Defining the role of p38^MAPK signaling during mammary epithelial acini development

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Mammary epithelial cells are critically dependent on integrin-mediated adhesion to a suitable basement membrane (BM) matrix for normal growth and survival. The stress-activated kinase, p38, has been identified as a potential tumor suppressor pathway involved in mediating suspension-induced cell death in epithelial cells. However, little is known about its function during normal mammary gland morphogenesis or in breast cancer development.

In the present study, we investigated whether alterations in cell-BM adhesion resulted in changes in p38 signaling and consequently the fate of normal vs. cancerous breast epithelial cells. We used “normal” non-transformed MCF-10A mammary epithelial cells and tumorigenic T47D breast carcinoma cells, cultured in adhered vs. suspension growth assays and 3D cultures. Immunoblots were used to detect BimEL, GADD153, phospho-p38 and ATF4. SB203580, shRNA and a dominant negative p38 construct were used to inhibit p38 MAPK pathway. A GADD153-EGFP reporter construct was used to monitor p38 signals.

Our data revealed a robust activation of p38 in MCF10A cells, as early as 2hrs and up to 48hrs in suspension, followed by the initiation of apoptosis at 24hrs in suspension. Further, blocking β1 integrin function strongly induced p38 activation comparable with suspension growth conditions. Apoptosis was p38-mediated, since inhibition of p38 signaling by SB203580 significantly reduced apoptosis, and over expression of a dominant- negative form of p38 resulted in suppression of GADD153-promoter activation, a pro-apoptotic p38 target. In contrast, T47D carcinoma cells showed a significant delay in p38 activation, reduced GADD153-promoter activation and resistance to anoikis after 72hrs in suspension. Preliminary studies suggest that p38 activation is required for luminal apoptosis and inhibition of proliferation in the basal layer during acini morphogenesis.

We conclude that p38 activation is an early response to suspension-induced stress and in cancer cells attenuation of this response correlates with their survival in suspension.

Interestingly, in early stages of breast carcinoma, i.e.: ductal carcinoma in situ, cells accumulate in the lumen where BM is absent. Future studies using MCF-10A stably expressing a p38-shRNA in 3D Matrigel™ cultures will allow us to elucidate the role of p38 during mammary acini morphogenesis.