Anissa Belkaid

The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression

BackgroundChlorogenic acid (CHL), the most potent functional inhibitor of the microsomal glucose-6-phosphate translocase (G6PT), is thought to possess cancer chemopreventive properties. It is not known, however, whether any G6PT functions are involved in tumorigenesis. We investigated the effects of CHL and the potential role of G6PT in regulating the invasive phenotype of brain tumor-derived glioma cells.ResultsRT-PCR was used to show that, among the adult and pediatric brain tumor-derived cells tested, U-87 glioma cells expressed the highest levels of G6PT mRNA. U-87 cells lacked the microsomal catalytic subunit glucose-6-phosphatase (G6Pase)-α but expressed G6Pase-β which, when coupled to G6PT, allows G6P hydrolysis into glucose to occur in non-glyconeogenic tissues such as brain. CHL inhibited U-87 cell migration and matrix metalloproteinase (MMP)-2 secretion, two prerequisites for tumor cell invasion. Moreover, CHL also inhibited cell migration induced by sphingosine-1-phosphate (S1P), a potent mitogen for glioblastoma multiform cells, as well as the rapid, S1P-induced extracellular signal-regulated protein kinase phosphorylation potentially mediated through intracellular calcium mobilization, suggesting that G6PT may also perform crucial functions in regulating intracellular signalling. Overexpression of the recombinant G6PT protein induced U-87 glioma cell migration that was, in turn, antagonized by CHL. MMP-2 secretion was also inhibited by the adenosine triphosphate (ATP)-depleting agents 2-deoxyglucose and 5-thioglucose, a mechanism that may inhibit ATP-mediated calcium sequestration by G6PT.ConclusionWe illustrate a new G6PT function in glioma cells that could regulate the intracellular signalling and invasive phenotype of brain tumor cells, and that can be targeted by the anticancer properties of CHL.

Silencing of the human microsomal glucose‐6‐phosphate translocase induces glioma cell death: Potential new anticancer target for curcumin

G6P translocase (G6PT) is thought to play a crucial role in transducing intracellular signaling events in brain tumor‐derived cancer cells. In this report, we investigated the contribution of G6PT to the control of U‐87 brain tumor‐derived glioma cell survival using small interfering RNA (siRNA)‐mediated suppression of G6PT. Three siRNA constructs were generated and found to suppress up to 91% G6PT gene expression. Flow cytometry analysis of propidium iodide/annexin‐V‐stained cells indicated that silencing the G6PT gene induced necrosis and late apoptosis. The anticancer agent curcumin, also inhibited G6PT gene expression by more than 90% and triggered U‐87 glioma cells death. Overexpression of recombinant G6PT rescued the cells from curcumin‐induced cell death. Targeting G6PT expression may provide a new mechanistic rationale for the action of chemopreventive drugs and lead to the development of new anti‐cancer strategies.

Development and Experimental Validation of a 20K Atlantic Cod (Gadus morhua) Oligonucleotide Microarray Based on a Collection of over 150,000 ESTs

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

1H NMR metabolomics combined with gene expression analysis for the determination of major metabolic differences between subtypes of breast cell lines

1H NMR analysis was performed on metabolic extracts from a selection of six breast cell lines, including normal-immortalized, invasive ductal carcinomas and adenocarcinomas. Metabolites with significant concentration differences between normal and cancerous cells as well as ER+ and ER− (estrogen receptor) cells were determined and their relation to the differentially expressed genes was explored. Major differences have been shown for many amino acids and this was linked to expression level changes of related genes. Observed changes in choline concentration were connected to expression level changes of the SCL44A1 transporter gene.

