Breast Cancer DOI 10.1007/s12282-014-0553-z

ORIGINAL ARTICLE

Inhibition of telomerase activity by dominant-negative hTERT retards the growth of breast cancer cells Yaojian Rao • Wei Xiong • Huijuan Liu • Chunxia Jia • Hongxing Zhang • Zesheng Cui Ya Zhang • Jiawei Cui



Received: 17 April 2014 / Accepted: 30 June 2014 Ó The Japanese Breast Cancer Society 2014

Abstract Background Telomerase, a ribonucleoprotein enzyme mainly consisted of a catalytic protein subunit human telomerase reverse transcriptase (hTERT) and a human telomerase RNA component, is responsible for maintaining telomeres. Telomerase over-expression correlates significantly with tumors and is a prognostic marker. However, telomerase over-expression in breast cancers and the effect of telomerase inhibition as a candidate cancer therapy are unknown. Methods We used the dominant-negative mutant of hTERT (DN-hTERT) to inhibit telomerase activity on human breast adenocarcinoma cell line MCF-7 by transfection. Telomeric repeat amplification protocol assays and real-time quantitative RT-PCR were performed to investigate telomerase activity as well as expression of hTERT. Telomere length was measured by the flow-fluorescence in situ hybridization assay. Cell proliferation was assessed by the WST-8 assay, and apoptosis was evaluated by flow cytometry. The tumor formation ability of MCF-7 cells was investigated by transplanting cells subcutaneously into BALB/c nude mice.

Yaojian Rao and Wei Xiong are jointly first authors contributed equally to this work. Y. Rao (&)  H. Liu  C. Jia  H. Zhang  Z. Cui  Y. Zhang  J. Cui Luoyang Orthopedic Hospital of Henan Province, Luoyang 471000, Henan, China e-mail: [email protected] W. Xiong The orthopedic department of tongji hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China

Results Ectopic expression of DN-hTERT caused dramatically inhibition of telomerase activity and reduction of telomere length. Telomerase inhibition induced growth arrest and apoptosis of MCF7 cells in vitro and loss of tumorigenic properties in vivo. Conclusion This study shows that telomerase inhibition by DN-hTERT can effectively inhibit the cell viability and tumorigenicity of MCF7 cells and is an attractive approach for breast cancer therapy. Keywords MCF-7  Telomerase activity  DN-hTERT  Breast cancer Abbreviations DN-hTERT Dominant negative-human telomerase reverse transcriptase hTR Human telomerase RNA hTERT Human telomerase reverse transcriptase PCR Polymerase chain reaction TRAP Telomeric repeat amplification protocol

Introduction Breast cancer is one of the most common female malignancies and is the second leading cause of cancer-related death all around the world [1]. The incidence of breast cancer are gradually increasing and breast cancer brings more and more economic burden to patients [2]. Although some advances had been made in early diagnosis and standard treatment, breast cancer is often diagnosed at the advanced stages due to lack of symptoms or nonspecific symptoms, leading to a poor prognosis [3]. Consequently,

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there is a pressing need to elucidate the molecular mechanisms underlying the development of breast cancer, which contribute to the development of new therapeutic methods and improve the survival of breast cancer patients. Telomeres are the natural caps of linear chromosomes that are composed of tandem hexameric repeats, 50 TTAGGG-30 [4]. Telomeres serve to protect the chromosomal ends from undergoing degradation and ligation with other chromosomes [5]. The abnormal chromosomes will break during mitosis, which results in severe damage to the genome and activation of DNA damage checkpoints and leads to cell senescence or the initiation of apoptotic cell death pathways [6]. Telomerase is a ribonucleoprotein enzyme composed of two essential components, a RNA template human telomerase RNA (hTR) which serves as a template for telomeric DNA synthesis, and a catalytically protein subunit human telomerase reverse transcriptase (hTERT), which is the catalytic component of the enzyme [7]. Telomerase is responsible for elongation of telomeric repeats at chromosomal ends and is obligatory for controlling cell survival by maintaining telomere length [8]. It has been shown that telomerase is highly expressed in more than 85 % of human tumors and in over 90 % of breast carcinomas whereas in normal tissues it is not active or detectable [9, 10]. Several recent studies have proven that high telomerase activity was associated with poor prognosis of breast cancer [11]. Therefore, the development of agents having activity against telomerase may be a productive approach to develop novel breast cancer therapy methods. However, up to now, our understanding of telomerase activity in breast cancer remains limited. In this study, we aimed to investigate the effect of telomerase inhibition on human breast adenocarcinoma cell lines MCF. Since the hTERT protein is the rate-limiting component of the telomerase, a catalytically inactive dominant negative form of hTERT (DN-hTERT) was created and ectopically expressed in MCF-7 cells, then the physiological effects of telomerase inhibition on cellular immortality and tumorigenicity was assessed.

