Bone morphogenetic protein 4 regulates microRNA expression in breast cancer cell lines in diverse fashion.

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.

BMP4 inhibits the proliferation of breast cancer cells and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells in 3D environment.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) belongs to the transforming growth factor ß (TGF-ß) family of proteins. BMPs regulate cell proliferation, differentiation and motility, and have also been reported to be involved in cancer pathogenesis. We have previously shown that BMP4 reduces breast cancer cell proliferation through G1 cell cycle arrest and simultaneously induces migration in a subset of these cell lines. Here we examined the effects of BMP4 in a more physiological environment, in a 3D culture system. METHODS: We used two different 3D culture systems; Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel. AlamarBlue reagent was used for cell proliferation measurements and immunofluorescence was used to determine cell polarity. Expression of cell cycle regulators was examined by Western blot and matrix metalloproteinase (MMP) expression by qRT-PCR. RESULTS: The MCF-10A normal breast epithelial cells formed round acini with correct apicobasal localization of α6 integrin in Matrigel whereas irregular structures were seen in PEG gel. The two 3D matrices also supported dissimilar morphology for the breast cancer cells. In PEG gel, BMP4 inhibited the growth of MCF-10A and the three breast cancer cell lines examined, thus closely resembling the 2D culture conditions, but in Matrigel, no growth inhibition was observed in MDA-MB-231 and MDA-MB-361 cells. Furthermore, BMP4 induced the expression of the cell cycle inhibitor p21 both in 2D and 3D culture, thereby partly explaining the growth arrest. Interestingly, MDA-MB-231 cells formed large branching, stellate structures in response to BMP4 treatment in Matrigel, suggestive of increased cell migration or invasion. This effect was reversed by Batimastat, a broad-spectrum MMP inhibitor, and subsequent analyses showed BMP4 to induce the expression of MMP3 and MMP14, that are thus likely to be responsible for the stellate phenotype. CONCLUSIONS: Taken together, our results show that Matrigel provides a more physiological environment for breast epithelial cells than PEG gel. Moreover, BMP4 partly recapitulates in 3D culture the growth suppressive abilities previously seen in 2D culture and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells.

The expression patterns of gremlin 1 and noggin in normal adult and tumor tissues.

Gremlin 1 and noggin are inhibitors of bone morphogenetic protein (BMP) signaling. They are vital during early development but their role in adult tissues has remained largely unresolved. The BMP signaling pathway has also been implicated in tumorigenesis, however with emphasis on the role of the ligands and receptors. We performed a concurrent survey of gremlin 1 and noggin protein expression in multiple normal and cancer samples, using immunohistochemistry on tissue microarrays containing 96 samples from 34 different normal organs/tissue sites and 208 samples of 34 different tumor types. In majority of both normal and tumor samples, gremlin 1 and noggin expression was negative or weak. However, normal stomach and skin demonstrated distinct gremlin 1 and noggin expression indicating a role in adult tissues. Likewise, strong expression of both antagonists was detected in Leydig cells of testis. In the tumor panel, the expression patterns were more variable but elevated BMP antagonist expression was detected for the first time in few cases, such as glioblastoma, hepatocellular carcinoma and diffuse B-cell lymphoma for gremlin 1 and renal granular cell tumor and thyroid papillary carcinoma for noggin. Even though gremlin 1 and noggin were not widely expressed in adult tissues, in a subset of organs their expression pattern indicated a potential role in normal tissue homeostasis as well as in malignancies.

Bone morphogenetic protein 4 expression in multiple normal and tumor tissues reveals its importance beyond development.

