Skip to main content
NCBI home page
Cell Cycle logoLink to Cell Cycle
. 2010 Dec 15;9(24):4900–4907. doi: 10.4161/cc.9.24.14158

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines

Anne Cooley 1,#, Stanislav Zelivianski 1,#, Jacqueline S Jeruss 1,2,✉
PMCID: PMC3047813  PMID: 21150326

Abstract

Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild-type (WT) Smad3 or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1) or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.

Key words: Smad3, breast cancer, cyclin E, CDK2, TGFβ

Introduction

Every year in the United States, approximately 200,000 women are diagnosed with breast cancer and 44,000 patients die of the disease. Previous work has implicated members of the TGFβ superfamily and their associated downstream signaling components, the Smads, in several aspects of breast cancer onset and disease progression.1,2 The role of Smad3 as a tumor suppressor in breast cancer is an emerging area of intense research. Smad3, together with transcriptional co-factors, induces expression of the cyclin dependent kinase inhibitors (cdki) p15 and p21. These cdkis facilitate G1 cell cycle arrest by inhibiting cyclin D/E mediated CDK4/2 phosphorylation of the retinoblastoma (Rb) protein.3–5 As a consequence of this cell cycle repression, the Rb protein remains unphosphorylated and the E2F-1 transcription factor inactive, and thus unable to actualize movement of cells into the S phase.6,7 Smad3 also represses expression of c-myc, a key cell cycle mitogen that is overexpressed in many human cancers and is thought to be involved with oncogenic progression in breast cancer cells.8

Members of the TGFβ superfamily of growth factors share significant structural and functional homology, and several have crucial roles in mammary gland physiology.9 Activin and TGFβ each signal through a specific set of type II and type I receptors, (activin: ActRIIA or ActRIIB with ActRIB; TGFβ: TβRII with TβRI), both type I receptors have very similar kinase domains, and both phosphorylate the regulatory Smads, Smad2 and Smad3, to mediate their action.9 Phosphorylated Smad2/3 interacts with Smad4 to facilitate the modulation of DNA transcription in the nucleus. While the signaling mechanisms of activin and TGFβ are nearly identical, and the actions of these ligands are closely related, ultimately, they are not the same. Prior work has shown that during murine mammary gland lactation and involution, activin and TGFβ are expressed in temporally distinct patterns, with activin/Smad3 signaling present during lactation and TGFβ involved in post-lactation involution.10 As the type I receptor is the primary initiator of ligand action, differences in the structure and activity of the relationship of either the TβRII:TβRI and ActRIIA/ActRIIB:ActRIB receptor complexes, or the relative expression of each set of receptors within an organ system, may confer the unique actions of these different ligands in vivo. Specificity of the activin or TGFβ signal may also be dependent on the particular DNA binding transcriptional co-factors present, aberrant expression of cell cycle proteins at the time when the Smads translocate into the nucleus, or through cross-talk with other signaling pathways.3,11

Non-canonical CDK4 and CDK2 phosphorylation sites have been found within the Smad3 protein.12 In mouse embryonic fibroblasts, phosphorylation of these CDK sites in Smad3 led to abrogation of Smad3 activity.12 The CDKs are serine/threonine protein kinases whose functional activity is mediated by cyclins; CDK2 activity is mediated by cyclin E. Overexpression of cyclin E has been identified in aggressive breast cancers, and is associated with poor prognosis.13 Patients with a particularly aggressive type of breast cancer, the basal subtype, can have tumors with high cyclin expression, and certain patients with hereditary breast cancer, harboring a BRCA1 mutation, can exhibit a basal-type cancer biology and high cyclin E levels.14 Although patients with aggressive, rapidly proliferative tumor biology tend to have a greater reduction in tumor size in response to chemotherapy, these patients tend to demonstrate poor survival outcomes when compared with patients who have more favorable breast cancer biology.15

A clear understanding of the mechanisms underlying Smad3-mediated cell cycle arrest is critical to efforts aimed at preventing or interrupting oncogenic insults to the Smad3 signaling pathway that favor breast cancer growth and dedifferentiation. Elucidation of the role of Smad3 signaling in breast cancer may reveal its potential as a therapeutic target and as a valuable clinical marker of cancer prognosis. Here, we tested the hypothesis that decreased Smad3 function mediated by cyclin E overexpression and CDK2 phosphorylation may contribute to tumorigenesis and resistance to Smad3 signal transduction in breast cancer cells, potentially uncovering an additional mechanism that contributes to the biologic aggressiveness of breast cancers that overexpress cyclin E.

Results

Characterization of cyclin E overexpressing breast cancer cell lines.

