Low-density microarray analysis of TGF 1-dependent cell cycle regulation in human breast adenocarcinoma MCF 7 cell line

Transforming growth factor 1 (TGF 1) is a growth regulator that has antiproliferative effects on a range of epithelial cells at the early stages and promoting tumorigenesis at the later stages of cancer progression. The molecular mechanisms of a duel role of TGF 1 in tumor growth regulation remain poorly understood. Aim. To analyze the TGF 1-dependent cell cycle regulation of tumorigenic breast epithelial cells. Methods. Our present study was designed to examine the regulatory effect of TGF 1 on the expression of a panel of 96 genes which are known to be critically involved in cell cycle regulation. GEArray Q series Human Cell Cycle Gene Array was applied to profile the gene expression changes in MCF7 human breast adenocarcinoma cell line treated with TGF 1. Results. The gene expression array data enabled us to reveal the molecular regulators that might connect TGF 1 signaling to the promoting of the tumor growth, e. g. retinoblastoma protein (pRB1), check-point kinase 2 (Chk2), breast cancer 1, early onset (BRCA1), DNA damage checkpoint protein RAD9, cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1). Conclusions. The uncovering of the key signaling modules involved in TGF 1dependent signaling might provide an insight into the mechanisms of TGF 1-dependent tumor growth and can be beneficial for the development of novel therapeutic approaches.


Introduction.
Transforming growth factor b1 (TGFb1) is a dimeric polypeptide growth factor with multiple physiological functions that has been first described as a stimulator of cellular tumorigenic transformation [1].TGFb1 initiates intracellular signaling through the binding to the specific receptors type I (TbRI) and type II (TbRII) on the cellular surface.TGFb1 receptors contain serine/threonine kinase domains and form a heterotetramer composed of two TbRI:TbRII heterodimers complex upon TGFb1 binding [2].The formation of the ligand-receptor complex triggers a number of TGFb1-dependent signaling pathways [3,4].The TGFb1-dependent signal transmission through the Smad transcriptional factors is considered to be the most important for TGFb1 cell response.However, over the last decade the TGFb1 -mediated activation of Smad-independent signal pathways has been also described.Among them, the Ras and mitogen-activated protein kinase (MAPK) pathways, p38, extracellular signal regulated kinases (ERK), cJun N-terminal kinase (JNK) have been shown to be activated in response to TGFb1 [5].Emergence of genomic and proteomic approaches advanced our understanding of the plasticity of TGF cellular response and provides a comprehensive overview of the role of TGFb1 regulatory effect on the maintenance of cell and tissue homeostasis and functions.A number of novel TGFb1 targets, which affect cell proliferation, death, DNA damage repair, differentiation, cytoskeleton rearrangement, and cellular metabolism have been identified in our previous reports [6][7][8][9][10].
A variety of studies have shown a dual role of TGFb1 in the normal tissue maintenance and cancer [11].TGFb1 is a negative growth regulator that has antiproliferative effects on a range of epithelial cells at the early stages and promoting tumorigenesis at the later stages of cancer progression.Discovery of the selected signaling molecules has provided an insight into some molecular mechanisms behind this dual role of TGFb1.However, a number of evidence suggests that an ability of cancer cells to overcome the growth inhibitory effects of TGFb1 is a result of the functional changing a host of the intracellular signaling components.
Therefore, a large-scale genomic and proteomic analysis has to be employed to explain a switch in the TGFb-dependent cell response during tumor development.Our comparative analysis for some of the identified gene targets (c-abl, CDKN2D, RAD9) has shown their distinct expression in the nontumorigenic epithelial cells MCF10A and human breast adenocarcinoma cells MCF7.
Expanding our knowledge of the differential employment of TGFb1 signaling network by premalignant and tumor cells might contribute to our understanding of breast cancer development and can be potentially employed for therapeutic benefit.Our present study was designed to examine the TGFb1 regulatory effect on the expression of 96 genes known to be critically involved in the cell cycle regulation.
Materials and methods.Cells and reagents.MCF7 cells were obtained from American Type Culture Collection (Manassas, USA), and cultured in DMEM supplemented with 10 % of foetal bovine serum («Sigma-Aldrich», USA).TGFb1 was added to the cells to final concentration of 5 ng/ml.At 18 h after treatment, cells were lysed and protein extracts were analyzed by immunoblotting.
