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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cervical cancer is the second most common cancer in women. Inactivation of tumor suppressor genes underlies the transformation and progression of cervical cancer. Previously, we reported MAFIP can inhibit the growth of human cervical cancer HeLa cells. In this study, MAFIP was found to be downregulated in cervical intraepithelial neoplasia tissues. Induced expression of MAFIP in HeLa cells strongly inhibited tumor formation in nude mice, confirming its tumor suppressor activity in vivo. Overexpression of MAFIP inhibited activation of the NF-κB pathway, a commonly active pathway in cancer cells, by preventing the phosphorylation of IKK and IκBα, degradation of IκBα and the nuclear localization of p65. Induction of c-myc, an oncogene controlled by NF-κB, was severely impaired in the cells overexpressing MAFIP. In contrast, knockdown of MAFIP by siRNA activated the NF-κB pathway and promoted cell proliferation. These data suggest MAFIP functions as a tumor suppressor in cervical cancer in part by inhibiting activation of the NF-κB pathway. (Cancer Sci 2011; 102: 2043–2050)

Cervical cancer is a malignant neoplasm of the cervix uteri or cervical area. Just behind breast cancer, cervical cancer is the second most frequent cancer among women. Globally, each year, approximately 490 000 new cases are diagnosed and nearly 270 000 women die from the disease.(1) Enormous cancer-related morbidity and mortality impose significance and urgency on the fundamental research of cervical cancer.

In the previous few decades, persistent human papillomavirus (HPV) infection and virus-induced transcription have been proven to play a necessary role in cervical carcinogenesis.(2) However, HPV infection alone is not sufficient to drive the transformation and progression.(3) Recurrent genetic alterations, typically inactivation of tumor suppressor genes (TSG), are required for carcinogenesis.(4) For example, downregulation of LMX-1A and ZBRK1 contribute to the invasion and metastasis of tumor.(5,6) A decreased level of CCS-3 helps to prevent apoptosis,(7) and those of DKK3 and NOL7 are involved in the proliferation of tumor cells.(8,9)

NF-κB belongs to a family of transcription factors that control the expression of numerous genes, including those encoding cytokines, chemokines, cell adhesion molecules, growth factors, oncogenes and pro-/anti-apoptotic proteins.(10–12) NF-κB is typically a heterodimeric complex consisting of Rel family proteins p65 and p50. The heterodimer is sequestered in the cytoplasm through its association with IκBα, which is known as the inhibitor of NF-κB. Stimulation of cells by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), results in the phosphorylation of the IκB kinase (IKK) complex, whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase. ΙΚΚγ(ΝΕΜΟ) serves as the regulatory subunit.(13) Ser177 and Ser181 in the activation loop of IKKβ (176 and 180 in IKKα) are the specific sites whose phosphorylation causes conformational changes resulting in kinase activation.(14) Activated IKK then phosphorylates IκBα at Ser32 and Ser36. Phosphorylated IκBα is ubiquitinated and then degraded by 26S proteasome. The liberated NF-κB translocates to the nucleus where it binds to specific promoters and regulates the expression of target genes.(15)

The link between chronic inflammation and cancer has become evident in recent years.(16) The NF-κB pathway has been recognized as a potential link between inflammation and cancer. It is a critical factor conferring the ability of both pre-neoplastic and malignant cells to resist apoptosis-based tumor-surveillance mechanisms, as demonstrated in colitis-associated cancer and liver cancer.(17) As far as cervical cancer is concerned, constitutive activation of NF-κB with nuclear accumulation of p65 and p50 during cervical cancer progression has been described.(18–20)

