Sine oculis homeobox homolog 1 promotes DNA replication and cell proliferation in cervical cancer

  • Authors:
    • Dan Liu
    • Xiao-Xue Zhang
    • Bi-Xin Xi
    • Dong-Yi Wan
    • Li Li
    • Jin Zhou
    • Wei Wang
    • Ding Ma
    • Hui Wang
    • Qing-Lei Gao
  • View Affiliations

  • Published online on: June 20, 2014     https://doi.org/10.3892/ijo.2014.2510
  • Pages:1232-1240
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Abstract

Malignant proliferation is the fundamental trait of tumor cells. The initiation of DNA replication represents a key process for cell proliferation, and has a marked impact on tumorigenesis and progression. Here we report that Sine oculis homeobox homolog 1 (SIX1) functions as a master regulator in DNA replication of cervical cancer cells. The expression of SIX1 was induced by the E7 oncoprotein of human papillomaviruses in cervical intraepithelial neoplasia and cervical cancer. The increase of SIX1 expression resulted in the upregulation of multiple genes related to the initiation of DNA replication, including the genes coding for the proteins in minichromosome maintenance complex (MCM2, MCM3, MCM6), DNA polymerase α-primase complex (POLA1, PRIM1, PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA polymerase δ complex (POLD3) and DNA polymerase ε complex (POLE2). In line with this, the increase of SIX1 expression enhanced DNA synthesis, accelerated G1 to S phase progression, and promoted the proliferation of cervical cancer cells and the growth of cervical cancer. Consistently, knockdown of SIX1 could hamper DNA synthesis, slow down G1 to S phase progression, and suppress tumor cell proliferation and tumor growth. Importantly, SIX1 could more efficiently promote anchorage-independent cell growth. These results suggest that the increase of SIX1 expression could promote tumorigenesis, progression and invasive growth of cervical cancer by promoting DNA replication, and that targeting SIX1 may have significant therapeutic value in cervical cancer treatment.

Introduction

Cervical cancer is ranked as the second cause of cancer deaths in women worldwide (13). Human papillomaviruses (HPV) are considered as the causative agents of most cervical cancers (13). The binding of HPV E6 and E7 oncoproteins to tumor-suppressor genes p53 and RB could result in impaired tumor-suppressor-gene function and eventual cell immortalization (2). Although recent studies have identified other equally important molecular events for malignant transformation of cervical epithelial cells, including the activation of certain tumor-promoting genes and pathways (1), E6 and E7 oncoproteins are widely recognized as the driving force in carcinogenesis of cervical epithelium. However, it is not clear whether and how E6 and E7 could promote the progression of cervical cancer after the tumor formation.

The immortalized cells resulting from transfection with HPV DNA have been found to express significantly higher levels of Sine oculis homeobox homolog 1 (SIX1), suggesting that HPV might be able to promote tumor cell proliferation by increasing SIX1 expression (4). SIX1 gene is one of homeobox genes which code for transcription factors functioning as master regulators in proliferation, apoptosis, migration and invasion (5). Similar to many cell cycle proteins, the expression of SIX1 is regulated in a cell cycle-specific manner, where it is transcriptionally upregulated at the G1/S boundary, highly expressed in S and G2/M phases, then degraded via the ubiquitin-proteasome pathway (6). SIX1 is necessary in early embryogenesis, but its expression is lost in most well differentiated tissues. However, SIX1 has been found to be overexpressed in a variety of malignancies, showing the ability to promote tumor growth and metastasis (7). Moreover, SIX1 was reported to abolish the G2 cell cycle checkpoint and upregulate cyclin A1 to promote cell proliferation in breast cancer (8,9). These results implied that SIX1 might play an important role in the proliferation of tumor cells and the development of tumor.