Low-density microarray experiment was performed with RNA extracted from MCF7 cells, which were treated or non-treated with TGFb1.RNA was extracted using a RNAsy mini kit («QIAGEN», USA), and RNA without any signs of degradation was used for analysis.Preparation of the probes (GEArray Probe synthesis kit, SuperArray Bioscience, USA), and hybridization with the membranes of Human Cell Cycle Gene array (HS-001-4 GE array Q series; SuperArray Bioscience) were performed, as recommended by the manufacturer.After hybridization, the membranes were exposed in a FujiX 2000 PhosphorImager, and the acquired images were analysed using ScanAlyze software (http://rana.lbl.gov/EisenSoftware.htm).
Two-fold changes were considered as a threshold for significant TGFb1-dependent changes in gene expression level.
Pathway analysis.Functional and signalling pathway analysis was done using Ingenuity Pathway Analy-sis, a commercial database for identifying networks and signalling pathways of interest in global genomic data.Dataset containing identified genes and corresponding expression values was uploaded into the Ingenuity Pathway Analysis application and TGFb1 dependent network regulating cell growth and proliferation was generated.Fischer's exact test was used to calculate a p-value determining the network connectivity.
Luciferase reporter assay.Reporter assays with CAGA(12)-luc reporter was performed as described previously [12].293T cells were used, because they are responsive to TGFb treatment and allow for efficient exogenous protein expression.
Results and discussion.TGFb1 dependent expression of genes regulating cell proliferation in MCF7 cells.In the current study we explored changes in expression of genes involved in cell cycle regulation in MCF7 cells in response to TGFb1 stimulation.To assess long-term changes in TGFb1-dependent expression, MCF7 cells were analyzed after incubation with TGF b1 for 18 h and compared with nontreated cells.Phosphorylation of Smad2 on C-terminal serine residue upon TGFb1 treatment was used as an indicator for TGFb1 signalling activation (Fig. 1, A). Human Cell Cycle Gene Array Q series has been applied to evaluate transcription level of 96 genes which are known to be regulators of cell cycle and response to DNA damage.We found that expression of 41 genes was altered at least twofold (p < 0.05) after the treatment of the cells with TGFb1 (Table 1).Among identified genes 17 were already known TGFb1 target genes, i. e., transcription factors DP-1, DP-2, cyclins D1, D2, F, G1 and G2, cyclin dependent kinases cdk2, cdk4, cdk6, cdk7, cyclin-dependent kinase inhibitors CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN2D, metallopeptidase inhibitor TIMP3 [9,10].However, most of the differentially regulated genes have not been previously implicated in TGFb1 signaling.Semi-quantitative RT-PCR and Western blotting analysis has been performed to validate the TGFb1 dependent changes in the expression of some of the identified genes (Fig. 1, B, C).The obtained results were consistent with those from gene expression array supporting the validity of the microarray data.We revealed a distinct TGFb1-dependent regulation of some of the identified genes (c-abl, CDKN2D, RAD9) in MCF10A nontumorigenic epithelial cells as compared to the MCF7 human breast adenocarcinoma cells that can be explained, at least in part, by switching the responsiveness of the mammary epithelial cells to TGFb1 during tumor progression (Fig. 1, D).
Ingenuity Pathway analysis.To shed light on the signaling pathways contributing to the regulation of cell cycle by TGFb1, all 41 genes differentially expressed in the TGFb1 and untreated cells were subjected to pathway analysis by using Ingenuity Pathway Analysis.The Ingenuity Pathways Knowledge Base, a comprehensive knowledge base for identifying signalling networks for genes of human, rat and mouse was used to build signalling networks and make functional analysis of an entire dataset.Among the 96 genes that have been annotated as cell cycle regulators, expression of 41 genes was changed significantly upon TGFb1 treatment in MCF7 human breast adenocarcinoma cell line (Table 1).All 41 overlaid genes (100 %) were found in the pathway map.By focusing on these 41 genes, we constructed a TGFb1dependent cell cycle regulating network that included all known interaction between dataset genes.The signaling networks, which include TGFb1-responsive genes are listed in Table 2. Upstream regulator analysis revealed Smad2, Smad3 and TGFb1 as regulators of transcription in this gene network (Fig. 2, A).According to generated signaling network, we have identified a few signaling modules which might be considered as key transmitters of TGFb-signaling, which control cell cycle, e. g. estrogen receptor (ER), retinoblastoma protein (pRB1), checkpoint kinase 2 (Chk2), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), breast cancer 1, early onset (BRCA1), platelet-derived growth factor (PDGF), cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1).An overlapping of the canonical pathways involving identified genes revealed, among others, a preferential activation of the aryl hydrocarbon receptor (AhR) pathway, the growth arrest and DNA damage (GADD45) pathway and p53 signaling rout (Fig. 2, B).