Bone morphogenetic proteins (BMPs) are extracellular signaling molecules that belong to the transforming growth factor ß (TGFß) superfamily and are known to regulate cell proliferation, differentiation and motility, especially during development. BMP4 has an indispensable role in vertebrate development while limited information on BMP4 expression and function exists in adult tissues. Nevertheless, its contribution to cancer development and progression has gained increasing interest in recent years. Functional studies, especially in breast cancer, have implicated BMP4 both in inhibition of cell proliferation and in promotion of cell migration and invasion. To gain an insight into the function of BMP4 in normal and cancer tissues, BMP4 protein expression levels were analyzed by immunohistochemistry in 34 different normal organs/tissues, 34 different tumor types and finally in 486 breast cancer samples where possible associations between BMP4 and clinicopathological parameters were statistically evaluated. In over 20% of normal and malignant tissues, BMP4 was expressed at high level. Strong expression was observed particularly in some normal epithelial cells, such as bladder and stomach, and in squamous cell carcinomas. In breast cancer, strong BMP4 expression was detected in 25% of patients, and was associated with low proliferation index and increased frequency of tumor recurrence. Taken together, BMP4 is expressed in a subset of normal adult tissues and is likely to contribute to tissue homeostasis. However, in tumors, BMP4 expression levels vary considerably, implying diverse roles in different tumor types. This role is biphasic in breast cancer as BMP4 expression is linked to reduced proliferation and increased recurrence, thus corroborating our previous in-vitro functional data.

Analysis of BMP4 and BMP7 signaling in breast cancer cells unveils time-dependent transcription patterns and highlights a common synexpression group of genes.

BACKGROUND: Bone 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. RESULTS: Both 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. CONCLUSIONS: All 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 and -5 in pancreatic cancer--novel bidirectional players.

Bone morphogenetic proteins (BMPs) are multifunctional signaling molecules that have gained increasing interest in cancer research. To obtain a systematic view on BMP signaling in pancreatic cancer we first determined the mRNA expression levels of seven BMP ligands (BMP2-BMP8) and six BMP specific receptors in pancreatic cancer cell lines and normal pancreatic tissue. BMP receptor expression was seen in all cancer and normal samples. Low expression levels of BMP5 and BMP8 were detected in cancer cells compared to the normal samples, whereas BMP4 expression was elevated in 25% of the cases. The impact of BMP4 and BMP5 signaling on cell phenotype was then evaluated in five pancreatic cancer cell lines. Both ligands suppressed the growth of three cell lines (up to 79% decrease in BMP4-treated PANC-1 cells), mainly due to cell cycle changes. BMP4 and BMP5 concurrently increased cell migration and invasion (maximally a 10.8-fold increase in invaded BMP4-treated PANC-1 cells). The phenotypic changes were typically associated with the activation of the canonical SMAD pathway, although such activation was not observed in the PANC-1 cells. Taken together, BMP4 and BMP5 simultaneously inhibit the growth and promote migration and invasion of the same pancreatic cells and thus exhibit a biphasic role with both detrimental and beneficial functions in pancreatic cancer progression.

Bone morphogenetic proteins in breast cancer: dual role in tumourigenesis?

The human bone morphogenetic protein (BMP) family consists of over 20 growth factor proteins that are involved in bone formation and developmental processes. BMPs are extracellular signalling molecules that are able to regulate various cellular functions, proliferation, differentiation, apoptosis and migration. For the last 10 years, these powerful cytokines have increasingly been studied in several cancers, and aberrant expression patterns of BMPs have been reported. Functional studies have suggested that BMPs are involved in both cancer promotion and inhibition. The role these signalling molecules play in breast cancer is only starting to emerge: thus far, studies have been even contradictory. Different BMP ligands have been shown to decrease as well as increase cancer cell growth and migration. Furthermore, they are involved in bone metastases, which are a common feature in breast cancer. In this sense, BMPs resemble a closely related protein transforming growth factor beta, which possesses a bidirectional role in cancer cell regulation. In this review, we focus on the current knowledge of BMP expression, functional roles and involvement in bone metastasis in breast cancer.

Parallel inhibition of cell growth and induction of cell migration and invasion in breast cancer cells by bone morphogenetic protein 4.

Bone morphogenetic proteins (BMP) are extracellular signaling molecules that belong to the transforming growth factor ß (TGFß) 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 TGFß, it possesses both tumor suppressive and oncogenic properties in breast cancer.

BMP7 influences proliferation, migration, and invasion of breast cancer cells.