Materials and methods Cell culture The human breast adenocarcinoma cell line MCF-7 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) in a humidified incubator at 37 °C under a 5 % CO2 atmosphere. Cells were

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washed, trysinized and resuspended in 10 ml of growth media at each cell passage. Retroviral vectors and infections The retroviral plasmids, pBABE-puro (carrying puromycin resistance gene) and pBABE-puro-DN-hTERT (carrying the dominant-negative catalytically inactive mutant of hTERT) were kindly provided by Dr. Robert Weinberg (Massachusetts Institute of Technology, Boston). DNhTERT was created by substituting the aspartic acid and valine residues at positions 710 and 711 with alanine and isoleucine residues by site-directed mutagenesis, then the resulting mutant was completely sequenced and subcloned into the vector pBABE-puro [12]. For virus production, the pBABE-puro or DN-hTERT vectors were transfected into the 293T competent packaging cell lines along with two additional vectors encoding critical packaging proteins (pCl and VSVG) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) [13]. Retroviral supernatant was collected at 48 h after transfection, and then filtered with a 0.45-lm filter to remove packaging cells. The MCF-7 cells were infected with retroviral supernatants for 24 h. The cells were then allowed to recover for 24 h in normal growth medium followed by puromycin (Roche, CA, USA, 500 lg/mL) selection for 5 days. Single cell-derived clones were established by limiting dilution, followed by selective trypsinization using sterile cloning cylinders. Cells were then maintained in growth medium with low level of puromycin (2 lg/ml). About 3 weeks after the clone establishment, the cells were used for the following experiments. Real-time quantitative PCR analysis of hTERT expression The expression of ectopic DN-hTERT transcripts was quantified by real-time quantitative reverse transcriptase (RT)PCR using the LightCycler technology. Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized from 2 lg of total RNA using Moloney murine leukemia virus reverse transcriptase (MMLV) (Promega, Madison, WI, USA). The sequences of the specific primers were: forward 50 -CGGAAG AGTGTCTGGAGCAA-30 , reverse 50 -CTCCCACGACGTA GTCCATG-30 for hTERT [14]; forward 50 -GTTGCGTTACACCCTTTCTTG-30 , reverse 50 -GACTGCTGTCACCTTC ACCGT-30 for internal control b-actin. Real-time quantitative PCR using SYBR Premix (DRR041A, TaKaRa, Japan) was performed as described previously [15]. Amplifications were performed in a LightCycler machine (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions. All samples were assayed in triplicate. hTERT levels

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were normalized to the expression of b-actin and then to the expression of endogenous hTERT untransfected control cells. Telomerase repeat amplification protocol (TRAP) assay Telomerase activity of cell populations was determined using the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. For each sample, 0.5 lg of total protein was added to the PCR reaction. Relating the enzyme-linked immunosorbent assay (ELISA) signal of the sample to the one obtained by a control template with a known number of telomeric repeats, relative telomerase activity values were calculated and translated in fold of telomerase activity of the 293T cell line-based phoenix ampho cells carried along with each test. RNase-treated cellular lysates were used as negative controls for each sample. In addition, 20 ll of PCR product was resolved by a 12 % polyacrylamide gel and visualized on a Biotin Luminescent Detection Kit (Roche) according to the manufacturer’s protocol. The telomerase activity for the MCF-7/DN-hTERT cells was assessed and presented as a percentage of that measured in the control untransfected cells. Flow-fluorescence in situ hybridization (flow-FISH) assay for telomere length measurement To determine average telomere length in individual cells, cells were hybridized in situ with a fluorescent telomerespecific peptide nucleic acid probe, as has been described recently [16]. Telomere length was expressed in telomere fluorescence units based on calibration experiments using TRF (telomere restriction fragment) length analysis by Southern blotting. Cell proliferation and viability assay Cell proliferation assays were performed by using a CCK 8 assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 1 9 104 cells per well were plated in 96-well plates and cultured with growth medium. At the indicated time points (24, 48, 72, 96 h), the medium was aspirated. Then each well was added with 100 ll serum-free RPMI-1640 and 10 ll WST-8 and incubated at 37 °C for 1.5 h. Absorbance was measured at 450 nm with a reference wavelength of 630 nm on a spectrophotometer (Molecular Devices, Sunnyvale, CA). All experiments were repeated in five times. Evaluation of apoptosis by flow cytometry Apoptosis analysis was performed by using an Annexin V-FITC KIT (Bender, Burlingame, CA, USA) according