17β-estradiol-induced ACSL4 protein expression promotes an invasive phenotype in estrogen receptor positive mammary carcinoma cells

Long chain acyl-CoA synthase-4 (ACSL4) expression has been associated with an aggressive phenotype in breast carcinoma cells, whereas its role in ERα-positive breast cancer has not been studied. ACSL4 prefers 20-carbon polyunsaturated fatty acid (PUFA) substrates, and along with other ACSLs has been associated with cellular uptake of exogenous fatty acids. 17β-estradiol induces proliferation and invasive capacities in ERα+ve breast carcinoma that is associated with modifications of cellular lipid metabolism. In this study, treatment of steroid-starved ERα-positive MCF-7 and T47D mammary carcinoma cells with 17β-estradiol resulted in increased cellular uptake of the PUFA arachidonic acid (AA) and eicosapentaenoic acid (EPA), important building blocks for cellular membranes, and increased ACSL4 protein levels. There was no change in the expression of the ACSL1, ACSL3 and ACSL6 protein isotypes. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life compared to untreated cells. ERα silencing reversed the impact of 17β-estradiol on ACSL4 protein levels and half-life. Silencing of ACSL4 eliminated the 17β-estradiol-induced increase in AA and EPA uptake, as well as the 17β-estradiol-induced cell migration, proliferation and invasion capacities. ASCL4 silencing also prevented the 17β-estradiol induced increases in p-Akt and p-GSK3β, and decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. Taken together, these results demonstrate that ACSL4 is a target of 17β-estradiol-stimulated ERα and is required for the cellular uptake of exogenous PUFA and the manifestation of a more malignant phenotype in ERα+ve breast carcinoma cells.

Metabolic Effect of Estrogen Receptor Agonists on Breast Cancer Cells in the Presence or Absence of Carbonic Anhydrase Inhibitors

Metabolic shift is one of the major hallmarks of cancer development. Estrogen receptor (ER) activity has a profound effect on breast cancer cell growth through a number of metabolic changes driven by its effect on transcription of several enzymes, including carbonic anhydrases, Stearoyl-CoA desaturase-1, and oncogenes including HER2. Thus, estrogen receptor activators can be expected to lead to the modulation of cell metabolism in estrogen receptor positive cells. In this work we have investigated the effect of 17β-estradiol, an ER activator, and ferulic acid, a carbonic anhydrase inhibitor, as well as ER activator, in the absence and in the presence of the carbonic anhydrase inhibitor acetazolamide on the metabolism of MCF7 cells and MCF7 cells, stably transfected to express HER2 (MCF7HER2). Metabolic profiles were studied using 1D and 2D metabolomic Nuclear Magnetic Resonance (NMR) experiments, combined with the identification and quantification of metabolites, and the annotation of the results in the context of biochemical pathways. Overall changes in hydrophilic metabolites were largest following treatment of MCF7 and MC7HER2 cells with 17β-estradiol. However, the carbonic anhydrase inhibitor acetazolamide had the largest effect on the profile of lipophilic metabolites.

Abstract 4419: HER2 expression and 17β-estradiol induce ACSL4 expression in estrogen receptor positive mammary carcinoma cells

Breast cancer is a multifactorial disease involving several molecular changes and a high proliferation rate often under the activation of hormonal receptors. Therefore, cancer cells require the metabolic machinery for membrane synthesis and to promote signaling pathways. Amongst biomolecules required for efficient cell growth are polyunsaturated fatty acids, like arachidonic acid, that are essential nutrients whose cellular uptake is partly governed by long chain acyl-CoA synthetases (ACSL). In this study, we investigated the impact of 17β-estradiol and Human Epidermal growth factor Receptor 2 (HER2) on cellular uptake of polyunsaturated fatty acids and on the expression of ACSLs. The administration of 17β-estradiol in steroid-starved MCF-7 and T47D mammary carcinoma cells induced a significant increase in cellular uptake of arachidonic acid (AA) and eicosapentaenoic acid (EPA), and in ACSL4 protein content (p Citation Format: Maroua Mbarik, Anissa Belkaid, Marc E. Surette. HER2 expression and 17β-estradiol induce ACSL4 expression in estrogen receptor positive mammary carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4419. doi:10.1158/1538-7445.AM2017-4419

