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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Characterization of Bone Morphogenetic protein 7 in Breast Cancer

Tässä väitöskirjassa tutkittiin luun morfogeneettisen proteiini 7:n (BMP7) aktivaatiota, ilmentymistä, kliinistä merkitystä ja tehtäviä rintasyövässä. Rintasyöpä on naisten yleisin syöpä länsimaissa ja koskettaa joka kymmenettä naista tämän elinaikana. Luun morfogeneettiset proteiinit (BMP) ovat solunulkoisia signaalimolekyylejä, jotka toimivat hyvin kattavasti selkärankaisten alkiokehityksessä. Ne säätelevät solujen kasvua, erilaistumista ja apoptoosia, sekä voivat nimensä mukaisesti aiheuttaa luun muodostumista. Viime vuosina on saatu enenevästi tietoa siitä, että syövän syntyprosessin aikana erilaiset, normaalin yksilönkehityksen aikana toimivat geenit ja signalointireitit aktivoituvat uudelleen. Luun morfogeneettisten proteiinien keskeinen rooli kehityksen säätelyssä onkin johtanut niiden lisääntyneeseen tutkimukseen myös syövän saralla. Tähänastisten tulosten perusteella BMP-perheenjäsenillä on selvästi merkitystä syövässä, mutta tutkimustuloksia on rajoitetusti ja ne ovat olleet myös osin ristiriitaisia. Väitöskirjan ensimmäisen ja toisen osatyön tulokset osoittivat, että BMP7 ilmenee suuressa osassa kasvainnäytteistä hyvin voimakkaasti verrattuna normaaliin rintakudokseen, mikä viittaa syövälle spesifiseen ilmentymiseen. BMP7:n lisäksi toinen geeniperheen jäsen BMP4 ilmenee laajasti solulinjoissa ja kasvaimissa. Lisäksi BMP:ille spesifisten reseptorien ilmentyminen osoittaa, että rintasyövässä on toimiva signaalireitti BMP:ille. BMP7 on selvästi osallinen luustometastaasien muodostumiseen. Kolmannessa osatyössä selvitettiin, miten BMP7:n ilmentyminen liittyy erilaisiin rintasyövän kliinispatologisiin ominaisuuksiin, potilaiden taudin uusiutumiseen ja eliniän ennusteen laajan potilasaineston avulla. Tulosten mukaan BMP7 ilmenee merkittävästi useammin lobulaarista tyyppiä olevissa rintasyövissä kuin duktaalisissa. Yllättävää kyllä, BMP7 ilmeni paikallisuusimissa huomattavasti vähemmän kuin saman potilaan primaarikasvaimessa. Kuitenkin potilaat, joilla oli BMP7 positiivinen primaarikasvain, kärsivät nopeammin kehittyvistä luumetastaaseista. Monimuuttuja-analyysi osoitti, että BMP7 on itsenäinen ennustekijä varhaiselle luumetastaasille. BMP7:n avulla voidaan mahdollisesti tunnistaa potilaita, joilla on lisääntynyt riski saada luustometastaasi. Viimeisen osatyön tulosten perusteella BMP7 vaikuttaa rintasyöpäsolujen käyttäytymiseen hyvinkin dramaattisesti, mutta solutyyppikohtaisesti. BMP7 mahdollisia vaikutuksia rintasyöpäsolujen ilmiasuun, kuten kasvuun, apoptoosiin ja invasiivisuuteen tutkittiin joko estämällä BMP7 ilmentyminen sitä ilmentävistä rintasyöpäsolulinjoista tai lisäämällä BMP7:ää sitä ilmentämättömiin solulinjoihin. BMP7 lisäsi kasvua kahdella solulinjalla viidestä joko solusyklin tai apoptoosin kautta. Lisäksi BMP7 lisäsi voimakkaasti rintasyöpäsolujen (MDA-MB-231) migraatiota ja invaasiota. Tässä väitöskirjassa saatujen tulosten perusteella BMP7 ilmenee laajasti rintasyövässä ja vaikuttaa syöpäsolujen ilmiasuun solutyyppikohtaisesti. BMP7 on siten huomioonotettava tekijä rintasyövässä ja lisäksi altistaa erityisesti varhaiselle luustometastaasille. BMP-signaalireitti tarjoaa monia mahdollisuuksia signaalinvälityksen säätelyyn ja on siten myös lupaava kohde kliinisille sovelluksille.Bone morphogenetic proteins (BMP) are extracellular signaling molecules that regulate vertebrate development and bone formation. Due to their pleiotropic actions, they have been an object of growing interest in cancer research in the last decade. The purpose of this study was to characterize the activation, expression, clinical relevance and function of bone morphogenetic protein 7 (BMP7) in breast cancer. A previous report indicated that BMP7 may be a putative amplification target gene based on a microarray study