Even in a simplified model of epithelial morphogenesis—in vitro three-dimensional MCF10A cell culture—integration of several signaling pathways is required to form a hollow acinus. Previous studies revealed that integrin-dependent signaling, which allows cells to recognize their position relative to ECM (contacting ECM or trapped within a multicellular structure) is an essential regulator of cell survival or death. In the present study, we describe quite unexpected roles of cAMP and the PKA-dependent pathway in MCF10A morphogenesis.
According to the current model of MCF10A acinus formation, proliferation, polarization, and death of luminal cells are the result of integrin-dependent interaction with ECM. After initial proliferation, the cells that contact ECM become polarized. Integrin-dependent signaling in these cells ensures their survival. In contrast, lack of integrin–ECM interaction in inner cells of the acinus prevents polarization and leads to down-regulation of EGFR with a subsequent decrease in ERK activity (Reginato et al., 2003
). This deprives these cells of important survival signals, increases expression of proapoptotic protein BIM, and leads to cell death. However, this view may be not complete. First, we have observed that polarization of MCF10A acini is delayed in conditions in which cAMP is not elevated. cAMP is well known to facilitate polarization of neurons (Shelly et al., 2010
; Cheng et al., 2011
). One of the mechanisms is switching ubiquitin E3 ligase Smurf1 substrate preference from Par6 to RhoA due to PKA-dependent phosphorylation (Cheng et al., 2011
). Because Smurf1, Par6, and RhoA are also involved in establishing of polarity in epithelial cells (Wang et al., 2003
), it remains to be tested whether PKA regulates polarization of epithelial cells via Smurf1 phosphorylation. An alternative mechanism revealed in the present study may be facilitation of integrin redistribution. cAMP facilitates redistribution of α6-integrin to the acinar periphery. The polarizing effect of cAMP could be mimicked by EGFR or ERK inhibition and prevented by inhibitory antibody against α6-integrin. Because cAMP decreases pERK levels in MCF10A three-dimensional culture, we hypothesized that cAMP exerts its polarizing effect through ERK-dependent regulation of α6-integrin.
Importantly, in mammary epithelial cells, detachment from ECM does not inevitably result in down-regulation of ERK signaling. In primary mouse mammary epithelial cells, detachment did not lead to a loss of ERK phosphorylation (Wang et al., 2004
). Similarly, in primary cultured HMEC, inhibition of β1-integrin did not affect pERK levels, despite down-regulation of EGFR. On the other hand, cAMP decreased pERK in HMEC. Thus, in MCF10A and other mammary epithelial cells, decreased ERK signaling may result from a combination of integrin- and cAMP-dependent effects, with integrins regulating the levels of EGFR and cAMP acting downstream of the receptor level.
cAMP not only accelerates polarization of outer cells of the acinus, but also increases the rate of luminal apoptosis, which is required to hollow the lumen. Although the decrease in ERK activity may be a reason for the increase of BIM and apoptosis, our data demonstrate that the cAMP effect is at least partially ERK-independent. First, cAMP does not increase BIM mRNA level as detachment and ERK inhibition do (Reginato et al., 2003
; Hughes et al., 2011
). Second, in MCF10A cells overexpressing EGFR, which have much higher ERK activity and in which cAMP had no effect on ERK activity, cAMP still increased the protein levels of BIM. Third, in NMuMG cells, activation of PKA increased both ERK phosphorylation and BIM protein levels. While this study was in progress, a report describing a posttranslational PKA-dependent stabilization of BIM was published. PKA phosphorylates the largest isoform of BIM, BIMEL
, and thus prevents its proteasomal degradation in MCF7 cells (Moujalled et al., 2011
). It is likely that the same mechanism is operating in MCF10A cells as well.
Both polarization of the MCF10A acini and intraluminal apoptosis are delayed but not completely prevented in the absence of cAMP. We speculate that the early onset of polarization and apoptosis is important in morphogenesis for elimination of inner cells before they can proliferate, while without this early apoptosis, cells proliferate, more cells occupy the lumen, and the later apoptosis is not able to quickly eliminate them. It is also important to note that lumen formation was not completely prevented but was drastically delayed by the absence of cAMP. It is very likely that MCF10A acini would eventually clear. We were not able to test this hypothesis experimentally, due to matrix destruction and disorganization of three-dimensional structures under prolonged treatment in the absence of cAMP.
Whether cAMP and PKA play a role during normal mammary morphogenesis in vivo in not clear. During puberty, pregnancy, lactation, and involution, mammary glands undergo a complex remodeling that involves multiple hormones and growth factors. At least some of these hormones and growth factors may activate cAMP synthesis (Clegg and Mullaney, 1985
; Matsuda et al., 2004
; Stull et al., 2007
; Pai and Horseman, 2008
). We demonstrated here that activation of β-adrenergic receptor is sufficient to mimic effects of cAMP analogues in MCF10A three-dimensional culture.
It is also an interesting question whether deficient cAMP- and PKA-dependent signaling may contribute to epithelial carcinogenesis, since both loss of cell polarity and resistance to apoptosis are hallmarks of carcinogenesis (Debnath and Brugge, 2005
In summary, in the present study we have demonstrated a novel role for cAMP- and PKA-dependent signaling in regulation of mammary acinus formation. In MCF10A three-dimensional culture, cAMP accelerates polarization of outer acinus cells and apoptosis of inner cells. cAMP exerts its effects both through inhibition of ERK signaling and through ERK-independent elevation of the proapoptotic protein BIM.