Introduction
Breast cancer is the leading cause of cancer death in women worldwide. Despite advances in detection and chemotherapy, many women with breast cancer continue to die of this malignancy [
1]. Therefore, an understanding of the molecular mechanisms involved in breast cancer formation and progression should be helpful in developing more effective treatments for breast cancer.
c-Myc is believed to participate in most aspects of cellular function, including replication, growth, metabolism, differentiation, and apoptosis [
2]. Previous studies indicate that c-Myc activates a variety of known genes as part of a heterodimeric complex with Max [
2]. A frequent genetic abnormality seen in breast cancer is the elevated expression of c-Myc [
3,
4]. The importance of c-Myc expression in breast cancer is demonstrated both by studies of transgenic mice and by clinical research [
3,
5]. Abnormal expression of c-
myc transgenes in the mouse mammary gland is associated with an increased incidence of breast carcinomas [
5]. Moreover, clinical studies have indicated that c-Myc is important in the development and progression of breast cancer, in that overexpression of c-Myc was found in most breast cancer patients and was correlated with poor prognosis in those patients [
3].
The role of c-Myc in breast cancer has been extensively examined in many studies for the past decade [
6]; however, specifically reducing its level by genetic means in established breast cancer cell lines is still helpful for a better understanding of its role in maintaining the malignant phenotype. Thus, in this study, we investigated whether specifically decreasing the protein level of c-Myc in a breast cancer cell line in which this protein was overexpressed might result in the inhibition of cell growth
in vitro and
in vivo. For this purpose, RNA interference (RNAi) directed against c-
myc was used.
RNAi is the sequence-specific gene silencing induced by double-stranded RNA (dsRNA). This phenomenon is conserved in a variety of organisms:
Caenorhabditis elegans,
Drosophila, plants, and mammals. RNAi is mediated by short interfering RNAs (siRNAs) that are produced from long dsRNAs of exogenous or endogenous origin by an endonuclease of the ribonuclease-III type, called Dicer. The resulting siRNAs are about 21–23 nucleotides (nt) long and are then incorporated into a nuclease complex, the RNA-inducing silencing complex, which then targets and cleaves mRNA containing a sequence identical to that of the siRNA [
7]. Rapid progress has been made in the use of RNAi [
8]. More recently, a technical breakthrough came from the demonstration that dsRNA of 19–29 nt expressed endogenously with RNA polymerase III promoter induced target gene silencing in mammalian cells [
9]. The expression of siRNA from DNA templates offers several advantages over chemically synthesized siRNA delivery. Hairpin siRNAs transcribed from a vector are thought to suppress the expression of targeted genes more efficiently, less expensively and more easily than synthesized siRNA [
10].
Here we used a plasmid-based polymerase III promoter system to deliver and express siRNA targeting c-myc to determine whether this technique could be used for the specific inhibition of oncogene overexpression and whether this inhibition resulted in antitumor effects. We showed in our study that specific downregulation of c-Myc by RNAi was sufficient to inhibit the growth of MCF-7 cells in vitro and in vivo, and that c-myc might serve as a therapeutic target for human breast cancer.
Method
Plasmid construction
To generate c-Myc knockdown vector, one annealed set of oligonucleotides encoding short hairpin transcripts corresponding to nt 1906–1926 of c-
myc mRNA (GenBank accession no. NM-002467) [
11] were cloned into p
Silencer 1.0_U6 (Ambion; hereafter abbreviated to p
Silencer). In brief, the short-hairpin-RNA-encoding complementary single-stranded oligonucleotides, which hybridized to give overhangs compatible with
ApaI and
EcoRI, were designed with a computer program available on the Internet
http://www.ambion.com/techlib/misc/psilencer_converter.html. Oligonucleotides encoding short hairpin RNAs were then ligated into p
Silencer. Bacterial colonies were pooled and used for plasmid preparation. The positive clones were confirmed by sequencing. The resulting plasmid was designated as p
Silencer–c-Myc.
Cell line and cell culture
The breast cancer adenocarcinoma cell line MCF-7 was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco BRL), 50 units/ml penicillin, and 50 μg/ml streptomycin. The MCF-7 cells were maintained in a humidified 37°C incubator with 5% CO2, fed every 3 days with complete medium, and subcultured when confluence was reached.