Previously, we reported the cloning of a novel human gene MAFIP (GenBank Accession No. AF289559, also known as pp5644 or MIP).(21,22) MAFIP, consisting of 124 amino acids, interacts with hMafF and functions as a co-factor for hMafF-mediated transcription.(21) Overexpression of MAFIP inhibited the in vitro growth of HeLa human cervical cancer cells.(22) In the present study, we further investigated the role of MAFIP in cervical cancer. We found that MAFIP was downregulated in cervical intraepithelial neoplasia (CIN) tissues. Consistently, induced expression of MAFIP significantly inhibited tumor formation in a nude mouse xenograft model of HeLa cells. Overexpression of MAFIP repressed TNF-α-induced activation of NF-κB signaling in HeLa cells. In contrast, knockdown of MAFIP by siRNA activated the NF-κB pathway and promoted cell proliferation. Taken together, our data suggest MAFIP functions as a tumor suppressor in cervical cancer, in part by inhibiting activation of the NF-κB pathway.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Clinical specimens.  Eighteen pairs of clinical specimens of CIN and their corresponding para-CIN (PCIN) were obtained, with informed consent from patients. Specimens were collected by the loop electrosurgical excision procedure with the aid of a colposcope from the Department of Gynecology, Changhai Hospital of Second Military Medical University between July and October 2009. The diagnoses were confirmed by histopathological study. After excision, specimens were immediately frozen at −80°C until use. Before RNA extraction, tissue was ground in liquid nitrogen.

Real-time PCR.  RNA extracted from the cells were reverse transcribed into cDNA. Real-time PCR were performed using SYBR Premix Ex Taq II (Takara, Dalian, China) in an iCycler iQ real-time PCR system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instruction. As a control, each cDNA sample was simultaneously subjected to amplification of the human hypoxanthine guanine phosphoribosyl transferase gene (h-HPRT). Primers used in the real-time PCR were: 5′-CTGCCCTAGAGCCGCTTTCCTGGTA-3′ and 5′-CGGTCGATGAAGACACTGTTGGTCA-3′ for MAFIP; 5′-GCTGTCGCAGAGGGGCTACGAGTGG-3′ and 5′-TCCTCCCCCAGTTCACCCCGTCCCT-3′ for p21; 5′-CTCGGTGCAGCCGTATTT-3′ and 5′-CGGGTCGCAGATGAAACTC-3′ for c-myc; 5′-GCTGGTGGTTGACTTTCTCTCCTAC-3′ and 5′-GAGTTCATTCACTACCTGTTCAAAG-3′ for bcl-xl; and 5′-CCTGCTGGATTACATCAAAGCACTG-3′ and 5′-TCCAACACTTCGTGGGGTCCT-3′ for h-HPRT. The threshold cycle (CT) of each sample was determined by the real-time PCR system and then normalized to the values for h-HPRT using the following equation: ΔCT = CT(gene of interest)− CT(h-HPRT). The relative level was calculated as 2−ΔCT. The reaction for each sample was performed in triplicates.

Flow cytometry analysis.  The entire coding region of MAFIP was cloned into eukaryotic expression vector pCMV-myc (Clontech, Mountain View, CA, USA). HeLa cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). For apoptosis analysis, cells were transfected with pCMV-myc or pCMV-myc-MAFIP by lipofectamine (Invitrogen, Carlsbad, CA, USA) and cultivated for 48 h. Then, 4 × 105 cells were double stained with FITC-conjugated annexin V and propidium iodide (PI) using the Annexin V-PE Apoptosis Detection kit (BD). Cells were measured by FACSCalibur flow cytometer and the data were analyzed using CellQuest software (Becton-Dickinson, Mountain View, CA, USA). For cell cycle analysis, cells were harvested 36 h post-transfection and then subjected to the procedure as described previously.(23) Data were analyzed using flowjo 2.0 with a Watson-Pragmatic model. To count the viable cells, cells were cultured in 24-well plates and transfected with the indicated siRNA. Transfected cells were stained with PI and viable cells (PI negative) were counted using flow cytometry.

Generation of a stable cell line.  The entire coding region of MAFIP with N terminal myc-tag was cloned into eukaryotic expression vector pCDNA4/TO (Invitrogen). pCDNA4/TO-myc-MAFIP and pCDNA4/TO plasmid were linearized by Sca I (Takara) and transfected into T−REx HeLa cells (Invitrogen) separately. Stable transfectants were selected in Minimum Essential Medium with Earle’s salt (EMEM; Invitrogen) containing 5 μg/mL Blasticidin (Invitrogen), 100 μg/mL Zeocin (Invitrogen) and 10% FBS. At least 10 foci were picked and expanded to test tetracycline-inducible gene expression by western blot analysis using myc antibody (Sigma-Aldrich, St Louis, MO, USA; M4439).

