ORIGINAL RESEARCH published: 27 July 2017 doi: 10.3389/fphys.2017.00544 β-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells Daniele Vergara 1, 2 †, Eleonora Stanca 1, 2 †, Flora Guerra 1, Paola Priore 3, Antonio Gaballo 3, Julien Franck 4, Pasquale Simeone 5, Marco Trerotola 6, Stefania De Domenico 7, Isabelle Fournier 4, Cecilia Bucci 1, Michel Salzet 4, Anna M. Giudetti 1* and Michele Maffia 1, 2* 1 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy, 2 Laboratory of Clinical Proteomic, “Giovanni Paolo II” Hospital, Lecce, Italy, 3 CNR NANOTEC - Institute of Nanotechnology, Lecce, Italy, 4 University of Lille, Institut national de la santé et de la recherche médicale, U-1192 - Laboratoire Protéomique, Réponse Edited by: Inflammatoire et Spectrométrie de Masse-PRISM, Lille, France, 5 Unit of Cytomorphology, CeSI-MeT and Department of Andrei Surguchov, Medicine and Aging Sciences, School of Medicine and Health Sciences, University “G. d’Annunzio,” Chieti, Italy, 6 Unit of Kansas University of Medical Center Cancer Pathology, CeSI-MeT and Department of Medical, Oral and Biotechnological Sciences, University “G. d’Annunzio,” Research Institute, United States Chieti, Italy, 7 C.N.R. Unit of Lecce, Institute of Food Production Sciences, Lecce, Italy Reviewed by: Kamal Datta, β-catenin plays an important role as regulatory hub in several cellular processes including Georgetown University, United States Silvana Gaetani, cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved Sapienza Università di Roma, Italy by its dual role as structural component of cadherin-based adherens junctions, and as Clizia Chinello, University of Milano-Bicocca, Italy a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins *Correspondence: are differentially regulated via β-catenin dependent mechanisms. Here, we applied a Anna M. Giudetti liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins [email protected] modulated after β-catenin knockdown in the breast cancer cell line MCF-7. We used a Michele Maffia michele.maffi[email protected] label free analysis to compare trypsin-digested proteins from CTR (shCTR) and β-catenin †Co-first authors. 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 Specialty section: morphological changes and a significant modulation of the expression levels of proteins This article was submitted to Lipidology, associated with primary metabolic processes. In detail, proteins involved in carbohydrate a section of the journal metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas Frontiers in Physiology proteins associated to lipid metabolism were found up-regulated in shβcat compared Received: 26 May 2017 to shCTR. A loss of mitochondrial mass and membrane potential was also assessed Accepted: 12 July 2017 Published: 27 July 2017 by fluorescent probes in shβcat cells with respect to the controls. These data are Citation: consistent with the reduced expression of transcriptional factors regulating mitochondrial Vergara D, Stanca E, Guerra F, biogenesis detected in shβcat cells. β-catenin driven metabolic reprogramming resulted Priore P, Gaballo A, Franck J, Simeone P, Trerotola M, De also in a significant modulation of lipogenic enzyme expression and activity. Compared to Domenico S, Fournier I, Bucci C, controls, β-catenin knockout cells showed increased incorporation of [1-14C]acetate and Salzet M, Giudetti AM and Maffia M decreased utilization of [U-14C]glucose for fatty acid synthesis. Our data highlight a role (2017) β-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid of β-catenin in the regulation of metabolism and energy homeostasis in breast cancer Metabolism in Breast Cancer Cells. cells. Front. Physiol. 8:544. doi: 10.3389/fphys.2017.00544 Keywords: β-catenin, proteomics, LC-MS/MS, mitochondria, lipid metabolism, MYC Frontiers in Physiology | www.frontiersin.org 1 July 2017 | Volume 8 | Article 544 Vergara et al. β-catenin Knockdown in Breast Cancer Cells INTRODUCTION proliferation and invasion. However, the substantial concept of β-catenin as regulator of development and stemness has now β-catenin is a multifunctional protein localized at multiple expanded, and β-catenin is found to be involved in a large variety subcellular regions, including adherents junctions, cytoplasm of cellular processes. Analysis of KEGG pathways/functions and/or nucleus. β-catenin plays an essential role in the revealed that up- or down-regulations of β-catenin lead maintenance of adult tissue homeostasis. In normal epithelial to altered regulation of actin cytoskeleton, insulin signaling cells, β-catenin is mainly localized at the plasma