The mouse mammary gland is an excellent model system with which to study both the regulation of development and the functional differentiation of an organ. Most of the development occurs postnatally, when the gland undergoes a highly regulated cascade of invasive growth, branching, differentiation, secretion, apoptosis and remodelling during each pregnancy cycle [1,2]. Terminal differentiation of the alveolar epithelium is completed at the end of gestation with the onset of milk secretion (lactation). APR = acute-phase response; C/EBP = CCAAT/enhancer-binding protein; H&E = haematoxylin and eosin; IHC = immunohistochemistry; IL = interleukin; LIF = leukaemia inhibitory factor; LPS = lipopolysaccharide; OSM = oncostatin M; RT-PCR = reverse transcriptase polymerase chain reaction; SOM = self-organising map; TNF = tumour necrosis factor; WAP = whey acidic protein. Abstract Introduction: Involution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process.

Purpose: Microarray studies have linked Annexin A8 RNA expression to a ''basal cell^like'' subset of breast cancers, including BRCA1-related cancers, that are characterized by cytokeratin 5 (CK5) and CK17 expression and show poor prognosis. We assessed Annexin A8's contribution to the overall prognosis and its expression in normal, benign, and cancerous tissue and addressed Annexin A8's physiologic role in the mammary gland. Experimental Design: Using microarrays and reverse transcription-PCR, the Annexin A8 expression was studied during mouse mammary gland development and in isolated mammary structures. Reverse transcription-PCR on cultured human luminal and basal cells, along with immunocytochemistry on normal and benign breast tissues, was used for cellular localization. Annexin A8's prognostic relevance and its coexpression with CK5 were assessed on tissue arrays of 1,631cases of invasive breast cancer. Coexpression was further evaluated on a small cohort of 14 BRCA1-related breast cancers. Results: Annexin A8 was up-regulated during mouse mammary gland involution and in pubertal ductal epithelium. Annexin A8 showed preferred expression in cultured basal cells but predominant luminal expression in normal human breast tissue in vivo. Hyperplasias and in situ carcinomas showed a strong staining of basal cells. Annexin A8 expression was significantly associated with grade (P < 0.0001), CK5 (P < 0.0001), and estrogen receptor status (P < 0.0001); 85.7% BRCA1-related breast tumors coexpressed Annexin A8 and CK5. Conclusion: Annexin A8 is involved in mouse mammary gland involution. In humans, it is a luminally expressed protein with basal expression in cell culture and in hyperplasia/ductal carcinoma in situ. Expression in invasive breast carcinomas has a significant effect on survival (P = 0.03) but is not independent of grade or CK5.

Mammary morphogenesis in the mouse is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEB). The mechanisms controlling the precise branching and spacing of the ducts are, as yet, unknown. To identify genes that are associated with migration of TEB and differentiation of the subtending ducts, we developed a novel method of isolating TEB and ducts free of stroma, and compared the gene expression profiles of these two isolates using oligonucleotide microarrays. Ninety one genes were upregulated in TEB compared to ducts. Three of these genes, Sprr1A, Sema3B, and BASP1, are associated with axonal growth and guidance. Two additional members of the Sprr family, Sprr2A and 2B, not previously associated with axonal growth, were also highly expressed in TEB. Expression of these genes was confirmed by RT-PCR and Western blotting, and the cellular distribution of Sprr1A and BASP1 was demonstrated by immunohistochemistry. Other semaphorins, including Sema3C, 4A, 4F and the cancer invasion associated Sema 4D were also expressed in the mouse mammary gland along with the semaphorin receptors, Plexins A2, A3, B2, and D1, and Neuropilins 1 and 2. These results are discussed in the context of other proteins expressed in the developing gland that are known to be downstream effectors of these signaling molecules. We suggest that these genes may influence ductal growth and morphogenesis in the developing mammary gland.