Chk2 cooperates with Smad3 in transcriptional regulation.To explore the possible mechanism for the involvement of some of identified genes in TGFb1-initiated signal transduction, we performed a luciferase reporter assay with TGFb1-responsive CAGA(12)-luc reporter.This reporter contains multiple CAGA boxes which are specific binding site for Smad3 and Smad4 -TGFb1 activated transcriptional factors.We used 293T cells because they are TGFb1 responsive and allow high level of ectopic expression of target proteins.We observed that protein product of RAD53 gene -Chk2 kinase, which expression is found to be downregulated upon TGFb1 treatment, cooperated with Smad3 in activation of CAGA( 12)-luc reporter (Fig. 3, A).To explore whether Chk2 and Smad proteins could form a complex, we   Genes involved in TGFb1-dependent cell cycle regulation performed a co-immunoprecipitation assay of Smad3 cotransfected with Chk2.We revealed that Smad3 interacts with Chk2, and these Smad3/Chk2 complexes have been formed even in the absence of treatment with TGF b1 ligand (Fig. 3, B).Therefore, we found that Smad3 and the protein product of RAD53 gene Chk2 kinase exerted a cooperative effect in activation of the TGFb1/ Smad3-responsive transcription.This transcription regulation may be an example of a feedback mechanism, which includes TGFb1-dependent inhibition of RAD53 gene expression and simultaneous restrain of Smad3dependent transcriptional regulation.In contrast, we found that other checkpoint Rad proteins Rad9, which has been also identified in the gene expression array experiment, did not show any effect on Smad3 dependent transcription and is not involved in the interaction with Smad3 protein (Fig. 4, A, B).
In our previous study we have showed that TGFb1 is counteracting the BRCA1-dependent DNA repair process [13] and is implicated in the maintenance of genome stability via regulation of RAD51 expression [14].Another report suggests a functional link between TGF b1 signaling and the ATM-mediated genotoxic stress response [15].In this study we have also observed that, in addition to Chk2 and RAD9, TGFb1 is effecting an expression a few other proteins implicated in DNA repair, cell cycle checkpoint control, apoptosis and maintenance of the genomic integrity including c-Abl, ATM, MRE11B (Meiotic recombination (S. cerevisiae) 11 homolog B).Downregulation of the expression of c-Abl, ATM, RAD9 and RAD53 genes in malignant cells upon TGFb1 treatment suggests an additional mechanism of increasing genomic instability, which can potentially contribute to the cancer development.
TGFb1 is a ubiquitous cytokine that switches its roles from tumor suppressor to tumor promoter as the tumor progresses through the multiple mechanisms, including mutational inactivation of TGFb1 receptors and Smad proteins, loss of selective cytostatic gene response, and activating tumor promoter genes [1][2][3][4][5]).Given the integral role of TGFb1 in the tumor progression, it follows that TGFb1 signaling offers an attractive target for cancer therapy.
