The Effect of Mammary Extracellular Matrix in Controlling Oral and Mammary Cancer Cells

Extracellular matrix (ECM) plays an important role in the normal physiology of tissues and progression to disease. Earlier studies and our external microarray data analysis indicated that mammary matrix from involuting tissue showed upregulation of genes involved in ECM remodeling. The present study examines the fate of mammary and oral cancer cells grown in the ECM from lactating mammary gland. Our findings show that non-tumorigenic cells, MCF10A and DOK cells did not proliferate but the tumorigenic and metastatic cells, SCC25 and MDA-MB-231, underwent apoptosis when grown on mammary ECM isolated from lactating mice. In addition, the cytokinesis marker, CEP55, was repressed in the oral and breast cancer cells. In contrast, these cells proliferated normally on mammary ECM isolated from mice undergoing involution. External microarray data analysis of mammary tissue further revealed over expression (~16 fold) of QSOX1 gene, which promotes cellular quiescence, in lactating mammary gland. A recent study has indicated that QSOX1 overexpression in breast cancer cells led to reduced proliferation and tumorigenic properties. This extracellular protein in mammary ECM may be responsible for reduced cellular proliferation. The present study has shown that ECM from lactating mammary gland can regulate signals to oral and breast cancer cells to halt cell division. This preliminary observation provided insights into the potential role of ECM factors present in lactating mammary gland as therapeutic targets to control cancer cell division. This preliminary study is an attempt to understand not only the requirement of ECM remodeling factors essential for the growth and survival of cancer cells but also the factors present in the lactation matrix that simultaneously halts cell division and selectively inhibits the growth of cancer cells.

Transcriptome analysis of mammary epithelial cell gene expression reveals novel roles of the extracellular matrix.

BACKGROUND: The unique lactation strategy of the tammar wallaby (Macropus eugeni) has been invaluable in evaluating the role of lactogenic hormones and the extracellular matrix (ECM) in the local control of mammary gland function. However molecular pathways through which hormones and ECM exert their effect on wallaby mammary gland function remain unclear. This study undertakes transcriptome analysis of wallaby mammary epithelial cells (WallMEC) following treatment with mammary ECM from two distinct stages of lactation. METHODS: WallMEC from MID lactation mammary glands were cultured on ECM from MID or LATE lactation and treated for 5 days with 1 µg/ml cortisol, 1 µg/ml insulin, 0.2 µg/ml prolactin, 650 pg/ml triodothyronine and 1 pg/ml estradiol to induce lactation. WallMEC RNA from triplicate ECM treatments was used to perform RNAseq. RESULTS: ECM from MID and LATE lactation differentially regulated key genes in sugar and lipid metabolism. Seven pathways including galactose metabolism, lysosome, cell adhesion molecules (CAM), p53 signaling, the complement and coagulation and Nod-like receptor signaling pathways were only significantly responsive to ECM in the presence of hormones. The raw RNA-seq data for this project are available on the NCBI Gene Expression Omnibus (GEO) browser (accession number GSE81210). CONCLUSIONS: A potential role of ECM in regulation of the caloric content of milk, among other functions including apoptosis, cell proliferation and differentiation has been identified. GENERAL SIGNIFICANCE: This study has used a non-eutherian lactation model to demonstrate the synergy between ECM and hormones in the local regulation of mammary function.

Dimeric but not monomeric α-lactalbumin potentiates apoptosis by up regulation of ATF3 and reduction of histone deacetylase activity in primary and immortalised cells.

α-lactalbumin is a protein of dual function found in milk of most mammals. α-lactalbumin binds ß-1,4-galactosyltransferase to form the regulatory subunit for lactose synthesis and has also been shown to cause cell death. This study shows, for the first time, that α-lactalbumin isolated in a rare 28kDa dimeric form induces cell death, while 14kDa monomeric α-lactalbumin is inactive. In contrast to the casein derived and chemically induced α-lactalbumin variants, MAL and HAMLET/BAMLET, the effects of 28kDa α-lactalbumin are calcium independent and, unlike MAL and HAMLET, 28kDa α-lactalbumin dimer causes cell death of primary mammary cells and a variety of immortalised cell lines, which are committed to cell death pathways within 1-4h of exposure. Microarray analysis confirmed that cell death was the result of an apoptotic response. Functional assays determined that the mechanism by which 28kDa α-lactalbumin kills cells involved inhibition of histone deacetylase activity mediated by NF-kB. We also show that 28kDa α-lactalbumin occurs naturally in the milk of cows, goats and sheep, is low in concentration during mid-lactation, but accumulates during milk stasis, consistent with a role in involution.

Essential role for a novel population of binucleated mammary epithelial cells in lactation.

