Silencing of FOXM1 transcription factor expression by adenovirus-mediated RNA interference inhibits human hepatocellular carcinoma growth

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The Forkhead Box M1 (FOXM1) transcription factor has been considered as a potential target for the prevention and/or therapeutic intervention in human carcinomas because of its roles in tumorigenesis and tumor progression through regulating the expression of genes relevant to cell proliferation and transformation. In this study, FOXM1 was found to express strongly in both clinical tissue specimens and human hepatocellular carcinoma (HCC) cell lines such as Huh-6, Huh-7 and HepG2. The knockdown of FOXM1 expression through an adenovirus vector (named AdFOXM1shRNA), which expresses a short hairpin RNA to downregulate FOXM1 expression specifically, diminished the proliferation of Huh-7 and HepG2 cells and anchorage-independent growth of Huh-7 cells. Furthermore, we assessed the efficacy of AdFOXM1shRNA for tumor gene therapy with the Huh-7 cell xenograft mouse model and found that 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 HCC and AdFOXM1shRNA may be an additional gene therapeutic intervention for HCC treatment.


Hepatocelluar carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death.1 HCC is diagnosed in more than half a million people worldwide every year. Most of the burden of HCC (85%) is borne in developing countries, with the highest incidence rates reported in regions infected with hepatitis B virus (HBV).2 HCC is commonly treated by surgical resection, transplantation, percutaneous ablation and chemoembolisation, depending on the stage of the disease.3 Until lately, no effective treatment was available for patients diagnosed at advanced stage or who progressed into an advanced stage after other treatments failed.1 Increasing knowledge of molecular events that govern tumor progression and dissemination has allowed development of targeted treatments to overcome this problem.4, 5

Transcription factor FOXM1 belongs to the fork head/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.14, 15, 16 Furthermore, we and others have demonstrated that FOXM1 is involved in counteracting 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 glioblastoma,22 prostate cancer,23 basal cell carcinoma,24 breast cancer,25, 26 pancreatic cancer,27 gastric cancer28 and squamous cell carcinoma.29 The inactivation of FOXM1 leads to the 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.30, 31, 32, 33 FOXM1 can be induced by HBV and contributes to the progression of HBV-associated HCC.34 Recently, FOXM1 has been proposed to be a prognostic molecular marker for HCC.35 Furthermore, knockdown of FOXM1 by siRNA inhibits proliferation and invasion of human HCC cells in vitro.36

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

Materials and methods

Cell culture

Human hepatocellular carcinoma cell lines Huh-6, Huh-7 and HepG2 (Chinese Academy of Sciences Cell Bank, Shanghai, China) and Ad-purification 293 A cell line (Invitrogen, Carlsbad, CA, USA) were used in this study. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% FBS (Invitrogen) and 1% penicillin streptomycin (Invitrogen) in a 5% CO2-humidified atmosphere at 37 °C.

Immunohistochemical analysis

Human hepatocellular carcinoma paraffin sections were obtained from the Department of Pathology, Hunan Provincial Tumor Hospital, China. The slides of samples of patients were stained with a mouse antiFOXM1 antibody (1:100; Abcam ab55006, Cambridge, UK), followed by the incubation of a HRP-conjugated antimouse secondary antibody. Color was detected with DAB and pictures were taken at × 200 magnification using a TE2000 microscope (Nikon, Japan).

Western blot assay

Whole-cell lysates prepared from HCC cell lines and tumor tissues were separated using SDS-PAGE and transferred onto PVDF membrane for western blotting as described earlier.19 The antibodies and dilutions used for western blotting were rabbit antiFOXM1 (1:1000; Abcam ab47808, UK) and mouse antiβ-actin (1:5000; Beyotime AA128, Haimen, China). The signals from the primary antibody were amplified by horseradish peroxidase (HRP)-conjugated antirabbit immunoglobulin (Ig) G (1:10 000; GE LNA934VAE, Fairfield, CT, USA) or antimouse IgG (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, USA) according to the manufacturer’s protocols. The cDNAs were synthesized with M-MLV Reverse Transcriptase (Promega, Fitchburg, WI, USA) 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′-IndexTermGCT TGC CAG AGT CCT TTT TGC-3′ and hFOXM1-AS, 5′-IndexTermCCA CCT GAG TTC TCG TCA ATG C-3′ (Ta:60 °C, N:30); hCyclinB1-S, 5′-IndexTermGGT CTG GGT CGG CCT CTA CCT-3′ and hCyclinB1-AS, 5′-IndexTermAGC CAG GTG CTG CAT AAC TGG AA-3′ (Ta:60 °C, N:30); hPLK1-S, 5'-IndexTermCCT GCA CCT CAG CAA CGG CA-3' and hPLK1-AS, 5'-IndexTermCCA TAG TGC GGG CGT AGC GG-3' (Ta: 59_C, N: 30); hCENPB-S, 5′-IndexTermATT CAG ACA GTG AGG AAG AGG ACG-3′ and hCENPB-AS, 5′-IndexTermCAT CAA TGG GGA AGG AGG TCA G-3′ (Ta:60 °C, N:30); hSKP2-S, 5′-IndexTermGCT GTT TGT AAG AGG TGG TAT CGC-3′ and hSKP2-AS, 5′-IndexTermCAC GAA AAG GGC TGA AAT GTT C-3′ (Ta:59 °C, N:30); hCyclinD1-S, 5′-IndexTermGAC CCC GCA CGA TTT CAT TG-3′ and hCyclinD1-AS, 5′-IndexTermCTC TGG AGA GGA AGC GTG TG-3′ (Ta:60 °C, N:30); hGAPDH-S, 5′-IndexTermGGA GCG AGA TCC CTC CAA AAT-3′ and hGAPDH-AS, 5′- IndexTermGGC TGT TGT CAT ACT TCT CAT GG-3′ (Ta:60 °C, N:20). qPCR was performed with SYBR Green (Toyobo, Osaka, Japan) in the realplex2 qPCR system (Eppendorf, Hamburg, Germany).

