Overexpression of Bcl-2 Inhibits Alveolar Cell Apoptosis during Involution and Accelerates c-myc-induced Tumorigenesis of the Mammary Gland in Transgenic Mice

Summary
Expression of the apoptosis-inhibitory protein Bcl-2 has frequently been detected in human cancer including mammary carcinoma. The functional significance of its expression has been well established in experimental tumors of the lymphoid system, however, remains to be elucidated for the epithelial tumors. In order to assess the role of Bcl-2 in mammary tumorigenesis we have generated WAP-bcl-2 transgenic mice. The strong overexpression of Bcl-2 in lactating mammary glands was preserved during early postlactational involution and apoptosis of alveolar epithelial cells was prevented without influencing the dedifferentiation of the milk-producing epithelium. Although Bcl-2 overexpression was not sufficient to induce spontaneous tumors it, however, led to an accelerated development of MMTVmyc transgene-induced mammary tumors. In the mammary glands of MMTVmyc transgenic mice, a high proportion of apoptotic cells was detected which was significantly reduced in the mammary glands of WAP-bcl-2/MMTVmyc double transgenic mice. Taken together, these results suggest that Bcl-2 contributes to mammary tumorigenesis by inhibiting apoptosis.

Citations

Richard Jager, Ute Herzer, Johannes Schenkel and Hans Weiher, Oncogene (1997) 15:  1787-1795.

Background
Carcinogenesis is a multistep process involving the activation of proto-oncogenes and the loss of tumor suppressor genes. During lymphomagenesis, activation of the proto-oncogene bcl-2 by the t(14;18) chromosomal translocation represents one of these steps. The encoded 26 kDa Bcl-2 protein is a potent inhibitor of apoptotic cell death in many experimental systems, and this property is thought to account for its oncogenic potential in lymphoma. Generating transgenic mice which overexpress Bcl-2 in their mammary gland epithelium during lactation and early involution we adressed the following questions: (i) Does Bcl-2 overexpression interfere with the apoptosis of mammary epithelial cells normally seen during involution? (ii) Does Bcl-2 overexpression predispose to mammary carcinoma similar to the situation in lymphoma?

Transgene Knockout
The human bcl-2 alpha cDNA was placed under the control of the 1.6 kb Bgl II/ Kpn I WAP promoter fragment.

Mouse Strain
DBA/2

Phenotype:
(i) Involution: The mammary glands of transgenic mice were histologically indistinguishable from wildtype mice during involution, suggesting that Bcl-2 has no influence on (de-) differentiation of alveolar epithelial cells. However, the number of apoptotic cells was significantly reduced during the first three days of involution (Figure 1). By contrast, at the fourth day of involution, when alveoli collapsed, transgenic mammary glands displayed even higher numbers of apoptotic cells. Based on whole-mount analysis, no cell accumulation took place after multiple pregnancies. (ii) Tumorigenesis: Neither wildtype nor transgenic mice developed mammary carcinoma during 1.5 years of investigation, even after multiple pregnancies. However, when crossed to MMTVmyc-transgenic mice, resulting bigenic WAP-bcl-2/MMTVmyc female mice developed tumors earlier than their single-transgenic MMTVmyc sisters. In addition, the fulminant apoptosis of mammary epithelial cells induced by the MMTVmyc transgene was significantly reduced in mammary glands of bigenic mice (Figure 2). Of note, both MMTVmyc transgenic and WAP-bcl-2/MMTVmyc bigenic mice were unable to lactate.

Gene Expression
The transgene was expressed at high levels during lactation and the first three days of involution (Figure 3). In one of the transgenic lines expression was also detected in the brain.

Mechanistic Implications
(i) Involution: Since Bcl-2 expression was inhibiting the apoptosis of alveolar cells during involution without preventing the remodeling of the mammary gland architecture it can be concluded that both processes are independent. This is consistent with the finding that the peak in number of apoptotic cells (day 3) precedes the alveolar collapse (day 4). However, at day 4, when transgene expression dropped, we observed an even higher number of apoptotic cells in the transgenic than in the wildtype mammary glands. This could be explained by an additional survival signal provided by the ECM, which is disrupted at that stage. An alternative explanation could be that also in the transgenic mammary glands the cells have already been commited to suicide and now simply fulfill this programm once the "survival transgene" is no longer expressed. In either case, the additionally surviving cells in the transgenic mammary epithelium become finally deleted.
(ii) Tumorigenesis: That Bcl-2 overexpression unlike the situation in lymphoma did not predispose to mammary carcinoma in a wildtype background may be due to the deletion of the initially surviving cells later in involution. However, Bcl-2 cooperated with c-Myc in inducing mammary carcinoma, most likely by preventing the c-Myc-induced apoptosis of mammary epithelial cells. This finding points towards a model of multistep carcinogenesis wherein the disruption of the apoptotic machinery is a necessary step (Figure 4).

Submitted by:

Richard Jager
Department Klinische Forschung
Universitaet Bern
Tiefenaustrasse 120
CH-3004 Bern, Switzerland

Richard Jaeger
tel:41-31-308 8020
FAX: 41-31-308 8028