In vivo xenograft model.  Thirty-two 6-week-old female Balb/c-nu mice (Shanghai Laboratory Animal Center, Chinese Academy Sciences, Shanghai, China) were distributed randomly into two groups (16 animals each) for injection of pCDNA4/TO or pCDNA4/TO-myc-MAFIP stable cell line. Then, 5 × 106 cells were resuspended in 200 μL EMEM and implanted subcutaneously on the dorsal flank of each mouse. One day after inoculation, natural mineral water containing 0.2% (w/v) doxycycline (Sigma) was supplied to the mice. Tumor-bearing animals were killed 5 weeks after injection. Tumor weight and incidence were measured. Tumor issues were subjected to immunohistochemical staining by using the anti-Myc antibody as previously described.(24) Tumor tissues were lysed using tissue lysis buffer (Pierce, Rockford, IL, USA) and the lysates were subjected to western blot analysis using anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-126) and anti-p21 (Santa Cruz; sc-6246) antibodies. β-actin (Abcam, Cambridge, MA, USA; ab6276) was detected as an internal control.

Luciferase assay.  Analysis of NF-κB activation was carried out in a pNF-κB-luc and pRL-TK reporter system (Promega, Madison, WI, USA; E2241). HeLa cells, 1 × 104, were grown in 96-well plates, co-transfected with a total of 100 ng plasmid containing 60 ng pCMV-myc or pCMV-myc-MAFIP, 30 ng pNF-κB-luc plasmid and 10 ng pRL-TK plasmid using lipofectamin 2000 (Invitrogen; 11668-019). At 48 h post-transfection, cells were grown in serum-free DMEM containing TNF-α (50 ng/mL) and then harvested at the indicated time. The activity of firefly luciferase expressed from pNF-κB-luc was measured and normalized to the activity of renilla luciferase expressed from the control plasmid pRL as described previously.(25) The experiments were performed in triplicate and repeated three times.

Detection of IKK, IκBα and p65.  HeLa cells were plated in six-well plates and transfected with pCMV-myc or pCMV-myc-MAFIP. At 48 h post-transfection, cells were grown in serum-free DMEM containing TNF-α (50 ng/mL) and harvested at the indicated times. Whole cell extract, fractions of nuclear and cytoplasm were prepared using a Nuclear Extract kit (Active Motif, Carlsbad, CA, USA; 40010) and then subjected to western blot analysis. To detect phosphorylated IκBα, cells were lysed with the existence of 50 μM MG132 (Sigma). IκBα was immunoprecipitated using mouse anti-IκBα antibody (Biovision, Palo Alto, CA, USA; 3315-100) and detected using rabbit anti-IκBα (Cell Signaling Inc., Beverly, MA, USA; 4812). Additional antibodies used in the present study include: anti-p65 (Biovision; 3012-100), anti-Histone H3 (Millipore, Bedford, MA, USA; 06-755), anti-Phospho-IKKα (Ser176)/IKKβ(Ser177) (Cell Signaling; 2078), anti-Phospho-IκBα (Ser32/36) (Cell Signaling; 9246), anti c-myc (Chemicon, Temecula, CA, USA; CBL434) and anti Bcl-xL (Cell Signaling; 2762).

MAFIP small interfering RNA.  HeLa cells were transfected with MAFIP siRNA (CCU CUC CGU AAA CCU AUG UTT, referred to as Si-1 or GAC CAG AGC UAA GAG ACA ATT, referred to as Si-2) or negative control siRNA (UUC UCC GAA CGU GUC ACG U, Shanghai GenePharma Co., Shanghai, China) at a final concentration of 100 nM using Fugene HD (Hoffmann-La Roche Inc., Nutley, NJ, USA).

3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.  HeLa cells, 5 × 104, were cultured in 96-well plates in 150 μL medium for 1 day and transfected with the MAFIP siRNA or control siRNA. MTT (Sigma) was added to a final concentration of 50 μg/well after culturing for 24, 48 and 72 h. After 4 h of incubation at 37°C, the reaction was stopped by adding 200 μL DMSO. The reaction product was quantified by measuring the absorbance at 570 nm using an ELISA reader (SpectraMax 190, Molecular Devices Inc., Menlo Park, CA, USA). All samples were assayed in triplicate and repeated three times.

