title: Paradoxical E-cadherin increase in 5FU-resistant colon cancer is unaffected during mesenchymal-epithelial reversion induced by γ-secretase inhibition.
abstract: OBJECTIVE
  Presenilin-1 (PS1), the main component of γ-secretase activity support a key role during Epithelial-Mesenchymal Transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells.
  
  
  METHODS
  HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and α-SMA expression were determined using Western blot technique. β-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. γ-Secretase activity was inhibited by DAPT, a γ-secretase inhibitor.
  
  
  RESULTS
  Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8.
  
  
  CONCLUSIONS
  High E-cadherin concentration in presence of enhanced γ-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.
authors.affiliation: Department of Clinical and Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy; Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Dinicola
authors.affiliation: Azienda Policlinico Umberto I, viale del Policlinico 155, 00161 Rome, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Pasqualato
authors.affiliation: Department of Clinical and Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy; Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Proietti
authors.affiliation: Department of Clinical and Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy; Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Masiello
authors.affiliation: Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy; Department of Experimental Medicine, Sapienza University of Rome, Systems Biology Group Lab, viale Regina Elena 324, 00161 Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Palombo
authors.affiliation: Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy; Azienda Policlinico Umberto I, viale del Policlinico 155, 00161 Rome, Italy.
authors.forename: Pierpaolo
authors.initials: P
authors.lastname: Coluccia
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Canipari
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Catizone
authors.affiliation: Department of Experimental Medicine, Second University of Naples, Via Santa Maria di Costantinopoli 16, 80138 Naples, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Ricci
authors.affiliation: Department of Zoology, College of Science, King Saud University, PO Box 2455, 11451, Riyadh, Saudi Arabia.
authors.forename: Abdel Halim
authors.initials: AH
authors.lastname: Harrath
authors.affiliation: Department of Zoology, College of Science, King Saud University, PO Box 2455, 11451, Riyadh, Saudi Arabia.
authors.forename: Saleh H
authors.initials: SH
authors.lastname: Alwasel
authors.affiliation: Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy; Azienda Policlinico Umberto I, viale del Policlinico 155, 00161 Rome, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Cucina
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Systems Biology Group Lab, viale Regina Elena 324, 00161 Rome, Italy; Systems Biology Group Lab, Sapienza University of Rome, Rome, Italy. Electronic address: mariano.bizzarri@uniroma1.it.
authors.forename: Mariano
authors.initials: M
authors.lastname: Bizzarri
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D015820:Cadherins / Q000378:metabolism*; D045744:Cell Line, Tumor; D003106:Colon / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D019008:Drug Resistance, Neoplasm; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D053764:Presenilin-1 / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D015820:Cadherins; D053764:Presenilin-1; D053829:Amyloid Precursor Protein Secretases; D005472:Fluorouracil
keywords: Chemoresistance; E-cadherin; EMT; Presenilin-1; SNAI1; γ-Secretase activity