Stephan Chevalier

Fatty acids and breast cancer: sensitization to treatments and prevention of metastatic re-growth.

Lifestyle and nutritional factors have been recognized to influence breast cancer survival, irrespective of genomic alterations that are the hallmarks of the disease. The biological and molecular mechanisms involved in the effects of dietary polyunsaturated fatty acids and breast cancer response to treatments in clinical and preclinical studies have been reviewed. Among nutrients, rumenic acid, a naturally occurring CLA isomer and n-3 docosahexaenoic acid (DHA) a highly unsaturated fatty acid, have emerged due to their potential to increase cancer treatment efficacy without additional side effects. In this review, we analyze the literature evidence that breast cancer treatment and outcome could be improved through an adjuvant dietary supplementation. Such an original approach would involve two successive phases of breast cancer treatment: an initial sensitization of residual tumor cells to chemotherapy and to radiation therapy with dietary DHA; then a prevention of metastatic re-growth with a prolonged rumenic acid supplementation. Safety is not anticipated to be a critical issue, although it has to be assessed in the long term. Dietary supplements, used in combination to anti-cancer agents, should be provided under medical prescription. Such an original use of fatty acids in breast cancer treatment could provide the lipid field with a new avenue to impact public health.

Exploration of global gene expression in human liver steatosis by high-density oligonucleotide microarray.

Understanding the molecular mechanisms underlying fatty liver disease (FLD) in humans is of major importance. We used high-density oligonucleotide microarrays (22.3 K) to assess the mechanisms responsible for the development of human liver steatosis. We compared global gene expression in normal (n=9) and steatotic (n=9) livers without histological signs of inflammation or fibrosis. A total of 34 additional human samples including normal (n=11), steatosis (n=11), HCV-related steatosis (n=4) or steatohepatitis associated with alcohol consumption (n=4) or obesity (n=4) were used for immunohistochemistry or quantitative real-time PCR studies. With unsupervised classification (no gene selection), all steatotic liver samples clustered together. Using step-down maxT multiple testing procedure for controlling the Family-Wise Error-Rate at level 5%, 110 cDNAs (100 over- and 10 underexpressed) were found to be differentially expressed in steatotic and normal livers. Of them were genes involved in mitochondrial phosphorylative and oxidative metabolism. The mean ratio of mitochondrial DNA to nuclear DNA content was higher in liver steatosis compared to normal liver biopsies (1.12±0.14 vs 0.67±0.10; P=0.01). An increased expression of genes involved in inflammation (IL-1R family, TGFB) was also observed and confirmed by quantitative RT-PCR or immunochemistry. In steatohepatitis, an increase of the protein expression of mitochondrial antigens, IL-1R1, IGF2 and TGFB1 was also observed, interleukin 1 receptor being always strongly expressed in steatohepatitis linked to alcohol or obesity. In conclusion, mitochondrial alterations play a major role in the development of steatosis per se. Activation of inflammatory pathways is present at a very early stage of steatosis, even if no morphological sign of inflammation is observed.

NaV1.5 Na+ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia

Summary The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na+ channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na+/H+ exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4–7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Prospective evaluation of blood concentration of mitochondrial DNA as a marker of toxicity in 157 consecutively recruited untreated or HAART-treated HIV-positive patients.

Highly active antiretroviral therapy (HAART) can cause mitochondrial toxicity. The concentration of mitochondrial DNA (mtDNA) in peripheral blood cells has been reported to be a marker of this toxicity. However, these observations are controversial and were drawn from small series. Thus, we analysed the value of blood mtDNA as a marker of mitochondrial toxicity in a large cohort of human immunodeficiency virus (HIV)-infected out-patients during routine clinical evaluations. Real-time quantitative PCR was used to determine the mtDNA to nuclear DNA (nDNA) ratio in peripheral blood mononuclear cells from 157 consecutive HIV-1-infected patients (13 naive, 144 receiving HAART) and 30 HIV-1-uninfected patients. The mtDNA to nDNA ratio was significantly lower in both groups of HIV-infected patients than in the control group. No significant difference was observed between treated and naive HIV-infected patients. Lactataemia was significantly lower in controls than in the group of HIV-treated patients. None of the treated patients had lactataemia >5 mmol/l or bicarbonates <20 mmol/l. Triglyceride levels were significantly higher in the HAART-treated patients than in the nontreated patients. Clinical symptoms of lipodystrophy were observed in 62 HAART-treated patients. These symptoms were not associated with an abnormal mtDNA to nDNA ratio or plasma triglyceride concentration. The mtDNA to nDNA ratio was lower in DDI/D4T-treated patients than in AZT/3TC-treated patients. In conclusion, there are no obvious links between the mtDNA to nDNA ratio in peripheral mononuclear cells and any clinical symptoms or lactate level. Thus, the mtDNA to nDNA ratio in leukocytes does not seem to be an accurate marker of mild and/or long-term mitochondrial toxicity.

Efficiency of oxidative phosphorylation in liver mitochondria is decreased in a rat model of peritoneal carcinosis.