Adenovirus purification and infection

The AdFOXM1shRNA and control virus (AdGFP) were preserved in our lab, and the large-scale adenovirus purification was performed as described previously.32, 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 PBS. Cells were fixed in 70% ethanol and collected by centrifugation 3,000 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 a Quanta SC flow cytometer (Beckman, Brea, CA, USA).

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

All animal experiments were conducted in accordance with institutional animal care and use guidelines, following approval by the Laboratory Animal Center of Hunan, China (Protocol No. SYXK [Xiang] 2008-0001). BalB/c nude mice (male, 4 week old) were purchased from Slac Experimental Animal Company, China. To generate xenograft tumor models, 5 × 106 Huh-7 cells were injected subcutaneously to right back of each mouse. The mice were separated into three groups randomly for injecting cells with different treatments. While the tumor volume reached 30–50 mm3, the nontreated group was injected with PBS (100 μl), the AdGFP group was injected AdGFP (5 × 108 p.f.u. in 100 μl), and the AdFOXM1shRNA group was injected AdFOXM1shRNA (5 × 108 p.f.u. in 100 μl) into the tumors. The injections 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.


The expression of FOXM1 in clinical tissues and HCC cell lines

FOXM1 was found to be overexpressed in clinical samples of HCC.12, 34, 35 To confirm the published findings, we evaluated FOXM1 expression in the primary specimens obtained from 13 patients diagnosed with hepatocellular carcinoma. By using quantitative real-time PCR (qPCR) analysis, we found significantly elevated levels of FOXM1 mRNA in HCC samples (n=13) compared with normal liver tissues (n=5) (P-value=0.042) (Figure 1a). The elevated expression of FOXM1 mRNA in the five representative clinical HCC samples was validated by semi-quantitative RT–PCR (Figure 1a). We immunostained the tissue sections with an antiFOXM1 antibody and observed positive FOXM1 staining predominantly in the nuclei of cells of HCC samples but not in the normal liver tissues (Figure 1b). Then with qPCR analysis, we found that FOXM1 mRNA was expressed at relatively high levels in malignant Huh-6, Huh-7 and HepG2 HCC cells (Figure 1c). As shown by western blot analysis, all of the HCC cell lines exhibited high levels of FOXM1 protein expression (Figure 1d). These results indicated that FOXM1 was commonly expressed in clinical tissue specimens and cell lines of human HCC.

Figure 1

The expression of FOXM1 in HCC. (a) The FOXM1 mRNA expression levels in clinical human HCC samples were measured by qPCR, and the FOXM1 expression of five representative cases was visualized by semi-quantitative RT–PCR resolved with gel electrophoresis. The levels of FOXM1 expression in HCC samples were significantly higher than that of normal liver tissues (P-value=0.042). (b) Tissue sections of clinical samples were immunostained with an antiFOXM1 antibody, and the pictures were taken at × 200 magnification. Typical FOXM1 immunostaining was observed in HCC tissues. Bar, 100 μm. (c, d) FOXM1 expression in malignant Huh-6, Huh-7 and HepG2 HCC cell lines. FOXM1 mRNA levels were measured by qPCR (c), and FOXM1 protein levels were measured by western blotting (d) in the three HCC cell lines.

