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Inhibition of FOXM1 transcription factor suppresses cell proliferation and tumor growth of breast cancer

Abstract

The forkhead box M1 (FOXM1) transcription factor regulates the expression of genes essential for cell proliferation and transformation and is implicated in tumorigenesis and tumor progression. FOXM1 has been considered as a potential target for the prevention and/or therapeutic intervention in human carcinomas. In this study, we observed a strong expression of FOXM1 in clinical tissue specimens and cell lines of human breast cancer and a correlation between FOXM1 levels and the proliferation ability in the tested MCF-7, MDA-MB-231 and ZR-75-30 cells. By using an adenovirus vector (named AdFOXM1shRNA) that expresses a short hairpin RNA (shRNA) to downregulate FOXM1 expression specifically, we found that the knockdown of FOXM1 expression diminished the proliferation and anchorage-independent growth of the breast cancer cells. The FOXM1 silencing in ZR-75-30 cells dramatically prevented the tumorigenicity of the AdFOXM1shRNA-treated cells in vitro and in vivo. Furthermore, the efficacy of AdFOXM1shRNA for tumor gene therapy was assessed with the breast cancer xenograft mouse model and the tumor growth was significantly suppressed when inoculated mice were injected with AdFOXM1shRNA in the tumors. Together, our results suggest that FOXM1 is a potential therapeutic target for breast cancer and AdFOXM1shRNA may be an additional gene therapeutic intervention for breast cancer treatment.

Introduction

Breast cancer is an important cause of mortality among women and remains a public-health issue on a global scale.1 According to the assessment of breast cancer data during the period of 1980–2010 in 187 countries, the global breast cancer incidence increases by an annual rate of 3.1%, from 641 000 cases in 1980 to 1 643 000 cases in 2010, and there are 425 000 deaths caused by breast cancer in 2010.1 Breast cancer is commonly treated by various combinations of surgery, radiation therapy, chemotherapy and hormone therapy, and the selection of therapy is influenced by the clinical and pathology features based on conventional histology, immunohistochemistry and molecular profiling of the cancer.2, 3, 4 Combinatorial therapies that use novel agents targeting growth factor receptors, signal transduction pathways and tumor angiogenesis are investigated in clinical trials.5

Transcription factor forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors6 and is ubiquitously expressed in proliferating and regenerating mammalian cells.7, 8 FOXM1 is a key cell cycle regulator of both the transition from G1 to S phase and the progression to mitosis by regulating transcription of cell cycle genes.9, 10, 11, 12, 13, 14, 15 Loss of FOXM1 expression causes diminished DNA replication, mitotic spindle defects and mitotic catastrophe.13, 14, 16 Furthermore, along with others, we have shown that FOXM1 is involved in conteracting stresses induced by cytotoxic or genotoxic signals, such as oxidative stress or DNA damage.17, 18, 19 Moreover, we have characterized that FOXM1 has an essential role in maintenance of stem cell pluripotency and its expression is absent from differentiated cells.20 These observations suggest that altered expression of FOXM1 is associated with tumorigenesis through its critical roles in cell proliferation, prevention of differentiation and malignant transformation of undifferentiated cells. This notion is apparently supported by the fact that FOXM1 is highly expressed in various types of human malignancies, such as lung cancer,21 glioblastomas,22 prostate cancer,23 basal cell carcinomas,24 hepatocellular carcinoma,15 pancreatic cancer,25 gastric cancer26 and squamous cell carcinoma.27 The inactivation of FOXM1 leads to inhibition of progression and/or invasion of these cancers, suggesting that FOXM1 appears to be an attractive target for the development of novel anticancer therapies.28, 29, 30

Elevated expression or activity of FOXM1 is associated with the development and progression of breast cancer. It was found that FOXM1 was overexpressed in breast cancer in comparison with normal breast tissue both on the RNA and protein level.31 FOXM1 was one of the markers that might allow early detection of both the ductal carcinoma in situ and invasive ductal carcinoma of the mammary gland.32 Interestingly, there was a strong and significant positive correlation between ERalpha and FOXM1, and FOXM1 was shown to stimulate the transcription of ERalpha in breast cancer cells.33 On the other hand, the expression of FOXM1 could be regulated by ERalpha and/or ERbeta.34, 35 Furthermore, there was a significant correlation between FOXM1 expression and the HER2 status31 and FOXM1 was a downstream target and marker of HER2 overexpression in breast cancer,36 pointing to a potential role of FOXM1 as a new drug target in HER2-resistant breast cancer. As the depletion of FOXM1 limits proliferation of breast cancer cells, inhibiting FOXM1 represents a therapeutic strategy to target breast cancer.

In this study, we observed strong expression of FOXM1 in clinical tissue specimens and cell lines of human breast cancer. We found that the downregulation of FOXM1 expression inhibited the breast cancer cell proliferation. To develop innovative strategies for breast cancer gene therapy, we developed an efficient adenovirus-mediated short hairpin RNA (shRNA) expression system to specifically knockdown FOXM1 expression in breast cancer cells. Using this system, we investigated the function of FOXM1 in breast cancer tumorigenesis and evaluated the potential of FOXM1 as a therapeutic target for breast cancer treatment.

Materials and methods

Cell culture

The human breast cancer cell lines MCF-7, MDA-MB-231 and ZR-75-30 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained according to ATCC (Manassas, VA) instructions. Adenovirus-purification 293A (Invitrogen, Grand Island, NY) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin streptomycin (Invitrogen). All cells were cultured in a 5% CO2-humidified atmosphere at 37 °C.

Immunohistochemical analysis

Human breast ductal tumor paraffin sections were obtained from the Department of Pathology, Hunan Provincial Tumor Hospital, Hunan, China. The slides of samples of patients and nude mouse xenograft tumors were stained with a mouse anti-FOXM1 antibody (1:100; Abcam, Cambridge, UK; ab55006), followed by the incubation of a horseradish peroxidase-conjugated anti-mouse secondary antibody. Color was detected with 3,3'-diaminobenzidine and pictures were taken at × 200 magnification using a TE2000 microscope (Nikon, Tokyo, Japan).

