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[Cancer Research 66, 7948-7956, August 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

HuR Contributes to Cyclin E1 Deregulation in MCF-7 Breast Cancer Cells

Xun Guo and Rebecca S. Hartley

Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico

Requests for reprints: Rebecca S. Hartley, Department of Cell Biology and Physiology, MSC08 4750, 1 University of New Mexico, Albuquerque, NM 87131-0001. Phone: 505-272-4009; Fax: 505-272-9105; E-mail: rhartley{at}salud.unm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t1/2) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G1-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t1/2 and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer. (Cancer Res 2006; 66(16): 7948-56)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The E-type cyclin family consists of two members, cyclins E1 and E2. Cyclin E1 (formerly termed cyclin E) is a G1 cyclin, which complexes with cyclin-dependent kinase (Cdk) 2 and propels progression through the G1-S transition by controlling the initiation of DNA synthesis and other S-phase functions during the mammalian cell cycle (1–3). In normal proliferating cells, cyclin E1 expression is limited to a short window from G1 into S phase. Cyclin E1-Cdk2 activity is highest when cyclin E1 levels peak (4). Cyclin E1-Cdk2 activity is precisely regulated by multiple layers of control, including transcriptional and post-transcriptional control of cyclin E1 abundance, the binding of Cdk inhibitors (CKI), such as those from the Cip/Kip family, and modification of Cdk2 activity by inhibitory and activating phosphorylation (5). Regulated expression of cyclin E1 plays a crucial role in cell proliferation (6, 7).

Because the major regulatory events leading to cell proliferation occur in the G1 phase of the cell cycle, the deranged expression of cyclins and Cdks active in G1 phase and consequently the deregulation of the G1-S transition may be key to loss of growth control, a hallmark of cancer. Because cyclin E1 is a crucial regulator of the G1-S transition, any alteration in its expression could result in inappropriate G1 cyclin function and have a critical effect on oncogenesis. This has been established by several independent studies showing aberrant expression of cyclin E1 in many human cancers, including colorectal, ovarian, breast, and gastric cancers (8–11), and in several carcinogen-induced animal tumor models (12, 13). A contributing role for cyclin E1 in mammary tumorigenesis was revealed in cyclin E1 transgenic mice, which exhibited an induction of mammary gland hyperplasia and carcinomas (14). In addition, constitutive overexpression of cyclin E1, but not cyclin D1 or A, in both immortalized rat embryo fibroblasts and human breast epithelial cells results in chromosome instability (15). Lastly, several tumor cohort studies have documented a strong correlation between cyclin E1 overexpression and poor patient outcome (16, 17). In breast cancer, not only the full-length cyclin E1 (50 kDa) but also its low molecular weight (LMW) isoforms ranging from 49 to 33 kDa are overexpressed. The LMW isoforms are fully functional and even hyperactive regarding in vitro Cdk2 kinase activity and are resistant to inhibition by the CKIs p21 and p27 (18–20). Recent studies showed that the tumor-specific LMW isoforms of cyclin E1 are strong independent prognostic indicators in breast cancer due to significant correlation with poor outcome in patients with breast cancer (21), further supporting the relevance for cyclin E1 as a marker for tumor aggressiveness.

HuR, a ubiquitously expressed mRNA-binding protein, regulates the stability and translation of mRNAs bearing AU- and U-rich elements (collectively termed AREs) in their 3'-untranslated regions (3'UTR; ref. 22). A growing body of evidence shows that HuR plays a central role in malignant transformation by binding to mRNAs encoding proteins important for cell growth and proliferation. HuR enhances the levels of these proteins via mRNA stabilization and/or altered translation. These target mRNAs include cell cycle regulators, such as cyclins A and B1, proliferation-associated genes, such as c-myc, as well as other factors that influence tumor growth, such as vascular endothelial growth factor, cyclooxygenase-2, and ß-catenin (23–26). Recently, a report showed that high cytoplasmic HuR level is associated with high histologic grade, large primary tumor size, and poor survival of patients with invasive ductal breast cancer. In addition, a consistent correlation has emerged between HuR expression levels and advancing stage of malignancy in cancers of the breast, colon, lung, and ovary (25, 27–30).

