SFRP1 Expression is Inversely Associated With Metastasis Formation in Canine Mammary Tumours.

BACKGROUND: Canine mammary tumours (CMTs) are the most frequent tumours in intact female dogs and show strong similarities with human breast cancer. In contrast to the human disease there are no standardised diagnostic or prognostic biomarkers available to guide treatment. We recently identified a prognostic 18-gene RNA signature that could stratify human breast cancer patients into groups with significantly different risk of distant metastasis formation. Here, we assessed whether expression patterns of these RNAs were also associated with canine tumour progression. METHOD: A sequential forward feature selection process was performed on a previously published microarray dataset of 27 CMTs with and without lymph node (LN) metastases to identify RNAs with significantly differential expression to identify prognostic genes within the 18-gene signature. Using an independent set of 33 newly identified archival CMTs, we compared expression of the identified prognostic subset on RNA and protein basis using RT-qPCR and immunohistochemistry on FFPE-tissue sections. RESULTS: While the 18-gene signature as a whole did not have any prognostic power, a subset of three RNAs: Col13a1, Spock2, and Sfrp1, together completely separated CMTs with and without LN metastasis in the microarray set. However, in the new independent set assessed by RT-qPCR, only the Wnt-antagonist Sfrp1 showed significantly increased mRNA abundance in CMTs without LN metastases on its own (p = 0.013) in logistic regression analysis. This correlated with stronger SFRP1 protein staining intensity of the myoepithelium and/or stroma (p < 0.001). SFRP1 staining, as well as ß-catenin membrane staining, was significantly associated with negative LN status (p = 0.010 and 0.014 respectively). However, SFRP1 did not correlate with ß-catenin membrane staining (p = 0.14). CONCLUSION: The study identified SFRP1 as a potential biomarker for metastasis formation in CMTs, but lack of SFRP1 was not associated with reduced membrane-localisation of ß-catenin in CMTs.

Antibiotic profiling of wild-type bacilli led to the discovery of new lanthipeptide subtilin-producing Bacillus spizizenii strains whose 16S rDNA sequences differ from the B. spizizenii typing strain / El perfil antibiótico de los bacilos de tipo salvaje condujo al descubrimiento de nuevas cepas de Bacillus spizizenii productoras de lantipéptido subtilina cuyas secuencias de ADNr 16S difieren de la cepa de tipificación de B. spizizenii

Two dozen field-collected Bacillus and a dozen Bacillus spizizenii wild-type strains from strain collections were selected on the basis of their antagonistic properties against the Gram-positive strain Micrococcus luteus. Based on their genetic and antibiotic profiles, they were characterized (subtilin encoding spaS gene sequences, mass spectrometric, and quantitative-reversed phase liquid chromatographic analyses, as well as the presence of the lanthionine cyclase protein SpaC by western blotting), seven novel producers of the lanthipeptide subtilin. Phylogenetic analyses of the subtilin-producing wild-type strains based on their 16S rRNA sequences showed that all seven strains could be classified as B. spizizenii: The field-collected strains HS and N5, as well as strains DSM 618, 1087, 6395, 6405, and 8439 from the German Collection of Microorganisms and Cell Cultures. To the best of our knowledge, all B. spizizenii strains described so far are characterized by the fact that they can produce a lanthipeptide of the subtilin family. Both the lanthipeptide structures and the organization and sequences of the 16S rRNA-encoding genes suggest a subdivision of B. spizizenii into subspecies: The subtilin-producing B. spizizenii strains are distinctly different from the entianin-producing B. spizizenii typing strain TU-B-10 T (DSM 15029 T).(AU)

Systematic analysis of different degrees of haemolysis on miRNA levels in serum and serum-derived extracellular vesicles from dogs.

BACKGROUND: Circulating microRNAs (miRNAs) are described as promising non-invasive biomarkers for diagnostics and therapeutics. Human studies have shown that haemolysis occurring during blood collection or due to improper sample processing/storage significantly alters the miRNA content in plasma and serum. Nevertheless, no similar research has been performed in dogs so far. We therefore investigated the effects of different degrees of haemolysis on the levels of selected miRNAs in serum and serum-derived extracellular vesicles (EVs) from dogs, by inducing a controlled in vitro haemolysis experiment. RESULTS: The abundance of miR-16, miR-92a, miR-191, miR-451 and miR-486 was significantly sensitive to haemolysis in serum and serum-derived EVs, while other selected miRNAs were not influenced by haemolysis. Furthermore, we found that the abundance of some canine miRNAs differs from data reported in the human system. CONCLUSIONS: Our results describe for the first time the impact of haemolysis on circulating miRNAs not only in whole serum, but also in serum-derived EVs from dogs. Hence, we provide novel data for further analyses in the discovery of canine circulating biomarkers. Our findings suggest that haemolysis should be carefully assessed to assure accuracy when investigating circulating miRNA in serum or plasma-based tests.

