title: Proteomic characterization of peroxisome proliferator-activated receptor-γ (PPARγ) overexpressing or silenced colorectal cancer cells unveils a novel protein network associated with an aggressive phenotype.
abstract: Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor of the nuclear hormone receptor superfamily implicated in a wide range of processes, including tumorigenesis. Its role in colorectal cancer (CRC) is still debated; most reports support that PPARγ reduced expression is associated with poor prognosis. We employed 2-Dimensional Differential InGel Electrophoresis (2-D DIGE) followed by Liquid Chromatography (LC)-tandem Mass Spectrometry (MS/MS) to identify differentially expressed proteins and the molecular pathways underlying PPARγ expression in CRC progression. We identified several differentially expressed proteins in HT29 and HCT116 CRC cells and derived clones either silenced or overexpressing PPARγ, respectively. In Ingenuity Pathway Analysis (IPA) they showed reciprocal relation with PPARγ and a strong relationship with networks linked to cell death, growth and survival. Interestingly, five of the identified proteins, ezrin (EZR), isoform C of prelamin-A/C (LMNA), alpha-enolase (ENOA), prohibitin (PHB) and RuvB-like 2 (RUVBL2) were shared by the two cell models with opposite expression levels, suggesting a possible regulation by PPARγ. mRNA and western blot analysis were undertaken to obtain a technical validation and confirm the expression trend observed by 2-D DIGE data. We associated EZR upregulation with increased cell surface localization in PPARγ-overexpressing cells by flow cytometry and immunofluorescence staining. We also correlated EZR and PPARγ expression in our series of CRC specimens and the expression profiling of all five proteins levels in the publicly available colon cancer genomic data from Oncomine and Cancer Genome Atlas (TCGA) colon adenocarcinoma (COAD) datasets. In summary, we identified a panel of proteins correlated with PPARγ expression that could be associated with CRC unveiling new pathways to be investigated for the selection of novel potential prognostic/predictive biomarkers and/or therapeutic targets.
authors: Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy.
authors: Maria Rita
authors: MR
authors: Milone
authors: Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy.
authors: Biagio
authors: B
authors: Pucci
authors: Department of Sciences and Technologies, University of Sannio, Benevento, Italy.
authors: Tommaso
authors: T
authors: Colangelo
authors: Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy.
authors: Rita
authors: R
authors: Lombardi
authors: Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy.
authors: Federica
authors: F
authors: Iannelli
authors: Department of Sciences and Technologies, University of Sannio, Benevento, Italy.
authors: Vittorio
authors: V
authors: Colantuoni
authors: Department of Sciences and Technologies, University of Sannio, Benevento, Italy. Electronic address: sabatino@unisannio.it.
authors: Lina
authors: L
authors: Sabatino
authors: Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy; Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy. Electronic address: a.budillon@istitutotumori.na.it.
authors: Alfredo
authors: A
authors: Budillon
mesh_terms: D015153:Blotting, Western; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology*; D019295:Computational Biology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional; D020868:Gene Silencing; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D047495:PPAR gamma / Q000378:metabolism*; D010641:Phenotype; D000091402:Prohibitins; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000378:metabolism; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D047495:PPAR gamma; D000091402:Prohibitins; D012333:RNA, Messenger; D000099149:Ezrin; C000717328:PHB protein, human
keywords: 2-D DIGE; Colorectal cancer; Ingenuity pathway analysis; Mass spectrometry; Peroxisome proliferator-activated receptor gamma; Proteomics