The study panel of cells included parental, vector control (A1) and cyclin E overexpressing (EL1) MCF7 breast cancer cell lines. Immunoblotting confirmed that EL1 cells expressed the highest amount of cyclin E protein relative to the parental and vector control cells (Fig. 1A). Whereas Smad3 levels appeared higher in EL1 cells, protein expression of phosphorylated Smad3 (phospho-Smad3) and CDK2 was similar for all the study cells. A kinase assay was performed to determine the relative amount of CDK2 activity in the study cells. As expected, EL1 cells showed the highest amount of CDK2 activity relative to the parental and A1 cells (Fig. 1B). Immunofluorescent staining for Smad3 showed that all the MCF7 study cells contained both cytoplasmic and nuclear Smad3; thus all study cells had the capacity for nuclear translocation of Smad3 (Fig. 2).

Figure 1.

Figure 1

Open in a new tab

Characterization of MCF7 stably transfected cells. (A) Protein expression levels were measured in extracts from parental cells (MCF7) or MCF7 clones stably transfected with empty vector (A1) or cyclin E (EL1). Total protein (30 µg/lane) was separated on an SDS/PAGE gel and subjected to immunoblot analysis with the indicated antibodies. (B) CDK2 activity in cyclin E overexpressing MCF7 cells. CDK2 kinase assay radiographs and densitometric quantification in MCF7 study cell lines (parental, MCF7; vector control, A1; cyclin E overexpressing, EL1). CDK2 kinase activity was quantified relative to the amount of activity in the parental line.

Figure 2.

Figure 2

Open in a new tab

Localization of Smad3 in the MCF7 study cell lines. Study cell lines underwent immunofluorescent staining to detect Smad3. DAPI indicates nuclei, FITC indicates Smad3 protein. The overlay demonstrates that Smad3 is localized to both the nuclear and cytoplasmic compartments (arrowheads) in the parental, vector control (A1) and cyclin E overexpressing (EL1) cell lines.

Cyclin E affects Smad3 transcriptional activity.

To determine the effect of cyclin E overexpression and Smad3 phosphorylation on cell cycle component mRNA levels, the MCF7 breast cancer cell lines were transfected with vector control, WT Smad3 and the Smad3 5M CDK phosphorylation site mutant, and expression levels of p15INK4B, p21 and c-myc were determined by real-time quantitative RT-PCR (Fig. 3). Parental and A1 MCF7 cells transfected with the WT Smad3 or Smad3 5M constructs showed lower levels of c-myc transcripts, and higher amounts of p15INK4B and p21 transcripts, when compared with cells transfected with the vector control. By contrast, the cyclin E overexpressing EL1 cells transfected with the control vector had the highest amounts of c-myc, low levels of p21 and nearly undetectable levels of p15INK4B transcripts, when compared with the other cell lines. When the EL1 cells were transfected with WT Smad3, c-myc mRNA levels decreased and p15INK4B and p21 mRNA levels increased compared with control transfected cells. Transfection with the 5M construct resulted in a further decrease in c-myc and increase in p15 mRNA levels, whereas p21 levels were nearly equivalent to those of WT Smad3 transfected cells. Together, these findings suggest that restoration of Smad3 function by overexpression of the Smad3 5M CDK phosphorylation site mutant resulted in some repression of CDK2-mediated inhibition and, consequently, higher cdki expression and lower c-myc expression.

Figure 3.

Figure 3

Open in a new tab

Expression of Smad3-regulated genes in study cell lines. MCF7 study cells were transfected with wild-type Smad3 (WT) or the 5M Smad3 CDK phosphorylation site mutant and transcript levels of c-myc, p15 and p21 were measured by real-time quantitative RT-PCR. * denotes significant difference from cells transfected with CS2 control for each cell line.

Inhibition of Smad3 phosphorylation by CDK2 increases Smad3 transcriptional activity in MCF7 cells.

Transfection experiments were performed to determine the transcriptional activity of the various Smad3 CDK phosphorylation site mutant constructs in the MCF7 study cells (Fig. 4). The study cells were co-transfected with each of the Smad3 expression plasmids, the Smad3-responsive CAGA-luc reporter, and a pRenilla luciferase construct. Compared with cells overexpressing WT Smad3, overexpression of the Smad3 T179 mutant or the multiple site mutants (3M, 4M or 5M) resulted in relatively higher CAGA-luc reporter activity in all three study cell lines. In the study cells, the serine mutants were also associated with an increase in reporter activity, though less so than the individual T179 mutant. These studies confirm the inhibitory impact of CDK phosphorylation on Smad3 transcriptional activity. Based on these experiments, the Smad3 T179 and Smad3 5M constructs, which induced the most consistent increase in reporter activity among the study cells, were chosen for further examination.

Figure 4.

Figure 4

Open in a new tab

Relative transcriptional activity of various Smad3 constructs in cyclin E overexpressing MCF7 cells. Cells were co-transfected with the Smad3-responsive CAGA-luc reporter construct and Renilla luciferase reporter, in addition to the indicated Smad3 expression constructs. Data are shown as fold increase in normalized luciferase activity (firefly/Renilla) compared with empty vector-transfected MCF7 cells (CS2). Error bars indicate standard deviation from the mean of normalized luciferase activity for each study condition. * denotes significant difference from cells transfected with the WT Smad3 for each cell line.