The mammary gland represents a unique tissue to study organogenesis as it predominantly develops in the post-natal animal and undergoes dramatic morphogenetic changes during puberty and the reproductive cycle. The physiological function of the mammary gland is to produce milk to sustain the newborn. Here we view the lactating gland through three-dimensional confocal imaging of intact tissue. We observed that the majority of secretory alveolar cells are binucleated. These cells first arise in very late pregnancy due to failure of cytokinesis and are larger than mononucleated cells. Augmented expression of Aurora kinase-A and Polo-like kinase-1 at the lactogenic switch likely mediates the formation of binucleated cells. Our findings demonstrate an important physiological role for polyploid mammary epithelial cells in lactation, and based on their presence in five different species, suggest that binucleated cells evolved to maximize milk production and promote the survival of offspring across all mammalian species.

Coassembled nanostructured bioscaffold reduces the expression of proinflammatory cytokines to induce apoptosis in epithelial cancer cells.

The local inflammatory environment of the cell promotes the growth of epithelial cancers. Therefore, controlling inflammation locally using a material in a sustained, non-steroidal fashion can effectively kill malignant cells without significant damage to surrounding healthy cells. A promising class of materials for such applications is the nanostructured scaffolds formed by epitope presenting minimalist self-assembled peptides; these are bioactive on a cellular length scale, while presenting as an easily handled hydrogel. Here, we show that the assembly process can distribute an anti-inflammatory polysaccharide, fucoidan, localized to the nanofibers within the scaffold to create a biomaterial for cancer therapy. We show that it supports healthy cells, while inducing apoptosis in cancerous epithelial cells, as demonstrated by the significant down-regulation of gene and protein expression pathways associated with epithelial cancer progression. Our findings highlight an innovative material approach with potential applications in local epithelial cancer immunotherapy and drug delivery.

Analysis of human breast milk cells: gene expression profiles during pregnancy, lactation, involution, and mastitic infection.

The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step toward understanding why some women display poor lactation outcomes. Here, we demonstrate the utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from five time points (24 h prepartum during the colostrum period, midlactation, two involutions, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (e.g., CEL, OLAH, FOLR1, BTN1A1, and ARG2), while milk cells collected during involution showed a significant downregulation of milk synthesis genes and activation of involution associated genes (e.g., STAT3, NF-kB, IRF5, and IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, while maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MECs within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland.

Comparative analysis of caveolins in mouse and tammar wallaby: role in regulating mammary gland function.

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the ß-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.

In vitro response to functionalized self-assembled peptide scaffolds for three-dimensional cell culture.

Nanomaterials are rich in potential, particularly for the formation of scaffolds that mimic the landscape of the host environment of the cell. This niche arises from the spatial organization of a series of biochemical and biomechanical signals. Self-assembling peptides have emerged as an important tool in the development of functional (bio-)nanomaterials; these simple, easily synthesized subunits form structures which present the properties of these larger, more complex systems. Scaffolds based upon these nanofibrous matrices are promising materials for regenerative medicine as part of a new methodology in scaffold design where a "bottom-up" approach is used in order to simulate the native cellular milieu. Importantly, SAPs hold the potential to be bioactive through the presentation of biochemical and biomechanical signals in a context similar to the natural extracellular matrix, making them ideal targets for providing structural and chemical support in a cellular context. Here, we discuss a new methodology for the presentation of biologically relevant epitopes through their effective presentation on the surface of the nanofibers. Here, we demonstrate that these signals have a direct effect on the viability of cells within a three-dimensional matrix as compared with an unfunctionalized, yet mechanically and morphologically similar system.

Bioactive Functions of Milk Proteins: a Comparative Genomics Approach.

The composition of milk includes factors required to provide appropriate nutrition for the growth of the neonate. However, it is now clear that milk has many functions and comprises bioactive molecules that play a central role in regulating developmental processes in the young while providing a protective function for both the suckled young and the mammary gland during the lactation cycle. Identifying these bioactives and their physiological function in eutherians can be difficult and requires extensive screening of milk components that may function to improve well-being and options for prevention and treatment of disease. New animal models with unique reproductive strategies are now becoming increasingly relevant to search for these factors.

The extracellular matrix locally regulates asynchronous concurrent lactation in tammar wallaby (Macropus eugenii).