Cyclin E1 mRNA contains several AREs in its 3'UTR, suggesting that its mRNA may be regulated by ARE-binding proteins (ARE-BP), such as HuR. Here, we investigated the potential role of HuR in regulating cyclin E1 in MCF-7 breast cancer cells. We compared cyclin E1 protein level, mRNA adenylation state and stability, as well as HuR abundance and subcellular localization between MCF-7 breast cancer cells and MCF10A normal breast epithelial cells. We also investigated the influence of HuR on cyclin E1 mRNA stability and protein level in MCF-7 cells using small interfering RNA (siRNA) and in MCF10A cells by overexpressing HuR. Finally, we overexpressed a LMW isoform of cyclin E1 in HuR siRNA-treated MCF-7 cells to see if it could rescue the effects of HuR knockdown on the cell cycle. We found that cyclin E1 was present at a high level throughout the cell cycle in MCF-7 cells. Cyclin E1 mRNA was more stable in MCF-7 cells, which was mediated in part by HuR binding the cyclin E1 3'UTR. Knockdown of HuR with siRNA decreased cyclin E1 mRNA stability as well as the abundance of full-length cyclin E1 and its LMW isoforms in MCF-7 cells. HuR siRNA also delayed G1-S phase transition and inhibited cell proliferation, which was partially recovered by cyclin E1 overexpression. HuR overexpression in MCF10A cells significantly increased cyclin E1 mRNA half-life (t1/2) and protein level. These findings implicate HuR in deregulation of cyclin E1 in MCF-7 breast cancer cells and reinforce the pivotal role of cyclin E1, particularly its LMW isoforms, in malignant transformation of breast epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. MCF10A cells [American Type Culture Collection (ATCC), Manassas, VA] were maintained in complete growth medium of DMEM/F12 mixture (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum (FBS), 20 ng/mL epidermal growth factor (Sigma), 0.01 mg/mL insulin (Sigma), 500 ng/mL hydrocortisone (Sigma), 100 units/mL penicillin, and 100 µg/mL streptomycin. MCF-7 cells (ATCC) were grown in complete growth medium of DMEM (Invitrogen, Carlsbad, CA) containing 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin.

Synchronization and cell cycle analysis. Cells of 50% to 60% confluence were synchronized at G0/early G1 by serum and growth factor deprivation for 72 hours (serum free) or synchronized at late G1 by serum and growth factor deprivation for 48 hours followed by 24 hours of incubation with aphidicolin (5 µg/mL). Synchronized cells were stimulated with complete growth medium and then harvested at indicated times. Samples were analyzed using Becton Dickinson (San Jose, CA) FACScan flow cytometry and CellQuest software at the University of New Mexico Shared Flow Cytometry Resource (Albuquerque, NM).

Subcellular fractionation and Western blot analysis. Cytoplasmic and nuclear fractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL). Total, cytoplasmic, or nuclear lysates were resolved on a 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Cyclins E1, E2, and HuR were detected with mouse monoclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). To control sample loading and protein transfer, the membranes were stripped and reprobed to assess ß-actin for total and cytoplasmic extracts (Sigma) or {alpha}-histone deacetylase 1 (HDAC1) for nuclear fractions (Santa Cruz Biotechnology).

Real-time PCR. Total cellular RNA was isolated from MCF10A and MCF-7 cells at the indicated times using Trizol reagent (Invitrogen). RNA was reverse transcribed using random hexamers. The resulting cDNAs were amplified by real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). PCR was carried out with the following conditions: 5 minutes at 94°C followed by 40 cycles of 30 seconds at 94°C, 1 minute at 60°C. Primers used for amplification were as follows: cyclin E1 (Genbank accession no. NM-001238), 5'-CGGCTCGCTCCAGGAA-3' (forward) and 5'-TCATCTGGATCCTGCAAAAAAA-3' (reverse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Genbank accession no. XM-006959), 5'-GGCCTCCAAGGAGTAAGACC-3' (forward) and 5'-AGGGGTCTACATGGAAACTG-3' (reverse). Threshold cycles (Ct values) were normalized to GAPDH, and the data were expressed as relative mRNA levels. For mRNA stability measurements, cells synchronized at late G1 phase and exhibiting the highest level of cyclin E1 mRNA were treated with actinomycin D (5 µg/mL). Total RNA was extracted at indicated time points following addition of actinomycin D. Real-time PCR was done to compare the rate of cyclin E1 mRNA decay in MCF-7 and MCF10A cells.

Plasmid constructs. The full-length cyclin E1 3'UTR and the cyclin E1 coding region (E1CR) were PCR amplified from MCF10A cDNA using the following primers: cyclin E1 3'UTR, 5'-CTCATCTAGACCACCCCATCCTTCTCCACC-3' (forward) and 5'-GTTCGATATCGTCTCAAAAACAGTATTATC-3' (reverse) and E1CR, 5'-CATCATGCCGAGGGAGCGCAGGGAGC-3' (forward) and 5'-CAGGCGCGCAACTGTCTTTGGTGGAG-3' (reverse). The PCR products were gel purified and ligated directly into the TA cloning vector pGEM-T Easy (Invitrogen). The E1CR378 construct (coding region from 301 to 678) was then generated by PCR from E1CR using T7 promoter sequence-flanked (italicized) forward (5'-TAATACGACTCACTATAGGGCAGGATCCAGATGAAGAAATGG-3') and reverse (5'-GGTCTCCCTGTGAAGTTTATAG-3') primers. All constructs were confirmed by DNA sequencing. [32P]UTP-radiolabeled in vitro–transcribed transcripts were used for UV cross-link competition and UV cross-link immunoprecipitation experiments.