Smad3 activity is restored through CDK2 inhibition in breast cancer cells.

To study the effect of direct CDK2 inhibition on Smad3 transcriptional activity, the study cells were transfected with WT Smad3 and the CAGA-luc and pRenilla reporter constructs, and then treated with increasing concentrations of CDK2 inhibitor. A dose-dependent increase in Smad3 transcriptional activity was seen in MCF7 cells treated with increasing concentrations of the CDK2 inhibitor (Fig. 5A).

Figure 5.

Figure 5

Open in a new tab

Effect of CDK2 inhibition on Smad3 transcriptional activity in MCF7 cells. (A) Dose-dependent increase in Smad3 transcriptional activity in MCF7 cells transfected with the Smad-responsive CAGA-luc reporter construct, Renilla luciferase reporter and wild-type (WT) Smad3 expression vector, then treated with the indicated concentrations of CDK2 inhibitor (CDK2i). (B) Restoration of Smad3 transcriptional activity with inhibition of CDK4 activity. MCF7 study cell lines were transfected with the Smad3-responsive CAGA-luc reporter construct, Renilla luciferase reporter, and either wild-type Smad3 (WT), T179 Smad3 (T179) or 5M Smad3 (5M) expression vectors. The cells were treated with vehicle or 240 nM CDK2 inhibitor (CDK2i). Data are shown as fold increase in normalized luciferase activity (firefly/Renilla) compared with empty vector-transfected MCF7 cells (CS2). Error bars indicate standard deviation from the mean of normalized luciferase activity for each study condition. * denotes significant difference from cells transfected with WT Smad3 for each cell line.

We then examined the impact of both CDK2 inhibition and abrogation of CDK phosphorylation sites within Smad3 on Smad3 transcriptional activity. Study cells were transfected with WT Smad3 or Smad3 mutant constructs and the CAGA-luc and pRenilla reporter constructs, and then treated with vehicle or 240 nM Cdk2 inhibitor (Fig. 5B). Confirming the suppressive effect of CDK phosphorylation on Smad3 activity, transfection with the Smad3 T179 or Smad3 5M CDK phosphorylation site mutants resulted in higher CAGA-luc reporter activity compared with cells transfected with WT Smad3. Transfection of these cells with WT Smad3 or Smad3 T179 and treatment with the Cdk2 inhibitor resulted in a further increase in CAGA-luc reporter activity. Transfection with the Smad3 5M construct and treatment with the Cdk2 inhibitor did not result in a further increase in CAGA-luc reporter activity, when compared with transfection with the 5M construct alone, indicating that the five CDK sites mutated in the Smad3 5M construct account for the totality of CDK phosphorylation affecting Smad3 transcriptional activity.

siRNA knockdown of CDK2 restores Smad3 activity in MCF7 cell lines.

To further establish the impact of CDK2 inhibition on Smad3 transcriptional activity, the study cells were transfected with scrambled or cdk2-specific siRNA. MCF7 cells transfected with cdk2 siRNA showed a 76% decrease in CDK2 levels compared with cells treated with a control vector or scrambled siRNA, confirming the specificity of the cdk2 siRNA knockdown (Fig. 6A). Co-transfection of the MCF7 study cells with cdk2 siRNA and WT Smad3 or the T179 mutant construct and the Smad3-responsive CAGA-luc and pRenilla reporter constructs resulted in increased reporter activity when compared with cells transfected with the WT Smad3 or T179 and the respective scrambled control siRNA. While the trends were the same for all study cells, the overall increase in Smad3 reporter activity found upon transfection of the cdk2 siRNA was slightly lower in the cyclin E overexpressing EL1 cells when compared with the parental and A1 vector control cells. This finding suggests that breast cancer cells with high cyclin E levels sustain some degree of suppression of Smad3 activity despite CDK2 inhibition. Taken together, these data further support the direct role of CDK2 in the inhibition of Smad3 action in cyclin E overexpressing breast cancer cells.

Figure 6.

Figure 6

Open in a new tab

Restoration of Smad3 transcriptional activity with siRNA knockdown of CDK2. (A) MCF7 cells were transfected with scrambled (SC) or cdk2 siRNA (siRNA) for 48 h, then the cells were lysed and the level of CDK2 protein was determined in untransfected cells (UC) and transfected cells by immunoblot analysis using anti-CDK2 antibody. GAPDH was used as a loading control. (B) MCF7 study cell lines were co-transfected with the Smad3-responsive CAGA-luc reporter construct and Renilla luciferase reporter; siRNA or cdk2 siRNA; and either empty vector (CS2), wild-type Smad3 (WT) or T179 Smad3 mutant (T179) expression vectors. Data are shown as fold increase in normalized luciferase activity (firefly/Renilla) compared with empty vector-transfected MCF7 cells. Error bars indicate standard deviation from the mean of normalized luciferase activity for each study condition. * denotes significant difference from cells transfected with WT Smad3 and scrambled siRNA for each cell line.