Asynchronous concurrent lactation (ACL) is an extreme lactation strategy in macropod marsupials including the tammar wallaby, that may hold the key to understanding local control of mammary epithelial cell function. Marsupials have a short gestation and a long lactation consisting of three phases; P2A, P2B and P3, representing early, mid and late lactation respectively and characterised by profound changes in milk composition. A lactating tammar is able to concurrently produce phase 2A and 3 milk from adjacent glands in order to feed a young newborn and an older sibling at heel. Physiological effectors of ACL remain unknown and in this study the extracellular matrix (ECM) is investigated for its role in switching mammary phenotypes between phases of tammar wallaby lactation. Using the level of expression of the genes for the phase specific markers tELP, tWAP, and tLLP-B representing phases 2A, 2B and 3 respectively we show for the first time that tammar wallaby mammary epithelial cells (WallMECs) extracted from P2B acquire P3 phenotype when cultured on P3 ECM. Similarly P2A cells acquire P2B phenotype when cultured on P2B ECM. We further demonstrate that changes in phase phenotype correlate with phase-specific changes in ECM composition. This study shows that progressive changes in ECM composition in individual mammary glands provide a local regulatory mechanism for milk protein gene expression thereby enabling the mammary glands to lactate independently.

Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene: new insights into the function of the protein.

Whey acidic protein (WAP) belongs to a family of four disulfide core (4-DSC) proteins rich in cysteine residues and is the principal whey protein found in milk of a number of mammalian species. Eutherian WAPs have two 4-DSC domains, whereas marsupial WAPs are characterized by the presence of an additional domain at the amino terminus. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. We have characterized the genomic structure of tammar WAP (tWAP) gene, identified its chromosomal localization and investigated the potential function of tWAP. We have demonstrated that tWAP and domain III (DIII) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of DIII from tWAP abolishes this proliferative effect. However, tWAP does not induce proliferation of human embryonic kidney (HEK293) cells. DNA synthesis and expression of cyclin D1 and cyclin-dependent kinase-4 genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DIII. These data suggest that DIII is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy.

Molecular analysis of tammar (Macropus eugenii) mammary epithelial cells stimulated with lipopolysaccharide and lipoteichoic acid.

The immunological function of the metatherian mammary gland plays a crucial part in neonatal survival of the marsupial young. Marsupial pouch young do not develop adult like immune responses until just prior to leaving the pouch. The immune components of the maternal milk secretions are important during this vulnerable early post-partum period. In addition, infection of the mammary gland has not been recognized in metatherians, despite the ready availability of pathogens in the pouch. Regardless of which, little is known about the immunobiology of the mammary gland and the immune responses of mammary epithelial cells in metatherians. In this study, a molecular approach was utilized to examine the response of tammar (Macropus eugenii) mammary epithelial cells to Escherichia coli derived lipopolysaccharide (LPS) and Staphylococcus aureus derived lipoteichoic acid (LTA). Using custom-made cDNA microarrays, candidate genes were identified in the transciptome, which were involved in antigen presentation, inflammation, cell growth and proliferation, cellular damage and apoptosis. Quantification of mRNA expression of several of these candidate genes, along with seven other genes (TLR4, CD14, TNF-alpha, cathelicidin, PRDX1, IL-5 and ABCG2) associated with innate immunity in LPS and LTA challenged mammary epithelial cells and leukocytes, was assessed for up to 24 h. Differences in genes associated with cellular damage and pro-inflammatory cytokine production were seen between stimulated mammary epithelial cells and leukocytes. LTA challenge tended to result in lower level induction of pro-inflammatory cytokines, increased PRDX1 mRNA levels, suggesting increased oxidative stress, and increased CD14 expression, but in a non-TLR4-dependent manner. The use of functional genomic tools in the tammar identified differences in the response of tammar mammary epithelial cells (MEC) and leukocytes to challenge with LPS and LTA, and validates the utility of the approach. The results of this study are consistent with a model in which tammar mammary epithelial cells have the capacity to elicit a complex and robust immune response to pathogens.

Uteroplacental insufficiency alters the mammary gland response to lactogenic hormones in vitro.

Adequate mammary development and coordinated actions of lactogenic hormones are essential for the initiation of lactation. Pregnancies compromised by uteroplacental insufficiency impair mammary development and lactation, further slowing postnatal growth. It is not known whether the initiation of lactation or galactopoesis is compromised. Uteroplacental insufficiency induced in rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on Day 18 of gestation preceded collection of mammary tissue on Day 20 of pregnancy. Mammary explants were cultured with combinations of insulin, cortisol and prolactin and analysed for alpha-lactalbumin and beta-casein gene expression. Mammary tissue from late pregnant Restricted rats had elevated alpha-lactalbumin, but not beta-casein, mRNA, which is consistent with premature lactogenesis resulting from an early decline in peripheral maternal progesterone. Explants from Restricted rats were more responsive to hormone stimulation after 3 days in culture, indicating that compromised galactopoesis, not lactogenesis, most likely leads to the reduced growth of suckled pups.

