title: 3-BrPA eliminates human bladder cancer cells with highly oncogenic signatures via engagement of specific death programs and perturbation of multiple signaling and metabolic determinants.
abstract: BACKGROUND
  Urinary bladder cancer is one of the most fatal and expensive diseases of industrialized world. Despite the strenuous efforts, no seminal advances have been achieved for its clinical management. Given the importance of metabolic reprogramming in cancer cell survival and growth, we have herein employed 3-BrPA, a halogenated derivative of pyruvate and historically considered inhibitor of glycolysis, to eliminate bladder cancer cells with highly oncogenic molecular signatures.
  
  
  METHODS
  Bladder cancer cells were exposed to 3-BrPA in the absence or presence of several specific inhibitors. Cell viability was determined by MTT and flow-cytometry assays; cell death, signaling activity and metabolic integrity by Western blotting and immunofluorescence; mutant-gene profiling by DNA sequencing; and gene expression by RT-sqPCR.
  
  
  RESULTS
  3-BrPA could activate dose-dependent apoptosis (type 1 PCD) and regulated necrosis (type 3 PCD) of T24 (grade III; H-Ras(G12V); p53(ΔY126)), but not RT4 (grade I), cells, with PARP, MLKL, Drp1 and Nec-7-targeted components critically orchestrating necrotic death. However, similarly to RIPK1 and CypD, p53 presented with non-essential contribution to 3-BrPA-induced cellular collapse, while reactivation of mutant p53 with PRIMA-1 resulted in strong synergism of the two agents. Given the reduced expression of MPC components (likely imposing mitochondrial dysfunction) in T24 cells, the suppression of constitutive autophagy (required by cells carrying oncogenic Ras; also, type 2 PCD) and derangement of glucose-homeostasis determinants by 3-BrPA critically contribute to drug-directed depletion of ATP cellular stores. This bioenergetic crisis is translated to severe dysregulation of Akt/FoxO/GSK-3, mTOR/S6, AMPK and MAPK (p44/42, p38 and SAPK/JNK) signaling pathways in 3-BrPA-treated T24 cells. Sensitivity to 3-BrPA (and tolerance to glucose deprivation) does not rely on B-Raf(V600E) or K-Ras(G13D) mutant oncogenic proteins, but partly depends on aberrant signaling activities of Akt, MAPK and AMPK kinases. Interestingly, MCT1- and macropinocytosis-mediated influx of 3-BrPA in T24 represents the principal mechanism that regulates cellular responsiveness to the drug. Besides its capacity to affect transcription in gene-dependent manner, 3-BrPA can also induce GLUT4-specific splicing silencing in both sensitive and resistant cells, thus dictating alternative routes of drug trafficking.
  
  
  CONCLUSIONS
  Altogether, it seems that 3-BrPA represents a promising agent for bladder cancer targeted therapy.
authors.affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou 15784, Athens, Greece. eumokon@biol.uoa.gr.
authors.forename: Eumorphia G
authors.initials: EG
authors.lastname: Konstantakou
authors.affiliation: Laboratory of Environmental Mutagenesis and Carcinogenesis, Institute of Biosciences and Applications, NCSR Demokritos, Athens, Greece. mvoutsin@bio.demokritos.gr.
authors.forename: Gerassimos E
authors.initials: GE
authors.lastname: Voutsinas
authors.affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou 15784, Athens, Greece. tveletz@biol.uoa.gr.
authors.forename: Athanassios D
authors.initials: AD
authors.lastname: Velentzas
authors.affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou 15784, Athens, Greece. stefania.basogianni@stud.ki.se.
authors.forename: Aggeliki-Stefania
authors.initials: AS
authors.lastname: Basogianni
authors.affiliation: Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. eparonis@bioacademy.gr.
authors.forename: Efthimios
authors.initials: E
authors.lastname: Paronis
authors.affiliation: Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. vbalafas@bioacademy.gr.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Balafas
authors.affiliation: Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. nkostom@bioacademy.gr.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Kostomitsopoulos
authors.affiliation: Oncology Unit GPP, Sotiria General Hospital, Athens School of Medicine, Athens, Greece. ksyrigos@med.uoa.gr.|Yale School of Medicine, New Haven, Connecticut, USA. ksyrigos@med.uoa.gr.
authors.forename: Konstantinos N
authors.initials: KN
authors.lastname: Syrigos
authors.affiliation: Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. anastasiadou@bioacademy.gr.
authors.forename: Ema
authors.initials: E
authors.lastname: Anastasiadou
authors.affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou 15784, Athens, Greece. dstravop@biol.uoa.gr.
authors.forename: Dimitrios J
authors.initials: DJ
authors.lastname: Stravopodis
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics*; D001343:Autophagy / Q000187:drug effects / Q000235:genetics; D001372:Aza Compounds / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004734:Energy Metabolism / Q000187:drug effects; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics; D006801:Humans; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010873:Pinocytosis / Q000187:drug effects; D021381:Protein Transport; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology*; D012326:RNA Splicing; D015398:Signal Transduction / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: C543636:3-bromo-2-oxopropionic acid; D001372:Aza Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D051275:Glucose Transporter Type 4; D011773:Pyruvates; D016159:Tumor Suppressor Protein p53; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras); C451219:2,2-bis(hydroxymethyl)-1-azabicyclo(2,2,2,)octan-3-one; D005947:Glucose