Statistical analysis.  Statistical analysis was performed using SPSS 13.0 for Windows (SPSS, Chicago, IL, USA). The paired student’s test was applied and a P-value < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Decreased mRNA level of MAFIP in cervical intraepithelial neoplasia.  Cervical intraepithelial neoplasia is the potentially premalignant transformation and abnormal growth of squamous cells on the surface of the cervix. It is well recognized as a potential precursor of cervical cancer.(26) To evaluate the correlation between MAFIP and the initiation of cervical cancer, we analyzed the mRNA level of MAFIP in CIN tissue and the corresponding para-CIN tissue using real-time PCR. As shown in Figure 1(A), all 18 CIN tissues expressed a significantly reduced amount of MAFIP compared with the corresponding PCIN tissues. The average mRNA level of MAFIP in CIN tissues was approximately 29.4% of that in PCIN tissues (Fig. 1B, P < 0.05). This result suggests that reduced expression of MAFIP is involved in the early stage of cervical carcinogenesis.

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Figure 1. MAFIP mRNA levels in cervical intraepithelial neoplasia (CIN) and para-CIN (PCIN) tissues. (A) Eighteen clinical specimens, including CIN and their corresponding PCIN, were collected. mRNA levels of MAFIP were quantified by real-time PCR and the level in the corresponding PCIN was designated as 1. (B) Comparison of average MAFIP levels in CIN or PCIN of 18 samples (= 0.036).

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Elevated p21 level in HeLa cells overexpressing MAFIP.  Previous work has shown that overexpression of MAFIP inhibited the growth of HeLa human cervical cancer cells.(22) We investigated whether apoptosis or cell cycle arrest contributed to the inhibitory effect of MAFIP. As shown in Figure 2(A), overexpression of MAFIP had no significant effect on cell apoptosis. Meanwhile, it caused a mild but reproducible arrest at the G1 phase (Fig. 2B). G1 delay is commonly linked with an elevated level of p21, an inhibitor of G1 cyclin-dependent kinase.(27) mRNA analysis and western blot revealed a significant increase of p21 in the cells overexpressing MAFIP (Fig. 2C,D), suggesting MAFIP induces G1 arrest by increasing expression of p21. It is well known that p21 can be upregulated by the tumor suppressor p53 to cause G1 arrest.(28) However, the level of p53 remained unchanged (Fig. 2C,D). Transcriptional activity of p53 could be induced by increased DNA binding activity or post-translation modification without a change in protein level.(29,30) Thus, it is possible that MAFIP induces p53-dependent upregulation of p21 through these pathways.

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Figure 2.  Effects of overexpressing MAFIP on apoptosis and cell cycle. (A) HeLa cells were transfected with pCMV-myc-MAFIP or pCMV-myc. Percentages of apoptotic cells from one typical experiment are shown. (B) The percentage of each phase in the transfected HeLa cells was calculated (n = 3). (C) mRNA levels of p21 and p53 in the transfected HeLa cells were quantified by real-time PCR (n = 3). (D) Protein levels of p21 and p53 were analyzed by western blot and β-actin was detected as a loading control. PI, propidium iodide.

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Inhibition of tumor growth by overexpressing MAFIP in a xenograft model.  In the next step, we investigated the effect of overexpressing MAFIP on tumorigenesis in a HeLa-based nude mouse xenogragft model. A stable cell line in which overexpression of MAFIP was induced by tetracycline was first established in T−REx HeLa cells, and the stable cell line transfected with void vector pCDNA4/TO was used as a negative control (Fig. 3A). Both stable cell lines were injected into nude mice separately. Five weeks later, tumors formed in 15 of the 16 mice in the void vector group. The tumorigenic incidence was 94%, and average tumor weight was 0.48 (mean) ± 0.21 (SD) g. In contrast, only one mouse formed a tumor in the group stably expressing MAFIP. The tumorigenic incidence was 6% and the weight of the only tumor was 0.016 g (Fig. 3B,C). Immunohistochemical analysis confirmed the expression of MAFIP in the tumor (Fig. 3D). Similar to the results in transient transfected HeLa cells (Fig. 2C), MAFIP induced by tetracycline in the stable cell line also increased the mRNA level of p21 (Fig. 3F). Consistently, there was a significant increase in the p21 protein level in the tumor stably expressing MAFIP (Fig. 3E).

