Glycosylation in breast cancer progression and mammary development: Molecular connections and malignant transformations.

INTRODUCTION: The cellular behavior in normal mammary gland development and the progression of breast cancer is like the relationship between an object and its mirror image: they may appear similar, but their essence is completely different. Breast cancer can be considered as temporal and spatial aberrations of normal development in mammary gland. Glycans have been shown to regulate key pathophysiological steps during mammary development and breast cancer progression, and the glycoproteins that play a key role in both processes can affect the normal differentiation and development of mammary cells, and even cause malignant transformation or accelerate tumorigenesis due to differences in their type and level of glycosylation. KEY FINDINGS: In this review, we summarize the roles of glycan alterations in essential cellular behaviors during breast cancer progression and mammary development, and also highlight the importance of key glycan-binding proteins such as epidermal growth factor receptor, transforming growth factor ß receptors and other proteins, which are pivotal in the modulation of cellular signaling in mammary gland. Our review takes an overall view of the molecular interplay, signal transduction and cellular behaviors in mammary gland development and breast cancer progression from a glycobiological perspective. SIGNIFICANCE: This review will give a better understanding of the similarities and differences in glycosylation between mammary gland development and breast cancer progression, laying the foundation for elucidating the key molecular mechanisms of glycobiology underlying the malignant transformation of mammary cells.

The mesenchymal property of mouse mammary anlagen repopulating cell population is associated with its stemness.

During early embryogenesis, mammary glands are derived from surface ectoderm and their morphogenesis is controlled by mammary stem cells (MaSCs) and epithelial-mesenchymal transition (EMT). Mammary anlagen stage (E13.5-15.5) is an important stage for fetal mice to achieve EMT dependent mammary morphogenesis. And the characteristics of mammary anlagen repopulating cell population (MaRC) should be identified for understanding its stemness at earlier embryonic stage. Here we quantify and characterize MaSCs proportion at mammary anlagen stage. Compared with adult mouse mammary gland, our data revealed that E14.5 mammary anlagen exhibit higher stem cell activities. Then we purified mammary anlagen cell populations depending on the expression levels of CD24 and CD49f in mouse mammary anlagen, and identified an unique MaRC population (Lin-CD24medCD49f+) by real-time PCR, transplantation and mammosphere forming assays. In addition, by comparing with adult MaSC (Lin-CD24+CD29hi) and differentiated mammary anlagen cells, we find that E14.5 mouse MaRC population exhibit gene expression programs related to mesenchymal properties. To further identify the cell types of E14.5 mouse MaRC population, the expressions of K8, K14, K18, e-cadherin, n-cadherin and vimentin in mammary anlagen Lin-CD24medCD49f + cells were detected by immunofluorescence assay. These findings verified that the undifferentiated E14.5 mouse MaRC population is a heterogeneous population with mesenchymal property, which is associated with cell stemness and mammary duct morphogenesis.

A novel immunochromatographic strips assay for rapid and simple detection of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex multi-system autoimmune disease. Detection of anti-nuclear antibodies (ANA) is fundamental for the diagnosis of SLE. In the present study, we found that the level of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) is markedly up-regulated on immunoglobulin G (IgG) in the sera of SLE patients detected by Aspergillus oryzae lectin (AOL) blot. In sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA), the core fucosylation level was also found significantly increased in the sera from SLE patients with a higher ANA titer. To establish a rapid and sensitive laboratory test for the diagnosis of SLE, we prokaryotically expressed AOL and C3-D1-C3-D2-C3 of protein G (SpG3), and generate AOL-conjugated colloid gold immunochromatographic strips (ICS). The detection limit of core fucosylated IgG was 10 µg/mL for AOL-conjugated colloid gold ICS. As well as indirect immunofluorescence, the AOL-conjugated colloid gold ICS showed reliable results in the serum of 39 SLE patients. Our results indicated that the AOL-conjugated colloid gold ICS could serve as a rapid test for the detection of ANA and suspected cases of SLE.

[Corrigendum] c­Myc regulates the CDK1/cyclin B1 dependent­G2/M cell cycle progression by histone H4 acetylation in Raji cells.

Subsequently to the publication of this article, the authors have noticed that the published version of Fig. 5E contained incorrect data showing the cyclin E1 expression in the Raji­KD tumor (bottom row, rightmost panel; the data erroneously incorporated into the figure were the same as those correctly showing cyclin E1 expression in the Raji tumor experiment). The revised version of Fig. 5, showing the correct data for cyclin E1 expression in the Raji­KD tumor in Fig. 5E, is presented opposite. This error did not affect either the results or the conclusions reported in this paper. The authors apologize to the Editor of International Journal of Molecular Medicine and to readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 41: 3366­3378, 2019; DOI: 10.3892/ijmm.2018.3519].