A population of mammary epithelial cells do not require hormones or growth factors to survive.

Hormonal stimulation of mammary explants mimics many of the biochemical changes observed during lactogenesis. Previous studies using eutherian species conclude that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. The present study examines the survival of mammary explants from the wallaby and mouse using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants from pregnant tammars and mice showed that milk protein gene expression was significantly elevated after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. Surprisingly, mammary explants remained hormone responsive and expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. Furthermore, the alveolar architecture was maintained. Global functional microarray analysis showed that classic involution markers were not differentially expressed, although two stress-induced survival genes were significantly up-regulated. We report that a population of mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. We propose that the mammary explant culture model uncouples the first phase of involution, as milk accumulation that normally provides involution stimuli is absent in this culture model allowing a population of cells to survive.

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii).

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.

Identification and transcript analysis of a novel wallaby (Macropus eugenii) basal-like breast cancer cell line.

BACKGROUND: A wide variety of animal models have been used to study human breast cancer. Murine, feline and canine mammary tumor cell lines have been studied for several decades and have been shown to have numerous aspects in common with human breast cancer. It is clear that new comparative approaches to study cancer etiology are likely to be productive. RESULTS: A continuous line of breast carcinoma cells (WalBC) was established from a primary breast cancer that spontaneously arose in a female tammar wallaby (Macropus eugenii). The primary tumor was 1.5 cm3 and although large, did not appear to invade the stroma and lacked vimentin expression. The WalBC cell line was cultured from the primary tumor and passaged for 22 months. WalBC cells displayed an epithelial morphology when grown on plastic, were not EGF responsive, stained strongly for cyto-keratin and negatively for vimentin. WalBC cells were shown to be non-invasive within a Matrigel invasion assay and failed to produce tumors following transplantation into nude mice. Gene expression profiling of WalBC cells was performed using a cDNA microarray of nearly 10,000 mammary gland cDNA clones and compared to normal primary mammary cells and profiles of human breast cancer. Seventy-six genes were down-regulated and sixty-six genes were up-regulated in WalBC cells when compared to primary mammary cells. WalBC cells exhibited expression of known markers of basal invasive human breast cancers as well as increased KRT17, KRT 14 and KRT 19, DSP, s100A4, NDRG-1, ANXA1, TK1 and AQP3 gene expression and decreased gene expression of TIMP3, VIM and TAGLN. New targets for breast cancer treatment were identified such as ZONAB, PACSIN3, MRP8 and SUMO1 which have human homologues. CONCLUSION: This study demonstrates how novel models of breast cancer can provide new fundamental clues regarding cancer etiology which may lead to new human treatments and therapies.

Molecular evolution of monotreme and marsupial whey acidic protein genes.

Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

The fur seal-a model lactation phenotype to explore molecular factors involved in the initiation of apoptosis at involution.

Mammary gland involution requires co-ordination of milk production, immune responses, apoptosis and remodeling. Initiation and progression of each of these components involves integral control by the mammary gland. Although cell-based culture models and genetically manipulated animals have shed light on these processes, the factors controlling each step in the involution cascade are still poorly understood. The fur seal displays a unique lactation phenotype. During the lactation cycle the mammary gland downregulates milk production and initiates an immune response but fails to initiate the apoptotic phase of involution, allowing the female fur seal to undertake long foraging trips of up to 28 days between suckling bouts. Upon return to shore the female continues feeding her pup following resumption of lactation and milk production. Expression profiling of genes involved in this lactation cycle provides valuable tools for investigation of the factors responsible for the initiation of apoptosis at involution.

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus).

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10-12 month lactation period, female fur seals suckle pups on shore for 2-3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 microm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (alphaS2-casein, beta-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell-matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Hormone-dependent milk protein gene expression in bovine mammary explants from biopsies at different stages of pregnancy.

A method for the collection of mammary biopsies developed previously was refined and used to study the endocrine regulation of bovine milk protein gene expression. Our surgical biopsy method used real-time ultrasound imaging and epidural analgesia to enable recovery of a sufficient quantity of mammary tissue from late-pregnant dairy cows for explant culture in vitro. The time of biopsy was critical for prolactin-dependent induction of milk protein gene expression in mammary explants, as only mammary tissue from cows nearing 30 d prepartum was hormone-responsive. This suggests that during the later stages of pregnancy a change in the responsiveness of milk protein gene expression to endocrine stimuli occurred in preparation for lactation. This may relate to the diminution of a putative population of undifferentiated cells that were still responsive to prolactin. Alternatively, the metabolic activity of the tissue had increased to the level whereby the response of the tissue was no longer assessable using this model in vitro.