The ability of tumor cells to sustain the uncontrolled proliferation might be influenced by the factors that could modulate DNA replication, cell cycle, apoptosis-resistance, metabolic capacity, etc. In this study we investigated the mechanism underlying the effect of SIX1 on the proliferation of cervical cancer cells. The resultant data showed that SIX1 functioned as a master regulator in DNA replication. SIX1 upregulated multiple genes affecting the initiation of DNA replication, accelerated G1 to S phase progression, and promoted tumor cell proliferation. These results implied that SIX1 might play a crucial role in tumorigenesis and progression of cervical cancer, and also suggested that SIX1 might be a therapeutic target in cervical cancer treatment.

Materials and methods

Cells and transfection

Human cervical cancer cell lines C33a (HPV-negative), Siha and Caski (HPV-positive) were purchased from the American Type Culture Collection (Manassas, VA). Cells were transduced with CMV-Fluc-IRES-RFP lentiviral particles (GeneChem, Shanghai, China). The cells with stable transfection (RFP+) were isolated by FACS. RFP/luciferase-expressing cells were used in living imaging. In cell cycle analysis by flow cytometry, RFP-free cells were used.

The expression vectors for E6 and E7 were constructed as previously described (10). Briefly, the cDNAs coding for E6 and E7 were amplified from total RNA isolated from Caski cell lines using following primers: E6, 5′-GCGGCCGCC ACCATGTTTCAGGACCACAG-3′ (sense) and 5′-CTGCGG CCGCGATTACAGCTGGGTTTTCTCT-3′ (antisense); E7, 5′-GCGGCCGCCACCATGGCATGGCATGGAGATAC ACCT-3′ (sense) and 5′-AGGCGGCCGCGATTATGGTTT CTGAGAACA-3′ (antisense). The cDNAs were inserted into expression vector pcDNA3.1. C33a cells were transfected with plasmid pcDNA3.1-SIX1 (a kind gift from Kong-Ming Wu, Thomas Jefferson University, Philadelphia, PA), pcDNA3.1-E6, pcDNA3.1-E7 or pcDNA3.1 using Lipofectamine 2000 (Invitrogen, Life Technologies). ShSIX1(1) and shSIX1(2) shRNA lentiviral particles (GeneChem) were used to knockdown SIX1 expression, targeting 5′-CCAGCTCAGAAGAGG AATT-3′ and 5′-CACGCCAGGAGCTCAAACT-3′, respectively. Shcon, the shRNA not targeting any known gene, was used as control. Caski cells were transduced with SIX1 shRNA lentiviral particles, or control shRNA lentiviral particles. The cells with stable transfection were selected with G418 (C33a) and puromycin (Caski). The transfected cells were designated as C33a-3.1, C33a-E6, C33a-E7, C33a-SIX1, Caski-shcon, Caski-shSIX1(1) and Caski-shSIX1(2), respectively.

Immunohistochemistry

A tissue microarray (TMA) (CIN481) was purchased from Alenabio (Xi-an, China), containing cervical intraepithelial neoplasia (CIN) samples. Tumor samples were acquired by surgery from cancer patients without preoperative treatment, which was approved by the Ethics Committee of the Medical Faculty of Tongji Medical College. Informed consent was obtained from all subjects. The tumor samples were processed into TMA at Outdo Biotech Company (Shanghai, China). TMAs and tissue sections were subjected to immunohistochemical analysis as described previously (11), using the Avidin-Biotin Complex (ABC) Vectastain Kit (Zsgb-Bio, Beijing, China). Rabbit anti-human SIX1 antibody (Sigma-Aldrich) and rabbit anti-human Ki67 antibody (Santa Cruz Biotechnology) were used as primary antibodies. For semi-quantitative evaluation of SIX1 expression in tissue, staining intensity was graded (0, absence; 1, weak; 2, moderate; 3, strong), and assessed using an immunoreactivity-scoring system, HSCORE (12). The HSCORE was calculated using the following formula: HSCORE = ∑Pi(i + 1), where i is the staining intensity of tumor cells and Pi is the percentage of tumor cells at each level of intensity. The percentage of Ki67-positive tumor cells was measured as mean of five hot-spots on one slide from each tumor. Hot-spots were defined as areas with the highest Ki67 intensity. The slides were read by two pathologists and each data point represents the mean of the scores from two pathologists.