to the manufacturer’s instructions. Briefly, cells were harvested, washed with PBS and resuspended in 100 ll of annexin V binding buffer (10 mM Hepes, pH 7.4, 5 mM CaCl2 and 140 mM NaCl). Then cells were incubated with FITC-labeled annexin V and propidium iodide (PI) (50 lg/ml) for cellular staining in binding buffer at room temperature for 15 min in the dark. Stained cells were then analyzed for the apoptosis by a FACSAria Cell Cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed with CellQuest software (BD Biosciences). Each experiment was performed in triplicate. The percentage of cells that were annexin V positive but PI negative was considered as apoptotic cells and compared among the different treatment groups.

Mouse tumor model All animal experiments were approved by the Animal Care and Use Committee of our university and were carried out in compliance with the ‘Guide for the Care and Use of Laboratory Animals, 8th edition’ published by the National Institutes of Health [17]. Female specific pathogen-free BALB/c nude mice (6–8 weeks old and weighed 18–22 g) were purchased from Weitonglihua Company (Beijing, China). The mice were housed in laminar flow cabinets under a specific pathogen-free environment. The animals were acclimatized for 1 week before use, and maintained throughout at standard conditions: 25 ± 2 °C temperature, 40–60 % relative humidity, and 12-h light/12-h dark cycle. After disinfecting the nude mice skin with 75 % ethanol, 1 9 106 cells were injected subcutaneously into the neck back using a sterile syringe. Mice were monitored to check for the appearance of signs of disease, such as subcutaneous tumors or weight loss due to potential tumor growth in internal sites. The growth of tumors was monitored throughout the experiment and tumor size was measured with calipers every 3 days. Tumor volume was determined as pls2/6, where l long side and s short side.

Statistical analysis GraphPad Prism version 5.03 (GraphPad, San Diego, CA, USA) was used for all statistical analysis. All data are expressed as mean ± SEM. Differences between groups were analyzed by a 2-tailed Student’s paired t test for single comparisons and by one-way ANOVA with LSD post hoc test for multiple comparisons. Bonferroni’s correction was used to adjust multiple comparisons. A P value \0.05 was considered to be statistically significant.

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Results Ectopic expression of DN-hTERT in MCF-7 cells inhibited the levels of telomerase activity and shortened the telomere length To investigate the consequence of telomerase inhibition in breast cancer cells by long-term expression of by the catalytically inactive dominant-negative (DN)-hTERT, DNhTERT or a control vector expressing only a puromycinresistant marker was introduced into the human breast adenocarcinoma MCF-7 cells by retroviral infection. After puromycin selection, successfully transfected cells were clonally isolated. The expression of hTERT mRNA was assessed by quantitative real-time RT-PCR. As shown in Fig. 1a, hTERT transcripts were significantly higher in the DN-hTERT transduced cells with respect to that of nontransduced or empty vector-transduced cells (P \ 0.01).

Fig. 1 Ectopic expression of DN-hTERT in MCF-7 cells reduces telomerase activity and shortens telomere length. a The levels of endogenous hTERT and exogenous DN-hTERT transcripts were analyzed by real-time quantitative PCR, bactin was used as an internal control to normalize the expression levels of target gens. Data show mean ± SEM of representative experiments performed in triplicate. b The telomerase activity was analyzed using the telomerase repeat amplification protocol ELISAplus assay. TRAP related ELISA absorbances were translated in relative telomerase activity values, which were then expressed as a percentage of that detected in the parental (uninfected) cells. c After infection with DN-hTERT or the empty vector, the average telomere lengths in MCF-7 cells were assessed by FISH assay (#P \ 0.01, *P \ 0.01)

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Western blot analysis revealed a decrease in overall hTERT protein levels in MCF-7 cells expressing DNhTERT compared with the nontransduced or vector-transduced cells (data not shown), indicating that DN-hTERT expression results in a decline in endogenous hTERT protein levels. To determine whether DN-hTERT exerted a dominant-negative effect on endogenous telomerase enzyme, TRAP assay was performed to assess telomerase activity of cell extracts. DN-hTERT transduced MCF-7 cells showed a dramatically reduced telomerase activity (decrease about 86 %), whereas vector transduced cells exhibited no change in the level of telomerase activity compared with the control untransduced cells (Fig. 1b). We next investigated whether telomerase activity inhibition affected telomere length in MCF-7 cells. As predicted, transfection of MCF-7 cells with DN-hTERT resulted in a remarkable decrease in telomere length compared with parental MCF-7 or pBABE-puro control (P \ 0.01,