Antibiotic profiling of wild-type bacilli led to the discovery of new lanthipeptide subtilin-producing Bacillus spizizenii strains whose 16S rDNA sequences differ from the B. spizizenii typing strain.

Two dozen field-collected Bacillus and a dozen Bacillus spizizenii wild-type strains from strain collections were selected on the basis of their antagonistic properties against the Gram-positive strain Micrococcus luteus. Based on their genetic and antibiotic profiles, they were characterized (subtilin encoding spaS gene sequences, mass spectrometric, and quantitative-reversed phase liquid chromatographic analyses, as well as the presence of the lanthionine cyclase protein SpaC by western blotting), seven novel producers of the lanthipeptide subtilin. Phylogenetic analyses of the subtilin-producing wild-type strains based on their 16S rRNA sequences showed that all seven strains could be classified as B. spizizenii: The field-collected strains HS and N5, as well as strains DSM 618, 1087, 6395, 6405, and 8439 from the German Collection of Microorganisms and Cell Cultures. To the best of our knowledge, all B. spizizenii strains described so far are characterized by the fact that they can produce a lanthipeptide of the subtilin family. Both the lanthipeptide structures and the organization and sequences of the 16S rRNA-encoding genes suggest a subdivision of B. spizizenii into subspecies: The subtilin-producing B. spizizenii strains are distinctly different from the entianin-producing B. spizizenii typing strain TU-B-10 T (DSM 15029 T).

A matrisome RNA signature from early-pregnancy mouse mammary fibroblasts predicts distant metastasis-free breast cancer survival in humans.

BACKGROUND: During pregnancy, the mouse mammary ductal epithelium branches and grows into the surrounding stroma, requiring extensive extracellular matrix (ECM) and tissue remodelling. It therefore shows parallels to cancer invasion. We hypothesised that similar molecular mechanisms may be utilised in both processes, and that assessment of the stromal changes during pregnancy-associated branching may depict the stromal involvement during human breast cancer progression. METHODS: Immunohistochemistry (IHC) was employed to assess the alterations within the mouse mammary gland extracellular matrix during early pregnancy when lateral branching of the primary ductal epithelium is initiated. Primary mouse mammary fibroblasts from three-day pregnant and age-matched non-pregnant control mice, respectively, were 3D co-cultured with mammary epithelial cells to assess differences in their abilities to induce branching morphogenesis in vitro. Transcriptome analysis was performed to identify the underlying molecular changes. A signature of the human orthologues of the differentially expressed matrisome RNAs was analysed by Kaplan-Meier and multi-variate analysis in two large breast cancer RNA datasets (Gene expression-based Outcome for Breast cancer Online (GOBO) und Kaplan-Meier Plotter), respectively, to test for similarities in expression between early-pregnancy mouse mammary gland development and breast cancer progression. RESULTS: The ECM surrounding the primary ductal network showed significant differences in collagen and basement membrane protein distribution early during pregnancy. Pregnancy-associated fibroblasts (PAFs) significantly enhanced branching initiation compared to age-matched control fibroblast. A combined signature of 64 differentially expressed RNAs, encoding matrisome proteins, was a strong prognostic indicator of distant metastasis-free survival (DMFS) independent of other clinical parameters. The prognostic power could be significantly strengthened by using only a subset of 18 RNAs (LogRank P ≤ 1.00e-13; Hazard ratio (HR) = 2.42 (1.8-3.26); p = 5.61e-09). The prognostic power was confirmed in a second breast cancer dataset, as well as in datasets from ovarian and lung cancer patients. CONCLUSIONS: Our results describe for the first time the early stromal changes that accompany pregnancy-associated branching morphogenesis in mice, specify the early pregnancy-associated molecular alterations in mouse mammary fibroblasts, and identify a matrisome signature as a strong prognostic indicator of human breast cancer progression, with particular strength in oestrogen receptor (ER)-negative breast cancers.

Deregulation of miR-27a may contribute to canine fibroblast activation after coculture with a mast cell tumour cell line.

The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer-associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2-derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha-smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR-27a and let-7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR-27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR-27a in canine fibroblasts and involved in their activation. Importantly, miR-27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin-6 (IL-6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR-27a may play an important role in the tumour-microenvironment crosstalk.