Discussion

The objective of this work was to examine the connection between cyclin E overexpression and CDK2 phosphorylation of Smad3 as a possible mechanism for breast oncogenesis. Overexpression of cyclin E inhibited WT Smad3 transcriptional activity, and this activity was restored with the overexpression of Smad3 containing single or multisite mutations at the CDK phosphorylation sites. The restoration of Smad3 activity was also found to positively correlate with increased cdki (p21 and p15) levels and decreased c-myc levels in the study cells. Direct inhibition of CDK2, either by treatment with Cdk2 inhibitor or transfection with siRNA, also restored Smad3 transcriptional activity. Collectively, these findings support our hypothesis that Smad3 function is negatively impacted by cyclin E overexpression and CDK2 phosphorylation in breast cancer cells.

The G1 to S phase transition is a key check point in cell cycle control. Multiple proteins are responsible for regulating the progression of cells through this transition, including cyclin D and cyclin E.13,16 Cyclin E is overexpressed in approximately 25% of breast cancers.13 The overexpression of cyclin E in breast cancer is mediated via gene amplification, mRNA stabilization and post-translational modification.17 Consequences of aberrant cyclin E overexpression include genetic instability in vitro, and mammary gland hyperplasia and malignant tumor formation in cyclin E overexpressing transgenic mice.18,19 Furthermore, overexpression of cyclin E has been linked to the early transformation of human mammary cells along the continuum of benign to invasive disease.20 Additionally, high levels of cyclin E have been shown to strongly correlate with resistance to endocrine and anthracycline-based therapy and thus poor outcomes for breast cancer patients.13,21,22

Mechanistically, it has been demonstrated in breast cancer cell lines that overabundant cyclin E can function independently of cyclin D to phosphorylate the Rb protein and drive the cell cycle forward.23 Cyclin E overexpression has also been associated with genomic instability leading to p53 loss of heterozygosity.24 Furthermore, aberrant cyclin E expression occurring throughout the cell cycle leads to S-phase deregulation and has been linked to malignant cellular transformation.24,25 In fact, the MCF7 breast cancer cell line was characterized as having increased stability of cyclin E1 mRNA and elevated cyclin E levels, mediated by the presence of the mRNA binding protein HuR.26 HuR binding has been associated with oncogenic progression through stabilization and expression of cell cycle mitogens.26 Low molecular weight isoforms of cyclin E have also been characterized, and these isoforms have been associated with breast malignancy.21 These low molecular weight isoforms are thought to exert a potent mitogenic effect in breast cancer cells through several mechanisms, including prolonged duration of action, resistance to cdkis p21 and p27, and inactivation of p53.27,28 Based on these collective findings, cyclin E overexpression appears to have a significant impact on tumorigenesis through the exploitation of several different cellular processes.

The cell cycle-related implications of cyclin E/CDK2-regulated Smad3 repression were shown through the impact of transfection with the Smad3 5M construct and subsequent measurement of the levels of cdkis, p15 and p21, and c-myc. In control cells, transfection with WT Smad3 led to an increase in the levels of cdkis and a decrease in levels of c-myc as compared to baseline. Upon transfection with the 5M construct, cdki levels were further augmented in the MCF7 cells. At baseline, MCF7-EL1 cells were found to have elevated levels of c-myc, low levels of cdki p21 and undetectable levels of p15 mRNA, as compared to parental MCF7 cells. When the EL1 cells were transfected with WT Smad3, c-myc levels decreased and p15 and p21 levels increased. A trend toward a further decrease in c-myc and increase in p15 mRNA levels was found when the EL1 cells were transfected with the 5M construct, though the increase in p21 levels reached a plateau that was nearly equivalent to results obtained with transfection of WT Smad3. These findings suggest that inhibition of CDK2 phosphorylation in MCF7 cells restores Smad3 activity related to cell cycle control, even when cyclin E is overexpressed.

In a mouse mammary tumor model mediated by the Myc oncogene, cyclin E transcription was found to be directly linked to c-myc activity.29 Thus, CDK2 inhibition and rescue of Smad3 action may be part of a negative feedback mechanism to downregulate c-myc action and potentially decrease cyclin E transcription. With regard to p21 activity, full-length cyclin E activity was found to be squelched by p21, thus pointing to an additional means by which restoration of Smad3 action and consequent p21 production may deter cyclin E-mediated oncogenesis.30 Additional work has described a nuclear Smad complex that includes Smad3 and the transcription factor Sp1.4 Acting in concert, Smad and Sp1 mediate transcription of p15, whose promoter contains both Sp1 and Smad3 binding sites. More specifically, it is thought that Sp1-driven transcription is Smad dependent.4 c-myc also has the ability to join the Smad/Sp1 complex and render it ineffectual, thereby blocking p15 transcription.4 Thus, Smad3 may act in a dual capacity to both block c-myc transcription and stimulate p15 production to actualize G1 arrest. In this way, it would be possible for CDK2-mediated repression of Smad3 action to induce a very thorough and potent cell cycle release in cyclin E overexpressing cells.

