We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen.

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.

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...

The molecular events regulating the elimination of cells to create a hollow lumen during tissue development are poorly understood. By using an in vitro morphogenesis model in which MCF-10A human mammary epithelial cells form hollow acini-like structures, we have observed both caspase-mediated apoptosis and autophagy associated with cells that are lost during lumen formation. Here, we show that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates induction of autophagic processes associated with lumen formation. TRAIL is up-regulated during morphogenesis of MCF-10A mammary epithelial cells in 3D basement-membrane cultures and inhibition of TRAIL signaling during morphogenesis blocks the formation of autophagic vacuoles. In addition, treatment with exogenous TRAIL induces extensive autophagy in monolayer and 3D cultures. When combined with inhibition of caspase 3 activity (by Bcl-X L overexpression), inhibition of TRAIL-induced autophagy results in luminal filling. Thus, TRAIL regulates an autophagic program during acinar morphogenesis, which together with caspase-mediated apoptotic events, results in lumen formation during MCF-10A morphogenesis. P rogrammed cell death is important for the formation and maintenance of tissue architecture in multicellular organisms. Programmed cell death regulates the sculpting of structures, deletion of vestigial organs, and maintenance of proper cell number (reviewed in refs. 1 and 2). Cell death is involved in cavitation during the development of the inner-cell mass (3) and some glandular organs (4, 5). The specific molecular events that characterize and drive programmed cell-death processes associated with cavitation remain poorly understood. We have used an in vitro 3D cell-culture model employing the immortalized human mammary epithelial cell line MCF-10A to examine the molecular events involved in lumen formation. When seeded in a matrix-rich environment, MCF-10A cells form acinar-like structures that are notable for the presence of a hollow lumen (6, 7).Lumen formation in MCF-10A spheroids is associated with the death of centrally located cells (6); furthermore, caspase 3 activation is observed in these dying cells, indicating that classical apoptosis is involved. In addition, overexpression of Bcl-2 or Bcl-X L , which abrogates caspase 3 activation, delays lumen formation significantly (6). Recently, we have found that the proapoptotic BH3-only protein Bim is an important regulator of apoptotic processes and lumen formation during MCF-10A morphogenesis (M.R., K.R.M., J.D., D. Lynch, and J.S.B., unpublished data). Apoptosis has been implicated also in the clearance of central cells in several other 3D spheroid-culture systems (8)(9)(10)(11)(12). Similarly, studies of mouse mammary-gland development indicate that apoptosis is involved in terminal end bud morphogenesis (13).The evidence that inhibition of apoptotic events by overexpression of Bcl-2 or Bcl-X L delays, but does not inhibit, lumen formation in MCF-10A structures indicated that additional ...