Core fucosylation of copper transporter 1 plays a crucial role in cisplatin-resistance of epithelial ovarian cancer by regulating drug uptake.

Core fucosylation catalyzed by core fucosyltransferase (Fut8) contributes to the progressions of epithelial ovarian cancer (EOC). Copper transporter 1 (CTR1), which contains one N-glycan on Asn15 , mediates cellular transport of cisplatin (cDDP), and plays an important role in the process of cDDP-resistance in EOC. In the present study, we found that the core fucosylation level elevated significantly in the sera of cDDP-treated EOC patients. The in vitro assays also indicate that core fucosylation of CTR1 was significantly upregulated in cDDP-resistant A2780CP cells compared to the cDDP-sensitive A2780S cells. Intriguingly, the hyper core fucosylation suppressed the CTR1-cDDP interactions and cDDP-uptake into A2780CP cells. Conversely, contrast to the Fut8+/+ mouse ovarian epithelial cells, the Fut8-deleted (Fut8-/- ) cells obviously showed higher cDDP-uptake. Furthermore, the recovered core fucosylation induced the suppression of cDDP-uptake in Fut8-restored ovarian epithelial cells. In addition, the core fucosylation could regulate the phosphorylation of cDDP-resistance-associated molecules, such as AKT, ERK, JNK, and mTOR. Our findings suggest that the core fucosylation of CTR1 plays an important role in the cellular cDDP-uptake and thus provide new strategies for improving the outcome of cDDP based chemotherapy of EOC.

Prolactin stimulation affects the stem cell-dependent mammary repopulating ability of embryonic mammary anlagen.

The function of prolactin in mammary gland development and lactation has been demonstrated in previous studies. However, the potential action of prolactin on mammary duct morphogenesis at mammary anlagen stage (E13.5˜15.5) has not been elucidated. Transplantation of mammary cells or tissue is an effective approach for the regeneration of damaged mammary gland. Defining the actions of prolactin stimulation on embryonic mammary anlagen has important implications for defining the role of prolactin in the poorly understood processes of mammary duct morphogenesis and regeneration. Here, we have employed the GFP-transgenic mouse model to examine the effects of prolactin on stem cell activity. Using in vitro prolactin stimulation and transplantation of E14.5 mouse mammary anlagen, we identified the influence of prolactin on their mammary repopulating ability. Furthermore, using immunofluorescence staining and microarray analysis, the influence of prolactin on cell characteristics and gene expression in mammary anlagen were also profiled. We also demonstrate that E14.5 mouse mammary anlagen possess mammary stem cells (MaSCs) which achieve mammary repopulation. Furthermore, we found that in vitro prolactin stimulation elevates MaSC activity and promotes the mammary repopulating ability of mammary anlagen. Finally, microarray and pathway analysis showed that MAPK, Akt and JAK-STAT signaling was activated by prolactin stimulation, and drove the mammary repopulation of mammary anlagen. We conclude that prolactin stimulation elevates the stem cell-dependent mammary repopulating ability of embryonic mammary anlagen.

CD24 and CD49f expressions of E14.5 mouse mammary anlagen cells define putative distribution of earlier embryonic mammary stem cell activities.

Stem cell biology offers promise for understanding the origins of the mammary gland. However, the distribution of mammary stem cell (MaSC) activities at earlier embryonic stages has not been fully identified. The markers for sorting adult MaSC, CD24, CD29, and CD49f have been applied to analyze fetal MaSCs. Here we explored mammary anlagen MaSCs by investigating the expression of CD24 and CD49f. According to the comparative analysis between adult mammary gland and fetal mammary anlagen, we found that fetal mouse mammary anlagen may possess a high percentage of potential MaSCs. Flow cytometry analysis revealed 2 distinct mammary anlagen populations: Lin-CD24med and Lin-CD24high. Sphere-forming and mammary repopulating assays confirmed that the stem cell activity of E14.5 mouse mammary anlagen was restricted to the Lin-CD24med cell population. Furthermore, CD24med mammary anlagen cells were separated into Lin-CD24medCD49f+ and Lin-CD24medCD49f- populations and identified, respectively. The results proved that the mammary anlagen Lin-CD24medCD49f+ cell population possesses more stem cell activities than the Lin-CD24medCD49f- cell population. However, a limited numbers of stem cells and large numbers of stromal cells were identified in mammary anlagen in the Lin-CD24med cell population.

c-Myc regulates the CDK1/cyclin B1 dependent­G2/M cell cycle progression by histone H4 acetylation in Raji cells.