Western blot assay

The lysates of clinical tumor tissues or breast cancer cell lines were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane for western blotting as described earlier.19 The following antibodies and dilutions were used for western blotting: rabbit anti-FOXM1 (1:1000; Abcam ab47808), mouse anti-β-actin (1:5000; Beyotime AA128, Beyotime, Shanghai, China). The signals from the primary antibody were amplified by horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1:10 000; GE, Fairfield, CT LNA934VAE) or anti-mouse immunoglobulin G (1:10 000; GE LNA931VAD), and detected with Enhanced Chemiluminescence Plus (Beyotime).

Isolation RNA, reverse transcription-PCR and quantitative real-time PCR

The total RNA was isolated by Total RNA Kit (Omega, Norcross, GA) according to the manufacturer’s protocols. The complementary DNAs were synthesized with M-MLV Reverse Transcriptase (Invitrogen) from total RNA samples. PCR amplification was performed with Taq DNA polymerase mix (Biotech, Qingdao, China) with following sense (S) and antisense (AS) primers, annealing temperature (Ta) and number of PCR cycles (N): hFOXM1-S, 5′-IndexTermGCTTGCCAGAGTCCTTTTTGC-3′ and hFOXM1-AS, 5′-IndexTermCCACCTGAGTTCTCGTCAATGC-3′ (Ta:56 °C, N:30); hCyclinB1-S, 5′-IndexTermGGTCTGGGTCGGCCTCTACCT-3′ and hCyclinB1-AS, 5′-IndexTermAGCCAGGTGCTGCATAACTGGAA-3′ (Ta:59 °C, N:30); hCENPB-S, 5′-IndexTermATTCAGACAGTGAGGAAGAGGACG-3′ and hCENPB-AS, 5′-IndexTermCATCAATGGGGAAGGAGGTCAG-3′ (Ta:60 °C, N:30); hβ-actin-S, 5′-IndexTermAGCGAGCATCCCCCAAAGTT-3′ and hβ-actin-AS, 5′-IndexTermGGGCACGAAGGCTCATCATT-3′ (Ta:54 °C, N:23). Quantitative real-time PCR was performed with SYBR Green (Roche, Basel, Switzerland) in the realplex2 quantitative real-time PCR system (Eppendorf, Hamburg, Germany).

Adenovirus purification and infection

The AdFOXM1shRNA and control virus (AdLacZ) were preserved in our lab and the large-scale adenovirus purification was performed as described previously.30, 37, 38 Infection was induced by the addition of the virus directly to serum-free medium for 1 h at 37 °C. The viral infection was stopped by replacing with the culture medium and the cells were cultured for additional different time courses.

Flow cytometry analysis

The tested cells were collected and washed twice with phosphate-buffered saline. Cells were fixed in 70% ethanol and collected by centrifugation 3000 r.p.m. for 8 min. The cell pellets were resuspended in propidium iodide (0.05 mg ml−1) plus RNase (0.02 mg ml−1) and incubated in the dark at room temperature for 30 min. The cells were filtered and analyzed for DNA content on Quanta SC flow cytometer (Beckman, Brea, CA).

Soft agar colonization assay

The tested cells were mixed with 1 ml culture medium containing 0.35% (w/v) agar and layered over a basal layer of 0.7% (w/v) agar with culture medium in six-well plates. The cells were allowed to grow for 10–14 days. Experiments were carried out three times and the results are representative of the three independent observations.

Xenograft tumor models in nude mice and ad injection

BalB/c nude mice (female, 4-week old) were purchased from SLAC Laboratory Animal Company (Shanghai, China), China. The mice were maintained under standard conditions according to the institutional guidelines for animal care. To study the effect of FOXM1 silencing on tumor growth in vivo, two separate experiments were performed. In the first experiment, the mice were divided into three groups randomly (four mice per group) and injected in the flank with 3 × 106 ZR-75-30 cells transfected with serum-free medium, AdLacZ or AdFOXM1shRNA (10 plaque-forming unit (p.f.u.) per cell), respectively. In the second experiment, the tumor model in nude mice involved subcutaneous inoculation of 3 × 106 ZR-75-30 cells. Although the tumor volume reached 50–100 mm3, we injected 5 × 108 p.f.u. AdLacZ or AdFOXM1shRNA in two groups, respectively. The infections were repeated three times at 3-day intervals. The tumor volume in each group was measured at certain time points according to the method described earlier.39 All the mice were killed at the end of the procedure and the tumor samples were collected.

Statistical analysis

We used Microsoft Excel Program to calculate s.d. and statistically significant differences between samples. The asterisks in each graph indicate statistically significant changes with P-values calculated by Student’s T-test: *P<0.05, **P0.01 and ***P0.001. P-values <0.05 were considered statistically significant.

Results

The expression of FOXM1 in clinical tissues and malignant cell lines of breast cancer

The expression of FOXM1 was found to be elevated in clinical samples of primary breast cancer.16, 31, 32, 40 We first confirmed the published findings by measuring the expression levels of FOXM1 with western blotting in the lysates of clinical human breast ductal carcinoma samples, in which 14 out of 16 analyzed cases showed FOXM1 positive (Figure 1a). We immunostained the tissue sections with an anti-FOXM1 antibody and observed positive FOXM1 staining predominantly in the samples of breast ductal carcinoma but not in the normal breast tissues (Figure 1b). By using quantitative real-time PCR analysis, we found that FOXM1 mRNA was expressed at relatively high levels in malignant MCF-7, MDA-MB-231 and ZR-75-30 breast cancer cells (Figure 1c). The expression of FOXM1 in the tested breast cancer cell lines was further confirmed by western blot analysis, in which MCF-7 cells showed lower levels of FOXM1 protein than that of the other two cell lines (Figure 1d). Interestingly, growth curves of these three cell lines showed statistically difference on the rate of cell proliferation and MCF-7 cells proliferated slower than MDA-MB-231 or ZR-75-30 cells (Figure 1e). These results indicated that FOXM1 was commonly expressed in clinical tissue specimens and cell lines of human breast cancer and the levels of FOXM1 expression correlated with the ability of cell proliferation.