Abstract A09: ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines

Polyunsaturated fatty acids (PUFA) are important building blocks for cellular membranes and are incorporated into membrane glycerophospholipids to sustain many cellular functions. Newly absorbed PUFA like arachidonic acid (AA) and eicosapentaenoic acid (EPA) are rapidly transformed to acyl-CoAs by members of the acyl-CoA synthetase family (ACSL1, 3, 4, 5 and 6) prior to their incorporation into membrane phospholipids. However, there is little information on the impact of estrogen on PUFA uptake and ACSL expression in estrogen receptor-positive (ER+ve) breast cancer cells. In the current study, the effect of 17 beta-estradiol on PUFA uptake and ACSL expression was measured in (ER+ve) mammary carcinomas cell lines. Stimulation of estrogen-starved ER+ve MCF-7 and T47D mammary carcinomas cells with 17 beta-estradiol resulted in a significant increase in the cellular uptake of exogenous AA and EPA. This was accompanied by a three-fold increase in the expression the ACSL4 isoform measured by western blot in both MCF-7 and T47D cells, but not in immortalized MCF-10A mammary epithelial cells. There was no measured change in the expression of the other ACSL isoforms. ER alpha silencing with siRNA reversed the 17 beta-estradiol-induced ACSL4 expression in both cells lines showing for the first time that hormonal stimulation of ER+ve breast cancer cells is a driver for ACSL4 expression. However, ACSL4 mRNA levels measured by qPCR were unchanged by 17 beta-estradiol treatment, and no change in mRNA stability was measured with 17beta-estradiol treatment as assessed in actinomycin D-treated cells. Conversely, the half-life of ACSL4 protein after treatment with 17 beta-estradiol was extended from 8±0.5 hours in untreated cells to 26±2 hours in 17 beta-estradiol treated cells as assessed following incubation in the absence or presence of cycloheximide. ER alpha silencing with specific siRNA reversed this half-life extension. SHP2 is a tyrosine phosphatase that regulates ACSL4 expression, however the investigation of possible SHP2-mediated changes in ACSL4 in 17 beta-estradiol-treated cells incubated or not with the SHP2 inhibitor NSC87877 showed no difference in ACSL4 expression. Importantly, ACSL4 silencing with siRNAs reversed the 17 beta-estradiol-induced uptake of AA and EPA in both MCF-7 and T47D cells, indicating that ACSL4 was responsible for the enhanced uptake of these PUFA in ER+ve breast cancer cells. Moreover, ACSL4 silencing completely reversed 17 beta-estradiol-induced cell migration as assessed by an in vitro wound healing assay. Overall, these results demonstrate that 17 beta-estradiol enhances the ability of ER+ve mammary carcinoma cells to incorporate PUFA that are important membrane building blocks, via the induction of ACSL4 protein stability. The requirement of enhanced ASCL4 expression for 17 beta-estradiol-induced cell migration suggests that it is a potential therapeutic target in ER+ve mammary carcinoma cells. This work was supported by the Canadian Breast Cancer Foundation, the Canadian Institutes of Health Research and the Canada Research Chairs Program. Citation Format: Anissa Belkaid, Rodney J. Ouellette, Marc E. Surette. ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A09.

Abstract 1706: Estrogen induces stearoyl-CoA desaturase expression in human MCF-7 breast carcinoma cells.

Approximately 95% of breast cancers are estrogen-dependant. Estrogen plays a crucial role in many cellular processes in estrogen-sensitive breast cancer such as carcinogenesis, production of growth factors, angiogenesis and growth stimulation. To sustain cell growth, many cancer cells exhibit an altered metabolism that is characterized by increased glycolysis and lipogenesis. We observed for the first time an up regulation of stearoyl-CoA desaturase-1 (SCD1) in response to estrogen in the hormone-dependent MCF7 human breast carcinoma cell line. SCD1 is a key enzyme in the production of monounsaturated fatty acids that are essential for membrane biogenesis. Using estrogen-starved MCF7 cells, treatment with 2 nM of estrogen induced cell proliferation, increased SCD-1 gene expression measured by qPCR (2.6±0.1 fold increase, p Overall, these results show for the first time that estrogen induces SCD-1 expression in breast carcinoma cells and suggests a crucial role for SCD1 in estrogen dependent cancer cell proliferation. SCD1 may therefore represent a potential therapeutic target for estrogen-dependent cancer. (This work was supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program). Citation Format: Anissa Belkaid, Rodney J. Ouellette, Marc E. Surette. Estrogen induces stearoyl-CoA desaturase expression in human MCF-7 breast carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1706. doi:10.1158/1538-7445.AM2013-1706