Techniques such as microarray hybridization allow a big-scale analysis of genes implicated in TGFb1dependent signal transduction.In conjunction with pathways analysis, transcriptional profiling might be beneficial for identification of the key signal transmitters and assessment of a functional load of the distinct signaling components.To identify the key cell cycle regulators of TGFb1signaling we performed low-density microarray analysis of human breast adenocarcinoma MCF7 cell line treated or non-treated with TGFb1.Among the 96 genes that have been annotated as cell cycle regulators, expression of 41 genes was changed significantly upon TGFb1 treatment in MCF7 human breast adenocarcinoma cell line.Some of identified genes have been reported before as TGFb-responsive targets (transcription factors DP-1, DP-2, cyclins CCND1, CCNF, CCNG1 and CCNG2, CDK2, CDK4, CDK6, CDK7, cyclin-dependent kinase inhibitors CDKI1C, CDKI2A, CDKI2B, CDKI2C, CDKI2D) [9].However, for the most of the differentially regulated genes identified in this study, little information is known on their role in the TGFb1-directed cell cycle regulation.
A lot of recent data suggest that regulation of the cell cycle depends on protein degradation by the ubiqui-  12)-luc reporter and DNA constructs expressing RAD9 and Smad3-myc proteins (cell were treated with TGFb1 (0,5 ng/ml) for 18 h, and luciferase activity has been measured); B -coimmunoprecipitation assay of cotransfected Smad3-myc and RAD9 has indicated that RAD9 is not involved in the interaction with Smad3 protein tin-proteasome machinery.On the other hand, the TGFb/ Smads-dependent signaling is known to be regulated by reversed ubiquitination and protein degradation process [16,17].The ubiquitination reaction requires the coordination of various classes of functional modules, and cullin complexes play an important role in the regulation of protein degradation through their ubiquitin-ligase activity.Human genome encodes five different cullins -Cul1, Cul2, Cul3, Cul4, and Cul5.All of them can compose the catalytic core of the different cullin-based ubiquitin-ligase, and, therefore, target a large number of substrates for ubiquitin-dependent degradation, including p27, p21, p57, p130, Cyclin E, E2F-1, Cdk9, c-Myc, which are involved in regulation of cell proliferation and apoptosis [18].We found that expression of two cullins 4A and 4B, as well as ubiquitin-conjugating enzyme, CDC34 were suppressed by TGFb1, suggesting that proteosomal degradation is one of the mechanisms trough which TGFb1 can regulate the cell cycle machinery.
Our previous proteomics studies indicate that TGFb1 can regulate transcription machinery via controlling gene expression and through the post-translation modification mechanisms [17,19].In this study we also observed that some of the identified TGFb1-responsive proteins are involved in the regulation of gene transcription, such as E2F6 (E2F transcription factor 6), TFDP1 (Homo sapiens E2F-related transcription factor, DP-1), TFDP2 (Transcription factor DP-2, E2F dimerization partner 2).Recent studies suggest that transcriptional repression of c-myc protooncogene is critical for the manifestation of TGFb1-dependent cytostatic program.The c-myc transcription is repressed through the E2F4/ 5, DP-1 and Smad3 complexes [20,21].Transcriptional factor c-Myc is one of the key regulators of cell growth, metabolism and apoptosis [22].TGFb1-dependent downregulation of DP-1 gene in MCF7 cells can contribute to the restraining of TGFb1-dependent transcription repression of c-myc.At the same time the E2F/ DP heteromeric transcription factor family is a very well characterized pRb interactor [23].A lot of studies have demonstrated that transcriptional activity of E2F proteins is linked to poor clinical outcome in a wide-variety of different types of human cancers [24][25][26][27].They have an oncogenic function, which has been attributed to the ability of the E2F proteins to induce S-phase of cell cycle through the transcriptional regulation of cyc-lins A and E, proto-oncogenes c-myc and c-myb, genes important for DNA replication, and Rb family genes (Rb and p107) [23,28].On the other hand, E2F proteins are known to be the inductors of p53-dependent apoptotic pathway.Plasticity of E2F dependent functional outcomes can provide the fine-tuned mechanism of TGFb1-dependent regulation of cell growth and inhibition.
