While netrin-1 is primarily thought of as an axon guidance cue, guidance is unlikely to be its only function, since expression studies have shown that many netrins are widely expressed outside the nervous system (Koch et al., 2000;Meyerhardt et al., 1999;Salminen et al.,

The product of the WT1 Wilms tumor suppressor gene controls the expression of genes encoding components of the insulin-like growth factor and transforming growth factor ␤ signaling systems. The role of these growth factors in breast tumor growth led us to investigate possible WT1 gene expression in normal and cancerous breast tissue. WT1 was detected by immunohistochemistry in the normal mammary duct and lobule, and the patterns of expression were consistent with developmental regulation. In a survey of 21 infiltrating tumors, 40% lacked immunodetectable WT1 altogether and an additional 28% were primarily WT1-negative. Cytoplasmic, but not nuclear, localization of WT1 was noted in some tumor cells and WT1 was detected, sometimes at high levels, in more-advanced estrogenreceptor-negative tumors. In this highly malignant subset, the tumor suppressor protein p53, which can physically interact with WT1, was also sometimes detected. WT1 mRNA was detected in normal and tumor tissue by reverse transcription-coupled PCR. Alternative splicing of the WT1 mRNA may regulate gene targeting of the WT1 protein through changes either in its regulatory or zinc-finger domains. The relative proportions of WT1 mRNA splice variants were altered in a random sample of breast tumors, providing evidence that different tumors may share a common WT1-related defect resulting in altered regulation of target genes.

The genes encoding Slits and their Robo receptors are silenced in many types of cancer, including breast, suggesting a role for this signaling pathway in suppressing tumorigenesis. The molecular mechanism underlying these tumor-suppressive effects has not been delineated. Here, we show that loss of Slits, or their Robo1 receptor, in murine mammary gland or human breast carcinoma cells results in coordinate up-regulation of the Sdf1 and Cxcr4 signaling axis, specifically within mammary epithelium. This is accompanied by hyperplastic changes in cells and desmoplastic alterations in the surrounding stroma. A similar inverse correlation between Slit and Cxcr4 expression is identified in human breast tumor tissues. Furthermore, we show in a xenograft model that Slit overexpression down-regulates CXCR4 and dominantly suppresses tumor growth. These studies classify Slits as negative regulators of Sdf1 and Cxcr4 and identify a molecular signature in hyperplastic breast lesions that signifies inappropriate up-regulation of key prometastatic genes.

Mammary ducts, and the highly mitotic terminal end buds from which they are derived, consist of two layers of ectodermally derived epithelium, forming a tube-within-a-tube structure. We investigated the role of Ca(2+)-dependent cell-cell adhesion molecules in maintaining the integrity of these layers. Immunostaining showed abundant E-cadherin on the lateral membranes of end bud body cells and ductal lumenal cells, but no P-cadherin. The basally located cap cells and their differentiated descendants, the ductal myoepithelial cells, displayed only P-cadherin. We investigated the functional significance of this pattern of cadherin expression in situ by surgically implanting small, slow-release plastic implants releasing function-blocking antibodies. End buds exposed to a monoclonal antibody to E-cadherin showed disruption of the body epithelium, with epithelial cells floating freely in the lumen. Epithelial DNA synthesis, which is normally very high in these growth buds, abruptly declined. That this reduction in growth was not due to cell damage was shown by spontaneous reaggregation of the cells into a normal epithelium, with resumption of DNA synthesis, when the blocking antibody was depleted. A monoclonal antibody to P-cadherin had no effect on the lumenal layer but partially disrupted the basally located cap cell layer. These data indicate that spatially selective expression of E- and P-cadherins is required for mammary tissue integrity, which is in turn a prerequisite for normal rates of DNA synthesis.