title: Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk.
abstract: BACKGROUND
  The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-β/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells.
  
  
  RESULTS
  Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling.
  
  
  CONCLUSIONS
  Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid) cells could result in a progression of these cells. Thus, not only the receptor (activation) status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.
authors.affiliation: Zentrum für Biomedizinische Ausbildung und Forschung der UWH (ZBAF), Institute of Immunology, Witten/Herdecke University, Stockumer Str, 10, 58448 Witten, Germany. thomas.dittmar@uni-wh.de.
authors.forename: Cem
authors.initials: C
authors.lastname: Ozel
authors.forename: Jeanette
authors.initials: J
authors.lastname: Seidel
authors.forename: Sönke
authors.initials: S
authors.lastname: Meyer-Staeckling
authors.forename: Burkhard H
authors.initials: BH
authors.lastname: Brandt
authors.forename: Bernd
authors.initials: B
authors.lastname: Niggemann
authors.forename: Kurt S
authors.initials: KS
authors.lastname: Zänker
authors.forename: Thomas
authors.initials: T
authors.lastname: Dittmar