Figure 1
figure1

The expression of forkhead box M1 (FOXM1) in breast ductal carcinoma. The expression levels of FOXM1 protein were measured with the lysates of clinical human breast ductal carcinoma samples by western blotting, and 14 out of 16 analyzed cases showed FOXM1 positive (a). Tissue sections of clinical samples were immunostained with an anti-FOXM1 antibody and the pictures were taken at × 200 and × 400 magnification. Typical FOXM1 immunostaining was observed in breast ductal carcinoma (b). (c, d) FOXM1 expression in malignant MCF-7, MDA-MB-231 and ZR-75-30 breast cancer cells. FOXM1 mRNA levels were measured by quantitative real-time reverse transcription (RT)-PCR (c), and FOXM1 protein levels were measured by western blotting (d). β-Actin was used as the loading control. (e) Growth curves of MCF-7, MDA-MB-231 and ZR-75-30 breast cancer cells. The cells (1 × 105) were seeded in each well (in triplicate) of six-well tissue culture plate and cell numbers were counted every day afterward. Bars, s.d.; ***P<0.001 indicates statistically difference between MCF-7 and MDA-MB-231/ZR-75-30 cells at the day 3 time point.

Knockdown of FOXM1 by AdFOXM1shRNA decreased the expression of cell cycle genes and inhibited the proliferation of breast cancer cells

FOXM1 is considered as an attractive target for the development of novel anticancer therapies.28, 29, 30 To develop innovative strategies for breast cancer gene therapy, we constructed an adenovirus-mediated shRNA expression system to knockdown FOXM1 expression and named this replication-defected adenovirus vector as AdFOXM1shRNA.30 AdLacZ control adenovirus infections determined that almost 100% of breast cancer cells were infected with the viral dosage at 10 p.f.u. per cell (Supplementary Figure S1). Western blot analysis further confirmed that the expression of FOXM1 protein was effectively suppressed in breast cancer cells at day 3 following infection with AdFOXM1shRNA (10 p.f.u. per cell) but not with AdLacZ (Figure 2a and Supplementary Figure S2). It is well known that FOXM1 regulates the transcription of cell cycle genes, such as cyclin B1 and CENPB, and knockdown of FOXM1 prevents the expression of these genes in multiple tumor cells.9, 10, 11, 12, 13, 14, 15 AdFOXM1shRNA infection decreased the expression of cyclin B1 and CENPB in MCF-7, MDA-MB-231 and ZR-75-30 breast cancer cells (Figure 2b), consistent with the published findings in other tumor cells.9, 10, 11, 12, 13, 14, 15 To determine the growth rate of FOXM1-depleted cells, breast cancer cells were infected with AdFOXM1shRNA or control AdLacZ and counted every day afterward. The growth curves showed that the cell growth of all the three breast cancer cell lines was inhibited dramatically by infection of AdFOXM1shRNA, compared with AdLacZ infection (Figure 2c). We observed no obvious difference in apoptosis among samples of the AdLacZ-infected, AdFOXM1shRNA-infected and non-treated cells (Supplementary Figure S3 and S4), indicating that the reduction in growth of the FOXM1-depleted cells was due to the inhibition of cell proliferation. This idea was further supported by the analysis of cell cycle progression with the MCF-7 and ZR-75-30 cell samples, in which the AdFOXM1shRNA-infected cells showed significant decrease in mitotic progression evidenced by a decrease in G1-phase cells and an increase in G2/M-phase cells compared with the untreated cells (Figure 2d). These data confirmed that AdFOXM1shRNA were effective in abolishing expression of FOXM1 and inhibiting the proliferation of breast cancer cells.

Figure 2
figure2

Knockdown of forkhead box M1 (FOXM1) by AdFOXM1shRNA decreased the expression of cell cycle genes and inhibited the proliferation of breast cancer cells. (a) The expression of FOXM1 was inhibited by AdFOXM1shRNA infection in breast cancer cells. The MCF-7, MDA-MB-231 and ZR-75-30 cells were infected with AdLacZ or AdFOXM1shRNA at dosage of 10 plaque-forming unit (p.f.u.) per cell. Total protein lysates were prepared at day 1 and day 3 following infection. The protein levels were measured by western blotting. (b) AdFOXM1shRNA infection decreased the expression of cell cycle genes in breast cancer cells. Total RNA was isolated at 48 h following viral infection. The levels of FOXM1 and various cell cycle-related genes were determined by reverse transcription (RT)-PCR. (c) The breast cancer cell growth was inhibited by the infection of AdFOXM1shRNA. Growth curves were calculated with MCF-7, MDA-MB-231 and ZR-75-30 cells or the cells infected with AdLacZ, or AdFOXM1shRNA at dosage of 10 p.f.u. per cell. 1 × 105 cells were seeded in each well (in triplicate) of six-well tissue culture plate and cell numbers were counted every day afterward. (d) Flow cytometry analysis of AdFOXM1shRNA-infected breast cancer cells shows the increased populations of G2/M cells. MCF-7 and ZR-75-30 cells were infected with AdFOXM1shRNA (10 p.f.u. per cell), collected at the day 3 post infection, and propidium iodide (PI) staining quantified the cycle of cells. Bars, s.d.; *P<0.05, ***P<0.001, relative to control at the same time point.