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

FOXM1 is considered as an attractive target for the development of novel anticancer therapies.30, 31, 32 To develop innovative strategies for HCC gene therapy, we constructed an adenovirus-mediated shRNA expression system to knockdown FOXM1 expression and named this replication-defected adenovirus vector as AdFOXM1shRNA.32 AdGFP control adenovirus infections determined that almost 100% of HCC cells were infected with the viral dosage at 10 plaque-forming units (p.f.u.) per cell (Supplementary Figure S1). Western blot analysis further confirmed that the expression of FOXM1 protein was effectively suppressed in HCC Huh-7 and HepG2 cells at day 3 following infection with AdFOXM1shRNA (10 p.f.u. per cell) but not with AdGFP (Figure 2a). It is well known that FOXM1 regulates the transcription of cell cycle genes, such as Cyclin B1, PLK1, CENPB, SKP2 and CyclinD1, 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, PLK1, CENPB, SKP2 and CyclinD1 in Huh-7 and HepG2 HCC cells (Figure 2b), consistent with the published findings.9, 10, 11, 12, 13, 14, 15 To determine the growth rate of FOXM1-depleted cells, HCC cells were infected with AdFOXM1shRNA or control AdGFP and counted every day afterward. The growth curves showed that the cell growth of all the two HCC cell lines was inhibited markedly by infection of AdFOXM1shRNA, compared with AdGFP infection (Figure 2c). This idea was further supported by the analysis of cell cycle progression with the Huh-7 and HepG2 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; Supplementary Figure S2). We observed no obvious difference in apoptosis among samples of the AdFOXM1shRNA-infected and the nontreated cells (Supplementary Figures S2 and S3), indicating that the reduction in growth of the FOXM1-depleted cells was due to the inhibition of cell proliferation. These data confirmed that AdFOXM1shRNA was effective in abolishing expression of FOXM1 and inhibiting the proliferation of HCC cells.

Figure 2

Knockdown of FOXM1 by AdFOXM1shRNA decreased the expression of cell cycle genes and inhibited the proliferation of HCC cells. (a) The expression of FOXM1 was inhibited by AdFOXM1shRNA infection in HCC cells. The Huh-7 and HepG2 cells were infected with AdGFP or AdFOXM1shRNA at dosage of 10 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 Huh-7 and HepG2 cells. Total RNA was isolated at 48 h following viral infection. The levels of FOXM1 and five cell cycle-related genes were determined by RT–PCR. (c) The HCC cell growth was inhibited by the infection of AdFOXM1shRNA. Growth curves were measured with Huh-7 and HepG2 cells, the cells infected with AdGFP or AdFOXM1shRNA at dosage of 10 p.f.u. per cell. A total of 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 showed a decreased S-phase cell population and an increased G2/M-phase cell population. Huh-7 and HepG2 cells were infected with AdFOXM1shRNA (10 p.f.u. per cell) and collected at the day 3 post infection. The PI staining quantified the cycle of the cells. Bars, s.d.; *P<0.05, **P<0.01, ***P<0.001, relative to the control at the same time point.

AdFOXM1shRNA inhibited the colony formation of HCC cells

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 HCC cells infected with AdGFP or AdFOXM1shRNA. Huh-7 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 marked decrease in colony formation of Huh-7 cells (Figure 3a). The quantitation of colony number (Figure 3b) demonstrated that depletion of FOXM1 expression by AdFOXM1shRNA inhibited the colony formation of the HCC cells.

Figure 3

The infection of AdFOXM1shRNA decreased the colony formation of HCC cells. Huh-7 cells were infected with AdGFP or AdFOXM1shRNA at dosage of 10 p.f.u. per cell. The soft agar assays were performed, and the colony formation of Huh-7 cells was shown by figures (a) and colony number counting (b). Bars, s.d.; ***P<0.001.

Tumor growth of HCC cells was prevented by AdFOXM1shRNA in nude mice xenografts

To determine the effect of AdFOXM1shRNA on HCC tumor growth kinetics in vivo, we first generated Huh-7 cell xenografts in nude mice by injecting the cells (5 × 106 per mouse) subcutaneously to BalB/c nude mice in groups of six. AdFOXM1shRNA intratumoral injections (5 × 108 p.f.u. per mouse) were started at day 7 after the cell injections and repeated three times with a 3-day interval. We continued to measure 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 compared with the control groups (nontreated or AdGFP-treated) (Figure 4a). In contrast to the large tumors produced by the nontreated and AdGFP-treated groups, the mice treated with AdFOXM1shRNA produced small tumors (Figure 4b). 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 4c), suggesting that the inhibition of tumor growth in the AdFOXM1shRNA-treated group was a consequence of FOXM1 depletion in the tumors. In addition, RNA samples from each group were tested with RT–PCR to measure the mRNA levels of FOXM1 and FOXM1 target genes, such as PLK1, CENPB and CyclinB1. As predicted, the FOXM1 mRNA level was significantly reduced in the AdFOXM1shRNA-treated group, and the downregulation of these FOXM1 target genes was also observed (Figure 4d). These results indicated that AdFOXM1shRNA targeting FOXM1 elicited a strong antitumor effect on HCC in vivo.

