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

Abstract

BackgroundWe 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.MethodsUsing 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.ResultsBlocking 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.ConclusionsPar6 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.

Par6 regulates cell cycle progression through enhancement of Akt/PI3K/GSK‐3β signaling pathway activation in glioma

“…We therefore determined the expression patterns of Par6 in the U87MG, U251, and HEB cell lines ( Figure 1D). 10,23,25,29,30 In addition, we found that Par6 expression was much higher in either U87MG or U251 cells than that in HEB cells. 10,23,25,29,30 In addition, we found that Par6 expression was much higher in either U87MG or U251 cells than that in HEB cells.…”

“…Our immunostaining results indicated that Par6 localized in either the cytoplasm or cell membrane in the glioma cell lines U87MG and U251, inconsistent with the localization in epithelial tumor cells reported previously. 10,23,25,29,30 In addition, we found that Par6 expression was much higher in either U87MG or U251 cells than that in HEB cells. Thus, this different expression pattern of Par6 between glioma cells and human malignant epithelial tumor cells suggests that the functions of Par6 in glioma might be different than those in malignant epithelial tumors.…”

“…31,32 In addition, the TGF-β-Par6 polarity pathway has been found to be associated with the EMT and with tumor progression in various human epithelial tumors. 10,23,24,26,30 We therefore examined whether the TGF-β-Par6 pathway played a role in Par6-mediated tumorigenicity in our cell lines. Our results indicated that the expression levels of Par6 were significantly higher in TGF-β-treated U87MG and U251 cells than in untreated cells at the transcriptional or translational level ( Figure 6A,B).…”

“…Malignant glioma cells are usually highly resistant to radiotherapy and chemotherapy, leading to recurrence. 10 In epithelia, it is a key polarity regulator at the apical surface and engages in intricate signaling networks with the Crumbs complex and with cell adhesion proteins, such as E-cadherin, to form and maintain cell junctions. Moreover, the majority of prognostic factors provide no insight into the molecular events that are responsible for tumor proliferation, apoptosis, or additional biological processes related to malignancy.…”

“…10 In epithelia, it is a key polarity regulator at the apical surface and engages in intricate signaling networks with the Crumbs complex and with cell adhesion proteins, such as E-cadherin, to form and maintain cell junctions. 10,[24][25][26] In this present study, our results showed abnormally high expression of Par6 protein in brain cancer tissues and cell lines, suggesting that Par6 might play a role in glioma tumorigenesis. [15][16][17][18] Accumulating evidences have indicated that Par6 and its complexes, members of an important protein family involved in the regulation of cellular polarity, are associated with the regulation of cancer cell proliferation, migration and invasion, as well as epithelial-mesenchymal transition (EMT).…”