Cyclin E1 NH2 terminally truncated FLAG-tagged constructs (Trunk1-3-FLAG constructs) were made as described previously (31), except that each truncated cyclin E1 PCR product was cloned directly into TA cloning vector pDrive (Qiagen, Valencia, CA). Each FLAG-tagged cyclin E1 was then cloned into the mammalian expression vector pTracer-CMV2 (Invitrogen) under the control of a cytomegalovirus promoter. This vector system was used to allow for constitutive, transient expression of high levels of FLAG-tagged cyclin E1 in MCF-7 cells. By comparing the mobility of the cyclin E1–truncated forms with MCF-7 cell–generated LMW cyclin E1 on a Western blot, we found that Trunk-2-FLAG construct synthesized a protein migrating at Mr ~40,000, which is the major cyclin E1 LMW isoform produced in MCF-7 cells. Therefore, we transfected this construct.

UV cross-link competition and UV cross-link immunoprecipitation analyses. For UV cross-link competition experiments, MCF10A and MCF-7 cytoplasmic extracts (20 µg) or purified glutathione S-transferase (GST)-HuR (50 ng) were preincubated for 15 minutes with 0, 10, or 50 molar excess of unlabeled cyclin E1 3'UTR or 50 molar excess of unlabeled nonspecific competitor (cyclin E1CR378) at room temperature before addition of 5 x 105 cpm 32P-labeled cyclin E1 3'UTR mRNA. RNA-protein complexes were UV cross-linked on ice in a Stratalinker 1800 and subsequently resolved on a 10% SDS-polyacrylamide gel. Gels were dried and analyzed on a phosphorimager (Molecular Dynamics, Sunnyvale, CA). For UV cross-link immunoprecipitation analysis, MCF10A and MCF-7 cytoplasmic extracts (100 µg) were incubated overnight at 4°C with mouse monoclonal HuR antibody or normal mouse IgG. The immunocomplexes were incubated with 500 fmol 32P-labeled cyclin E1 3'UTR or cyclin E1CR378 for 20 minutes at room temperature. RNA-protein complexes were UV cross-linked and then immunoprecipitated by incubation with 20 µL agarose conjugate suspension (protein G-agarose, Santa Cruz Biotechnology) for 3 hours at 4°C with gentle rotation. The beads were washed and resolved on a 10% SDS-polyacrylamide gel. Gels were dried and analyzed on a phosphorimager.

Polyadenosine tail analysis. MCF10A or MCF-7 cells were synchronized at late G1 phase. Total RNA was extracted at the indicated times after stimulation with complete growth medium. Reverse transcription was done using oligo(dT) and an oligo(dT) anchor 5'-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3'. The resulting cDNAs were used for PCR with the oligo(dT) anchor and a cyclin E1 3'UTR-specific primer 5'-GATGCTGCTATGGAAGGTGC-3'. PCR conditions were 1 cycle of 95°C for 15 min; 35 cycles of 94°C for 1 minute, 50°C for 1 minute and 72°C for 1 minute followed by 1 cycle of 72°C for 10 minutes. PCR products were resolved on 2% low-melting point agarose gels and visualized by ethidium bromide staining. The specificity of cyclin E1 PCR amplification products was verified by HindIII digestion.

Transfection of pcDNA3.1mycHuR. pcDNA3.1mycHuR, a kind gift from Dr. David Port (University of Colorado Health Sciences Center, Denver, CO), was transfected into MCF10A cells using electroporation as described previously (31, 32). Briefly, cells were grown to 70% confluence and 5 x 106 cells were suspended in 0.3 mL of medium with 1.25% DMSO and 10 µg linearized plasmid DNA (pcDNA3.1 vector or pcDNA3.1mycHuR) in a 0.4-cm gap cuvette. Conditions for transfection were 0.30 kV, 950 µF capacitance, and 100 {Omega}. Forty-eight hours after transfection, total cell lysates or total RNA was extracted for immunoblotting or real-time PCR, respectively.

Transfection of HuR siRNA. MCF-7 cells at 50% to 60% confluence in 12-well plates were transfected with HuR siRNA using siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer's protocol. Control siRNA (Santa Cruz Biotechnology), which does not target any known human gene, was used as a control. Seventy-two hours after siRNA transfection, total cell lysates or total RNA was extracted for immunoblotting or real-time PCR, respectively. Specific silencing was confirmed by Western blot using protein extracts. Fluorescently labeled nontargeting siRNA (siGLO RISC-Free siRNA, Dharmacon, Lafayette, CO) was used as a monitor of transfection efficiency. Under our experimental conditions, the transfection efficiency was 85% as determined 6 hours after transfection by quantifying the fraction of transfected cells in a cell population under a fluorescence microscope.

Cotransfection of HuR siRNA and pTracer-CMV2-Trunk2. MCF-7 cells at 50% to 60% confluence in 12-well or 6-well plates were first transfected with HuR siRNA, and 24 hours later, pTracer-CMV2-Trunk2 was transfected into MCF-7 cells using Lipofectamine (Invitrogen). Forty-eight hours after cotransfection, cells were harvested for cell proliferation, protein expression, and cell cycle analyses.