Activities of cyclin-dependent kinase (CDKs) and their activating subunits, the cyclins are critical for the function of cell cycle machinery [29,30].The pattern of cyclin expression defines the cell position within the cell cycle.Cyclin-dependent kinase inhibitors (CDKIs) bind and inhibit cyclin-associated kinase and serve as negative regulators of the cell cycle machinery.We have found that TGFb1 is regulating a number of cyclin genes (cyclin D1, D2, D3, E2, F, G1), cyclin-dependent kinase (CDK 2, 4, 6, 7), cyclin-dependent kinase inhibitors (CDKI1C, p57; CDKN2A, p16; CDKN2B, p15; CDKN2C, p18; CDKN2D, p19) suggesting that TGFb might regulate the cell cycle at the different stages and by the various molecular mechanisms.Some of these cell cycle regulatory genes have been previously reported as TGFb-responsive genomic targets.Previous investigation of the human umbilical vein endothelial cells (HU VEC) infected with TbRI-expressing adenovirus has shown that transcription a number of cell cycle regulators can be affected by TGFb signaling activation, including cyclin-dependent kinases CDK2, CDK4, CDK6, CDK7; cyclins CCND2, CCND3, CCNF, CCNG1; cyclin-dependent kinase inhibitors CDKN2B, CDKN2C, CDKN2D) [7].Some of these genes exhibited expression profiles similar to those stimulated by TGFb1 in our study, including CCND3, CDK2 (unregulated) and CCNF, CCNG1, CDKN2D (downregulated).However, some of identified genes have the different manner of TGFb1-dependent regulation for breast adenocarcinoma and nonmalignant human endothelial cells (CDKN2D, CDK4, CDK6, CDK7, CDKN2B, CDKN2C), suggesting the duel manner of TGFb1-dependent cell growth regulation in normal and cancer cells.To check a possibility of differential regulation of some other TGFbresponsive genes in normal and malignant cells, we have performed semi-quantitative RT-PCR analysis for MRE11B, c-Abl, CCND3, CDKN2D, RAD9, RAD53 gene expression in MCF10A non-tumorigenic epithelial cells and MCF7 human breast adenocarcinoma cell line.We found distinct TGFb1-dependent regulation of c-Abl, CDKN2D and RAD9 genes, suggesting that these genes can potentially contribute to the switching the responsiveness of the normal and tumor epithelial cells to TGFb1.
In order to search the TGFb1-dependent signaling modules connected to the regulation of cell cycle, we examined the identified genes in known pathways network with using of the Ingenuity Pathways Knowledge Base.An overlapping of the canonical pathways involving identified genes revealed, among others, a preferential activation of the aryl hydrocarbon receptor (AhR) pathway.
Recent study demonstrated that glioma pathogenesis involves altered AhR regulation of the TGFb/ Smad pathway suggesting AhR as a promising target for the treatment of human tumors associated with pathological TGFb activity [31].Nevertheless, the role of AhR for the TGFb-driven breast tumor development remains unknown.Finding of these signaling compounds might provide an insight into mechanism of TGFb1-dependent cell cycle control.
Taken together our study suggest that investigation of the TGFb-dependent gene expression regulation in breast cancer cells and their normal counterparts might contribute to the identification of molecular mechanisms critical for cancer development and can potentially be applied for the development of new therapeutic approaches.

Fig. 1 .
Fig. 1.Validation of microarray data: A -phosphorylation of Smad-2 on C-terminal serine residue 467 upon TGFb1 treatment was used as an indicator of the TGFb1 signaling activation in human breast adenocarcinoma MCF7 cell line.Semi-quantitative RT-PCR and Western blotting analysis for some of the identified genes has been performed to validate the TGFb1-dependent changes in gene expression (B, C).Semi-quantitative RT-PCR analysis revealed a distinct TGFb1 regulation for some of the identified genes (i. e. c-Abl, CDKN2D, RAD9) in MCF10A non-tumorigenic epithelial cells and MCF7 human breast adenocarcinoma cell line (D)

Fig. 3 .Fig. 4 .
Fig. 3. Chk2 kinase cooperates with Smad3 in activation of CAGA(12)-luc reporter: A -293T cells were transfected with TGFb1-responsive CAGA(12)-luc reporter and DNA constructs expressing Chk2-flag and Smad3-myc proteins (cell were treated with TGFb1 (0,5 ng/ml) for 18 h, and luciferase activity has been measured; a representative experiment out of three performed, is shown.*p < 0.05); B -to explore whether Chk2 and Smad3 proteins form a complex, we performed a co-immunoprecipitation assay of the Smad3-myc and Chk2-flag proteins which were overexpressed in 293T cells