Bone morphogenetic protein 7 (BMP7) is a signaling molecule originally identified based on its ability to form bone. It is essential during development, and more recently has also been implicated in cancer pathogenesis. We have recently shown that BMP7 is overexpressed in breast cancer, and that this increased expression is associated with early bone metastasis formation. In the present study, we explored the possible contribution of BMP7 function to the breast cancer cell phenotype. A two-way approach was applied in which BMP7 was silenced using RNA interference in three cell lines with high endogenous expression or, conversely, exogenous BMP7 was added to the growth medium of five cell lines with low or no BMP7 expression. These manipulations led to diverse cell line-specific phenotypic responses. BMP7 manipulation increased cell growth in two cell lines (BT-474, MDA-MB-231), and BMP7 treatment led to reduced growth in four cell lines (HCC1954, MDA-MB-361, T-47D, and ZR-75-30). Growth changes were due to distinct mechanisms since BMP7 silencing led to growth inhibition via G1 arrest in BT-474 cells, whereas BMP7 treatment protected MDA-MB-231 cells from apoptosis. Furthermore, BMP7 stimulation altered the MDA-MB-231 phenotype by inducing a distinct 2.3-fold increase in cell migration and an even more dramatic 3.9-fold increase in cell invasion. In conclusion, BMP7 can promote and inhibit cell growth in breast cancer cell lines and, in a suitable environment, can also considerably induce breast cancer cell migration and invasion.

PPM1D silencing by RNA interference inhibits proliferation and induces apoptosis in breast cancer cell lines with wild-type p53.

PPM1D is an oncogene that is amplified and overexpressed in many human tumors, including breast cancer. It functions as a negative regulator of the p38 MAP kinase-p53 signaling pathway and is also proposed to participate in other critical cell survival pathways. To define the functional significance of PPM1D specifically in breast cancer, we used RNA interference to inhibit PPM1D expression in BT-474, MCF7, and ZR-75-1 breast cancer cell lines harboring amplification and increased expression of PPM1D. Efficient downregulation of PPM1D resulted in significantly reduced cell proliferation in MCF7 and ZR-75-1 cells carrying wild-type p53 but not in BT-474 carrying mutant p53, which indicates that the antiproliferative effect of PPM1D silencing is dependent on the p53 status of the cells. This result is in excellent agreement with the notion that PPM1D activation is an alternative mechanism for p53 inactivation. Additionally, our data indicate that the reduced cell growth observed after PPM1D silencing is due at least in part to increased apoptotic cell death. Our findings demonstrate that PPM1D is involved in the regulation of cell proliferation in breast cancer in a p53-dependent manner and that overexpression of PPM1D contributes to malignant phenotype by promoting sustained cell growth and cell survival.

Identification of differentially expressed genes after PPM1D silencing in breast cancer.

Amplification and overexpression of PPM1D (protein phosphatase magnesium-dependent 1 delta) has been observed in various cancer cell lines and primary tumors and has also been associated with cancers of poor prognosis. In addition to the negative feedback regulation of p38-p53 signaling, PPM1D inhibits other tumor suppressor activities and is involved in the control of DNA damage and repair pathways. To elucidate the functional significance of PPM1D in breast cancer, we employed RNA interference to downregulate PPM1D expression in BT-474, MCF7, and ZR-75-1 breast cancer cell lines and then investigated the effects of PPM1D silencing on global gene expression patterns and signaling pathways using oligonucleotide microarrays. We identified 1798 differentially expressed (at least a two-fold change) gene elements with functions related to key cellular processes, such as regulation of cell cycle, assembly of various intracellular structures and components, and regulation of signaling pathways and metabolic cascades. For instance, genes involved in apoptosis (NR4A1, RAB25, PLK1), formation of nucleosome structure (HIST1H2AC, HIST1H2BF, HIST1H2BO, HIST1H1D), and hormone related activities (NR4A1, ESR1, STC1) were among the differentially expressed genes. Overall, our findings suggest that PPM1D contributes to breast cancer associated phenotypic characteristics by directly or indirectly affecting several important cellular signaling pathways.

A comprehensive expression survey of bone morphogenetic proteins in breast cancer highlights the importance of BMP4 and BMP7.