Tumorigenicity of breast cancer cells reduced by AdFOXM1shRNA

To investigate the effect of FOXM1 knockdown on tumorigenesis in vitro, we performed the soft agar assays to measure the ability of anchorage-independent growth of breast cancer cells infected with AdLacZ or AdFOXM1shRNA. ZR-75-30 cells were infected with Ad vectors (10 p.f.u. per cell) and plated in agar layers to grow for 10–14 days. AdFOXM1shRNA infection resulted in dramatic decrease in colony formation of ZR-75-30 cells (Figure 3a). Quantization of colony number demonstrated that depletion of FOXM1 expression by AdFOXM1shRNA inhibited the colony formation of the breast cancer cells (Figure 3b). To investigate the effect of FOXM1 knockdown on tumorigenesis in vivo, we inoculated the AdFOXM1shRNA-infected ZR-75-30 cells subcutaneously, and then compared the tumorigenicity of the FOXM1-silenced cells with that of the untreated or AdLacZ-infected cells. At 4 weeks post inoculation, tumor growth was nearly completely suppressed in mice inoculated with the FOXM1-silenced cells, whereas obvious tumors were formed in the control group mice inoculated with the untreated or AdLacZ-infected cells (Figure 4). As shown in Figures 4b and c, we observed only one small tumor formed in the four mice inoculated with the FOXM1-silenced cells. These observations suggested that AdFOXM1shRNA reduced the induction of the neoplastic phenotype of breast cancer cells in vivo.

Figure 3
figure3

The infection of AdFOXM1shRNA decreased the colony formation of breast cancer cells. ZR-75-30 cells were infected with AdLacZ or AdFOXM1shRNA at dosage of 10 plaque-forming unit (p.f.u.) per cell. The soft agar assays were performed according to a standard protocol. The colony formation of ZR-75-30 cells was shown by pictures (a) and colony number counting (b). Bars, s.d.; ***P<0.001.

Figure 4
figure4

Tumorigenicity was inhibited by knockdown of forkhead box M1 (FOXM1) using AdFOXM1shRNA in breast cancer ZR-75-30 cells. ZR-75-30 cells were infected with AdLacZ or AdFOXM1shRNA (10 plaque-forming unit (p.f.u.) per cell) and 1 day later the cells (3 × 106 per mouse) were injected subcutaneously to BalB/c nude mice in groups of four mice. The tumor volume in each group was measured at certain time points. (a) Tumor volumes of the nude mice in each group. Bars, s.d.; ***P<0.001. (b) The photographs of mice in each group at the week 4 post inoculation. Circles: the tumors formed in mice of the two control groups. Arrows: the location of subcutaneous injection of AdFOXM1shRNA-infected cells in mice. (c) The photographs of tumors isolated from each group at the week 4 post inoculation. Bar: 1 cm.

Tumor growth of breast cancer cells was prevented by AdFOXM1shRNA in nude mouse xenografts

To determine the effect of AdFOXM1shRNA on tumor growth kinetics in vivo, we first generated ZR-75-30 cell xenografts in nude mice by injecting the cells (3 × 106 per mouse) subcutaneously to BalB/c nude mice in groups of six. The following AdFOXM1shRNA intratumoral injections (5 × 108 p.f.u. per mouse) were started at day 14 after the cell injections and repeated with a 3-day interval till the end of the procedure. We continued to measured the tumor volume in each animal at certain time points during the procedure and found that tumor growth was significantly inhibited in the group treated with AdFOXM1shRNA as compared with the control group (AdLacZ-treated; Figure 5b). In contrast with the large tumors produced by the AdLacZ-treated group, the mice treated with AdFOXM1shRNA produced small tumors (Figure 5a). Total RNA and protein lysates were isolated from the tumors of the three groups. The FOXM1 protein was almost undetectable in the AdFOXM1shRNA-treated samples (Figure 5c), suggesting that the inhibition of tumor growth in the AdFOXM1shRNA-treated group was a consequence of FOXM1 depletion in the tumors. The levels of FOXM1 protein in the tumors were also measured by immunostaining with the FOXM1 antibody and typical FOXM1-positive staining in nuclei was detected in the tumor tissue sections of the AdLacZ-treated group but not in that of the AdFOXM1shRNA-treated group (Figure 5d). In addition, the pools of combined six RNA samples from each group were tested with reverse transcription-PCR to measure the mRNA levels of FOXM1 and FOXM1’s target genes, such as cyclin B1, CENPB and PLK1. As predicted, the FOXM1 mRNA level was significantly reduced in the AdFOXM1shRNA-treated group and the downregulation of these FOXM1’s target genes was also observed (Figure 5e). We observed no obvious difference in apoptosis among samples of the AdLacZ-infected and AdFOXM1shRNA-infected tumors (Supplementary Figure S5), implicating that the reduction in growth of the FOXM1-depleted tumors was due to the inhibition of cell proliferation. The tumor tissue taken from each group was also examined through hematoxylin and eosin staining. The tissue sections of the AdLacZ-infected group showed a condensed morphology compared with that of the AdFOXM1shRNA-treated group (Figure 5f). These results indicated that AdFOXM1shRNA targeting FOXM1 elicited a strong antitumor effect on breast cancers in vivo.

Figure 5
figure5

The tumor growth was suppressed by the injection of AdFOXM1shRNA. The breast cancer grafting animal model was created by subcutaneous inoculation of 3 × 106 ZR-75-30 cells. The formed tumors (50–100 mm3) were injected intratumorally with AdLacZ or AdFOXM1shRNA (5 × 108 plaque-forming unit (p.f.u.) per injection, six mice in each group) and the injections were repeated three times at 3-day intervals. (a) Representative photographs of tumors isolated at the week 3 after the first viral injection. Bar: 1 cm. (b) Tumor volumes were measured at the indicated time points and the mean tumor volume in each group of mice (n=6) was calculated. Bars, s.d.; **P<0.01. (c) Total protein lysates were isolated from the tumors of the non-treated group (ZR-75-30), the AdLacZ group (AdLacZ) and the AdFOXM1shRNA group (AdFOXM1shRNA) at the end of the procedure. The levels of FOXM1 protein were determined by western blotting. (d) The levels of forkhead box M1 (FOXM1) protein in the tumors were measured by immunostaining with the FOXM1 antibody. Arrows: typical FOXM1-positive staining in nuclei of tumor tissue sections. (e) Total RNA samples were isolated from the tumors of the three groups and the mRNA levels of FOXM1, cyclin B1, CENPB, PLK1 and β-actin were measured by reverse transcription (RT)-PCR. (f) Hematoxylin and eosin (H&E)-stained histological images of tumors.