Transfection of cells
A total of 2 × 105 cells were seeded into each well of a six-well tissue culture plate (Costar). The next day (when the cells were 70–80% confluent), the culture medium was aspirated and the cell monolayer was washed with prewarmed sterile phosphate-buffered saline (PBS). Cells were transfected with the appropriate plasmids by using LipofectAMINE reagent (Invitrogen) in accordance with the manufacturer's protocol. The cells were harvested at different time points. Western blot analysis or other experiments were performed.
Western blot analysis
Cells were harvested at different time points and lysed in mammalian cell lysis buffer, then western blot analysis was performed with the use of conventional protocols as described previously [
12]. In brief, the protein concentration was determined with a bicinchoninic acid kit with bovine serum albumin as a standard (Pierce). Equal amounts of total protein were then separated on 12% polyacrylamide gels by using standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) techniques, then transferred to nitrocellulose membranes (PROTRAN). The antibodies and dilutions used included anti-c-Myc (9E10; 1:1000 dilution; Santa Cruz) and anti-β-actin (AC-15; 1:5000 dilution; Sigma), and after being washed extensively the membranes were incubated with anti-mouse IgG–horseradish peroxidase conjugate antibody (Zhongshan Company) for 1 hour at room temperature and developed with a Luminol chemiluminescence detection kit (Santa Cruz). Membranes probed for c-Myc were reprobed for β-actin to normalize for loading and/or quantification errors and to allow comparisons of target protein expression to be made. Protein expression was quantified with a Gel EDAS analysis system (Cold Spring USA Corporation) and Gel-Pro Analyzer 3.1 software (Media Cybernetics).
Cell growth assay
At 2 days after transfection, MCF-7 cells transfected with indicated plasmids were harvested and replated at a density of 50 cells/mm2 in triplicate. The total cell number was quantified every 2 days with a hematocytometer and an Olympus inverted microscope. Cell viability was assessed by using trypan blue.
Soft agar colony assay
At 2 days after transfection, MCF-7 cells (300 cells per well) transfected with indicated plasmids were mixed with tissue culture medium containing 0.7% agar to result in a final agar concentration of 0.35%. Then 1 ml samples of this cell suspension were immediately plated in six-well plates coated with 0.6% agar in tissue culture medium (2 ml per well) and cultured at 37°C with 5% CO2. After 2 weeks the top layer of the culture was stained with 0.2% p-iodonitrotetrazolium violet (Sigma). The culture was analyzed in triplicate, and colonies larger than 100 μm in diameter were counted.
Tumor growth in nude mice
Equal numbers (106 or 2 × 106) of MCF-7 cells transfected with pSilencer–c-Myc or pSilencer were harvested by trypsinization 2 days after transfection, washed twice with 1 × PBS, and resuspended in 0.2 ml of saline. Two groups of five 4–6-week-old female nude mice were then given bilateral subcutaneous injections with control cells or cells transfected with plasmids against c-Myc. The mice were kept in pathogen-free environments and checked every 2 days. The date at which a palpable tumor first arose and the weight of the tumor were recorded.
Cell cycle analysis
Standard fluorescence-activated cell sorting analysis was used to determine apoptosis of the cells. In brief, MCF-7 cells were transfected with pSilencer–c-Myc or pSilencer; 24 hours later, cells were deprived of serum for 36 hours. Then cells were harvested, washed once in PBS and stained with propidium iodide (BD Biosciences). The apoptotic cells were assessed by flow cytometric detection of sub-G1 DNA content.
Apoptotic cells were confirmed with the in situ cell death detection kit, Alkaline Phosphatase (Roche Applied Science), in accordance with the manufacturer's instructions. In brief, MCF-7 cells were grown on coverslips. The next day, cells were transfected with pSilencer–c-Myc or pSilencer. At 24 hours after transfection, cells were deprived of serum for 36 hours. Coverslips with adherent cells were fixed in 4% paraformaldehyde for 1 hour at room temperature and permeabilized with 0.1% Triton X-100 for 2 min on ice. DNA fragments were labeled with the TdT-mediated dUTP nick end labelling (TUNEL) reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. The coverslips were then incubated with Converter alkaline phosphatase for 30 min at 37°C in a humidified chamber, rinsed in PBS, and incubated with nitro blue tetrazolium/5-bromo-4-chloroindol-3-yl phosphate (Roche Applied Science) for 10 min. Cells were mounted cell side downward on a microscope slide, and the apoptotic cells (dark blue staining) were counted under a microscope. Three fields were randomly counted for each sample.