Bone morphogenetic proteins (BMPs) regulate diverse cellular processes, such as proliferation, differentiation, and apoptosis. The BMPs have been studied in several cancers, but thus far contradictory results have been obtained and, especially in breast cancer, information on BMPs is still limited. We performed a systematic expression survey of BMPs and their receptors in breast cancer. mRNA expression was studied of seven BMP ligands (BMP2-BMP8) and six receptors (ACVR1, BMPR1A, BMPR1B, BMPR2, ACVR2A, and ACVR2B) that specifically mediate BMP signals. Expression levels were determined in 22 breast cancer cell lines, 39 primary breast tumors, normal human mammary epithelial cell line, and normal mammary gland using semiquantitative RT-PCR. The expression frequencies and expression levels of different BMPs varied considerably in breast cancer with BMP4 and BMP7 being most frequently expressed and showing highest expression levels. The BMP specific receptors were more uniformly expressed and indicated that breast cancer is fully capable of transmitting BMP signals. Expression frequencies and levels for both the ligands and the receptors were in good concordance between the breast cancer cell lines and primary tumors. We can conclude that breast cancers possess functional BMP signaling machinery on the cell surface with distinct differences in the expression of various BMP ligands. Our survey focuses the attention particularly toward BMP4 and BMP7 and suggests their importance in breast cancer. Breast cancer cell lines and the data generated here serve as a good resource for further studies on BMP function in breast cancer.

Bone morphogenetic protein 7 is widely overexpressed in primary breast cancer.

Bone morphogenetic proteins (BMP) make up a family of extracellular signaling molecules that play a critical role in vertebrate development and both inhibit and stimulate growth in cancer cells. BMP7 was recently identified in our genomewide copy number and expression survey as being activated through amplification in breast cancer cell lines. In the present study, we further explored BMP7 gene copy number and expression changes in 22 breast cancer cell lines and 146 primary breast tumors. FISH analysis revealed that BMP7 copy number varied greatly from one cell line to another, with three cell lines showing extremely high-level amplification. Among primary tumors, BMP7 copy number was increased in 16% of the cases. BMP7 mRNA expression was determined in the cell lines and in a subset of 44 tumor samples by RT-PCR or quantitative real-time RT-PCR, respectively. Despite elevated mRNA levels in cancer cells, there was no significant association between copy number increase and mRNA expression, even though the highest expression was seen in cell lines and tumors with increased BMP7 copy number. Most interestingly, immunohistochemical analysis revealed BMP7 protein staining in all 11 breast cancer cell lines examined and strongly elevated BMP7 protein expression in 71.4% of the tumor samples as compared to normal mammary epithelium. Our results illustrate the frequent involvement of BMP7 alterations in breast cancer and especially highlight overexpression of the BMP7 protein in a very large fraction of primary breast tumors, thus suggesting a possible functional role for BMP7 in breast cancer development.

The serine-threonine protein phosphatase PPM1D is frequently activated through amplification in aggressive primary breast tumours.

The serine-threonine protein phosphatase PPM1D is likely to play an important role in tumorigenesis. Through inactivation of p38 MAPK, PPM1D acts as a negative feedback regulator of p53 tumour suppressor gene and controls the expression of other cell cycle regulatory proteins, such as CCND1. In addition, recent knock-out mouse studies implicated PPM1D in the regulation of p16 expression and the RB tumour suppressor pathway. Here we explored the role of PPM1D aberrations in primary breast cancer. PPM1D copy number analysis showed amplification in 11% (13/117) of the tumours and quantitative real-time RT-PCR revealed a significant correlation (p = 0.0148) between PPM1D amplification and increased expression. PPM1D amplification occurred almost exclusively in tumours with wild-type p53 suggesting that these events are mutually exclusive and further confirming the role of PPM1D as a negative regulator of p53. Interestingly, PPM1D amplification was associated with ERBB2 expression (p = 0.0001) thus implying that PPM1D aberrations occurs in tumours with poor prognosis. We also explored the expression levels of two possible downstream targets of PPM1D. However, immunohistochemical analyses revealed no differences in the staining patterns of CCND1 and p16 proteins in tumours with or without PPM1D aberrations, thus suggesting that previous data from animal model experiments is not directly transferable to primary human tumours. On the other hand, these key cellular proteins are likely to be regulated through a complex fashion in breast cancer and apparently PPM1D represents only one of these mechanisms. Taken together, our findings substantiate an important role for PPM1D in breast cancer.