Discussion

FOXM1 expression is abnormally activated in many human malignancies. As a result of its involvement in regulating tumor cell proliferation, differentiation, DNA damage responses and migration, the attenuation of FOXM1 expression and activity has been found to increase therapeutic sensitivity of breast cancer therapeutic reagents. In human breast cancer samples, the overexpression of FOXM1 is related to poor prognosis and confers resistance to Herceptin and paclitaxel and targeting FOXM1 relieves therapeutic resistance in breast cancer.41 FOXM1 also confers acquired cisplatin resistance42 and epirubicin resistance43 in breast cancer cells. On the other hand, silencing FOXM1 expression leads breast cancer cells to become more sensitive to doxorubicin because of protective roles of FOXM1 in the cells facing doxorubicin-caused DNA damage.44 Furthermore, thiostrepton, a natural thiazole antibiotic with a potential druggability for breast cancer treatment, selectively induces cell cycle arrest and cell death in breast cancer cells through interacting directly with FOXM1 protein to inhibit FOXM1 functions.45, 46 In this study, we investigated the expression levels of FOXM1 in clinical samples and cell lines of breast cancer. We found that downregulation of FOXM1 elicited a dramatic effect on the inhibition of breast cancer cell proliferation. Our results provide additional evidence in support of the idea that FOXM1 is an attractive target for the development of novel anti-breast cancer therapies.

Cancer gene therapy, whose number of clinical trials takes up to 64.7% among all types of gene therapy clinical trials (the 2012 data of Journal of Gene Medicine Clinical Trial website (www.wiley.com//legacy/wileychi/genmed/clinical/)), is rapidly emerging as a possible therapeutic intervention for treatment of cancers. Among the strategies developed for cancer gene therapy such as blockage of cancer-related gene function, RNA interference (RNAi) technology attracts numerous attentions because of its robustness of gene silencing.47 RNAi describes the sequence-specific silencing of gene expression triggered by short double-stranded RNAs such as endogenous microRNAs or synthesized small interfering RNAs (siRNAs).48 Besides being utilized as a powerful technique in studying the biological function of genes, RNAi possesses a huge potential for its application in treatment of human diseases such as cancers.49, 50 The possibility of suppressing FOXM1 by FOXM1-specific siRNA in breast cancer cells has been tested in vivo. The expression levels of FOXM1 and its transcriptional targets such as cdc25B or aurora B kinase were reduced in subcutaneous MDA-MB-231 breast cancer tumors injected with a polyethylimine-based delivery agent encapsulating FOXM1 siRNA.51 Furthermore, FOXM1 downregulation in MDA-MB-231 and SUM149 cells by siRNA approach leads to inhibition of proliferation, migration and invasion of breast cancer cells through the modulation of extracellular matrix degrading factors.52

The successful application of RNAi in the disease treatment depends on effective and targeted delivery of specific siRNAs to cells and tissues. One approach is to use chemical or physical carriers for transfer of siRNAs.50, 51, 53 The other more popular approach is carried out by genetically modified viral vector delivery through the promoter-driven expression of shRNAs, which mimic endogenously processed microRNAs and are able to engage RNAi.54 Among the viral vectors of choice for RNAi expression, adenovirus is an efficient natural gene delivery system and is the most popular viral gene therapy vector in practice (www.wiley.com//legacy/wileychi/genmed/clinical/). In this study, we chose the adenovirus vector as our shRNA delivery vehicle. Adenoviruses are nonenveloped, double-stranded DNA viruses and >50 human Ad serotypes are identified to date. Ad serotype 5 is the most commonly used Ad serotype for gene delivery applications.55 Popularity of Ad vectors for therapeutic gene delivery is based on several advantages such as efficient transgene delivery and expression, transduction of both dividing and non-dividing cells, ease of propagation to high titers, episomal persistence of the Ad genome within the nucleus with minimal risk of genomic insertional mutagenesis, relative stability in blood following systemic administration, high capacity to accommodate foreign DNA and significant progress in our understanding of the biology of Ad.56, 57 The combination of the advantages of Ad as a gene delivery vector and RNAi technology potentiates the power of the approach for gene therapies, especially for cancer gene therapy. Ad-mediated RNAi technology has been evaluated for treatment of various types of human cancers, such as hepatocellular carcinoma, lung cancer, colon cancer, gastrointestinal stromal tumor58, 59, 60, 61, 62, 63 and offers a promising option for cancer gene therapy in the future.

In this study, we developed an efficient adenovirus-mediated shRNA expression system to specifically knockdown FOXM1 expression in breast cancer cells. We confirmed that this AdFOXM1shRNA vector was able to infect the cells with high infection efficiency and abolish the FOXM1 expression dramatically. As a consequence of the downregulation of FOXM1, the infection of AdFOXM1shRNA in the cancer cells significantly inhibited the proliferation and anchorage-independent growth of the cells. Furthermore, we conducted an in vivo experiment to confirm an effective suppression of the breast tumor growth through knockdown of FOXM1 gene expression by AdFOXM1shRNA intratumoral injection in an established breast cancer xenograft mice model. These results indicated that FOXM1 has an important role in breast cancer growth and the downregulation of FOXM1 expression possesses the therapeutic value for breast cancer treatment.

