Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium.

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8's association with this breast cancer subgroup we established ANXA8's cellular distribution in the mammary gland and ANXA8's effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (-ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8's association with their specific cells of origin.

Involvement of axonal guidance proteins and their signaling partners in the developing mouse mammary gland.

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.

Annexin A8 is up-regulated during mouse mammary gland involution and predicts poor survival in breast cancer.

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 1,631 cases 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.

Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3.

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. METHODS: We used Affymetrix oligonucleotide microarrays to perform a detailed transcript analysis on the mechanism of controlled involution after withdrawal of the pups at day seven of lactation. Some of the results were confirmed by semi-quantitative reverse transcriptase polymerase chain reaction, Western blotting or immunohistochemistry. RESULTS: We identified 145 genes that were specifically upregulated during the first 4 days of involution; of these, 49 encoded immunoglobulin genes. A further 12 genes, including those encoding the signal transducer and activator of transcription 3 (STAT3), the lipopolysaccharide receptor (CD14) and lipopolysaccharide-binding protein (LBP), were involved in the acute-phase response, demonstrating that the expression of acute-phase response genes can occur in the mammary gland itself and not only in the liver. Expression of LBP and CD14 was upregulated, at both the RNA and protein level, immediately after pup withdrawal; CD14 was strongly expressed in the luminal epithelial cells. Other genes identified suggested neutrophil activation early in involution, followed by macrophage activation late in the process. Immunohistochemistry and histological staining confirmed the infiltration of the involuting mammary tissue with neutrophils, plasma cells, macrophages and eosinophils. CONCLUSION: Oligonucleotide microarrays are a useful tool for identifying genes that are involved in the complex developmental process of mammary gland involution. The genes identified are consistent with an immune cascade, with an early acute-phase response that occurs in the mammary gland itself and resembles a wound healing process.

Detection of cytoplasmic CD antigens within normal human peripheral blood leucocytes.

Polymorphonuclear neutrophils (PMNs) are capable of synthesizing various pro-inflammatory cytokines which may indirectly influence specific immune responses. PMNs may also have the capacity to present foreign peptides to helper T cells (Th cells). In support of this hypothesis, recent studies have shown that neutrophils, when activated by the correct combination of cytokines, can be induced to express cell surface major histocompatibility complex (MHC) Class II (DR) antigen, CD80 (B7.1) and CD86 (B7.2): molecules required for antigen presentation and subsequent T-cell activation. In this study we have used normal "resting" human peripheral blood neutrophils and demonstrated, using a mild fixation and permeabilization protocol, significant cytoplasmic "stores" of these molecules known to be important in antigen presentation. Cytoplasmic MHC Class II antigen was found with two out of 20 normal donors tested whereas cytoplasmic CD80 and CD86 were found to a variable extent within all normal donors. Surprisingly, we also found several other neutrophil cytoplasmic CD antigens more commonly associated with B cells, i.e. CD20, CD21 (CR2/EBV-R) and CD22 (BL-CAM). All of these antigens were confined to the "resting" cell cytoplasm and were never found to be expressed on the cell surface. To exclude the possibility that these antigens were absorbed from plasma and to provide evidence for active synthesis, we used a novel whole blood in situ hybridization flow cytometry assay method to detect mRNA specific for these antigens within normal PMNs. We also conducted real-time polymerase chain reactions to confirm these findings using CD22 as a good example of an "inappropriately expressed" CD antigen. These observations therefore provide support for the hypothesis that human PMNs have the potential to express molecules required for antigen presentation and cell signalling.