Statistics. Values were expressed as mean ± SD and assessed by Student's t test. Ps < 0.05 were considered to be statistically significant. All experiments were repeated at least thrice except 2A was repeated twice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of cyclin E1 levels in synchronized MCF10A and MCF-7 cells. Cyclin E1 expression is periodic and peaks at the G1-S boundary in normal cells (1). To determine the relative levels of cyclin E1 in MCF-7 breast cancer cells compared with MCF10A immortalized breast epithelial cells, we first needed highly synchronized cells to provide us with an accurate description of the temporal changes in cyclin E1 protein levels. MCF-7 and MCF10A cells were synchronized at G0/early G1 by serum and growth factor deprivation for 72 hours (serum free) or synchronized at late G1 by serum and growth factor deprivation for 48 hours followed by a 24-hour incubation with aphidicolin. Aphidicolin, a potent antimitotic agent and an inhibitor of DNA polymerase, arrests cells at the G1-S boundary. Cells were then released synchronously from the arrest and harvested at the indicated times for up to 24 hours. Cell synchrony was confirmed by analyzing DNA content using flow cytometry. Cell cycle analysis of MCF10A and MCF-7 cells is shown in Fig. 1A and B , respectively. Notably, MCF-7 cells had a longer S phase (4 hours) than MCF10A cells (2 hours).


Figure 1
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  		Figure 1. Cyclin E1 levels in synchronized MCF10A and MCF-7 cells. MCF10A (A) and MCF-7 (B) cells were synchronized as described and harvested after growth factor deprivation for 72 hours (serum free) or at the indicated time points after release from the block with aphidicolin (0 hour). The cell cycle phase was determined by measuring DNA content using fluorescence-activated cell sorting (FACS). C, top, whole-cell extracts were prepared from cells at the indicated times, and cyclin E1 level was determined by Western blotting with a cyclin E1–specific antibody; middle, cyclin E1 levels in MCF10A and MCF-7 cells compared on the same blot. Immunoblotting for ß-actin indicated that similar amounts of proteins were loaded in each lane. Bottom, cyclin E2 levels in MCF10A and MCF-7 cells show no LMW isoforms. The relative quantity of protein was calculated after normalization to ß-actin. Asyn, asynchronous; SF, serum free; FL, full-length cyclin E1; LMW, low molecular weight isoforms of cyclin E1.

 
Cyclin E1 protein was detected by immunoblotting MCF10A and MCF-7 cells as they progressed through the cell cycle. MCF10A cells (Fig. 1C, top left) showed the expected expression pattern of cyclin E1, which was highest in S phase (4 hours) and lowest at G0/early G1 phase (serum free). In contrast, MCF-7 cells had higher overall levels of the full-length cyclin E1 in all phases of the cell cycle (Fig. 1C, top right). Furthermore, LMW isoforms of cyclin E1 were present in MCF-7 cells, in agreement with previous studies showing that processing of cyclin E differs between normal and breast cancer cells (10). The differences in cyclin E1 expression between MCF10A and MCF-7 cells were more clearly seen when we analyzed cell extracts of these two cell lines on the same blot (Fig. 1C, middle). Immunoblotting for ß-actin indicated that similar amounts of protein were loaded in each lane. Immunoblotting for cyclin E2 (Fig. 1C, bottom) shows that no LMW isoforms are present in MCF-7 cells, suggesting that LMW cyclin E1 forms are not artifacts produced on cell lysis due to higher lysosomal proteases. These results clearly show that the full-length cyclin E1 and its LMW isoforms are overexpressed in MCF-7 cells throughout the cell cycle.

Cyclin E1 mRNA is more stable in MCF-7 cells. The human cyclin E1 3'UTR contains several AREs, suggesting that its mRNA stability may be regulated by ARE-BPs, such as HuR (22). To investigate if a change in mRNA stability underlies the overexpression of cyclin E1 in MCF-7 cells, we first measured the level and t1/2 of cyclin E1 mRNA during the cell cycle by real-time reverse transcription-PCR (RT-PCR; Fig. 2 ). Similar to cyclin E1 protein, cyclin E1 mRNA level also fluctuated during the cell cycle, with a peak at late G1 in both MCF10A and MCF-7 cells (0 hour; Fig. 2A). We next analyzed cyclin E1 mRNA t1/2 using cells synchronized at late G1 phase (0 hour). Actinomycin D was added to the synchronized cells to inhibit new RNA transcription. Total RNA was extracted at the indicated times following the addition of actinomycin D. Real-time RT-PCR was done to compare the rate of cyclin E1 mRNA decay in MCF-7 and MCF10A cells. As shown in Fig. 2B, the t1/2 of cyclin E1 mRNA in MCF10A and MCF-7 is 2.06 ± 0.09 and 2.44 ± 0.26 hours, respectively (P < 0.05). Thus, cyclin E1 mRNA is more stable in MCF-7 cells.


