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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.

Both ErbB1 and ErbB2 are overexpressed or amplified in breast tumours. To examine the effects of activating ErbB receptors in a context that mimics polarized epithelial cells in vivo, we activated ErbB1 and ErbB2 homodimers in preformed, growth-arrested mammary acini cultured in threedimensional basement membrane gels. Activation of ErbB2, but not that of ErbB1, led to a reinitiation of cell proliferation and altered the properties of mammary acinar structures. These altered structures share several properties with early-stage tumours, including a loss of proliferative suppression, an absence of lumen, retention of the basement membrane and a lack of invasive properties. ErbB2 activation also disrupted tight junctions and the cell polarity of polarized epithelia, whereas ErbB1 activation did not have any effect. Our results indicate that ErbB receptors differ in their ability to induce early stages of mammary carcinogenesis in vitro and this three-dimensional model system can reveal biological activities of oncogenes that cannot be examined in vitro in standard transformation assays.The mammary epithelium of an adult breast is organized into ducts and lobules. The ducts end in a highly branched structure referred to as the terminal ductal lobular unit (TDLU). A TDLU is comprised of multiple individual units referred to as mammary acini. Each acinus has a central lumen, a single layer of polarized luminal epithelial cells surrounded by myoepithelial cells, and a basement membrane.We have previously shown that human mammary epithelial cells (MECs) form acini-like structures containing a single layer of polarized, growth-arrested cells when grown within a matrix rich in laminin and collagen IV (Matrigel, derived from the Englebreth-Holm Swarm (EHS) tumour) 1,2 . The epithelial cells within acini in vivo and in culture have an apico-basal distribution of polarity markers such as ZO-1, E-cadherin and α 6 β 4 integrins. They also deposit collagen IV and secrete sialomucin in their basal and apical surfaces, respectively 1,2 , indicating that the acinar structures formed in culture closely mimic the acini in an adult breast.Early stages of breast cancer (hyperplasia and ductal carcinoma in situ (DCIS)) are characterized by an increased proliferation of epithelial cells, a loss of acinar organization and § Correspondence and requests for materials should be addressed to J.S.B. joan_brugge@hms.harvard.edu. † Present addresses: Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 11724, USA (S.K.M.); Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907, USA (S.L.)Supplementary information is available on Nature Cell Biology's website (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 October 11. filling of the luminal space 3 . However, a lack of acinar organization and the acquisition of invasive behaviour are later events involved...