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Figure 3.  Effect of MAFIP on tumorigenesis of HeLa cells in a nude mice xenograft model. (A) pCDNA4/TO-myc-MAFIP (MAFIP) and pCDNA4/TO (Control) stable transfected cell lines were treated with tetracycline (5 μg/mL) for 24 h. The level of myc-MAFIP was analyzed using western blot with anti-myc antibody. (B) Average weight of tumors that formed on the mice injected with the stable cell line. (C) Photographs of the tumor-bearing mice. (D) Tumor samples were subjected to immunohistochemical staining using anti-myc antibody. (E) Protein levels of p21 and p53 in tumors were analyzed using western blot and β-actin was detected as a loading control. (F) mRNA levels of p21 and p53 in stable cell lines were quantified using real-time PCR (n = 3).

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Inhibition of the NF-κB signaling pathway by overexpressing MAFIP.  As mentioned previously, constitutive activation of NF-κB with nuclear accumulation of p65 and p50 during cervical cancer progression has been reported.(18–20) Also, repression of NF-κB activity in p53+ cells is able to increase the level of p21.(31) It raises the possibility that MAFIP might inhibit the growth and progression of cervical cancer cells by downregulating the NF-κB pathway.

First we tested the effect of overexpressing MAFIP on the activity of NF-κB in the luciferase reporter system. As shown in Figure 4(A), after TNF-α treatment for a period of 4–10 h, expression of luciferase increased significantly in HeLa cells transfected with pCMV-myc plasmid. In contrast, in cells transfected with pCMV-myc-MAFIP, expression of luciferase was substantially reduced after TNF-α treatment. The results suggest that expression of MAFIP can efficiently inhibit activation of NF-κB by TNF-α.

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Figure 4.  Influence of MAFIP on NF-κB signaling and cell proliferation. (A) HeLa cells co-transfected with pNF-κB-luc, pRL-TK and the indicated plasmid were treated with TNF-α for the indicated times. Relative levels of luciferase expressed from pNF-κB-luc were measured (n = 3). (B) HeLa cells were transfected with MAFIP siRNA (Si-1 or Si-2) or control siRNA (negative). Cells without transfection served as a mock control. Endogenous mRNA level of MAFIP in the transfected cells was analyzed by RT-PCR. (C) HeLa cells transfected with indicated siRNA were treated with TNF-α for 8 h or not treated. Relative luciferase activity was measured (n = 3). The difference between activity of the knockdown cells and that of the corresponding control cells was statistically significant (< 0.05) (D) HeLa cells transfected with indicated siRNA and cell viabilities were analyzed using MTT assay on the indicated days post-transfection (n = 3). (E) HeLa cells were transfected with indicated siRNA and viable cells were counted by flow cytometry on the indicated days post-transfection (n = 4).

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Activation of the NF-κB pathway and improved proliferation in MAFIP-knockdown cells.  To further address the role of MAFIP in NF-κB activation and proliferation of tumor cells, two MAFIP-specific siRNA (Si-1, Si-2) and one control siRNA oligonucleotide were transiently transfected into HeLa cells separately. Knockdown was confirmed by RT-PCR analysis of the endogenous MAFIP mRNA level (Fig. 4B). As shown in Figure 4(C), expression of NF-κB-driven luciferase increased in the MAFIP-knockdown cells compared with those in the negative control or mock cells, regardless of TNF-α treatment. Proliferation of MAFIP-knockdown HeLa cells was quantified by MTT assay or flow cytometry. After 24 h of transfection, a robust increase in cell viability was observed in HeLa cells transfected with either Si-1 or Si-2 (Fig. 4D,E). Therefore, data suggest MAFIP could suppress the NF-κB pathway, as well as the proliferation of cervical cancer cells.