membrane, and metabolism (Herbst et al., 2014), though the molecular where it interacts with the cytoplasmic tail of E-cadherin to form mechanisms underlying these associations appear still unclear. a cell adhesion complex at the intercellular junctions (Huber In this scenario, the complexity of the β-catenin network can be and Weis, 2001; Tian et al., 2011). In normal breast tissues, fully addressed by experimental approaches that allow managing β-catenin is highly expressed at the basolateral surface of the thousands of molecules simultaneously. luminal epithelium, as compared to the lateral surface of the Here, we performed a liquid chromatography-mass myoepithelial cells, where it is expressed at much lower levels spectrometry (LC-MS/MS) analysis to characterize the proteomic (Hashizume et al., 1996). modifications associated with the stable knockdown of β-catenin Dysregulation of the β-catenin/E-cadherin complex can in the breast cancer cell line MCF-7. By integrating label-free MS, induce profound defects in the organization of epithelial layers bioinformatics pathway analysis, and cell-based experimental leading to development of mammary tumors, among them approaches we identified 98 significantly modulated proteins adenocarcinoma and squamous metaplasia (Teulière et al., associated with distinctive molecular functions and cellular 2005). Disruption of cadherin-mediated cell adhesion has been processes. Specifically, we gained insight into the mechanisms proposed to drive the epithelial-mesenchymal transition (EMT) that link β-catenin to modifications of metabolic proteins, program, a process occurring in development and cancer, and we described in our model a metabolic reprogramming whereby epithelial cells lose cell-cell contacts and apical-basal accompanied by alterations in mitochondrial function and lipid polarity, and acquire a mesenchymal-like morphology (Thiery metabolism. and Sleeman, 2006; Lamouille et al., 2014). The Wnt pathway regulates β-catenin signaling, and nuclear MATERIALS AND METHODS localization of β-catenin is a hallmark of the activation of Wnt pathway. In the absence of Wnt ligands, the cytoplasmic Cell Culture and Reagents pool of β-catenin protein undergoes proteasomal degradation Human tumor cells were purchased from Banca Biologica and (MacDonald et al., 2009), thus preventing β-catenin localization Cell Factory (IRCCS Azienda Ospedaliera Universitaria San into the nucleus. In this way, Wnt/β-catenin target genes are Martino-IST Istituto Nazionale per la ricerca sul cancro, Genova, repressed by the DNA-bound T cell factor/lymphoid enhancer Italy). The human cancer cell line MCF-7 was cultured in factor (TCF/LEF) and transducing-like enhancer (TLE/Groucho) high glucose DMEM (Euroclone) supplemented with 10% FBS protein complex (MacDonald et al., 2009). In the canonical (Euroclone), 100 U/ml penicillin and 100 µg/ml streptomycin at ◦ Wnt pathway, binding of secreted Wnt ligands to Frizzled 37 C in an atmosphere of 5% CO2. The cells were maintained in receptors and lipoprotein receptor-related proteins (LRP) co- an exponential growth phase during experiments. receptors stimulates the stabilization of the cytosolic β-catenin Epidermal growth factor (EGF) was from Prospec. Primary pool by phosphorylation, leading to β-catenin translocation and antibodies were from Santa Cruz: β-Catenin (sc-1496), α- transcriptional regulation of target genes (Tetsu and McCormick, Tubulin (sc-23948), Cofilin (sc-33779), phospho-Cofilin (sc- 1999; Cong et al., 2004). Aberrant activation of the canonical 21867-R); or Cell Signaling: β-Actin (#8457), c-Myc (#13987), Wnt/β-catenin pathway is frequently observed in several human fatty acid synthase (FASN, #3180), and Acetyl CoA Carboxylase cancers. Wnt pathway components, including Axin, β-catenin (ACC, #3676). Secondary antibodies (HRP-conjugated) were and Adenomatous polyposis coli (APC), are frequently mutated from Santa Cruz Biotechnology (goat anti-mouse IgG-HRP, in colorectal adenocarcinoma but this is uncommon in breast sc-2005; goat anti-rabbit IgG-HRP, sc-2004) or Cell Signaling cancer samples (Zhan et al., 2017), where β-catenin activation (anti-rabbit IgG-HRP, #7074). Phalloidin-tetramethylrhodamine is not driven by β-catenin gene (CTNNB1) mutations (Geyer B isothiocyanate (TRITC) and Fluoroshield were from Sigma. All et al., 2011). β-catenin aberrant nuclear signaling was observed other reagents were from standard commercial sources and were in Her2 and triple-negative/basal-like breast carcinomas and of the highest grade available. associated with cancer progression and poor clinical outcome