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

ABSTRACT: ?-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 ?-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after ?-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 ?-catenin knockout cells (sh?cat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of ?-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 sh?cat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in sh?cat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in sh?cat cells. ?-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, ?-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 ?-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

SUBMITTER: Vergara D

PROVIDER: S-EPMC5529387 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Vergara Daniele D Stanca Eleonora E Guerra Flora F Priore Paola P Gaballo Antonio A Franck Julien J Simeone Pasquale P Trerotola Marco M De Domenico Stefania S Fournier Isabelle I Bucci Cecilia C Salzet Michel M Giudetti Anna M AM Maffia Michele M

Frontiers in physiology 20170727

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) appr ...[more]

PMID: 28798698

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Project description:Background Bone is a rigid organ that provides physical protection and support to vital organs of the body. Bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors that can differentiate into osteoblasts, adipocytes, and chondrocytes. Cell division cycle 20 (CDC20) is a co-activator of anaphase promoting complex/cyclosome (APC/C), and is required for ubiquitin ligase activity. Our previous study showed that CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we found that knockdown of CDC20 promoted adipogenic differentiation of BMSCs by modulating β-catenin, which suggested a link between adipogenesis and osteogenesis. Methods Lentivirus containing a CDC20 shRNA was used for CDC20 knockdown in human BMSCs (hBMSCs). Primary mouse BMSCs (mBMSCs) were isolated from Cdc20f/f and Sp7-Cre;Cdc20f/f mice. Adipogenesis was examined using quantitative real-time reverse transcription PCR (qRT-PCR) and western blotting analysis of adipogenic regulators, Oil Red O staining, and transplantation into nude mice. CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR, and western blotting of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections. Exploration of the molecular mechanism was determined through western blotting, Oil Red O staining, and qRT-PCR. Results CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. CDC20 knockdown enhanced hBMSC adipogenic differentiation in vitro. CDC20-knockdown hBMSCs showed more adipose tissue-like constructs upon hematoxylin and eosin (H&E) and Oil Red O staining. Sp7-Cre;Cdc20f/f mice presented increased adipocytes in their bone marrow compared with the control mice. mBMSCs from Sp7-Cre;Cdc20f/f mice showed upregulated adipogenic differentiation. Knockdown of CDC20 led to decreased β-catenin levels, and a β-catenin pathway activator (lithium chloride) abolished the role of CDC20 in BMSC adipogenic differentiation. Conclusions Our findings showed that CDC20 knockdown enhanced adipogenesis of hBMSC and mBMSCs adipogenesis in vitro and in vivo. CDC20 regulates both adipogenesis and osteogenesis of BMSCs, and might lead to the development of new therapeutic targets for “fatty bone” and osteoporosis. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-03062-0.

Project description:BackgroundEstrogen receptor β (ERβ) has been repeatedly suggested to play important roles in hormone-dependent cancer like in tumors of the breast, ovary or prostate. In this study, we intended to further elucidate its role in endometrial cancer.MethodsFor this purpose, we knocked down ERβ expression in two endometrial cancer cell lines, the ERα-negative/ERβ-positive line HEC-1A and the ERα/β-positive cell line RL95/2, by means of siRNA transfection. Cell proliferation after transfection was assessed using the fluorescent CTB Assay (Promega). In order to elucidate possible molecular mechanisms which might underlie the effect on proliferation, we performed transcriptome analyses by means of human Affymetrix Human Gene Chip 2.0. Additionally, we treated the employed cell lines with different ERβ modulators to examine their effect on proliferation.ResultssiRNA-mediated knockdown of ERβ significantly increased proliferation of both endometrial cancer cell lines. In HEC-1A cells, proliferation was significantly increased 4, 5 and 6 days after transfection, with a maximum of about 1.7-fold (p < 0.05) on day 6. Endometrial RL95/2 cells with an ERβ knockdown exhibited a clearly enhanced proliferation on day 3 and days 4 to 8, when even 2.4-fold higher numbers of viable cells were detected (p < 0.01). Transcriptome analysis revealed that this was accompanied by increased expression of several genes being known to be upregulated in cancer, including proliferation-associated genes and oncogenes, and by repression of genes associated with differentiation, apoptosis or growth inhibition. Corroborating the observed knockdown effects, treatment with the ERβ antagonists PHTTP and (R, R) THC was also able to induce proliferation of both cell lines.ConclusionsOur data clearly support the putative role of ERβ as tumor suppressor in endometrium as previously suggested in studies on other tissues and encourage further studies to find out to what extent this molecule might be a potential therapy target in this cancer entity.

Dataset Information

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

Publications

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

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