Inhibition of phosphorylation of IKK and IκBα, degradation of IκBα and nuclear translocation of p65 in cells overexpressing MAFIP.  Activation of the canonical NF-κB pathway results from IKK activation, phosphorylation and degradation of IκBα in cytoplasm and translocation of p65 from cytoplasm to nucleus. To check whether MAFIP inhibits the NF-κB pathway through this process, we tracked the levels of phosphorylated IKK, IκBα and p65 in the HeLa cells transfected with MAFIP using western blot. After TNF-α treatment, at each corresponding time point, overexpression of MAFIP significantly inhibited accumulation of p65 in the nucleus and degradation of IκBα in the cytoplasm (Fig. 5A,B). Inhibition of IκBα degradation was also confirmed by immunoprecipitation (Fig. 5C). Moreover, overexpressing MAFIP also suppressed the phosphorylation of IKK and IκBα (Fig. 5D). Therefore, MAFIP-mediated inhibition of the NF-κB pathway might occur in the cytoplasm.

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Figure 5.  Inhibition of the NF-κB pathway by overexpressing MAFIP. (A) HeLa cells transfected with pCMV-myc or pCMV-myc-MAFIP were exposed to tumor necrosis factor-α (TNF-α) for the indicated time. The nuclear and cytoplasm fractions were extracted, and the levels of p65 and IκBα were examined using western blot. Histone H3 was used as a loading control for the nuclear extract and β-actin for the cytoplasm. (B) The bands in (A) were scanned and analyzed using Bandscan. The relative amount of p65 was represented by the ratio of p65 to H3, and that of I-κBα by the ratio of IκBα to β-actin (n = 3). (C) HeLa cells were transfected and treated with TNF-α as in (A). IκBα was immunoprecipitated by mouse anti-I-κBα antibody from the whole cell lysates and detected using rabbit anti-IκBα. β-actin was detected as a loading control. (D) HeLa cells were transfected and treated with TNF-α as in (A). Levels of phosphorylated IKK (p-IKK) and IκBα (p-IκBα) were detected using western blot. (E) HeLa cells were transfected and treated with TNF-α for 24 h. The mRNA levels of c-myc and Bcl-xL were quantified using real-time PCR (n = 3). The mRNA level of the target gene in the void vector group before TNF-α treatment was designated as 1. (F) Protein levels of c-myc and Bcl-xL in transfected cells were analyzed using western blot.

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Inhibition of NF-κB targeted gene by overexpressing MAFIP.  Next, we analyzed the expression levels of NF-κB-targeted genes, including carcinogenesis-related c-myc and anti-apoptosis-related Bcl-xL,(32,33) by real-time PCR and western blot. As shown in Figure 5(E,F), overexpression of MAFIP almost totally abolished the induction of c-myc after TNF-α treatment. However, no apparent repressive effect was observed for the induction of Bcl-xL. Our data suggest MAFIP is able to inhibit the induction of the oncogene targeted by NF-κB, such as that of c-myc.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cervical cancer continues to be a worldwide health problem for women. It is well known that most cervical cancer is initiated from CIN upon HPV infection. However, the driving force behind the stepwise progression from CIN to invasive cervical cancer remains largely unknown. Inactivation of TSG is a well-documented occurrence in the process of carcinogenesis.(34) Not surprisingly, many TSG in cervical cancer have been identified.(4) In the present study, we add a new candidate, MAFIP, to this growing list. The expression level of MAFIP is significantly downregulated in CIN. Knockdown of MAFIP improved the proliferation of HeLa cells, while overexpression of MAFIP inhibits tumor formation in a nude mouse xenograft model of HeLa cells. The data suggest MAFIP functions as a TSG not only in the early stage of cervical carcinogenesis but also in the progressive cancer cells. MAFIP is actively expressed in a broad range of normal tissues, including brain, kidney, ovary, liver and thymus.(22) It will be intriguing to check whether MAFIP functions as TSG in the progression of cancers originating from these tissues.