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Fig. 1c). Thus, the expression of a catalytically inactive hTERT mutant results in telomerase activity inhibition and telomere length shortening. Inhibition of hTERT impaired the proliferation of MCF-7 cells As decreased telomerase activity is generally associated with cell growth arrest, the influence of telomerase inhibition in modulating proliferation of MCF-7 cells was evaluated by MTT assay about 3 weeks after the clone establishment. As shown in Fig. 2, the growth kinetics of MCF-7 cells carrying a control vector did not differ substantially from parental MCF-7 cells. However, MCF-7 cells expressing DN-hTERT showed slow growth and eventually stopped proliferating. These data indicated that telomerase inhibition by the expression of catalytically inactive DN-hTERT resulted in loss of proliferative capacity of MCF-7 cells in vitro.

observed between control vector-transfected and the parental MCF-7 cells. These data indicated that the inhibition of telomerase activity lead to increased apoptosis, which contributed to growth arrest of MCF-7 cells. Tumor formation in MCF-7 cells was reduced with loss of telomerase activity To investigate the effect of DN-hTERT on tumorigenicity of MCF-7 cells, cells transfected with DN-hTERT or empty vector and nontransduced cells were injected subcutaneously into female BALB/c nude mice, respectively. No mice died during the experiment. As shown in Fig. 4, mice xenografted with DN-hTERT cells lost their ability to generate tumor, since no palpable tumors could be detected during the experiment period. There were no significant differences between tumor growth in animal xenografted with parental cells and with empty vector transduced cells. Thus, these findings indicated that the in vivo tumorigenic capacity of MCF-7 cell was lost by inhibition of hTERT.

DN-hTERT induced apoptosis in MCF-7 cells To determine whether the MCF-7 cell growth arrest induced by DN-hTERT was associated with apoptosis, cells were collected for apoptosis analysis by flow cytometry assay. Cells were stained with annexin V plus PI. Apoptotic cells were determined as that were annexin V positive and PI negative. As shown in Fig. 3a, b, the fraction of apoptotic MCF-7/DN-hTERT cells was significantly higher than that of control vector-transfected or the parental MCF-7 cells (P \ 0.01). No significant differences of apoptosis were

Fig. 2 Effects of DN-hTERT on cell growth of MCF-7 cells. Cell proliferation was assessed by CCK-8 assay. Data were presented as the mean ± SEM of each time point from five samples (*P \ 0.05, # P \ 0.01)

Discussion Telomeres are the molecular caps at the ends of chromosomes that are composed of repetitive TTAGGG sequences and associated proteins [4]. Telomeres possess a number of functions including protecting chromosome ends from endto-end fusions, recombination, and degradation [5]. The maintenance of telomeres throughout many cycles of cell division requires the enzyme telomerase, which is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at chromosome ends [18]. The components of telomerase include a reverse transcriptase catalytic subunit (human telomerase reverse transcriptase, hTERT) and a RNA component (human telomerase RNA, hTR) [19]. It has been proven that telomerase activity is absent in most somatic human cells, and programmed shortening of telomeres has been observed in dividing cells and with aging. In contrast, most tumor and embryonic stem cells showed elevated telomerase activity [20]. Studies have shown that telomerase is highly active in most types of human cancers including breast cancer, but remains inactive in adjacent normal tissues [21]. In this study, to investigate the role of hTERT in human breast adenocarcinoma MCF-7 cells, the hTERT expression was blocked by ectopic expression of a catalytically inactive hTERT mutant dominant-negative (DN) hTERT. MCF-7 cells stably expressing DN-hTERT resulted in telomerase activity inhibition. Telomerase inhibition then induced telomere shortening and eventual growth arrest and apoptosis in vitro and loss of tumorigenic properties in vivo.

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Breast Cancer Fig. 3 DN-hTERT induces apoptosis of MCF-7 cells. Apoptosis was detected by annexin V and PI staining using FACS analysis. Results visualized as a representative experiment (a) or mean ± SEM of three experiments (b) (*P \ 0.01)

Telomerase is inactive in most normal cells, whereas it is active in cells with unlimited proliferative potential, such as germ line, stem cells, and cancer cells including breast cancer cells [22, 23]. The almost universal presence of telomerase in human tumors suggests that targeting telomerase may be an efficient way to specifically block tumor cell growth with minor effects on normal cells. Thus, telomerase has increasingly been seen as an attractive target for cancer therapy. Since hTERT protein is the catalytic rate-limiting determinant subunit of telomerase, several approaches have been used to target telomerase hTERT, such as oligonucleotides, ribozymes, or small interfering RNA, which have been effective in knocking down expression without reaching 100 % efficiency [24]. Point mutations of hTERT protein have emerged as an additional mechanism of regulating telomerase activity [25]. Telomerase activity can be abolished by replacement of any of the three conserved aspartic acid residues of amino-acid motifs A and C to the hTERT subunits in budding yeasts