FOXM1 participates in the regulation of cell cycle in both the transition from G1 to S phase and the progression to mitosis.13, 14 It is well known that FOXM1 regulates the transcription of cell cycle genes, such as cyclin D1, Cdc25A, Skp2, cyclin B1, Cdc25B, PLK1, aurora B, survivin, CENPA, CENPB and knockdown of FOXM1 prevents the expression of these genes in multiple tumor cells.9, 10, 11, 12, 13, 14, 15 In this study, the downregulation of cyclin B1, CENPB and PLK1 observed in breast cancer cells by the FOXM1 knockdown is consistent with the published findings in other tumor cells. These data generally support the mechanisms to explain how silencing FOXM1 results in proliferation inhibition of breast cancer cells. In addition, FOXM1 has been shown to have roles in the regulation of angiogenesis in various types of solid tumors, such as gastric cancer,26 pancreatic cancer,25 clear cell renal cell carcinoma64 and glioblastoma,65 through stimulating the transcription of vascular endothelial growth factor,65 and the downregulation of FOXM1 is related to the inhibition of angiogenesis in these tumors. Given the evidence that the regulatory roles of FOXM1 are similar in various types of tumor cells, we imagine that the mechanism of FOXM1-mediated angiogenesis is involved in breast cancer development and may contribute to the inhibitory effects of AdFOXM1shRNA on breast tumor growth. This hypothesis will be elucidated by future studies.

On the whole, we found that the suppression of FOXM1 through an adenovirus RNAi system could suppress ex vivo cell proliferation and in vivo tumor growth of breast cancers. We concluded that the FOXM1 gene could be a therapy target for breast cancer and the potential antitumor effect of AdFOXM1shRNA may be tested for clinical development in the future.

References

  1. 1

    Forouzanfar MH, Foreman KJ, Delossantos AM, Lozano R, Lopez AD, Murray CJ et al. Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis. Lancet 2011; 378: 1461–1484.

    Article  PubMed  Google Scholar 

  2. 2

    Simpson JF, Gray R, Dressler LG, Cobau CD, Falkson CI, Gilchrist KW et al. Prognostic value of histologic grade and proliferative activity in axillary node-positive breast cancer: results from the Eastern Cooperative Oncology Group Companion Study, EST 4189. J Clin Oncol 2000; 18: 2059–2069.

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001; 98: 10869–10874.

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA et al. Molecular portraits of human breast tumours. Nature 2000; 406: 747–752.

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Benson JR, Jatoi I, Keisch M, Esteva FJ, Makris A, Jordan VC . Early breast cancer. Lancet 2009; 373: 1463–1479.

    Article  PubMed  Google Scholar 

  6. 6

    Kaestner KH, Knochel W, Martinez DE . Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 2000; 14: 142–146.

    CAS  PubMed  Google Scholar 

  7. 7

    Costa RH, Kalinichenko VV, Holterman AX, Wang X . Transcription factors in liver development, differentiation, and regeneration. Hepatology 2003; 38: 1331–1347.

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Ye H, Kelly TF, Samadani U, Lim L, Rubio S, Overdier DG et al. Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol 1997; 17: 1626–1641.

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Wang X, Kiyokawa H, Dennewitz MB, Costa RH . The forkhead box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver regeneration. Proc Natl Acad Sci USA 2002; 99: 16881–16886.

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Wang X, Quail E, Hung N-J, Tan Y, Ye H, Costa RH . Increased levels of forkhead box M1B transcription factor in transgenic mouse hepatocytes prevents age-related proliferation defects in regenerating liver. Proc Natl Acad Sci USA 2001; 98: 11468–11473.

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Krupczak-Hollis K, Wang X, Kalinichenko VV, Gusarova GA, Wang I-C, Dennewitz MB et al. The mouse forkhead box m1 transcription factor is essential for hepatoblast mitosis and development of intrahepatic bile ducts and vessels during liver morphogenesis. Dev Biol 2004; 276: 74–88.

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Ye H, Holterman A, Yoo KW, Franks RR, Costa RH . Premature expression of the winged helix transcription factor HFH-11B in regenerating mouse liver accelerates hepatocyte entry into S-phase. Mol Cell Biol 1999; 19: 8570–8580.

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Laoukili J, Kooistra MR, Bras A, Kauw J, Kerkhoven RM, Morrison A et al. FoxM1 is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol 2005; 7: 126–136.

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Wang IC, Chen YJ, Hughes D, Petrovic V, Major ML, Park HJ et al. Forkhead box M1 regulates the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase. Mol Cell Biol 2005; 25: 10875–10894.

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Kalinichenko VV, Major ML, Wang X, Petrovic V, Kuechle J, Yoder HM et al. Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor. Genes Dev 2004; 18: 830–850.

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Wonsey DR, Follettie MT . Loss of the forkhead transcription factor FoxM1 causes centrosome amplification and mitotic catastrophe. Cancer Res 2005; 65: 5181–5189.

    CAS  Article  Google Scholar 

  17. 17

    Tan Y, Raychaudhuri P, Costa RH . Chk2 mediates stabilization of the FoxM1 transcription factor to stimulate expression of DNA repair genes. Mol Cell Biol 2007; 27: 1007–1016.

    CAS  Article  Google Scholar 

  18. 18

    Li SKM, Smith DK, Leung WY, Cheung AMS, Lam EWF, Dimri GP et al. FoxM1c counteracts oxidative stress-induced senescence and stimulates Bmi-1 expression. J Biol Chem 2008; 283: 16545–16553.

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Tan Y, Chen Y, Yu L, Zhu H, Meng X, Huang X et al. Two-fold elevation of expression of FoxM1 transcription factor in mouse embryonic fibroblasts enhances cell cycle checkpoint activity by stimulating p21 and Chk1 transcription. Cell Prolif 2010; 43: 494–504.