Statistical analysis
SPSS for Windows (SPSS Inc.) was used to analyze the data and plot curves. A two-tailed unpaired t-test was used to compare the statistical significance of the differences in data from the two groups.
Discussion
Cancer cells often show alteration in the signal-transduction pathways, leading to proliferation in response to external signals. Oncogene overexpression is a common phenomenon in the development and progression of many human cancers. Oncogenes therefore provide a potential target for cancer gene therapy [
13].
The important oncogene c-
myc is expressed in a high proportion of most human cancers, including breast, prostate, gastrointestinal cancer, lymphoma, melanoma, and myeloid leukemia [
14]. In its physiological role, c-Myc is broadly expressed during embryogenesis and in tissue compartments of the adult that possess high proliferative capacity. Altered expression of c-Myc seems to define a common event associated with the pathogenesis of most human cancers [
6]. Previous studies demonstrated that the continued presence of c-Myc was required for cancer development and not just for initiation, and inactivation of c-Myc resulted in the sustained regression of tumors [
15‐
17]. Similar results were also observed in breast cancer. D'Cruz and colleagues demonstrated that overexpression of c-Myc by an inducible system in the mammary epithelium of transgenic mice resulted in the formation of invasive mammary adenocarcinomas, many of which regressed fully after c-Myc deinduction [
18].
Therefore, specific downregulation of c-Myc might be a potential therapeutic strategy against human cancers, including breast cancer. In fact, the antagonists of c-Myc, including full-length antisense mRNA [
19], oligonucleotides against c-
myc mRNA [
20] or a dominant-negative mutant [
21], were previously reported to inhibit proliferation of cancer cell lines
in vitro. However, it was only successful in some situations; these technologies have been difficult to apply universally [
22]. Recently the advent of RNAi-directed 'knock-down' has sparked a revolution in somatic cell genetics, allowing the inexpensive, rapid analysis of gene function in mammals, and might be exploited for gene therapy [
7,
8]. Some studies directly compared RNAi with antisense RNA and found that RNAi seemed to be quantitatively more efficient and durable in cell culture and in nude mice [
23].
By means of the RNAi method, in the present study, cellular growth assays, both
in vitro and
in vivo, were used to determine the functional consequences of RNAi-mediated decreases in of c-Myc in established breast cancer cells. Our results demonstrated that RNAi can effectively downregulate oncogene overexpression with great specificity. We showed that the plasmids endogenously expressing siRNA could successfully deplete up to 80% of c-Myc expression in MCF-7 cells at day 5 after transfection. Furthermore, the tumor inhibition effects persisted for at least 12 days after transfection in dishes and for 2 months in nude mice as shown by experiments
in vitro and
in vivo, even though the protein level of c-Myc in silenced clones expressing siRNA was back to almost the same level as in the control cells by day 12 after transfection. Our data were consistent with the results of Jain and colleagues [
17].
They showed that within 24 hours of c-
myc inactivation, the osteogenic sarcoma cells flattened and showed less cell division. And even after reactivation of c-
myc expression in these cultured cells, total cell numbers continued to be lower. Less than 1% of the tumor cells regained their neoplastic growth properties [
17]. All of these data indicated that brief inactivation of c-Myc could induce a sustained loss of neoplastic phenotypes. Moreover, other groups using chemically synthesized siRNAs to knock down their favored oncogenes also found that a transient decrease in oncogene expression could inhibit the growth of tumor cells
in vitro and/or
in vivo [
8]. Nevertheless, the underlying mechanism of this phenomenon in MCF-7 cells should be further investigated.