Figure 2
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  		Figure 2. Cyclin E1 mRNA is more stable in MCF-7 cells. A, real-time RT-PCR of cyclin E1 mRNA during the cell cycle in MCF10A and MCF-7 cells. MCF10A (top) and MCF-7 (bottom) cells were synchronized as described. mRNA was harvested after growth factor deprivation for 72 hours (serum free) or at the indicated time points after release from the block with aphidicolin (0 hour). Threshold cycles (Ct values) were normalized to GAPDH and expressed as relative mRNA levels. B, cyclin E1 t1/2 determination. MCF10A and MCF-7 cells were synchronized at late G1 phase (0 hour), and total RNA was extracted at the indicated time after the addition of actinomycin D. Real-time RT-PCR was used to analyze cyclin E1 mRNA level. Data were normalized to GAPDH mRNA and plotted on semilogarithmic scales. P < 0.05, MCF-7 versus MCF10A.

 
Cyclin E1 mRNA is stably adenylated in MCF-7 cells. Adenylation status decides the fate of most mRNAs, with adenylation resulting in stabilization and translation and deadenylation resulting in mRNA decay (33). We next asked whether the increased cyclin E1 mRNA t1/2 in MCF-7 cells (Fig. 2B) was due to a change in adenylation status. We evaluated cyclin E1 mRNA polyadenosine [poly(A)] tail length by ligase-mediated poly(A) tail analysis with a specific primer for the cyclin E1 3'UTR. As shown in Fig. 3 , in MCF10A cells, cyclin E1 mRNA poly(A) tail length increased from early to late G1 phase (serum free to 0 hour), decreased at S phase (2-4 hours), and increased again at G2-M phase (8 hours). In MCF-7 cells, poly(A) tail length of cyclin E1 mRNA was consistently higher throughout the cell cycle. This result indicates that, in MCF-7 cells, higher stability of cyclin E1 mRNA is associated with its stable adenylation.


Figure 3
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  		Figure 3. Cyclin E1 mRNA is stably adenylated in MCF-7 cells. MCF10A and MCF-7 cells were synchronized as described and harvested for RNA extraction after growth factor deprivation for 72 hours (serum free) or at the indicated time points after release from the block with aphidicolin (0 hour). Total RNA was used as a template for ligase-mediated poly(A) tail analysis with a cyclin E1 3'UTR-specific primer and oligo(dT) anchor.

 
The ratio of cytoplasmic to nuclear HuR is increased in MCF-7 cells. AREs located in the 3'UTR regulate adenylation, translation, and degradation of mRNAs encoding critical growth response genes, cytokines, and cell cycle regulatory proteins (34). In searching for RNA-BPs potentially involved in the regulation of cyclin E1 mRNA, we focused on HuR, which specifically binds ARE-containing mRNAs and inhibits their decay (35). We have shown that the Xenopus homologue of HuR, ElrA, binds to AREs in the 3'UTR of Xenopus cyclin E1 (submitted). First, we compared HuR levels in MCF10A and MCF-7 cells by immunoblotting. As shown in Fig. 4 , HuR was more abundant in total MCF-7 cell extracts compared with MCF10A cells (Fig. 4, top). The mRNA stabilizing function of HuR seems to be intimately linked to its cytoplasmic presence (25, 36); therefore, we compared the levels of HuR in cytoplasmic and nuclear fractions of MCF10A and MCF-7 cells. Cytoplasmic HuR levels were higher in MCF-7 cells, whereas nuclear HuR levels were higher in MCF10A cells (Fig. 4, middle and bottom).


Figure 4
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  		Figure 4. Higher ratio of cytoplasmic to nuclear HuR in MCF-7 compared with MCF10A cells. Total, cytoplasmic (Cyto), and nuclear extracts were resolved on a 10% SDS-polyacrylamide gel, transferred to membrane, and probed with an HuR-specific antibody. As a loading control, the blots were stripped and reprobed with ß-actin antibody for total and cytoplasmic extracts or with HDAC1 antibody for nuclear extracts. The relative quantity of protein was calculated after normalization to ß-actin or HDAC1. The ratio of cytoplasmic to nuclear HuR protein was calculated and expressed as fold difference after normalization to the ratio in asynchronous MCF10A cells.

 
HuR binds to the cyclin E1 3'UTR. Next, we carried out UV cross-link competition and immunoprecipitation assays to ask if HuR specifically binds the cyclin E1 3'UTR. As shown in Fig. 5A , UV cross-link competition analysis showed that both GST-HuR and an ~34-kDa protein in cytoplasmic extracts of MCF10A and MCF-7 cells bound the cyclin E1 3'UTR. Binding of both proteins was specifically competed by unlabeled cyclin E1 3'UTR but not by a nonspecific competitor, unlabeled mRNA consisting of a partial cyclin E1 coding region (cyclin E1CR378). We further verified that HuR in the cytoplasmic fraction contributed to the observed 34-kDa band by UV cross-link immunoprecipitation analysis. The 34-kDa-binding protein was specifically immunoprecipitated by a HuR-specific antibody but not by normal IgG, and the control mRNA did not form complexes with cytoplasmic proteins (Fig. 5B). These data indicate that endogenous HuR binds the cyclin E1 3'UTR.