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Xie Z, Tan G, Ding M, Dong D, Chen T, Meng X et al. Foxm1 transcription factor is required for maintenance of pluripotency of P19 embryonal carcinoma cells. Nucleic Acids Res 2010; 38: 8027–8038.

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Kim I-M, Ackerson T, Ramakrishna S, Tretiakova M, Wang I-C, Kalin TV et al. The forkhead box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer Res 2006; 66: 2153–2161.

    CAS  Article  Google Scholar 

  22. 22

    Liu M, Dai B, Kang S-H, Ban K, Huang F-J, Lang FF et al. FoxM1B Is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells. Cancer Res 2006; 66: 3593–3602.

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Kalin TV, Wang I-C, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res 2006; 66: 1712–1720.

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Teh MT, Wong ST, Neill GW, Ghali LR, Philpott MP, Quinn AG . FOXM1 is a downstream target of Gli1 in basal cell carcinomas. Cancer Res 2002; 62: 4773–4780.

    CAS  PubMed  Google Scholar 

  25. 25

    Wang Z, Banerjee S, Kong D, Li Y, Sarkar FH . Down-regulation of Forkhead Box M1 transcription factor leads to the inhibition of invasion and angiogenesis of pancreatic cancer cells. Cancer Res 2007; 67: 8293–8300.

    CAS  Article  Google Scholar 

  26. 26

    Li Q, Zhang N, Jia Z, Le X, Dai B, Wei D et al. Critical role and regulation of transcription factor FoxM1 in human gastric cancer angiogenesis and progression. Cancer Res 2009; 69: 3501–3509.

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Gemenetzidis E, Bose A, Riaz AM, Chaplin T, Young BD, Ali M et al. FOXM1 upregulation is an early event in human squamous cell carcinoma and it is enhanced by nicotine during malignant transformation. PLoS One 2009; 4: e4849.

    Article  PubMed  Google Scholar 

  28. 28

    Adami GR, Ye H . Future roles for FoxM1 inhibitors in cancer treatments. Future Oncol 2007; 3: 1–3.

    Article  PubMed  Google Scholar 

  29. 29

    Wang Z, Ahmad A, Li Y, Banerjee S, Kong D, Sarkar FH . Forkhead box M1 transcription factor: a novel target for cancer therapy. Cancer Treat Rev 2010; 36: 151–156.

    CAS  Article  Google Scholar 

  30. 30

    Chen H, Yang C, Yu L, Xie L, Hu J, Zeng L et al. Adenovirus-mediated RNA interference targeting FOXM1 transcription factor suppresses cell proliferation and tumor growth of nasopharyngeal carcinoma. J Gene Med 2012; 14: 231–240.

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Bektas N, Haaf A, Veeck J, Wild PJ, Luscher-Firzlaff J, Hartmann A et al. Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer. BMC Cancer 2008; 8: 42.

    Article  PubMed  Google Scholar 

  32. 32

    Kretschmer C, Sterner-Kock A, Siedentopf F, Schoenegg W, Schlag PM, Kemmner W . Identification of early molecular markers for breast cancer. Mol Cancer 2011; 10: 15.

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Madureira PA, Varshochi R, Constantinidou D, Francis RE, Coombes RC, Yao KM et al. The Forkhead box M1 protein regulates the transcription of the estrogen receptor alpha in breast cancer cells. J Biol Chem 2006; 281: 25167–25176.

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Millour J, Constantinidou D, Stavropoulou AV, Wilson MS, Myatt SS, Kwok JM et al. FOXM1 is a transcriptional target of ERalpha and has a critical role in breast cancer endocrine sensitivity and resistance. Oncogene 2010; 29: 2983–2995.

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Horimoto Y, Hartman J, Millour J, Pollock S, Olmos Y, Ho KK et al. ERbeta1 represses FOXM1 expression through targeting ERalpha to control cell proliferation in breast cancer. Am J Pathol 2011; 179: 1148–1156.

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Francis RE, Myatt SS, Krol J, Hartman J, Peck B, McGovern UB et al. FoxM1 is a downstream target and marker of HER2 overexpression in breast cancer. Int J Oncol 2009; 35: 57–68.

    CAS  PubMed Central  PubMed  Google Scholar 

  37. 37

    Tan Y, Costa RH, Kovesdi I, Reichel RR . Adenovirus-mediated increase of HNF-3 levels stimulates expression of transthyretin and sonic hedgehog, which is associated with F9 cell differentiation toward the visceral endoderm lineage. Gene Expr 2001; 9: 237–248.

    CAS  Article  PubMed  Google Scholar 

  38. 38

    Tan Y, Xie Z, Ding M, Wang Z, Yu Q, Meng L et al. Increased levels of FoxA1 transcription factor in pluripotent P19 embryonal carcinoma cells stimulate neural differentiation. Stem Cells Dev 2010; 19: 1365–1374.

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Naito S, von Eschenbach AC, Giavazzi R, Fidler IJ . Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice. Cancer Res 1986; 46: 4109–4115.

    CAS  PubMed  Google Scholar 

  40. 40

    Yau C, Wang Y, Zhang Y, Foekens JA, Benz CC . Young age, increased tumor proliferation and FOXM1 expression predict early metastatic relapse only for endocrine-dependent breast cancers. Breast Cancer Res Treat 2011; 126: 803–810.

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Carr JR, Park HJ, Wang Z, Kiefer MM, Raychaudhuri P . FoxM1 mediates resistance to herceptin and paclitaxel. Cancer Res 2010; 70: 5054–5063.

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Kwok JM, Peck B, Monteiro LJ, Schwenen HD, Millour J, Coombes RC et al. FOXM1 confers acquired cisplatin resistance in breast cancer cells. Mol Cancer Res 2010; 8: 24–34.

