title: Application of two-dimensional gel-based mass spectrometry to functionally dissect resistance to targeted cancer therapy.
abstract: OBJECTIVE
  The majority of gastric cancers are diagnosed at advanced stages, characterized by robust therapy resistance. The oncoprotein hypoxia-inducible factor 1 (HIF-1) is associated with therapy resistance, partly via activation of the DNA damage response. We have noted a robust ability of gastric cancer cells to functionally compensate the loss of HIF-1 in vitro. The purpose of this study was to identify molecular pathways that underlie this compensation.
  
  
  METHODS
  We performed 2DE to compare the nuclear proteome of wild-type and HIF-1-deficient gastric cancer cells. Differently expressed protein spots were identified via MS). After bioinformatic evaluation, functional validation of selected identified pathways was performed.
  
  
  RESULTS
  2DE displayed a total of 2523 protein spots, from which 87 were identified as regulated by HIF-1. Seventy of the identified spots were different proteins and 17 were isoforms. Bioinformatic analyses revealed that a significant amount of the identified proteins were related to cellular survival pathways. Specifically, members of the proteasome pathway were found upregulated upon loss of HIF-1. Combined inhibition of HIF-1 and the proteasome inflicted significant DNA damage, supporting the hypothesis that the proteasome is of functional importance to compensate the loss of HIF-1.
  
  
  CONCLUSIONS
  Our data show robust and functional changes of the nuclear proteome upon inactivation of the HIF-1 oncoprotein in gastric cancer cells. We propose that 2DE-MS represents a useful tool to functionally dissect resistance mechanisms to targeted therapy and to identify novel targets for antiproliferative combination therapy.
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany; Core Unit Proteomics, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany; Institute of Medical and Human Genetics, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Klein
authors.forename: Nadine
authors.initials: N
authors.lastname: Rohwer
authors.forename: Katja Freitag
authors.initials: KF
authors.lastname: de Molina
authors.forename: Stefan
authors.initials: S
authors.lastname: Mergler
authors.forename: Petra
authors.initials: P
authors.lastname: Wessendorf
authors.forename: Marion
authors.initials: M
authors.lastname: Herrmann
authors.forename: Joachim
authors.initials: J
authors.lastname: Klose
authors.forename: Thorsten
authors.initials: T
authors.lastname: Cramer
mesh_terms: D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D013058:Mass Spectrometry; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism
keywords: 2DE; Gastric cancer; HIF-1α; Targeted therapy; Therapy resistance