Figure 5
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  		Figure 5. HuR specifically binds the cyclin E1 3'UTR. A, UV cross-link competition analysis. GST-HuR or MCF10A and MCF-7 cytoplasmic extracts were incubated with 32P-labeled cyclin E1 3'UTR mRNA in the presence of 0, 10, or 50 molar excess specific (unlabeled E1 3'UTR) or 50 molar excess nonspecific competitor (unlabeled E1CR378). Arrow, binding to cyclin E1 3'UTR by GST-HuR and a 34-kDa protein that was competed by unlabeled E1 3'UTR but not by E1CR378. B, UV cross-link immunoprecipitation analysis. Cytoplasmic extracts from MCF10A and MCF-7 cells were preincubated with specific HuR antibody or nonspecific normal IgG overnight before addition of 32P-labeled E1 3'UTR mRNA or 32P-labeled E1CR378 mRNA. RNA-protein complexes were isolated with agarose-conjugated protein G and resolved on a 10% SDS-polyacrylamide gel. RNA-protein complexes were immunoprecipitated by an HuR-specific antibody but not by normal IgG, and HuR was not immunoprecipitated when cell extracts were incubated with E1CR378 mRNA.

 
HuR regulates cyclin E1 mRNA stability and protein level. To define if there is an association between elevated levels of cytoplasmic HuR and cyclin E1 mRNA stability, we first applied an RNA interference strategy to knockdown HuR protein in MCF-7 cells. SiGLO siRNA, a nonfunctional and nontargeting siRNA, was used as a transfection marker. As shown in Supplementary Data, 6 hours after transfection with siGLO siRNA, transfected MCF-7 cells exhibited nuclear and perinuclear localization of fluorescently labeled siGLO siRNA. Under our transfection conditions, the transfection efficiency was estimated as 85% by quantifying the fraction of transfected cells in a cell population. Compared with control siRNA, treatment with HuR siRNA for 72 hours suppressed HuR protein by >70% (Fig. 6A, left ). Importantly, knockdown of HuR protein significantly decreased cyclin E1 mRNA stability in MCF-7 cells (Fig. 6B, left). The t1/2 of cyclin E1 mRNA in cells transfected with HuR siRNA is 1.68 ± 0.04 hours and control siRNA is 2.6 ± 0.6 hours, respectively (P < 0.01). Concomitantly, knockdown of HuR protein decreased the abundance of both the full-length cyclin E1 and its LMW isoforms. However, HuR siRNA inhibited the LMW isoforms (80%) more profoundly than full-length cyclin E1 (22%; Fig. 6A, left). These results indicate that HuR critically contributes to cyclin E1 overexpression by stabilizing its mRNA in MCF-7 cells. To verify these findings, we overexpressed HuR in MCF10A cells by transfection of pcDNA3.1mycHuR and explored its effect on cyclin E1 mRNA stability and protein level. By Western blot analysis, total cell lysates revealed a 58% increase in HuR in MCF10A cells transfected with pcDNA3.1mycHuR when compared with pcDNA3.1 vector only (Fig. 6A, right). Myc-tagged HuR shows a higher molecular weight compared with endogenous HuR (Fig. 6A, arrow). HuR overexpression in MCF10A cells led to a doubling of cyclin E1 protein (Fig. 6A, right). Concomitantly, cyclin E1 mRNA was also stabilized. The t1/2 of cyclin E1 mRNA in cells transfected with pcDNA3.1mycHuR or pcDNA3.1 vector were 3.2 ± 0.6 and 2.15 ± 0.16 hours, respectively (P < 0.05; Fig. 6B, right). These data clearly show that HuR critically regulates cyclin E1 expression.


Figure 6
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  		Figure 6. HuR regulates cyclin E1 expression in MCF-7 and MCF10A cells. A, left, HuR and full-length cyclin E1 and its LMW isoforms are decreased in MCF-7 cells transfected with HuR siRNA; right, HuR and full-length cyclin E1 are increased in MCF10A cells transfected with pcDNA3.1mycHuR. Cells were transfected with control siRNA (Si-ctrl), HuR siRNA (Si-HuR), pcDNA3.1, or pcDNA3.1mycHuR. Total proteins were extracted and immunoblotted for HuR and cyclin E1. Equal loading of protein was determined by reprobing of blots for ß-actin. The relative quantity of protein was calculated after normalization to ß-actin. B, left, cyclin E1 mRNA t1/2 is decreased in MCF-7 cells transfected with HuR siRNA; right, cyclin E1 mRNA t1/2 is increased in MCF10A cells transfected with pcDNA3.1mycHuR. Cells were synchronized at late G1 phase after transfecting control siRNA, HuR siRNA, pcDNA3.1, or pcDNA3.1mycHuR. Total RNA was extracted at the indicated time points after treatment with actinomycin D. Data from real-time RT-PCR were expressed as relative mRNA levels and plotted on semilogarithmic scales. P < 0.01, HuR versus control siRNA. P < 0.05, pcDNA3.1mycHuR versus pcDNA3.1. C, cell numbers are decreased in MCF-7 cells transfected with HuR siRNA. MCF-7 cells were transfected with control and HuR siRNA. Cell numbers were counted using a Beckman Coulter Counter (Fullerton, CA), and data were expressed as relative values. **, P < 0.01 versus control siRNA. D, overexpression of cyclin E1 LMW isoform rescues G1-S phase transition and proliferation in HuR knockdown MCF-7 cells. Left, top, cyclin E1-Trunk2 restores the main cyclin E1 LMW isoform in HuR siRNA knockdown cells. MCF-7 cells cotransfected with HuR siRNA and pTracer-CMV2-Trunk2 were extracted and immunoblotted for HuR and cyclin E1. Equal loading of protein was determined by reprobing of blots for ß-actin. The relative quantity of protein was calculated after normalization to ß-actin. Right, overexpression of the main cyclin E1 LMW isoform rescued the delayed G1-S phase transition in HuR knockdown MCF-7 cells. MCF-7 cells were cotransfected with HuR siRNA and pTracer-CMV2-Trunk2. The cell cycle phase was determined by measuring DNA content using FACS. Left, bottom, overexpression of the main cyclin E1 LMW isoform rescues proliferation in HuR knockdown MCF-7 cells. **, P < 0.01 versus control siRNA; ##, P < 0.01 versus HuR siRNA.