Novel strategies for evaluating breast cancer are necessary to bring crucial refinement to disease prognosis and treatment regimens. The study of new tumor suppressors, such as Smad3, may help to expand the conventional staging and grading of breast cancer by contributing to an organized molecular staging of disease. Also, the study of tumor markers in a broad context may ultimately allow for the further development of individualized prognostic markers to implement patient-specific treatment strategies. Therapeutic approaches that target inhibition of CDKs or block cyclin/CDK activity may hold promise for patients with cyclin E overexpressing or basal-like breast cancers who have the disease sub-type with a poor prognosis. CDK2 inhibitors are currently in development and some have been used in clinical trials.31 A key issue regarding the design of these inhibitors is the complexity and redundancy that is inherent to the cell cycle, which makes resistance to these treatments a critical hurdle.31,32 This may be particularly true of targeted CDK2 inhibition, which may not independently arrest cancer cell cycle proliferation in all malignancies.33 Consequently, an accurate understanding of the markers of effective CDK2 inhibition, such as rescue of Smad3 action, in the malignant setting may be critical to the determination of the therapeutic potential of CDK2 inhibitors. Overall, the impact of CDK2 inhibition on Smad3 action and the upregulation of cdki mRNA and downregulation c-myc found in the current study, along with other recent data showing CDK2 knockdown resulted in G1 arrest in both hormone-sensitive and resistant breast cancer cells, points toward a potential therapeutic role for CDK2 inhibition in breast cancer.34

The presence of cyclin E in breast cancer has been linked to more aggressive tumor biology through several different mechanistic approaches. This work provides an additional perspective on cyclin E-mediated tumorigenesis that ties cyclin E overexpression to the TGFβ signaling cascade directly through Smad3. A mechanistic interaction between cyclin E overexpression and deregulation of Smad3 tumor suppression may contribute to the more malignant phenotype found in cyclin E overexpressing breast cancers. Based on these findings, the possibility of implementing the selective use of therapeutic CDK2 inhibition to help enhance Smad3 action, and thus restore cell cycle control in cyclin overexpressing breast cancer, will be further explored.

Materials and Methods

Cell lines and culture.

MCF7 cells were obtained from ATCC (Manassas, VA) and maintained in DMEM-F12 media supplemented with antibiotics and 10% FBS. A1 (vector control) and EL1 (cyclin E overexpressing) MCF7 cells were a kind gift from the Keyomarsi lab (University of Texas M.D. Anderson Cancer Center, Houston, TX) and were maintained in HyQ MEM alpha modification media supplemented with 10% FBS along with hydrocortisone (1 µg/ml), insulin (1 µg/ml) and EGF (1.25 µg/ml). The study cell lines were kept at 37°C in a humidified incubator containing 5% CO2.

Antibodies.

Anti-cyclin E, anti-CDK2, anti-GAPDH and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Smad3 and anti-pSmad3 antibodies were obtained from Cell Signaling Technology (Danvers, MA).

Immunoblotting.

Cells were rinsed with cold PBS, then scraped, collected and lysed in lysis buffer containing multiple protease and phosphatase inhibitors. All steps were performed on ice or at 4°C. Cell lysates were then centrifuged at 13,200x rpm for 10 min at 4°C. The supernatants were collected and protein concentration determined by the Bradford method using a protein assay (Bio-Rad, Hercules, CA). Immunoblotting was performed with 30 µg of protein combined with a loading buffer and a reducing agent, which was boiled for 5 min. It was then loaded and electrophoresed on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (Millipore, Bedford, MA), and blocked in 5% non-fat milk and TBS-T (pH 7.5) for 1 h at room temperature. The membranes were then incubated with the indicated primary antibodies in TBS-T and 5% non-fat milk or 5% BSA at 4°C overnight, rinsed with TBS-T, and incubated with secondary antibody in TBS-T and 5% BSA for 1 h at room temperature. Bands were visualized by SuperSignal (Thermo Scientific, Rockford, IL). For reblotting, membranes were placed in stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, 100 mM β-mercaptoethanol) for 30 min at 60°C, rinsed twice with TBS-T, reblocked in non-fat milk and incubated with primary and secondary antibodies as previously described. GAPDH or β-actin was used as a loading control.

Immunofluorescence.

Subcellular localization of Smad3 in parental and cyclin E overexpressing cells was determined by immunofluorescence using an anti-Smad3 antibody. After fixation of cells to the slides with 4% paraformaldehyde, slides were incubated with anti-Smad3 antibody at 4°C for 16 h. Slides were then washed 3 times with TNT buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20). After a 30-min incubation at room temperature with secondary anti-rabbit-HRP conjugated antibody, slides were then incubated with Fluorophore Tyramide (PerkinElmer, Boston, MA) for 10 min. Slides were mounted with Vectashield mounting medium containing DAPI, and the cells were observed with a fluorescence microscope.

CDK2 kinase assay.

