β-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells

Abstract

β-catenin plays an important role as regulatory hub in several cellular processes including cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved by its dual role as structural component of cadherin-based adherens junctions, and as a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins are differentially regulated via β-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after β-catenin knockdown in the breast cancer cell line MCF-7. We used a label free analysis to compare trypsin-digested proteins from CTR (shCTR) and β-catenin knockout cells (shβcat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of β-catenin induced morphological changes and a significant modulation of the expression levels of proteins associated with primary metabolic processes. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas proteins associated to lipid metabolism were found up-regulated in shβcat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in shβcat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in shβcat cells. β-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, β-catenin knockout cells showed increased incorporation of [1-¹⁴C]acetate and decreased utilization of [U-¹⁴C]glucose for fatty acid synthesis. Our data highlight a role of β-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

Keywords: LC-MS/MS; MYC; lipid metabolism; mitochondria; proteomics; β-catenin.

Figure 1

Evaluation of β-catenin silencing efficiency. β-catenin (β-cat) silencing efficiency was measured by (A) Real time PCR and (B) Western blotting. β-cat mRNA and protein levels were expressed as percentage of control. The protein content in the nuclear and cytoplasmic fraction was quantified by densitometric analysis of blots, α-Tub was used to exclude a contamination with cytoplasmatic proteins. HSP70 was used as a nuclear fraction loading control. Data represent the mean ± SD from 3 independent experiments, ***P < 0.001. (C) Upper panel: images taken with an Olympus IX51 inverted microscope (10× magnification) showing MCF-7 shCTR and MCF-7 shβcat cells (scale bars, 12.5 μm); lower panel: CLSM micrographs of β-cat (stained with β-cat, in green) in MCF-7 shCTR and MCF-7 shβcat cells (nuclei stained with DAPI, in blue; scale bars, 25 μm). (D) Real time PCR was used to determine mRNA expression levels of c-Myc and cyclinD1. Rplp0 was used as housekeeping gene and the levels were expressed as percentage of control. Data represent the mean ± SD from 3 independent experiments, and ***P < 0.001. (E) Nuclear fraction protein was immunoblotting with c-Myc and whole fraction protein was immunoblotting with cyclinD1. β-actin was used for signal normalization. (F) MCF-7 shCTR and MCF-7 shβcat cells were seeded at 1 × 10⁴/well in a 96-well plate and incubated for 24 h and 48 h before estimating the cell proliferation rate by MTT test (***P < 0.001). (G) The percentage (%) of open wound area at 24 and 48 h against zero time was calculated using the GraphPad PRISM software version 4.0. Data represent the mean ± SD from 3 wounds for each sample, ***P < 0.001.

Figure 2

Effect of β-catenin gene silencing on morphology of MCF-7 cells. (A) CLSM micrographs of organization of F-actin (stained with phalloidin, in red) and microtubules (stained with α-tub, in green) filaments in MCF-7 shCTR and MCF-7 shβcat cells (nuclei stained with DAPI, in blue). The individual blue, red and green channels are shown followed by merged images (scale bars, 25 μm). The images are representative of three independent experiments. (B) Averages of cell, cytosol and nucleus areas of MCF-7 shCTR and s MCF-7 hβcat cells. Columns represent mean ± standard deviation (n cell analyzed = 50). ***P < 0.001. The outlines of DIC images of single cells and single nuclei were used for morphometric analysis. ImageJ64 was used for entering and storing the cell and nucleus outlines and for calculating the cellular and nuclear areas. (C) Western blot of phospho-Cofilin (pCof) and Cof protein levels. β-actin was used for signal normalization. The images are representative of three independent experiments.

Figure 3

β-catenin gene silencing alters the organization of actin stress fibers. Z stacks of confocal images from the the basal to the apical sides (a–h) (depth interval = 0.21 μm) of MCF-7 shCTR (A) and MCF-7 shβcat cells (B) stained for F-actin. Scale bars, 50 μm (A) and 25 μm (B). The images are representative of three independent experiments.

Figure 4

LC-MS/MS analysis of β-catenin knockdown in MCF-7 cells. (A) Hierarchical clustering of genes among MCF-7 shCTR and MCF-7 shβcat cells. Heat map illustrating differential expression of 98 proteins in MCF-7 shCTR cell respect to MCF-7 shβcat for biological triplicate samples. Color scale ranges from red to green (highest to lowest relative expression). (B) Two main clusters extracted from (A). The windows contain the expression profiles of the proteins within clusters. β-catenin expression levels are highlighted in blue. The number of differentially expressed proteins in each cluster is also depicted.

Figure 5

Gene ontology analysis using PANTHER. (A) Molecular function and (B) Biological process classes assigned to all differentially regulated proteins identified after proteomic analysis in MCF-7 shCTR and MCF-7 shβcat cells. It should be pointed that PANTHER may attribute multiple classes to a given protein. A complete list of the proteins can be found in Table 4. (C) Distribution among Primary metabolic processes and Organelle localization of down- and up-regulated proteins in MCF-7 shβcat respect to MCF-7 shCTR cells using PANTHER analysis.

Figure 6

β-catenin controls mitochondrial biogenesis. (A) Staining in live imaging of MCF-7 shCTR and MCF-7 shβcat mitochondria. MitoTracker CMXRos (red; a,b) accumulation is dependent upon mitochondrial membrane potential while MitoTracker Green FM (green; c,d) stains mitochondria regardless membrane potential, scale bar: 10 μm. Quantification of different intensities was performed with ImageJ software and represented in histograms. Data represent the mean ± SD from 3 independent experiments, with a significant difference of **P < 0.005 and ***P < 0.001. (B) Quantitative Real time PCR was used to determine nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) contents in MCF-7 shCTR (black columns) and MCF-7 shβcat (white columns). The mtDNA level was expressed as the ratio of mtDNA to nDNA copy number (mtDNA/nDNA). (C) Quantitative Real time PCR was used to determine PGC1α, TFAM and NRF1 gene expression levels in MCF-7 shCTR and MCF-7 shβcat cells. Data represent the mean ± SD from 3 independent experiments. **P < 0.005, ***P < 0.001.

Figure 7

Metabolic effect of β-catenin silencing in MCF-7 cells. (A) Quantitative Real time PCR was used to determine mRNA expression levels of citrate carrier (CiC), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and Sterol Regulatory Element Binding Protein1c (SREBP1c), involved in lipogenesis; Caveolin 1 (CAV1), CD36 and monoacylglycerol lipase (MGL) involved in fatty acid transport and remodeling; and glucose transporter 1 (GLUT1) regulating glucose transport. (B) Western blot of FASN and Acetyl CoA Carboxylase (ACC) protein levels. α-tubulin (α-tub) was used for signal normalization. The images are representative of three independent experiments. (C) ACC specific activity measured in digitonin-permeabilized cells is expressed as nmoles [¹⁴C]Acetyl-CoA incorporated into fatty acids/min × mg protein. (D) [1-¹⁴C]acetate and [U-¹⁴C]glucose incorporation into fatty acids was followed by incubating cells with the labeled substrates for 1 h. After this time synthetized fatty acids were extracted and radioactivity counted. Data represent the mean ± SD from 4 independent experiments; asterisks indicate significant differences compared to shCTR. *P < 0.01, **P < 0.005, ***P < 0.001.