 
Restoration of cyclin E1 expression in HuR knockdown MCF-7 cells rescues cell cycle progression and cell proliferation. We next explored whether HuR mediates the growth-promoting function of cyclin E1. First, we examined the effect of HuR siRNA on MCF-7 cell proliferation by directly counting cells. The number of MCF-7 cells decreased by 28% after HuR siRNA treatment when compared with MCF-7 cells transfected with control siRNA (Fig. 6C). We did not see apoptotic or necrotic cells (data not shown), suggesting that increased cell death does not account for the decrease in cell numbers. Next, we restored cyclin E1 expression by transfecting a LMW isoform of cyclin E1 (cyclin E1-Trunk2) into HuR knockdown MCF-7 cells. We chose to express this 40-kDa isoform as it is identical in size to the major LMW cyclin E1 in MCF-7 cells before HuR knockdown. As shown in Fig. 6D, left, top, cotransfection of cyclin E1-Trunk2 restored LMW cyclin E1. Accordingly, cell cycle analysis showed that HuR siRNA treatment retained more cells at G0-G1 phase and delayed S phase entry, which was partially recovered by cotransfection of cyclin E1-Trunk2 (Fig. 6D, right). In agreement with the partial rescue of G1-S phase progression, cotransfection of cyclin E1-Trunk2 also partially recovered MCF-7 cell proliferation (Fig. 6D, left, bottom). These results show that HuR contributes to the stabilization of cyclin E1 mRNA, the overexpression of cyclin E1, and its growth-promoting function in MCF-7 breast cancer cells. They also suggest a direct association between increased cyclin E1 mRNA stability and elevated levels of cyclin E1 protein.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we establish a causal link between HuR and elevated cyclin E1 protein in MCF-7 breast cancer cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms throughout the cell cycle. Cyclin E1 mRNA t1/2 and adenylation, as well as cytoplasmic HuR levels, are increased in MCF-7 cells. Using UV cross-link competition and UV cross-link immunoprecipitation assays, we show that HuR specifically binds the cyclin E1 3'UTR. Knockdown of HuR with siRNA decreases cyclin E1 mRNA stability as well as full-length cyclin E1 and its LMW isoforms in MCF-7 cells. Concomitantly, proliferation of MCF-7 cells is also suppressed by HuR siRNA due to a delay in G1-S phase transition. Overexpression of the LMW cyclin E1 isoform preferentially decreased by HuR siRNA partially rescued the G1-S phase delay and cell proliferation. Partial rescue is consistent with HuR, having other known cell cycle targets, including cyclin D1. In addition, overexpression of HuR in MCF10A cells led to a doubling of cyclin E1 protein and an increased t1/2 of the cyclin E1 mRNA. Taken together, we conclude that HuR contributes to the deregulation of cyclin E1 expression in MCF-7 cells.

Regulation of mRNA stability is an important element of eukaryotic gene expression (37). AREs are one of the best-characterized cis-acting elements regulating turnover of many unstable transcripts (22, 34). Cyclin E1 mRNA is unstable, and its 3'UTR contains multiple AREs, raising the possibility that its mRNA is regulated via these elements and that this regulation may be altered in cancer cells overexpressing cyclin E1. In support of this, we find that cyclin E1 mRNA is more stable in MCF-7 than in MCF10A cells, and in parallel, enhanced adenylation is also seen in MCF-7 cells. Several ARE-BPs have been characterized, and among them, HuR is well studied and always stabilizes mRNAs (22–24). It has recently been shown that HuR can also inhibit translation (38, 39). HuR is predominantly nuclear, but it can translocate to the cytoplasm where it binds target mRNAs and prevents their decay (26, 36). Here, we found more cytoplasmic than nuclear HuR in MCF-7 cells compared with MCF10A cells, which have more nuclear HuR. Because binding of HuR to target mRNAs has been linked to enhanced mRNA stability, we hypothesized that elevated cytoplasmic HuR in MCF-7 cells may stabilize cyclin E1 mRNA. We first show that HuR specifically binds to the cyclin E1 3'UTR, and further supporting this hypothesis, knockdown of HuR with siRNA significantly decreases cyclin E1 mRNA t1/2 in MCF-7 cells. In contrast, overexpression of HuR leads to a significant increase in cyclin E1 mRNA t1/2 in MCF10A cells. These results establish a causal link between elevated levels of cytoplasmic HuR and enhanced cyclin E1 mRNA stability in MCF-7 cells.