Oxygen-Limiting Growth Conditions and Deletion of the Transition State Regulator Protein Abrb in Bacillus subtilis 6633 Result in an Increase in Subtilosin Production and a Decrease in Subtilin Production.

It has been recently shown, that certain strains/isolates of Bacillus subtilis can be used as a probiotic for humans. The production of the macrocyclic sactibiotic subtilosin in B. subtilis ATCC 6633 is highly regulated. To improve the subtilosin productivity of B. subtilis, different growth conditions were compared for maximal expression of the sbo promoter that regulates the expression of the subtilosin biosynthetic gene cluster. Oxygen-limiting conditions led to a strong increase of sbo promoter activities compared to aerobic conditions, and accordingly, the subtilosin amount determined by reversed phase HPLC (7.8 mg/L) was 15-fold superior to the amount of aerobic grown cultures (0.5 mg/L). A further promising enhancement of the subtilosin yield was achieved using a deletion mutant that is avoiding the general transition state regulator protein AbrB. The subtilosin titer of 42 mg/L produced by ΔabrB cells grown under oxygen-limiting conditions corresponds to an 84-fold increase compared to the subtilosin titer obtained from B. subtilis wild type cells propagated in aerobic conditions. Furthermore, evidence is provided that oxygen-limiting conditions led to a strong decrease in the productivity of the lantipeptide subtilin suggesting contrary regulatory mechanisms for the B. subtilis antimicrobials subtilin and subtilosin.

Expression profiling of key pathways in rat liver after a one-year feeding trial with transgenic maize MON810.

In a recent one-year feeding study, we observed no adverse effects on tissue level in organs of rats fed with the genetically-modified maize MON810. Here, we assessed RNA expression levels of 86 key genes of the apoptosis-, NF-кB-, DNA-damage response (DDR)-, and unfolded-protein response (UPR) pathways by RT-qPCR in the rat liver. Male and female rats were fed either with 33% MON810 (GMO), isogenic- (ISO), or conventional maize (CONV) and RNAs were quantified from eight rats from each of the six feeding groups. Only Birc2 transcript showed a significant (p ≤ 0.05) consistent difference of ≥1.5-fold between the GMO and ISO groups in both sexes. Unsupervised cluster analysis showed a strong separation of male and female rats, but no clustering of the feeding groups. Individual analysis of the pathways did not show any clustering of the male or female feeding groups either, though transcript levels of UPR pathway-associated genes caused some clustering of the male GMO and CONV feeding group samples. These differences were not seen between the GMO and ISO control or within the female cohort. Our data therefore does not support an adverse effect on rat liver RNA expression through the long-term feeding of MON810 compared to isogenic control maize.

Fibulin-2 is required for basement membrane integrity of mammary epithelium.

Fibulin-2 (FBLN2) is a secreted extracellular matrix glycoprotein which has been associated with tissue development and remodelling. In the mouse mammary gland, FBLN2 can be detected during ductal morphogenesis in cap cells and myoepithelial cells at puberty and early pregnancy, respectively. In an attempt to assign its function, we knocked down Fbln2 in the mouse mammary epithelial cell line EpH4. FBLN2 reduction led to an increase in the size of spheroidal structures when compared to scrambled control shRNA-transduced cells plated on Matrigel matrix. This phenotype was associated with a disruption of the collagen IV sheath around the epithelial spheroids and downregulation of integrin ß1, suggesting a role for FBLN2 in stabilizing the basement membrane (BM). In contrast to mice, in normal adult human breast tissue, FBLN2 was detected in ductal stroma, and in the interlobular stroma, but was not detectable within the lobular regions. In tissue sections of 65 breast cancers FBLN2 staining was lost around malignant cells with retained staining in the neighbouring histologically normal tissue margins. These results are consistent with a role of FBLN2 in mammary epithelial BM stability, and that its down-regulation in breast cancer is associated with loss of the BM and early invasion.

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis.

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here, we demonstrate that PTPRB negatively regulates branching morphogenesis in the mouse mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in oestrogen receptor-positive luminal cells. During mammary development, Ptprb expression is downregulated during puberty, a period of extensive ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of fibroblast growth factor receptor (FGFR) activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology.

Developmental Expression of Claudins in the Mammary Gland.