Figure 4

Knockdown of FOXM1 by AdFOXM1shRNA inhibited the growth of HCC-grafting tumors in vivo. The HCC grafting animal model was created by subcutaneous inoculation of 5 × 106 Huh-7 cells. The formed tumors (30–50 mm3) were injected intratumorally with PBS (100 μl), AdGFP or AdFOXM1shRNA (5 × 108 p.f.u. per injection, six mice in each group), and the injections were repeated three times at 3-day intervals. (a) The tumor volume in each animal was measured at the indicated time points after tumor cell injection, and the mean tumor volume in each group of mice (n=6) was calculated. Bars, s.d.; **P<0.01, ***P<0.001. (b) Representative tumor photos of HCC-grafting tumors in nude mice were taken for the PBS group (Huh-7), the AdGFP group (AdGFP) and the AdFOXM1shRNA group (AdFOXM1shRNA) at the end of the procedure. Bar, 1 cm. (c) Total protein lysates were isolated from the tumors of the three groups. The levels of FOXM1 protein (mixture of six samples of each group) were determined by western blotting. (d) Total RNA samples were isolated from the tumors of the three groups, and the mRNA levels (mixture of six samples of each group) of FOXM1, CyclinB1, PLK1, CENPB and GAPDH were measured by RT–PCR.


FOXM1 is overexpressed in almost all carcinomas40 and is considered to be a master regulator of tumor metastasis.25, 41 In human HCC, the overexpression of FOXM1 is related to poor prognosis, and FOXM1 is considered as a potential therapeutic target.35, 41 Actually, there are currently few treatment alternatives to surgical resection in HCC. With the increasing knowledge of hepatocyte-specific networks, including signaling pathways and transcription factors potentially dysregulated in HCC, targeted treatments that aim to abrogate these disrupted pathways are allowed to be developed.1, 42 Several drugs are under development, but the only one with proven survival benefit is Sorafenib, which is an inhibitor of multiple kinases in Raf signaling of VEGF, PDGF and c-Kit.43, 44 Interestingly, the nuclear translocation and transcriptional activity of FOXM1 are stimulated by the Raf/MEK/MAPK signaling pathway.45 In this study, we investigated the expression levels of FOXM1 in clinical samples and HCC cell lines. We found that downregulation of FOXM1 elicited a marked effect on the inhibition of HCC cell proliferation. Our results provide additional evidence in support of the idea that FOXM1 is an attractive target for the development of novel antiHCC therapies.

Although RNAi is one of the most popular molecular tools in gene therapy recently, the delivery of siRNA into mammalian cells is the critical factor for a successful application of RNAi in gene function study and cancer gene therapy. To overcome this rate-limiting step, we chose an adenovirus vector as our shRNA delivery vehicle. Adenoviruses are nonenveloped, double-stranded DNA viruses, and more than 50 human Ad serotypes are identified to date. Ad serotype 5 (Ad5) is the most commonly used Ad serotype for gene delivery applications.46 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 nondividing 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.47, 48 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 and gastrointestinal stromal tumor,49, 50, 51, 52, 53, 54 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 HCC cells. We confirmed that this AdFOXM1shRNA vector was able to infect HCC cells with the high infection efficiency and abolish the FOXM1 expression markedly. As a consequence of the downregulation of FOXM1, the infection of AdFOXM1shRNA in HCC cells significantly inhibited the proliferation and anchorage-independent growth of the cancer cells. Furthermore, we conducted an in vivo experiment to confirm an effective suppression of the HCC tumor growth through knockdown of FOXM1 gene expression by AdFOXM1shRNA intratumorical injection in an established HCC xenograft mouse model. These results indicated that FOXM1 has an important role in HCC tumor growth, and the downregulation of FOXM1 expression possesses the therapeutic value for HCC treatment.

On the whole, we have found that the suppression of FOXM1 through an adenovirus shRNA system can suppress ex vivo cell proliferation and in vivo tumor growth of HCC. We conclude that the FOXM1 gene can be a therapy target for HCC, and the potential antitumor effect of AdFOXM1shRNA may be tested for clinical development in the future.


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This work was supported by the Ministry of Science and Technology of China [grant number 2010DFB30300]; and Natural Science Foundation of China [grant number 81171949, 31161160558 to Y.T.].

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Correspondence to L Yu or Y Tan.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on Cancer Gene Therapy website

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  • FOXM1 transcription factor
  • hepatocellular carcinoma
  • tumor gene therapy
  • adenovirus vector
  • RNA interference

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