Although some studies previously revealed that the effects of inactivation of c-Myc in some cell lines were modest [
6], other groups using different approaches to reduce the protein level of c-Myc found that a decrease in c-Myc expression could inhibit the growth of these tumor cells, including breast tumor cells [
19‐
21]. Nevertheless there were still conflicting results on whether c-Myc expression was necessary to maintain tumorigenesis in different animal models from different laboratories [
15‐
18,
24,
25]. For example, some studies showed that the role of oncogenic c-Myc in tumor maintenance was essential and that all effects of c-Myc
in vivo were reversible, in that without continuous c-Myc activation there would even be regression of established tumors back to phenotypically normal in transgenic mouse models [
15,
16,
24]. Similar phenomena were observed by other groups focusing on other oncogenes, such as
bcr/
abl [
26] and
H-
ras [
27]. However, there were also conflicting reports, mainly showing that brief inactivation of c-Myc could induce sustained loss of neoplastic phenotypes in certain animal models [
17,
18,
25]. It was notable that without secondary oncogenic mutation, spontaneously or selectively, such as
Kras2 oncogene mutation, in nearly all breast tumors induced by conditionally expressing the human c-Myc in the mammary epithelium of a transgenic mouse model, deinduction of c-Myc protein could lead to full regression of tumors [
18]. Similar data were also obtained by Karlsson and colleagues that the inactivation of c-Myc alone was found to be sufficient to cause sustained tumor regression in c-Myc-induced hematopoietic tumors; in contrast, tumor cells that acquired novel chromosomal translocations relapsed independently of Myc to maintain their neoplastic phenotype [
25]. It was therefore not surprising for us to show here that a transient reduction of c-Myc protein level by RNAi could significantly inhibit the growth rate of MCF-7 cells and its ability to form colonies in soft agar. Additionally, the remarkable effect in nude mice supported the effectiveness of this treatment.
Our data also suggested that knockdown of c-Myc by RNAi in MCF-7 cells could increase the sensitivity of these cells to apoptotic stimuli, such as serum starvation. This was most probably one of the reasons for the anti-tumor effects. There have previously been conflicting reports about the role of c-Myc in apoptosis [
3]. Constant overexpression of c-Myc might induce apoptosis [
2,
6], and a decrease in c-Myc levels by techniques brought about by, for example, an antisense approach might also cause apoptosis of certain tumor cells [
28‐
30] or might increase the sensitivity of the cells to apoptotic stimuli [
31]. These conflicting observations suggested that c-Myc was capable of both inducing and suppressing apoptosis in different types of tumor cell, under different conditions, and in different systems. In MCF-7 cells, suppression of c-Myc expression in response to aromatase inhibitors or topoisomerase α inhibitors could induce these cells to apoptosis [
32,
33]. However, the pathways that c-Myc controls and/or that are involved in the observed apoptosis remain obscure. D'Agnano and colleagues suggested that in melanoma cells the downregulation of c-Myc by an antisense approach could activate apoptosis by increasing the levels of p27
Kip1 [
28]. Overexpression of the cyclin-dependent kinase inhibitor p27
Kip1 was able to promote apoptosis in several mammalian tumor cell lines [
34]. However, understanding the precise pathway by which a decrease in c-Myc in MCF-7 cells by RNAi was able to induce apoptosis upon serum deprivation needs further study.
So far, RNAi has been used to inhibit virus-induced diseases (for example HIV [
35] and influenza [
36]), oncogenic
K-ras,
H-ras-induced tumorigenesis [
37,
38], activation of oncogenes resulting from chromosomal translocations (for example
bcr/
abl in chromic myeloid leukemia [
39]), cancers caused by viral infections [
40], and so on. Recently, retroviral-based approaches to deliver siRNA into tissue-cultured mammalian cells have been proved to be powerful [
37,
38], and doxycycline-regulated inducible knockdown of gene expression by RNAi has been shown to be particularly useful for the analysis of genes that are essential for cellular survival [
41,
42]. These studies have marked a new era in the genetic manipulation of human cancer development by allowing oncogenes to be downregulated by RNAi.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YW designed the experiments, constructed the plasmids, performed transfections, and wrote the manuscript. SL constructed the growth curve and performed the nude mice experiments. GZ performed the western blots and the soft agar assay. CZ, HZ, and XZ performed the flow cytometry and TUNEL experiments. LQ and JB conducted the cell culturing. NX is the corresponding author. All authors read and approved the final manuscript.