Abstract
Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, we found that knockdown of ΔNp73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT.
Highlights
Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini [1,2]
We showed that knockdown of p53 or p73 in 3-D cultured MCF10A cells disrupts acinus structure coupled with decreased levels of p53 family targets p53 upregulated modulator of apoptosis (PUMA) and p21 [6,7]
We showed that in parental MCF10A cells, PUMA was induced upon treatment of doxorubicin (Figure 2A, compare lane 1 vs. 2), whereas in cells with PUMA knockdown (PUMA-KD), the levels of PUMA were decreased by shRNA at both the basal and stress conditions (Figure 2A, lanes 3–6)
Summary
Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini [1,2]. Hallmarks of breast cancer include loss of cell polarity, absence of a hollow lumen, and loss of control of cell proliferation and organization [4]. It is still largely unclear what signal pathways directly control the balance between cell proliferation and apoptosis during mammary morphogenesis and tumorigenesis. We found that in threedimensional (3-D) culture of MCF10A mammary epithelial cells, downregulation of wild-type p53 or p73 leads to partial clearance of the inner cells in the lumen due to decreased apoptosis [6,7]. Since Bim is not a target gene of p53 or p73, it is obvious that in addition to Bim, a p53 family target plays a role in the apoptotic clearance of the inner cells within developing acini
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