Cell extracts were obtained by lysing cells in lysis buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 1 mM PMSF, 1 mM sodium vanadate, 1x Halt Protease Inhibitor Cocktail [Pierce, Rockford, IL]) on ice for 30 min with occasional agitation. Cell debris was removed by centrifugation at 14,000 g for 10 min at 4°C. Protein concentration was determined using the BioRad protein assay (BioRad). A total 50 µg of protein lysate was incubated with 2 µg of anti-CDK2 IgG at 4°C for 1 h followed by immunoprecipitation with protein A/G plus agarose conjugate beads (Santa Cruz) at 4°C overnight. Beads were washed 3 times with kinase buffer (8 mM MOPS pH 7.0, 0.2 mM EDTA). CDK2 kinase reactions were performed at 30°C for 30 min in kinase buffer containing 2 µg recombinant Histone H1 protein (Millipore) as a substrate and 2 µCi of 32P γATP. The reaction was stopped by adding 5x SDS loading buffer and then boiling for 5 min before loading on a 10% SDS-PAGE gel. The gel was then exposed to a phosphor screen for 24 h and analyzed using the Storm imaging system (GE Healthcare, San Diego, CA). Quantitative analysis was performed with MultiGauge software (FujiFilm, Edison, NJ).

Transfection and luciferase assay.

The Smad3 expression plasmids have been previously described in reference 12. Smad3 single mutant (T8, T179, S204, S208 and S213) and multiple mutant 3M (T8/T179/S213), 4M (T179/S204/S208/S213) and 5M (T8/T179/S204/S208/S213) expression constructs were a kind gift from Dr. Fang Liu (Rutgers University, Piscataway, NJ). Cells were plated 24 h prior to transfection at a density of approximately 4.5 × 105 cells per well in 48-well plates. A reporter construct consisting of a Smad3-responsive promoter upstream of a firefly luciferase reporter (CAGA-luc) and a control reporter (pRenilla) were cotransfected with 200 ng of each of the indicated Smad3 expression plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per the manufacturer's instructions. Cells were lysed 48 h post-transfection with 5x passive lysis buffer (Promega, Madison, WI) for analysis in a dual luciferase assay (Promega). Luminescence was measured by a Synergy2 luminometer (BioTek Instruments Inc., Winooski, VT) and normalized to the Renilla luminescence. Data are presented as mean ± standard deviation (SD) from a representative experiment performed in triplicate.

Real-time quantitative RT-PCR.

Endogenous mRNA levels for c-myc, p15INK4 and p21 were determined by real-time quantitative RT-PCR using PerfeCTa SYBR Green Fast Mix (Quanta Bioscience, Gaithersburg, MD). Total RNA was isolated from cells using the RNeasy mini kit (Qiagen, Valencia, CA). Amplification of the samples (1 µg of total RNA per reaction) was carried out using qScript cDNA Super Mix (Quanta Bioscience) according to the manufacturer's instructions. All amplifications were run on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using the following conditions: initial denaturation 95°C for 15 min, followed by 40 cycles at 95°C for 1 s and 60°C for 30 s. Gene expression was expressed in terms of the threshold cycle (Ct) normalized to GAPDH (ΔCt). ΔCt values were then compared between control samples and transfected cells to calculate ΔΔCt. Final comparison of transcript ratios between samples was given as 2−ΔΔCt. Data are shown as representative mean ± SD from three replicates of three independent experiments.

RNA interference.

Transfection of cdk2 siRNA (Thermo Scientific) was carried out 24 h after plating cells in 96-well plates. Cells were incubated overnight with transfection mix containing 12.5 pM siRNA and 0.25 µl DharmaFECT Transfection Reagent (Thermo Scientific) in Opti-MEM medium (Invitrogen). The Smad3-responsive reporter (CAGA-luc) and pRenilla reporter were then co-transfected with 100 ng of the indicated Smad3 expression plasmid as described previously. The cells were lysed with passive lysis buffer (Promega) 48 h post-transfection for the dual luciferase assay. Data are presented as mean ± SD from a representative experiment performed in triplicate.

Cdk inhibitors.

After transfection, media was changed and cells were treated with Cdk2 inhibitor II [Catalog #219445; IC50 value of 60 nM; (Calbiochem, San Diego, CA)] for 48 h. Control cells were maintained in complete media treated with solvent alone.

Statistical analysis.

All values are expressed as the mean ± SD. ANOVA was used to assess differences among the study panel of cells and a dependent t-test was used to assess differences between treatment groups and control samples. p < 0.05 was considered statistically significant.

Acknowledgements

We thank Dr. Fang Liu (Rutgers, State University of New Jersey, Piscataway, NJ) for providing the mutant Smad3 constructs, Dr. Khandan Keyomarsi (MD Anderson Cancer Center, Houston, TX) for providing the A1 control and EL1 cyclin E overexpressing MCF7 cells, and Elizabeth Tarasewicz, A. June Gordon, and Dr. Stacey C. Tobin for editorial assistance.

