Bim Regulation of Lumen Formation in Cultured Mammary Epithelial Acini Is Targeted by Oncogenes

Abstract

Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture.Tissue homeostasis of multicellular organisms is established and maintained by the delicate interplay between cell growth and cell death signals that are often altered in disease states such as cancer. Apoptosis is fundamental in maintaining proper cell number, sculpting of structures, and other cellular process that are necessary for embryonic development and for the maintenance of tissue homeostasis in the adult organism (7). Apoptosis has been implicated in the process of cavitation, or lumen formation in a solid cell mass in several three-dimensional (3D) spheroid models (2,6,15,18). In addition, apoptosis accompanies clearing of the terminal end buds in the developing mammary gland and lumen formation in the salivary gland (19,20). Many types of early breast cancer lesions such as ductal carcinoma in situ are characterized by loss of acinar organization and filling of the luminal space (14). The molecular mechanisms responsible for creation of the luminal space in epithelial acini are not well defined. In addition, it is not known how oncogenes that induce filling of the luminal space can target these pathways.We have investigated processes associated with lumen formation in the MCF-10A line of immortalized human mammary epithelial cells. When cultured on a reconstituted basement membrane derived from Engelbreth-Holm-Swarm tumor (Matrigel), immortalized MCF-10A mammary epithelial cells undergo a series of morphogenetic processes resulting in the formation of acinus-like structures containing a single layer of polarized cells surrounded by a hollow lumen (28). Formation of the luminal space in this model follows the development of apico-basal polarity and involv...

Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration

“…Due to the fact that ERK-mediated protection from anoikis in other cell lines has previously been shown to involve targeting of the pro-apoptotic protein Bim-EL for degradation, 23 we investigated the protein levels of Bim-EL in IBC cells that have been treated with increasing doses of U0126. Much to our surprise, we found that, contrary to published reports in other cell types, inhibition of the ERK pathway did not result in dramatically increased levels of Bim-EL.…”

“…[18][19][20] Interestingly, previous studies have shown that ErbB2 and EGFR, which are hyperactivated in a substantial percentage of IBC patients, 21 can effectively antagonize the anoikis program to facilitate anchorage-independent growth. [22][23][24][25][26][27][28] However, a detailed examination of the molecular mechanisms underlying anoikis inhibition in IBC cells has yet to be completed. In this study, we demonstrate that signaling from EGFR and ErbB2 through ERK/MAPK has a major role in the ability of IBC cells to survive in the absence of ECM attachment.…”

“…Surprisingly, we have discovered that ERKmediated anoikis suppression in IBC cells is not due to targeting of the pro-apoptotic protein Bim-EL for degradation that has previously been observed in mammary epithelial cells. 23,27 Rather, ERK activation in IBC cells promotes the formation of a protein complex containing Bim-EL, Beclin-1, and LC8, which functions to sequester Bim-EL from the mitochondria and thereby block anoikis. In support of the importance of this signaling pathway in IBC patients, five of the seven IBC patient samples assayed showed discernible Bim-EL staining.…”

BAG-1 overexpression attenuates luminal apoptosis in MCF-10A mammary epithelial cells through enhanced RAF-1 activation

“…Enhanced phosphorylation of BIM EL in BAG-1-expressing acini BIM is a critical effector of luminal cell death in MCF-10A acini (Reginato et al, 2005). Its expression is regulated by ERK-mediated phosphorylation on serine 69 (S69), which subsequently targets it for proteasomal degradation (Ley et al, 2003;Luciano et al, 2003).…”

“…This phenotype typifies those seen in most breast carcinomas and preinvasive lesions such as ductal carcinoma in situ, as well as some precancerous states such as atypical ductal hyperplasia (Harris et al, 1999), suggesting that disruption of the normal apoptotic program may have an important role in the initiation and progression of breast cancer. Studies aimed at elucidating the molecular mechanisms governing the formation and maintenance of the luminal space have demonstrated that it may be influenced both by Akt-mediated survival signals in the matrix-associated outer cells (Debnath et al, 2002) and active, BIMmediated apoptotic signals in the centrally localized cells (Reginato et al, 2005;Mailleux et al, 2007). BIM is upregulated immediately before the initiation of apoptosis and is specifically inhibited by prosurvival oncoproteins through stimulation of MEK-ERK signaling (Reginato et al, 2005).…”