Par6 is an essential mediator of apoptotic response to transforming growth factor beta in NMuMG immortalized mammary cells

Cancer Cell International

Alicia M Viloria-Petit¹  Patricia Huether¹  Amy Richard¹  Mahmoud Youssef¹  Richard WD Gilbert¹  Meghan Doerr¹  Geordon Avery-Cooper¹ 

[1]Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Room 3647, 50 Stone Road East, Guelph N1G 2 W1, ON, Canada

关键词: Cell survival;    Integrins;    EMT;    Polarity;    Apoptosis;    Par6;    TGFbeta;   

Others  :  792296
DOI  :  10.1186/1475-2867-14-19

 received in 2013-07-30, accepted in 2014-02-20,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Avery-Cooper et al.; licensee BioMed Central Ltd.

【 图 表 】

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