Overexpression of c-Myc is involved in the tumorigenesis of B-lineage acute lymphoblastic leukemia (B­ALL), but the mechanism is not well understood. In the present study, a c­Myc­knockdown model (Raji­KD) was established using Raji cells, and it was indicated that c­Myc regulates the expression of genes associated with cell cycle progression in G2/M­phase, cyclin D kinase (CDK)1 and cyclin B1, by modulating 60 kDa Tat­interactive protein (TIP60)/males absent on the first (MOF)­mediated histone H4 acetylation (AcH4), which was then completely restored by re­introduction of the c­Myc gene into the Raji­KD cells. The expression of CDK1 and cyclin B1 was markedly suppressed in Raji­KD cells, resulting in G2/M arrest. In comparison to Raji cells, the proliferation of Raji­KD cells was significantly reduced, and it was recovered via re­introduction of the c­Myc gene. In the tumorigenesis assays, the loss of c­Myc expression significantly suppressed Raji cell­derived lymphoblastic tumor formation. Although c­Myc also promotes Raji cell apoptosis via the caspase­3­associated pathway, CDK1/cyclin B1­dependent­G2/M cell cycle progression remains the major driving force of c­Myc­controlled tumorigenesis. The present results suggested that c­Myc regulates cyclin B1­ and CDK1­dependent G2/M cell cycle progression by TIP60/MOF-mediated AcH4 in Raji cells.

PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling.

PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells.

A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin(-)CD24(med)CD29(+) cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin(-)CD24(+)CD29(hi)), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin(-)CD24(med)CD29(+) cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin(-)CD24(med)CD29(+) cell population possessed low proportion of stem cells. In summary, heterogeneous Lin(-)CD24(med)CD29(+) cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment.

Synchronous behaviors of CBP and acetylations of lysine 18 and lysine 23 on histone H3 during porcine oocyte first meiotic division.

As a transcriptional coactivator and acetyltransferase, CREB-binding protein (CBP) is widely characterized due to its functions in cell proliferation and development. However, the activities of CBP in oocyte meiosis are not completely clear. Here we showed that the localization and expression of CBP changed regularly with the progression of porcine oocyte meiosis. The emergence of CBP in chromosomal domains is temporally coincident with the establishments of acetylated lysine 18 (AcH3/K18), lysine 23 (AcH3/K23) and dimethylated arginine 17 (dime-H3/R17) of histone H3 at meiotic stages from germinal vesicle breakdown (GVBD) to metaphase I (MI). Both CBP expression and these three histone modifications persisted to telophase I (TI). When trichostatin A (TSA) was used to enhance histone acetylations in porcine oocytes, we found that hyperacetylations of H3K18 and H3K23 occurred at meiotic stage from GVBD to TI, together with advanced and enhanced expression of CBP in the nucleus. In addition, disturbance of CBP activity by treatment with 2-Naphthol-AS-Ephosphate (KG-501, a drug targeting the KIX domain of CBP that disrupts the formation of CBP functional complex) led to synchronous decreases of CBP expression, AcH3/K18 and AcH3/K23 in chromosomal domains during oocyte meiosis. Therefore, these results indicate that the synchronous changes of CBP expression, AcH3/K18 and AcH3/K23 occur during porcine oocyte meiosis.

Localization of putative stem cells and four cell populations with different differentiation degree in mouse mammary anlagen.

The precise localization of putative stem cells and other cells within the embryonic mammary gland would help to elucidate the molecular pathways that govern normal mammary development. The ultrastructural appearances and the antigen of Sca-1 were considered together as putative stem cell markers, and the antigens of cytokeratin, CD10, Muc-1 and CD34 as differentiation markers. Electron microscopy was performed to reveal the ultrastructure of cells in different site of the mammary anlagen. An immunofluorescence system was used to reveal the whole structure profile of the mammary anlagen using the anti-cytokeratin antibody to localize specific types of cell populations such as Sca-1, CD10, Muc-1 and CD34 positive cells within the anlagen, which distribute near the inside edge, distribute uniformly, distribute in the central region and distribute in the site of mesenchyme around the neck of the anlagen, respectively. We also observed under electron microscope that some pale cells like putative stem cells reported by prevenient scholars, which are mainly distributed in the Sca-1(+) cell population near the inside edge of the anlagen, have pale-stained nucleoplasm and cytoplasm, sparse organelles clustered close to their nucleus and have a lack of rough endoplasmic reticulums and cell polarization. The results indicate that the putative stem cells are localized near the edge of the mammary anlagen; the cell populations with different differentiation degree were localized in the central part and around the edge within the anlagen.