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Millour J, de Olano N, Horimoto Y, Monteiro LJ, Langer JK, Aligue R et al. ATM and p53 regulate FOXM1 expression via E2F in breast cancer epirubicin treatment and resistance. Mol Cancer Ther 2011; 10: 1046–1058.

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Park YY, Jung SY, Jennings NB, Rodriguez-Aguayo C, Peng G, Lee SR et al. FOXM1 mediates Dox resistance in breast cancer by enhancing DNA repair. Carcinogenesis 2012; 33: 1843–1853.

    CAS  Article  PubMed  Google Scholar 

  45. 45

    Kwok JM, Myatt SS, Marson CM, Coombes RC, Constantinidou D, Lam EW . Thiostrepton selectively targets breast cancer cells through inhibition of forkhead box M1 expression. Mol Cancer Ther 2008; 7: 2022–2032.

    CAS  Article  PubMed  Google Scholar 

  46. 46

    Hegde NS, Sanders DA, Rodriguez R, Balasubramanian S . The transcription factor FOXM1 is a cellular target of the natural product thiostrepton. Nat Chem 2011; 3: 725–731.

    CAS  Article  PubMed  Google Scholar 

  47. 47

    Grimm D, Kay MA . RNAi and gene therapy: a mutual attraction. Hematology Am Soc Hematol Educ Program 2007: 473–481.

    Article  Google Scholar 

  48. 48

    Hannon GJ . RNA interference. Nature 2002; 418: 244–251.

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Hannon GJ, Rossi JJ . Unlocking the potential of the human genome with RNA interference. Nature 2004; 431: 371–378.

    CAS  Article  PubMed  Google Scholar 

  50. 50

    Kim DH, Rossi JJ . Strategies for silencing human disease using RNA interference. Nat Rev Genet 2007; 8: 173–184.

    CAS  Article  PubMed  Google Scholar 

  51. 51

    Wang M, Gartel AL . The suppression of FOXM1 and its targets in breast cancer xenograft tumors by siRNA. Oncotarget 2011; 2: 1218–1226.

    Article  PubMed  Google Scholar 

  52. 52

    Ahmad A, Wang Z, Kong D, Ali S, Li Y, Banerjee S et al. FoxM1 down-regulation leads to inhibition of proliferation, migration and invasion of breast cancer cells through the modulation of extra-cellular matrix degrading factors. Breast Cancer Res Treat 2010; 122: 337–346.

    CAS  Article  PubMed  Google Scholar 

  53. 53

    de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J . Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007; 6: 443–453.

    CAS  Article  PubMed  Google Scholar 

  54. 54

    Brummelkamp TR, Bernards R, Agami R . A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–553.

    CAS  Article  PubMed  Google Scholar 

  55. 55

    Sharma A, Li X, Bangari DS, Mittal SK . Adenovirus receptors and their implications in gene delivery. Virus Res 2009; 143: 184–194.

    CAS  Article  PubMed  Google Scholar 

  56. 56

    Douglas JT . Adenoviral vectors for gene therapy. Mol Biotechnol 2007; 36: 71–80.

    CAS  Article  PubMed  Google Scholar 

  57. 57

    Majhen D, Ambriovic-Ristov A . Adenoviral vectors--how to use them in cancer gene therapy? Virus Res 2006; 119: 121–133.

    CAS  Article  Google Scholar 

  58. 58

    Li H, Fu X, Chen Y, Hong Y, Tan Y, Cao H et al. Use of adenovirus-delivered siRNA to target oncoprotein p28GANK in hepatocellular carcinoma. Gastroenterology 2005; 128: 2029–2041.

    CAS  Article  PubMed  Google Scholar 

  59. 59

    Sumimoto H, Yamagata S, Shimizu A, Miyoshi H, Mizuguchi H, Hayakawa T et al. Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference. Gene Ther 2005; 12: 95–100.

    CAS  Article  PubMed  Google Scholar 

  60. 60

    Osada H, Tatematsu Y, Yatabe Y, Horio Y, Takahashi T . ASH1 gene is a specific therapeutic target for lung cancers with neuroendocrine features. Cancer Res 2005; 65: 10680–10685.

    CAS  Article  PubMed  Google Scholar 

  61. 61

    Dai Y, Qiao L, Chan KW, Yang M, Ye J, Zhang R et al. Adenovirus-mediated down-regulation of X-linked inhibitor of apoptosis protein inhibits colon cancer. Mol Cancer Ther 2009; 8: 2762–2770.

    CAS  Article  PubMed  Google Scholar 

  62. 62

    Wang H, Zhao G, Liu X, Sui A, Yang K, Yao R et al. Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo. J Exp Clin Cancer Res 2010; 29: 123.

    Article  PubMed  Google Scholar 

  63. 63

    Wang TB, Huang WS, Lin WH, Shi HP, Dong WG . Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft. World J Gastroenterol 2010; 16: 5122–5129.

    CAS  Article  PubMed  Google Scholar 

  64. 64

    Xue YJ, Xiao RH, Long DZ, Zou XF, Wang XN, Zhang GX et al. Overexpression of FoxM1 is associated with tumor progression in patients with clear cell renal cell carcinoma. J Transl Med 2012; 10: 200.

    CAS  Article  PubMed  Google Scholar 

  65. 65

    Zhang Y, Zhang N, Dai B, Liu M, Sawaya R, Xie K et al. FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells. Cancer Res 2008; 68: 8733–8742.

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Ministry of Science and Technology of China (grant number 2010DFB30300); and Natural Science Foundation of China (grant number 30871244, 81171949 to YT).

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Yang, C., Chen, H., Yu, L. et al. Inhibition of FOXM1 transcription factor suppresses cell proliferation and tumor growth of breast cancer. Cancer Gene Ther 20, 117–124 (2013). https://doi.org/10.1038/cgt.2012.94

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Keywords

  • FOXM1 transcription factor
  • breast cancer
  • tumor gene therapy
  • adenovirus vector
  • RNA interference

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