We have analysed the differential effects of activating ErbB1 and ErbB2 receptors in preformed, growth-arrested, polarized acinar structures to mimic the conditions under which ErbB receptors are amplified and activated in vivo. Activation of ErbB2 in preformed mammary epithelial acini results in the reinitiation of proliferation, the loss of polarized organization and the formation of structures containing multiple acinar units. Each acinus within these structures had a filled lumen, surrounded by an intact basement membrane, and did not display any invasive properties. In addition, the activation of ErbB2 homodimers disrupted cell polarity in polarized epithelial monolayers. Thus, ErbB2 dimerization in differentiated acini induces a phenotype that displays some properties of a premalignant stage of breast cancer in vivo that is referred to as carcinoma in situ. In contrast, neither stimulation by EGF nor the activation of ErbB1 homodimers was sufficient to induce the reinitiation of proliferation, alterations in the organization of pre-formed mammary epithelial acini or the disruption of epithelial cell polarity. These results suggest that ErbB2 homodimers are uniquely able to disrupt normal regulation of the proliferation and organization of MECs. This activity of ErbB2 might contribute to the phenotype of comedo–ductal carcinoma in situ, in which ErbB2 expression is significantly amplified.
It is unclear why ErbB1 homodimers were unable to reinitiate proliferation in growth-arrested acinar structures because they were able to substitute for EGF in inducing the proliferation of MCF10A cells on plastic dishes (). In addition, previous studies have shown that ErbB1 homodimers are able to induce the serum-independent or interleukin-3-independent proliferation of fibroblasts and haematopoietic cells, respectively14,23
. It is possible that the events required for the re-stimulation of polarized growth-arrested cells are distinct from those required to support the proliferation of epithelial cells or fibroblasts grown under standard 2D culture conditions. Several lines of evidence suggest that the polarization of epithelial cells suppresses cell proliferation. Some of the most compelling data come from recent studies in Drosophila
showing that the disruption of proteins that control epithelial cell polarity such as Scribble, Discs large and Lethal giant larvae results in uncontrolled proliferation of the epithelial cells and hyperplastic growth22
. Conversely, the restoration of polarized structural organization to transformed MECs re-establishes growth suppression in three-dimensional cultures2
. These studies indicate that regulation of cell architecture might be important in growth control. It is possible that oncogenes need to disrupt cell polarity to induce the proliferation of polarized epithelia. Consistent with this possibility are the observations that the overexpression of oncogenes such as fos24
alter the polarity of epithelial cells in culture. Our results indicate that ErbB2 and ErbB1 receptors differ in their ability to disrupt cell polarity in epithelial monolayers. It will be of interest to understand the mechanism by which ErbB2, but not ErbB1, homodimers affect cell polarity and induce the reinitiation of cell proliferation in polarized epithelia.
It is likely that the reinitiation of proliferation is not the only step involved in the ErbB2-induced generation of multi-acinar structures with filled lumen. For example, MECs expressing human papilloma virus protein E7 form acinar structures that fail to growth arrest. However, these structures retain a lumen, indicating that uncontrolled proliferation might not be sufficient to repopulate the luminal space27
. Lumen formation during normal acinar morphogenesis involves the apoptosis of centrally localized cells that are not in contact with basement membrane28
(J.S. Debnath, K. Mills, J.S.B. and S.K.M, unpublished observations). The evidence that the cells induced to proliferate by ErbB2 homodimerization are able to survive in the lumen suggests that ErbB2 protects cells from apoptosis within the lumen. Our preliminary results indicate that the expression of anti-apoptotic proteins such as Bcl2 and Bcl-xl are not significantly upregulated by the activation of ErbB2 (data not shown). However, the cells in the middle of a filled acinus deposit collagen IV around their surface (), and this might protect the cells from undergoing apoptosis. Basement membrane components have been shown to protect epithelial cells from apoptosis29,30
. Thus, the ErbB2 induced generation of multi-acinar structures with filled lumen might involve both the reinitiation of proliferation and the prevention of apoptosis.
ErbB2 amplification is detected in a high frequency (80–85%) of comedo-type DCIS tumours, which are non-invasive, premalignant mammary tumours7,8
. The absence of invasive growth under conditions in which ErbB2 is expressed suggests that the amplification of ErbB2 is not sufficient to induce tumour invasion. Consistent with this possibility is the observation that activation of ErbB2 homodimers in 10A.ErbB2 cells failed to induce migratory or invasive behaviour in vitro
(Supplementary Information, Fig. S3
). Our results are consistent with previous findings with HB2 MECs31
and 32D haematopoietic cells32
but contrast with those obtained with MDCK47
and tumour-derived MCF-7 cells33
. It is likely that ErbB2 is not sufficient to induce invasive behaviour in MCF-10A cells and that additional genetic/epigenetic events or higher levels of ErbB2 expression are required for the cells to acquire invasive behaviour. Candidate cooperating genes include Rac, Cdc42 and PtdIns-3-OH kinase because their activation induces the invasion and migration of MECs34
. The cell–cell adhesion molecule E-cadherin is thought to regulate epithelial migration/invasion negatively35
. Although it was shown previously that expression of active ErbB2 can suppress E-cadherin gene expression in MECs36
, we did not see such regulation in our experiments (data not shown), indicating that additional events are required.
Our results describing the inability of ErbB2 homodimers to induce anchorage independence in normal MECs are consistent with some previous studies31,37–39
but contrast with others40–42
. It is not possible to compare our results directly with previous observations because earlier studies were not designed to activate ErbB homodimers without contributions from endogenous ErbB receptors or EGF ligands secreted in an autocrine manner37,43
. Moreover, the isolation of stable, non-inducible transfectants in other reports might have involved selection for altered phenotypes. The effect of inducible ErbB2 dimerization on acinar morphogenesis was examined with EPH4 mouse MECs expressing a Trk-ErbB2 chimaeric receptor that had been dimerized with NGF44
. The activated ErbB2 chimaera was able to induce normal morphogenesis with no reported perturbations in acinar structures. It is possible that the levels of expression of this receptor were below the threshold required for loss of polarity and uncontrolled proliferation.
Inducible activation of Fos-ER and Jun-ER fusion proteins in tubular structures composed of MECs demonstrated that activation of these proto-oncogenes results in a more marked phenotype involving not only a loss of polarized organization but also an induction of an epithelial–mesenchymal conversion24,25
. These studies together with ours indicate that analysing the effects of candidate oncogenes in polarized epithelial structures might provide insights into the biological activities involved in the early stages of carcinogenesis.
In summary, these studies provide evidence that ErbB1 and ErbB2 receptors have differential abilities to affect polarized and growth-arrested acini and also demonstrate that acute activation of ErbB2 results in the generation of multi-acinar structures that share properties with structures associated with carcinoma in situ. Because one class of DCIS (comedo) shows extremely high levels of ErbB2 amplification, it is possible that ErbB2 amplification in vivo induces a state similar to that observed in our cultured mammary acini. In addition, this in vitro model provides a useful system with which to explain the mechanisms involved in early stages of carcinogenesis associated with ErbB2 amplification.