The NF-κB pathway is known to be closely connected to the process of tumorigenesis based on a multiplicity of evidence.(35) It is implicated in the control of cellular growth and apoptosis in neoplasia. The target genes of NF-κB include anti-apoptotic genes,(32,36,37) cell cycle regulators(38) and adhesion molecules.(39,40) Our data showed that overexpression of MAFIP significantly inhibits activation of the NF-κB pathway by TNF-α in the luciferase reporter system. Consistently, induction of the natural target of NF-κB, c-myc, was severely impaired. c-myc encodes a basic helix-loop-helix-zipper (bHLH-ZIP) transcription factor that is frequently deregulated in tumors with diverse origins.(41) An incidence of 32–34% of c-myc activation in cervical cancers has been reported.(3) It is likely that downregulation of c-myc and other oncogenes controlled by NF-κB contribute to MAFIP-mediated tumor suppression. In contrast, the induction of Bcl-xL, an anti-apoptotic gene, was not affected and was correlated with the observation that overexpression of MAFIP did not cause apoptosis in HeLa cells. This suggests that the level of Bcl-xL in cells overexpressing MAFIP can be induced by other transcription factors besides NF-κB, like STAT and Ets.(42) NF-κB and p53 are known to compete for the binding of some common transcriptional co-activators such as p300 and CBP(43) and inhibit the transcriptional activity of each other.(44) Consistently, p53 and NF-κB competitively bind at the p21 promoter, and inhibition of the NF-κB pathway stimulates p53-dependent induction of p21.(31) Therefore, MAFIP might upregulate the p21 level by suppressing the NF-κB pathway. Given the substantially increased level of p21, only mild G1 arrest was observed in the cells overexpressing MAFIP, which might be explained by the crosstalk between p21 and other cell cycle genes controlled by NF-κB.(45,46) In contrast, induced expression of MAFIP in a stable cell line strongly inhibited tumor formation in nude mice. Comparing the in vitro transient transfection system, an in vivo xenograft model might provide an optimal physiological environment to enhance the inhibitory effect of MAFIP.

Inhibitions of the canonical NF-κB p65/50 pathway in vivo can take place at different stages, including disruption of interaction between receptor-interacting protein and the NEMO (IKKγ),(47) inhibiting degradation of IκBα in cytoplasm,(48) repressing nuclear localization of p65 and inhibiting DNA binding activity of NF-κB.(49,50) The present study showed that overexpression of MAFIP inhibits phosphorylation of the IKK complex. Because activation of the IKK complex by phosphorylation is required for occurrence of the following events in the NF-κB pathway,(14) it was expected that inhibited phosphorylation and degradation of IκBα and repressed nuclear translocation of p65 in the cells overexpressing MAFIP would be observed. Therefore, MAFIP might interfere with activation of NF-κB through IKK or other upstream components in the pathway. MAFIP is primarily localized in the cytoplasm,(22) thus the inhibition should be mediated by the interaction with other cytoplasmic proteins. Previously, transcription factor MafF has been identified as a binding partner of MAFIP.(21) Because MafF is predominantly localized in the nuclei, MAFIP-mediated repression of NF-κB is not likely to be exerted by hMAF. Another binding partner of MAFIP, FASP1, (phosphatidylinositol-four-phosphate adaptor protein1 associated protein-1) might be involved in the metabolism of phosphatidylinositol-4,5-bisphosphate (PIP2).(22) PIP2 is the precursor for two-second messengers, IP3 and DAG.(51) DAG activates protein kinase C (PKC) and PKC could activate NF-κB through phosphorylation of IκBα.(52,53) Whether MAFIP represses NF-κB through FASP1 needs further investigation in the future. Recently, Ye et al.(54) reported that MAFIP binds to IKKγ (NEMO). This suggests an intriguing model: that the interaction between MAFIP and NEMO might affect the activity of IKK kinase and then inhibit the following activation of the NF-κB pathway. Further assays, such as an in vitro IKK kinase assay, and behavior of MAFIP mutant that is defective in binding NEMO, are required to support this model.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This work was supported by grants from National Key Basic Research and Development Plan of China (973 program, 2009CB825601) and from National Nature Science Foundation of China (NSFC 30771145 and 30671175) to H. L.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References