title: Par6 is an essential mediator of apoptotic response to transforming growth factor beta in NMuMG immortalized mammary cells.
abstract: BACKGROUND
  We previously observed that the TGFbeta-Par6 pathway mediates loss of polarity and apoptosis in NMuMG cells. Here we investigate the contribution of Par6 versus TGFbeta receptor I activation to TGFbeta-induced apoptosis in association with changes in apico-basal polarity. We focus on the effect of Par6 activation on alpha6beta4 integrin expression and localization, and Nuclear Factor-kappaB (p65/RelA) activation, previously shown to mediate polarity-dependent cell survival.
  
  
  METHODS
  Using immunoblotting and/or immunofluorescence we investigated the effect of TGFbeta1 on apoptosis, alpha6, beta4 and beta1 integrin expression/localization, and p65/RelA phosphorylation/localization in monolayer and three-dimensional (3D) cultures of NMuMG cells with an overactive or inactive Par6 pathway. Results were quantified by band densitometry or as percent of 3D structures displaying a phenotype. Differences among means were compared by two-way ANOVA.
  
  
  RESULTS
  Blocking Par6 activation inhibits TGFbeta-induced apoptosis. Par6 overactivation enhances TGFbeta-induced apoptosis, notably after 6-day exposure to TGFbeta (p < 0.001), a time when parental NMuMG cells no longer respond to TGFbeta apoptotic stimuli. 48-hour TGFbeta treatment reduced beta4 integrin levels in NMuMG monolayers and significantly reduced the basal localization of alpha6 (p < 0.001) and beta4 (p < 0.001) integrin in NMuMG 3D structures, which was dependent on both Par6 and TGFbeta receptor I activation and paralleled apoptotic response. After 6-day exposure to TGFbeta, Par6-dependent changes to beta4 integrin were no longer apparent, but there was reduced phosphorylation of p65/RelA (p < 0.001) only in Par6 overexpressing cells. Differences in p65/RelA localization were not observed among the different cell lines after 48-hour TGFbeta exposure.
  
  
  CONCLUSIONS
  Par6 and TGFbeta receptor I activation are both necessary for TGFbeta-induced apoptosis in NMuMG cells. Importantly, Par6 overexpression enhances the sensitivity of NMuMG to TGFbeta-induced apoptosis, notably upon prolonged exposure to this growth factor, when NMuMG parental cells are usually apoptosis-resistant. Thus, endogenous Par6 level might be important in determining whether TGFbeta will function as either a pro-apoptotic or pro-survival factor in breast cancer, and potentially aid in predicting patient's prognosis and therapy response.
authors.forename: Geordon
authors.initials: G
authors.lastname: Avery-Cooper
authors.forename: Meghan
authors.initials: M
authors.lastname: Doerr
authors.forename: Richard Wd
authors.initials: RW
authors.lastname: Gilbert
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Youssef
authors.forename: Amy
authors.initials: A
authors.lastname: Richard
authors.forename: Patricia
authors.initials: P
authors.lastname: Huether
authors.affiliation: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Room 3647, 50 Stone Road East, Guelph N1G 2 W1, ON, Canada. aviloria@uoguelph.ca.
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Viloria-Petit