA expression was normalized to β-actin expression. Values represent the mean ± SEM relative to Scd1 mRNA expression in scr-treated U2OS cells at 24 h. *, p<0.05 vs. siRNA scr-treated cells by Anova analysis followed by Tuckey test. <b>B</b>) U2OS and SW480 cells were treated with oligofectamine (-), siRNA control (scr) or with siRNA against Scd1 (Scd1.A and Scd1.B). Cells were collected 24 h after transfection for Scd1 expression analysis by Western-Blotting. <b>C</b>) U2OS and SW480 cells treated 72 h with siRNA were incubated for further 6 h with [¹⁴C] stearic acid and total lipid extraction was performed. Scd activity was evaluated by HPLC as the rate of [¹⁴C] stearic acid conversion into [¹⁴C] oleic acid in cells treated with siRNA for 72 h. Scd activity was expressed as the % ratio of [¹⁴C] oleic acid to [¹⁴C] oleic and stearic acids. Values represent the mean ± SEM from at least two separate experiments. *, p<0.05 vs. siRNA scr-treated cells by Anova analysis followed by Tuckey test. A representative expression of Scd1 protein was shown for 72 h of siRNA treatment. D) U2OS cells were exposed to DMSO as vehicle, Scd1 inhibitors (CVT-11127 or MF-438) at 10 µM for 24 h and prepared as above C) for measuring Scd activity. Values represent the mean ± SEM from three experiments. *, p<0.05 vs. vehicle-treated cells by Anova analysis followed by Tuckey test.</p

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

Abstract 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-β receptors mediated by a lipid-droplet accumulation-dependent Smad2 acetylation. As expected, inhibition of TGF-β 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-β receptors, cell invasion, and lung metastasis triggered by Elovl5 knockdown. Altogether, we showed that Elovl5 is involved in metastasis through lipid droplets-regulated TGF-β receptor expression and is a predictive biomarker of metastatic ER + breast cancer

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

Abstract 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-β receptors mediated by a lipid-droplet accumulation-dependent Smad2 acetylation. As expected, inhibition of TGF-β 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-β receptors, cell invasion, and lung metastasis triggered by Elovl5 knockdown. Altogether, we showed that Elovl5 is involved in metastasis through lipid droplets-regulated TGF-β receptor expression and is a predictive biomarker of metastatic ER + breast cancer