Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.

Bone morphogenetic proteins (BMP) are extracellular signaling molecules that belong to the transforming growth factor β (TGFβ) superfamily. Bone morphogenetic proteins have diverse roles during development where they regulate proliferation, differentiation, and apoptosis in many different cell types by modulating the transcription of specific target genes. BMPs have also been implicated in both promotion and inhibition of cancer progression. We have recently shown that BMP4 is commonly expressed in breast cancer but its functional significance has not been previously explored. Our data demonstrate that in all nine breast cancer cell lines studied, BMP4 treatment leads to a dramatic growth suppression as a result of the induction of G1 arrest of the cell cycle. At the same time, BMP4 stimulates cell migration and invasion in a subset of these breast cancer cell lines. The BMP4-induced phenotypic changes were mediated through the activation of the canonical SMAD signaling pathway whereas no activation of MAP-kinases ERK1/2 or p38 was detected. Our results thus implicate that BMP4 is an important regulator of key phenotypic characteristics of cancer cells, cell growth, cell migration, and invasion, and that, similar to TGFβ, it possesses both tumor suppressive and oncogenic properties in breast cancer.

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BackgroundBone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors. They are known for their roles in regulation of osteogenesis and developmental processes and, in recent years, evidence has accumulated of their crucial functions in tumor biology. BMP4 and BMP7, in particular, have been implicated in breast cancer. However, little is known about BMP target genes in the context of tumor. We explored the effects of BMP4 and BMP7 treatment on global gene transcription in seven breast cancer cell lines during a 6-point time series, using a whole-genome oligo microarray. Data analysis included hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data.ResultsBoth ligands had a strong effect on gene expression, although the response to BMP4 treatment was more pronounced. The cellular functions most strongly affected by BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, with regulation of gene expression and signal transduction leading to changes in metabolism and cell proliferation. Hierarchical clustering revealed distinct differences in the response of individual cell lines to BMPs, but also highlighted a synexpression group of genes for both ligands. Interestingly, the majority of the genes within these synexpression groups were shared by the two ligands, probably representing the core molecular responses common to BMP4 and BMP7 signaling pathways.ConclusionsAll in all, we show that BMP signaling has a remarkable effect on gene transcription in breast cancer cells and that the functions affected follow a logical temporal pattern. Our results also uncover components of the common cellular transcriptional response to BMP4 and BMP7. Most importantly, this study provides a list of potential novel BMP target genes relevant in breast cancer.

Bone morphogenetic protein 4 (BMP4) is a remarkably powerful inhibitor of breast cancer cell proliferation, but it is also able to induce breast cancer cell migration in certain cellular contexts. Previous data demonstrate that BMP4 controls the transcription of a variety of protein-coding genes, but not much is known about microRNAs (miRNA) regulated by BMP4. To address this question, miRNA expression profiles following BMP4 treatment were determined in one mammary epithelial and seven breast cancer cell lines using microarrays. While the analysis revealed an extensive variation in differentially expressed miRNA across cell lines, four miRNAs (miR-16-5p, miR-106b-5p, miR-23a-3p, and miR-23b-3p) were commonly induced in a subset of breast cancer cells upon BMP4 treatment. Inhibition of their expression demonstrated an increase in BT-474 cell number, indicating that they possess tumor suppressive properties. However, with the exception of miR-106b-5p, these effects were independent of BMP4 treatment. Scratch assay with miR-16-5p and miR-106b-5p inhibitors on BMP4-treated MDA-MB-231 cells resulted in enhanced cell migration, suggesting that these miRNAs are engaged in BMP4-induced motility. Taken together, we have for the first time characterized the BMP4-induced miRNA expression profiles in breast cancer cell lines, showing that induced miRNAs contribute to the fine-tuning of proliferation and migration phenotypes.