HuR has been previously shown to bind cyclins A, B1, and D1 mRNAs, but not cyclin E mRNA, in colorectal cancer RKO cells (26). These investigators used supershift analysis to show RNA-HuR interaction. We also failed to see HuR-cyclin E1 mRNA supershift complexes (data not shown). However, UV cross-link immunoprecipitation analysis clearly shows that HuR specifically binds to cyclin E1 mRNA in MCF10A and MCF-7 breast epithelial cells.

As described above, HuR enhances cyclin E1 mRNA stability, which therefore should lead to elevated cyclin E1 protein level in MCF-7 cells. In agreement with this expectation, knockdown of HuR with siRNA decreased the abundance of full-length cyclin E1 and its LMW isoforms. Notably, HuR siRNA inhibited cyclin E1 LMW isoforms to a much greater extent than full-length cyclin E1. This finding is of great interest because LMW isoforms of cyclin E1 may be more oncogenic than the full-length form. Recent studies have shown that the LMW isoforms of cyclin E1 have higher associated Cdk2 activity, are resistant to CKIs p21 and p27, and can stimulate cell cycle progression more effectively than the full-length form (19). LMW isoforms are indicators of high grade, severity, and poor prognosis of cancer as well as premalignancy (21). Our data suggest that cyclin E1 is a key target of HuR and raise the question of how HuR differentially regulates the expression of full-length cyclin E1 and its LMW isoforms.

LMW isoforms of cyclin E1 are generated by intracellular processing of full-length cyclin E1 (18). Consistent with reports that nontumorigenic cells do not have the ability to process cyclin E1 into its LMW forms (31), overexpression of HuR in MCF10A cells results only in an increase in full-length cyclin E1. There is evidence that LMW isoforms of cyclin E1 are generated by proteolytic processing of full-length cyclin E1 by either elastase or calpain, and consistent with this, increased activities of these two enzymes have been observed in tumor cells (18, 40). Therefore, the appearance and abundance of the LMW isoforms is determined by the amount of full-length cyclin E and the activities of these enzymes. It is expected that a portion of full-length cyclin E1 is constantly cleaved to produce the LMW isoforms in tumor cells when its level is above the threshold needed for cleavage, resulting in a dynamic balance between full-length cyclin E1 and cleaved LMW isoforms. If the decrease of the LMW isoforms is simply secondary to the decrease of full-length cyclin E1, it is reasonable that the change in LMW isoforms is more profound due to the complex dynamic course of enzymatic activity. Another possible explanation for the differential decrease in full-length cyclin E1 and its LMW isoforms by HuR siRNA is that HuR may also up-regulate elastase and/or calpain by stabilizing their mRNAs, resulting in an increase in enzyme activity.

Regardless of the mechanism of the decrease of full-length cyclin E1 and its LMW isoforms by HuR siRNA, the presence of LMW isoforms of cyclin E1 is closely associated with high proliferation in cancer cells (18). Excitingly, we also find that knockdown of HuR significantly inhibits cell proliferation, stalling cells in G0-G1, as would be expected on a decrease in a positive G1-S regulator. Cotransfection of a LMW isoform of cyclin E1 and HuR siRNA partially rescues the G1-S phase delay and cell proliferation, showing that, whereas cyclin E1 is one target of HuR, other G1 regulators, such as cyclin D1, are probably also affected.

Taken together, the present study provides direct evidence that increased mRNA stability is closely associated with elevated protein levels of cyclin E1 in MCF-7 cells. Our data expand the important role of HuR in tumorigenesis by showing that HuR critically contributes to the deregulation of cyclin E1 in breast cancer cells, particularly to the appearance of its LMW isoforms. These data also show that HuR regulates the stability of cyclin E1 mRNA in nontumorigenic breast epithelial cells. We are currently pursuing the mechanism by which HuR regulates LMW cyclin E1.


   Acknowledgments
 
Grant support: National Cancer Institute-NIH grant R01CA095898-703 (R.S. Hartley). Technical and instrument support was provided by the University of New Mexico Shared Flow Cytometry Resource.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. David Port (UCHSC, Denver, CO) for providing GST-HuR plasmid and pcDNA3.1mycHuR and Drs. Port and Wenlan Liu (University of New Mexico Health Sciences Center) for critical reading of the article and for technical advice.

Received 12/ 6/05. Revised 6/ 5/06. Accepted 6/19/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. K. Slevin, F. Gourronc, and R. S. Hartley
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