BACKGROUND & AIMS Cancer cachexia is a dynamic process characterized by a negative energy balance induced by anorexia and hypermetabolism. The mechanisms leading to hypermetabolism are not totally elucidated. This study examines the efficiency of oxidative phosphorylation and energy wasting in liver mitochondria isolated from rats with cancer cachexia induced by peritoneal carcinosis (PC). METHODS PC was generated by an intraperitoneal injection of cancer cells (PROb) in BDIX rats. The efficiency of oxidative phosphorylation and energy wasting as well as the role played by reactive oxygen species (ROS) and cardiolipin (mitochondrial inner membrane phospholipid) in these processes were assessed in liver mitochondria of PC and pair-fed control rats. RESULTS The efficiency of oxidative phosphorylation decreased (-26%) while energy wasting increased (+22%) in liver mitochondria from PC compared to control rats. The increased energy wasting was associated with a higher cardiolipin content (+55%, p<0.05; R(2)=0.64, p<0.05) and with a lower n-6/n-3 polyunsaturated fatty acid ratio in cardiolipin (-45%, p<0.05; R(2)=0.21, p<0.05) in PC rats. ROS production was increased by 12-fold in liver mitochondria from PC rats. CONCLUSIONS The efficiency of ATP synthesis was reduced and energy wasting processes were increased in liver mitochondria of PC rats. This suggests that liver mitochondria from PC rats request more nutrients than liver mitochondria from control rats to maintain the same ATP production. These alterations were associated to the content and fatty acid composition of cardiolipin.

SR-BI: Linking Cholesterol and Lipoprotein Metabolism with Breast and Prostate Cancer.

Studies have demonstrated the significant role of cholesterol and lipoprotein metabolism in the progression of cancer. The SCARB1 gene encodes the scavenger receptor class B type I (SR-BI), which is an 82-kDa glycoprotein with two transmembrane domains separated by a large extracellular loop. SR-BI plays an important role in the regulation of cholesterol exchange between cells and high-density lipoproteins. Accordingly, hepatic SR-BI has been shown to play an essential role in the regulation of the reverse cholesterol transport pathway, which promotes the removal and excretion of excess body cholesterol. In the context of atherosclerosis, SR-BI has been implicated in the regulation of intracellular signaling, lipid accumulation, foam cell formation, and cellular apoptosis. Furthermore, since lipid metabolism is a relevant target for cancer treatment, recent studies have focused on examining the role of SR-BI in this pathology. While signaling pathways have initially been explored in non-tumoral cells, studies with cancer cells have now demonstrated SR-BIs function in tumor progression. In this review, we will discuss the role of SR-BI during tumor development and malignant progression. In addition, we will provide insights into the transcriptional and post-transcriptional regulation of the SCARB1 gene. Overall, studying the role of SR-BI in tumor development and progression should allow us to gain useful information for the development of new therapeutic strategies.

Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells

Cardiolipin (CL) is a unique mitochondrial phospholipid potentially affecting many aspects of mitochondrial function/processes, i.e. energy production through oxidative phosphorylation. Most data focusing on implication of CL content and mitochondrial bioenergetics were performed in yeast or in cellular models of Barth syndrome. Previous work reported that increase in CL content leads to decrease in liver mitochondrial ATP synthesis yield. Therefore the aim of this study was to determine the effects of moderate decrease in CL content on mitochondrial bioenergetics in human hepatocytes. For this purpose, we generated a cardiolipin synthase knockdown (shCLS) in HepaRG hepatoma cells showing bioenergetics features similar to primary human hepatocytes. shCLS cells exhibited a 55% reduction in CLS gene and a 40% decrease in protein expression resulting in a 45% lower content in CL compared to control (shCTL) cells. Oxygen consumption was significantly reduced in shCLS cells compared to shCTL regardless of substrate used and energy state analyzed. Mitochondrial low molecular weight supercomplex content was higher in shCLS cells (+60%) compared to shCTL. Significant fragmentation of the mitochondrial network was observed in shCLS cells compared to shCTL cells. Surprisingly, mitochondrial ATP synthesis was unchanged in shCLS compared to shCTL cells but exhibited a higher ATP:O ratio (+46%) in shCLS cells. Our results suggest that lowered respiratory chain activity induced by moderate reduction in CL content may be due to both destabilization of supercomplexes and mitochondrial network fragmentation. In addition, CL content may regulate mitochondrial ATP synthesis yield.

Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways

Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.

PPARβ mRNA expression, reduced by n − 3 PUFA diet in mammary tumor, controls breast cancer cell growth

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as docosahexaenoic acid (DHA, 22:6n-3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n-3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n-3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.

Cardiolipin content is involved in liver mitochondrial energy wasting associated with cancer-induced cachexia without the involvement of adenine nucleotide translocase

Cancer-induced cachexia describes the progressive skeletal muscle wasting associated with many cancers leading to shortened survival time in cancer patients. We previously reported that cardiolipin content and energy-wasting processes were both increased in liver mitochondria in a rat model of peritoneal carcinosis (PC)-induced cachexia. To increase the understanding of the cellular biology of cancer cachexia, we investigated the involvement of adenine nucleotide translocator (ANT) in mitochondrial energy-wasting processes in liver mitochondria of PC and pair-fed control rats and its interactions with cardiolipin in isolated liver mitochondria from healthy rats exposed to cardiolipin-enriched liposomes. We showed in this study that functional ANT content was decreased in liver mitochondria from PC rats but without any effects on the efficiency of ATP synthesis. Moreover, non-phosphorylating energy wasting was not affected by saturating concentrations of carboxyatractylate (CAT), a potent inhibitor of ANT, in liver mitochondria from PC rats. Decreased efficiency of ATP synthesis was found in normal liver mitochondria exposed to cardiolipin-enriched liposomes, with increased non-phosphorylating energy wasting, thus mimicking mitochondria from PC rats. However, the functional ANT content in these cardiolipin-enriched mitochondria was unchanged, although non-phosphorylating energy wasting was reduced by CAT-induced inhibition of ANT. Finally, non-phosphorylating energy wasting was increased in cardiolipin-enriched mitochondria with substrates for complexes 1 and 2, but not for complex 4. In conclusion, increased energy wasting measured in liver mitochondria from rats with cancer cachexia is dependent on cardiolipin but independent of ANT. Interactions between ANT and cardiolipin are modified when cancer cachexia occurs.