conducted on breast cancer cell lines. BMP7 gene copy number and expression were explored in a large panel of 22 breast cancer cell lines, 146 primary breast tumors, and in normal mammary gland tissue. BMP7 copy numbers were analyzed using FISH. BMP7 mRNA and protein expression levels were determined using quantitative RT-PCR and immunohistochemistry. Increased BMP7 copy number was detected in half of the cell lines and in 16% of the primary tumors. Variable BMP7 expression was seen in both cell lines and primary tumors. Although the highest expression levels were detected in specimens with increased copy number, there was no significant association between BMP7 copy number increase and elevated mRNA expression. However, strong BMP7 protein expression was observed in over 70% of primary breast tumors compared to the normal samples indicating cancer specific overexpression. Systematic expression survey was performed for seven BMP ligands (BMP2-BMP8) and six BMP transmembrane receptors capable of transmitting BMP signals. Expression levels were determined using semiquantitative RT-PCR in 22 breast cancer cell lines and 39 primary breast tumors as well as in normal samples of mammary epithelial cell line and mammary gland tissue. In general the expression patterns in the cell lines were comparable to the patterns obtained from the primary tumors. The expression frequencies and levels differed considerably from one ligand to another and in addition to BMP7, BMP4 had a wide, variable, and cancer-specific expression profile. BMP specific receptors were ubiquitously expressed suggesting that breast cancer can receive BMP signals. The clinical relevance of BMP7 overexpression in breast cancer was studied in a group of 483 breast cancer patients with complete clinicopathological information and up to 15 years of follow-up. Samples contained 241 lobular carcinomas, 242 ductal carcinomas and 40 corresponding local recurrent tumors. BMP7 protein was expressed in 47% of the primary tumors determined by immunohistochemistry. BMP7 expression was tumor subtype dependent, since it was detected more often in lobular (57%) than in ductal (37%) tumors. Interestingly, BMP7 expression was observed in only 13% of local recurrent tumors. BMP7 expression did not affect overall survival but was clearly and significantly associated with accelerated rate of metastasis formation in bone. A multivariant analysis confirmed that BMP7 was indeed an independent prognostic factor for early bone metastasis. Finally, the possible contribution of BMP7 overexpression to breast cancer cell line phenotypes was examined using a bidirectional approach. BMP7 expression was silenced using RNA interference in three cell lines (BT-474, MCF7, SK-BR-3) with high endogenous expression and exogenous BMP7 was given to two cell lines (MDA-MB-231, T-47D) with no expression. The consequences of the manipulations were determined using functional assays for proliferation, cell cycle, apoptosis, migration, and invasion. BMP7 influenced the growth of two breast cancer cell lines. BMP7 silencing reduced growth in BT-474 cells that was caused by G1 arrest. Exogenous BMP7 treatment increased MDA-MB-231 growth instead by reducing the number of apoptotic cells. Thus in these two cell lines BMP7 stimulated proliferation either by regulating cell cycle or apoptosis. BMP7 treatment also significantly induced migration and even more drastically invasion of MDA-MB-231 cells. BMP7 does clearly have an impact on breast cancer cell phenotype and this is evidently dependent on the cellular context. Taken together, BMP7 is widely overexpressed in breast cancer and has an impact on breast cancer cell behavior. The clinical data furthermore implies that BMP7 is involved in the bone metastasis process in breast cancer

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 β (TGF-β) 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.BioMed Central open acces