Claudins are a large family of membrane proteins whose classic function is to regulate the permeability of tight junctions in epithelia. They are tetraspanins, with four alpha-helices crossing the membrane, two extracellular loops, a short cytoplasmic N-terminus and a longer and more variable C-terminus. The extracellular ends of the helices are known to undergo side-to-side (cis) interactions that allow the formation of claudin polymers in the plane of the membrane. The extracellular loops also engage in head-to-head (trans) interactions thought to mediate the formation of tight junctions. However, claudins are also present in intracellular structures, thought to be vesicles, with less well-characterized functions. Here, we briefly review our current understanding of claudin structure and function followed by an examination of changes in claudin mRNA and protein expression and localization through mammary gland development. Claudins-1, 3, 4, 7, and 8 are the five most prominent members of the claudin family in the mouse mammary gland, with varied abundance and intracellular localization during the different stages of post-pubertal development. Claudin-1 is clearly localized to tight junctions in mammary ducts in non-pregnant non-lactating animals. Cytoplasmic puncta that stain for claudin-7 are present throughout development. During pregnancy claudin-3 is localized both to the tight junction and basolaterally while claudin-4 is found only in sparse puncta. In the lactating mouse both claudin-3 and claudin-8 are localized at the tight junction where they may be important in forming the paracellular barrier. At involution and under challenge by lipopolysaccharide claudins -1, -3, and -4 are significantly upregulated. Claudin-3 is still colocalized with tight junction molecules but is also distributed through the cytoplasm as is claudin-4. These largely descriptive data provide the essential framework for future mechanistic studies of the function and regulation of mammary epithelial cell claudins.

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

Overview of Mammary Gland Development: A Comparison of Mouse and Human.

The mouse mammary gland is widely used as a model for human breast cancer and has greatly added to our understanding of the molecular mechanisms involved in breast cancer development and progression. To fully appreciate the validity and limitations of the mouse model, it is essential to be aware of the similarities and also the differences that exist between the mouse mammary gland and the human breast. This introduction therefore describes the parallels and contrasts in mouse mammary gland and human breast morphogenesis from an early embryonic phase through to puberty, adulthood, pregnancy, parturition, and lactation, and finally the regressive stage of involution.

RNA Profiling of Non-cultured Fibroblasts Isolated from Pubertal Mouse Mammary Gland Sections.

The epithelium of the pubertal mouse mammary gland grows and invades the mammary fat pad to form a primary ductal network. This outgrowth is tightly controlled by epithelial and stromal factors that are present in the environment around the terminal end buds (TEB) at the growth front and the newly formed ducts. Identifying the contribution that each cell type makes to this regulation is a major challenge. To identify the role that fibroblasts play during this process we have optimised a fibroblast isolation procedure, followed by cell cleanup, RNA extraction, and amplification from non-cultured, freshly isolated fibroblasts from around the TEB as well as the subtending ducts. This was facilitated by the use of mice that constitutively expressed EGFP, which allowed the visualization of the growth front of the pubertal mammary tree under UV light. The isolated RNA is of sufficiently high quality, giving reproducible qRT-PCR results, for transcriptome analysis after RNA amplification.

Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud.

The terminal end bud (TEB) is the growing part of the ductal mammary epithelium during puberty, enabling the formation of a primary epithelial network. These highly proliferative bulbous end structures that drive the ductal expansion into the mammary fat pad comprise an outer cap cell layer, containing the progenitor cells of the ductal myoepithelium, and the body cells, which form the luminal epithelium. As TEB make up only a very small part of the whole mammary tissue, TEB-associated factors can be easily missed when whole-tissue sections are being analyzed. Here we describe a method to enzymatically separate TEB and ducts, respectively, from the surrounding stroma of pubertal mice in order to perform transcriptomic or proteomic analysis on the isolated structures and identify potential novel regulators of epithelial outgrowth, or to allow further cell culturing. This approach has previously allowed us to identify novel TEB-associated proteins, including several axonal guidance proteins. We further include protocols for the culturing of isolated TEB, processing of mammary tissue into paraffin and immunohistochemical/fluorescent staining for verification, and localization of protein expression in the mammary tissue at different developmental time points.

Atypical chemokine receptor ACKR2 controls branching morphogenesis in the developing mammary gland.

Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2-/- mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes.

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.

Human breast development.

This review is intended to give an overview of current knowledge on human breast development. It focuses on the limitations of our understanding on the origins of human breast cancer in the context of this mainly morphological and static assessment of what is known about human breast development. The world literature is very limited and caution is needed in drawing analogies with the mouse. There is an increasing emphasis on research to understand normal stem cells in the breast on the assumption that these are the targets for the initiation of breast cancer. It is thus a priority to understand normal human breast development, but there are major obstacles to such studies mainly due to ethical considerations and to tissue acquisition.

Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity.

Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.

Loss of reelin expression in breast cancer is epigenetically controlled and associated with poor prognosis.

Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Here we analyzed reelin expression by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2 status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P=0.01) and positive lymph node status (P<0.001). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, we established that reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. We conclude that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation.