The Wnt Gene Family in Mammary Gland Development

table

The exact timecourse of Wnt expression varies for each gene, allowing large numbers of 'potential' correlations to be drawn with phases of mammary development / changes in systemic hormones. Wnt-2, Wnt-5a, Wnt-7b and Wnt-10b are expressed during ductal development at the earliest times examined (5-6 weeks of age). The time at which expression of these genes is initiated is currently unknown and will require analyses of earlier (possibly fetal) timepoints. By contrast, the increase in expression of Wnt-4, Wnt-5b and Wnt-6 during pregnancy suggests an involvement of these genes in lobular development. During lactation, expression of most Wnt messages is down-regulated, while some but not all genes are re-induced during involution. Localisation studies show Wnt expression in both the stromal and epithelial compartments of the mammary gland, raising the possibility that Wnts could be involved in both stromal-epithelial and epithelial-stromal interactions [22, 23] . Expression of some Wnt genes is reduced in ovariectomised animals, but it is not clear whether these changes are the direct consequences of hormonal regulation or if the changes in Wnt expression mediate hormonal regulation of mammary development.

b. Investigating Function Through Ectopic Expression
Cell culture studies
C57MG mammary epithelial cells and 10T1/2 fibroblasts have been used to compare the consequences of ectopic expression of different members of the murine Wnt gene family. Expression of Wnt-1 in C57MG and 10T1/2 cells causes cells to 'morphologically transform' ie. to elongate and overgrow [24, 25] . In C57MG cells, expression of Wnt-1, Wnt-2, Wnt-3A, Wnt-5B, Wnt-7A and Wnt-7B induced morphological transformation (with different efficiencies), while Wnt-4, Wnt-5A and Wnt-6 were non-transforming. In 10T1/2 fibroblasts, expression of Wnt-1, Wnt-6 and Wnt-7B caused 'morphological transformation', while expression of Wnt-4 and Wnt-5B had no obvious effect. These data show that cell lines differ in their sensitivity to Wnts and that Wnts can be divided into at least two classes: transforming and non-transforming. C57MG cells respond to Wnt-1 expression by down-regulating endogenous Wnt-4 and Wnt-5a mRNA levels and altering levels of ß-catenin [26-28] . A potential role for Wnt-5A in mammary morphogenesis was implied by the observation that Wnt-5A message levels are strongly down-regulated in human luminal epithelial cells cultured in a collagen matrix in the presence of HGF/scatter factor [29].

Transgenic mice and the 'Transgenic' fat pad
Ectopic expression of Wnt-1 in vivo has been achieved using transgenic mice and recombinant retroviruses. In both systems, expression of Wnt-1 caused the formation of a characteristic hyperplasia with a feathery morphology that occupied the majority of the mammary fat pad in virgin animals [30, 31] . The molecular mechanisms which generated the Wnt-1 hyperplastic phenotype are not understood, but indirect arguments suggest that Wnt-1 enhances the proliferation of the mammary epithelial cells in vivo. Recombinant retroviruses have recently been used to show that Wnt-4 induces a 'pregnancy-like' hyperplasia in the glands of virgin mice, By contrast, retroviruses containing Wnt-5B, Wnt-6 and Wnt-7B cDNAs failed to give rise to gross morphological changes although failure of viral transfer could not be excluded as an alternative reason for the lack of phenotype ( [32] ,; Unpublished data J. Bradbury, PAW Edwards, T. C. Dale).

Knock out mice
Knockout mice have been described for the Wnt-1, Wnt-3A, Wnt-4 and Wnt-7A genes [8, 11, 33-35] . Of these, only Wnt-4 is normally expressed in the mammary gland and might be expected to give a mammary phenotype. Unfortunately the role of Wnt-4 in mammary development cannot be easily assessed as Wnt-4 mutant mice suffer from defective kidney tubulogenesis and die before birth. The phenotypes of further Wnt null phenotypes are eagerly awaited.

Implications for Mammary Development and Tumourigenesis

Wnt expression is maximal during periods of morphological change and is lost during the terminal differentiation that accompanies lactation, suggesting Wnts may control developmental and morphological changes in the gland. An apparent problem with this hypothesis is that expression of some genes remains high in the mature virgin gland after ductal end bud development has ceased. However, these observations may not be inconsistent because significant levels of cell proliferation (and presumably apoptosis) occur in the virgin gland [36, 37] . The developmental patterns of expression TGFß, FGF, EGF and HGF/Scatter mRNAs show a similar range to that of Wnt family members [38-41] , suggesting that both Wnt factors may co-operate with members of other ligand families during mammary development. This contention is supported by the observation that basic FGF can regulate the expression of Wnt-4 and Wnt-5A in C57MG cells [28] , while several members of the FGF family co-operate with MMTV-Wnt-1 in the generation of mouse mammary tumours [42-45].

The best current model to explain Wnt induced neoplasia is the theory that Wnt-1 and Wnt-3 functionally substitute for one or more endogenous Wnt genes and drive inappropriate cellular proliferation. As ectopic Wnt-1 expression decouples mammary growth from a dependence on estrogen and progesterone and the expression of some endogenous Wnts is dependent on ovarian function [23, 46] , the model for Wnt-1 function may be further refined:

image

The identity of the endogenous homologue(s) for Wnt-1/3 in the above model would be expected to include pregnancy-induced Wnts as the morphology of Wnt-1 hyperplasias mimic aspects of pregnancy development. Although ectopic expression of Wnt-4 also generates a pregnancy-like hyperplasia, the phenotype is clearly different from that induced by Wnt-1, suggesting that Wnt-1 does not simply substitute for the function of Wnt-4. Wnts that are normally expressed in the mammary gland may be involved in tumourigenesis. Studies have shown amplifications of the Wnt-2 gene in mammary tumours from GR mice [47] . Interestingly, we have recent data that suggests that a proportion of breast carcinomas express high levels of WNT-2 (T. C. Dale, S. Weber-Hall K.Smith, E. Huguet, B.A. Gusterson, A. Harris. In preparation). As murine Wnt-2 and human WNT-2 are both normally expressed in the stromal compartment, this suggests that tumourigenesis may involve WNT-2 switching compartments to the tumour epithelium, with the possible creation of an autocrine loop. Overexpression of Wnt-5A and Wnt-7B in benign and malignant mammary tumours has also been documented [48-50] . In the case of Wnt-5A, expression was increased by a mechanism that did not involve gene amplification. The overexpression of Wnt genes in human breast cancer raises the possibility that 'functional substitution' may also be involved in human tumourigenesis. More detailed studies of the role of Wnt function in vivo should shed light on these processes.

Future directions

In the long term, studies in the mammary gland should be able to identify the networks of gene products whose expression defines the ductal and lobular developmental motifs utilised in other organs (eg. lung and salivary gland). A particularly interesting aspect of these studies will be an understanding of how these processes are brought under hormonal control. In the short term, two technical approaches should be powerful in the analysis of Wnt function. The first is the characterisation of mammary development in 'knockout' mice. The second is the recent availability of the soluble Wnt factors which will allow the characterisation of Wnt signal transduction pathways in the mammalian context [51, 52].

Acknowledgements

Many thanks to Stuart Naylor and Dave Cook for reading the manusript before submission. My apologies to anyone who is missed or incorrectly quoted in this summary. To make the most of the electronic medium, I would be happy to include changes, comments and suggestions in the next revision.

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contributed by Trevor Dale
Institute of Cancer Research
Haddow Laboratories
15 Cotswold Rd.
Sutton, Surrey
SM2 5NG, UK

tel. 44-181-643-8901
FAX 44-181-643-0238
e-mail ( trevor@icr.ac.uk)

submitted: March 15, 1996 (revised June 1998)

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