Footnotes

Financial Support

J.S.J. is a Lynn Sage Scholar supported by the American Cancer Society-Illinois Division, the Dixon Foundation, the Association of Women Surgeons and an NIH K22 CA138776 research grant.

References

  • 1.Jeruss JS, Sturgis CD, Rademaker AW, Woodruff TK. Downregulation of activin, activin receptors and Smads in high-grade breast cancer. Cancer Res. 2003;63:3783–3790. [PubMed] [Google Scholar]
  • 2.Burdette JE, Jeruss JS, Kurley SJ, Lee EJ, Woodruff TK. Activin A mediates growth inhibition and cell cycle arrest through Smads in human breast cancer cells. Cancer Res. 2005;65:7968–7975. doi: 10.1158/0008-5472.CAN-04-3553. [DOI] [PubMed] [Google Scholar]
  • 3.Moustakas A, Pardali K, Gaal A, Heldin CH. Mechanisms of TGFbeta signaling in regulation of cell growth and differentiation. Immunol Lett. 2002;82:85–91. doi: 10.1016/s0165-2478(02)00023-8. [DOI] [PubMed] [Google Scholar]
  • 4.Feng XH, Liang YY, Liang M, Zhai W, Lin X. Direct interaction of c-Myc with Smad2 and Smad3 to inhibit TGFbeta-mediated induction of the CDK inhibitor p15(Ink4B) Mol Cell. 2002;9:133–143. doi: 10.1016/s1097-2765(01)00430-0. [DOI] [PubMed] [Google Scholar]
  • 5.Ten Dijke P, Goumans MJ, Itoh F, Itoh S. Regulation of cell proliferation by Smad proteins. J Cell Physiol. 2002;191:1–16. doi: 10.1002/jcp.10066. [DOI] [PubMed] [Google Scholar]
  • 6.Knudsen ES, Wang JY. Targeting the RB-pathway in cancer therapy. Clin Cancer Res. 16:1094–1099. doi: 10.1158/1078-0432.CCR-09-0787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Park C, Lee I, Kang WK. E2F-1 is a critical modulator of cellular senescence in human cancer. Int J Mol Med. 2006;17:715–720. [PubMed] [Google Scholar]
  • 8.Yagi K, Furuhashi M, Aoki H, Goto D, Kuwano H, Sugamura K, et al. c-myc is a downstream target of the Smad pathway. J Biol Chem. 2002;277:854–861. doi: 10.1074/jbc.M104170200. [DOI] [PubMed] [Google Scholar]
  • 9.Yue J, Mulder KM. Transforming growth factor-beta signal transduction in epithelial cells. Pharmacol Ther. 2001;91:1–34. doi: 10.1016/s0163-7258(01)00143-7. [DOI] [PubMed] [Google Scholar]
  • 10.Jeruss JS, Santiago JY, Woodruff TK. Localization of activin and inhibin subunits, receptors and SMADs in the mouse mammary gland. Mol Cell Endocrinol. 2003;203:185–196. doi: 10.1016/s0303-7207(02)00291-5. [DOI] [PubMed] [Google Scholar]
  • 11.Chen YG, Hata A, Lo RS, Wotton D, Shi Y, Pavletich N, et al. Determinants of specificity in TGFbeta signal transduction. Genes Dev. 1998;12:2144–2152. doi: 10.1101/gad.12.14.2144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Matsuura I, Denissova NG, Wang G, He D, Long J, Liu F. Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature. 2004;430:226–231. doi: 10.1038/nature02650. [DOI] [PubMed] [Google Scholar]
  • 13.Keyomarsi K, Tucker SL, Buchholz TA, Callister M, Ding Y, Hortobagyi GN, et al. Cyclin E and survival in patients with breast cancer. N Engl J Med. 2002;347:1566–1575. doi: 10.1056/NEJMoa021153. [DOI] [PubMed] [Google Scholar]
  • 14.Aaltonen K, Blomqvist C, Amini RM, Eerola H, Aittomaki K, Heikkila P, et al. Familial breast cancers without mutations in BRCA1 or BRCA2 have low cyclin E and high cyclin D1 in contrast to cancers in BRCA mutation carriers. Clin Cancer Res. 2008;14:1976–1983. doi: 10.1158/1078-0432.CCR-07-4100. [DOI] [PubMed] [Google Scholar]
  • 15.Guarneri V, Broglio K, Kau SW, Cristofanilli M, Buzdar AU, Valero V, et al. Prognostic value of pathologic complete response after primary chemotherapy in relation to hormone receptor status and other factors. J Clin Oncol. 2006;24:1037–1044. doi: 10.1200/JCO.2005.02.6914. [DOI] [PubMed] [Google Scholar]
  • 16.Kang YK, Kim WH, Jang JJ. Expression of G1-S modulators (p53, p16, p27, cyclin D1, Rb) and Smad4/Dpc4 in intrahepatic cholangiocarcinoma. Hum Pathol. 2002;33:877–883. doi: 10.1053/hupa.2002.127444. [DOI] [PubMed] [Google Scholar]
  • 17.Akli S, Zheng PJ, Multani AS, Wingate HF, Pathak S, Zhang N, et al. Tumor-specific low molecular weight forms of cyclin E induce genomic instability and resistance to p21, p27 and antiestrogens in breast cancer. Cancer Res. 2004;64:3198–3208. doi: 10.1158/0008-5472.can-03-3672. [DOI] [PubMed] [Google Scholar]