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and in humans [25]. Ectopic expression of these hTERT mutants acting as dominant negatives of wild-type hTERT appears to be effective tools for studying the characteristics of telomerase in human cancer cells [12]. In our study, we employed a dominant-negative hTERT mutation (DNhTERT), which results in an inactive hTERT. The DNhTERT transfection causes a reduction in telomerase activity and function in MCF-7 cells. The mechanism of the dominant-negative telomerase effect or how exogenous inactive mutants affect the wild-type function is still being debated. Our data showed that DN-hTERT was successfully expressed in the human breast cancer MCF-7 cells and telomerase activity was reduced about 86 % by DNhTERT introduction. These results suggest that telomerase activity can be dramatically inhibited by DN-hTERT in MCF-7 cells. Telomere length plays important roles in maintaining genome stability and regulating cell replication and death [19]. Most normal human cells lack telomerase activity and

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Fig. 4 DN-hTERT inhibits tumour growth in nude mice. Cells (1 9 106) were subcutaneously injected into BALB/c nude mice for a total of 0.1 mL, and tumor volume was recorded every 3 days. Tumors in mice inoculated with the DN-hTERT MCF-7 cells showed a significant growth inhibition as compared with those inoculated with parental cells or empty vector-transduced cells. Data were presented as the mean ± SEM of each time point from six samples (*P \ 0.05, **P \ 0.01)

their telomeres shorten with each cell division, until they enter replicative senescence. The progressive telomere shortening resulted in the limited lifespan of the somatic cells [5, 22]. However, the immortal cells can acquire unlimited growth capacity by lengthening telomeres induced by the reactivation or up-regulation of telomerase [22]. Recent studies showed that telomerase activity can be detected in approximately 85–90 % of tumor samples including breast cancer and inhibition of telomerase can lead to progressive telomeric shortening during successive cell cycles and subsequently results in growth arrest and apoptosis [12, 21]. In the present study, telomerase inhibition by the expression of catalytically inactive DN-hTERT resulted in telomere length shortening, loss of in vitro proliferative capacity and increased apoptosis of MCF-7 cells. These findings indicated that the inhibition of telomerase activity by DN-hTERT resulted in telomere shortening, which lead to increased apoptosis and growth arrest of MCF-7 cells. Our data suggest that telomerase activity is the dominant mechanism providing telomere maintenance and viability to human breast cancer MCF-7 cells. Telomere-driven genome instability plays an important role in carcinogenesis and is thought to be the crucial event in the development of breast carcinomas [26]. It was assumed that disruption of telomerase activity would affect the genome instability of tumor cells, which can influence the development of cancers. Some investigations of in vitro cell cultures and in vivo animal models have demonstrated

that telomerase inhibitors can reduce tumorigenicity and suppress breast cancer growth and metastasis [27, 28]. In this study, inhibition of cell growth and induction of apoptosis in vitro indicated that inhibition of telomerase activity would reduce the tumorigenicity of cells in vivo. MCF-7 cells are highly tumorigenic in nude mice [29], however, such cells are no longer tumorigenic in immunodeficient mice by telomerase inhibition due to the stable expression of a dominant-negative variant of hTERT (DNhTERT). Thus, these findings indicated that the in vivo tumorigenic capacity of MCF-7 cell was lost by inhibition of endogenous hTERT, and hTERT activity is required for the maintenance of the malignant growth phenotype of MCF-7 cells. Moreover, hTERT can be valued as an attractive target for the development of new anti-breast cancer therapeutics. In conclusion, our results showed that telomerase inhibition in MCF-7 cells due to the stable expression of a dominant-negative variant of hTERT (DN-hTERT) induced telomere shortening and eventual growth arrest and apoptosis in vitro and loss of tumorigenic properties in vivo. Thus, telomerase inhibition using DN-hTERT could be potential approaches for breast carcinoma treatment. Acknowledgments This work was financially supported by the National Science Foundation of China (No. 30500596). Conflict of interest peting interests.

The authors declare that they have no com-

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Inhibition of telomerase activity by dominant-negative hTERT retards the growth of breast cancer cells.

Telomerase, a ribonucleoprotein enzyme mainly consisted of a catalytic protein subunit human telomerase reverse transcriptase (hTERT) and a human telo...
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