  • 18.Enders GH. Cyclins in breast cancer: too much of a good thing. Breast Cancer Res. 2002;4:145–147. doi: 10.1186/bcr439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bortner DM, Rosenberg MP. Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E. Mol Cell Biol. 1997;17:453–459. doi: 10.1128/mcb.17.1.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Shaye A, Sahin A, Hao Q, Hunt K, Keyomarsi K, Bedrosian I. Cyclin E deregulation is an early event in the development of breast cancer. Breast Cancer Res Treat. 2009;115:651–659. doi: 10.1007/s10549-008-0266-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Akli S, Keyomarsi K. Low-molecular-weight cyclin E: the missing link between biology and clinical outcome. Breast Cancer Res. 2004;6:188–191. doi: 10.1186/bcr905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Sgambato A, Camerini A, Collecchi P, Graziani C, Bevilacqua G, Capodanno A, et al. Cyclin E correlates with manganese superoxide dismutase expression and predicts survival in early breast cancer patients receiving adjuvant epirubicin-based chemotherapy. Cancer Sci. 2009;100:1026–1033. doi: 10.1111/j.1349-7006.2009.01141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Gray-Bablin J, Zalvide J, Fox MP, Knickerbocker CJ, DeCaprio JA, Keyomarsi K. Cyclin E, a redundant cyclin in breast cancer. Proc Natl Acad Sci USA. 1996;93:15215–15220. doi: 10.1073/pnas.93.26.15215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Smith AP, Henze M, Lee JA, Osborn KG, Keck JM, Tedesco D, et al. Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland. Oncogene. 2006;25:7245–7259. doi: 10.1038/sj.onc.1209713. [DOI] [PubMed] [Google Scholar]
  • 25.Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol. 1994;14:1669–1679. doi: 10.1128/mcb.14.3.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Guo X, Hartley RS. HuR contributes to cyclin E1 deregulation in MCF-7 breast cancer cells. Cancer Res. 2006;66:7948–7956. doi: 10.1158/0008-5472.CAN-05-4362. [DOI] [PubMed] [Google Scholar]
  • 27.Wang L, Shao ZM. Cyclin E expression and prognosis in breast cancer patients: a meta-analysis of published studies. Cancer Invest. 2006;24:581–587. doi: 10.1080/07357900600894799. [DOI] [PubMed] [Google Scholar]
  • 28.Akli S, Van Pelt CS, Bui T, Multani AS, Chang S, Johnson D, et al. Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the disruption of the ARF-p53 pathway. Cancer Res. 2007;67:7212–7222. doi: 10.1158/0008-5472.CAN-07-0599. [DOI] [PubMed] [Google Scholar]
  • 29.Geng Y, Yu Q, Whoriskey W, Dick F, Tsai KY, Ford HL, et al. Expression of cyclins E1 and E2 during mouse development and in neoplasia. Proc Natl Acad Sci USA. 2001;98:13138–13143. doi: 10.1073/pnas.231487798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wingate H, Zhang N, McGarhen MJ, Bedrosian I, Harper JW, Keyomarsi K. The tumor-specific hyperactive forms of cyclin E are resistant to inhibition by p21 and p27. J Biol Chem. 2005;280:15148–15157. doi: 10.1074/jbc.M409789200. [DOI] [PubMed] [Google Scholar]
  • 31.Wadler S. Perspectives for cancer therapies with cdk2 inhibitors. Drug Resist Updat. 2001;4:347–367. doi: 10.1054/drup.2001.0224. [DOI] [PubMed] [Google Scholar]
  • 32.Dean JL, Thangavel C, McClendon AK, Reed CA, Knudsen ES. Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure. Oncogene. 29:4018–4032. doi: 10.1038/onc.2010.154. [DOI] [PubMed] [Google Scholar]
  • 33.Tetsu O, McCormick F. Proliferation of cancer cells despite CDK2 inhibition. Cancer Cell. 2003;3:233–245. doi: 10.1016/s1535-6108(03)00053-9. [DOI] [PubMed] [Google Scholar]
  • 34.Johnson N, Bentley J, Wang LZ, Newell DR, Robson CN, Shapiro GI, et al. Pre-clinical evaluation of cyclindependent kinase 2 and 1 inhibition in anti-estrogensensitive and resistant breast cancer cells. Br J Cancer. 102:342–350. doi: 10.1038/sj.bjc.6605479. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cell Cycle are provided here courtesy of Taylor & Francis

RESOURCES