Assay of gene expression by real-time RT-PCR

Total RNA was extracted from cells with TRIzol reagent (Invitrogen). The relative quantity of mRNA was determined by real-time RT-PCR. Gapdh and EEF1A1 were chosen as reference genes, which were reported as the most reliable combination in cervical cancer (13). The primer sequences are shown in Table I. The expression of genes was quantified using the comparative CT method. The expression level of each mRNA was normalized to that of GAPDH and EEF1A1 mRNAs, and expressed as n-fold difference relative to the control.

Table I

Primers for real-time RT-PCR.

Table I

Primers for real-time RT-PCR.

GeneSense primersAntisense primers
GAPDH GACAGTCAGCCGCATCTTCT TTAAAAGCAGCCCTGGTGAC
EEF1A1 TGCGGTGGGTGTCATCAAA AAGAGTGGGGTGGCAGGTATTG
MCM2 TTGGCGTGAGTTGCGTATTC GAGACTGAAAACGATTACAAACATC
MCM3 CCACAGATGATCCCAACTTT GTCCCATGTAGAAGGTTGTC
MCM4 TGAACCTCTATACATGCAACGAC CAGGGTAACGGTCAAAGAAGATT
MCM5 TCACCAAGCAGAAATACCC GTCCATGAGTCCAGTGAG
MCM6 AGTGTGTGAGTCGATGAATA CATTAAAGAGGAGCGAGCTT
MCM7 GGAAATATCCCTCGTAGTATCAC CTGAGAGTAAACCCTGTACC
POLA1 AAAGATCCATTGGAGCTTCACC TCAGCACGTTTAAGAGGAACAG
POLA2 AGGAGCTAGAGACATTGTTTCCA CTCGCTTCTGAGAACCCTTTG
PRIM1 ACATTCGCTACCAATCCTTCAAC AGCTCCCAGCTTCACTGTATT
PRIM2 TCTTCGAGAACAGGAGATTGTTG CAGAGCATCAGCAAAAGGGAT
RFC1 TTGTCATGGGTCGTGATAGTGG CCTGGCATAGTCCGAATCAGAT
RFC2 GTGAGCAGGCTAGAGGTCTTT TGAGTTCCAACATGGCATCTTTG
RFC3 GTGGACAAGTATCGGCCCTG TGATGGTCCGTACACTAACAGAT
RFC4 CCGCTGACCAAGGATCGAG AGGGAACGGGTTTGGCTTTC
RFC5 GACATGCGTAGGGCTCTGAAC GTGCAGGTGTAGACAGTCTCC
POLD1 ATCCAGAACTTCGACCTTCCG ACGGCATTGAGCGTGTAGG
POLD2 ATGTTTTCTGAGCAGGCTGCC TGGGGGAGCAGGTTGTGCTC
POLD3 AAGCCATGCTAAAGGACAGTG CATTGGTGGTCAGCTCATTGTT
POLD4 ATCACTGATTCCTACCCGGTT AGAGATGCCAGAGACTGCACT
POLE TTGCGACCAGAAAGGGTTGT TGATTTGGCAAGTCCAGATCCT
POLE2 TGAGAAGCAACCCTTGTCATC TCATCAACAGACTGACTGCATTC
POLE3 GGCCCGAGGACCTAAACCT ATGTGGCGTACAGCACGAAG
POLE4 AAGGTCTTCCACTGTTTCTGTCTG CCTTTATTCTGGTCCTCATCATTC
Western blot assay

Western blot assay was performed as described previously (14). The antibodies were purchased from Sigma-Aldrich, Biorbyt and Santa Cruz Biotechnology.

Bioinformatic analysis

The normalized RNA-sequencing data of 116 cases of cervical cancer samples was publicly available from TCGA database (https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp). Differential gene expression based on SIX1 expression category was determined using Bioconductor edgeR package (15) as described previously (16). Then, pathway analysis of differentially expressed genes was performed using Onto-Express from Onto-Tools package (17).

To further analyze the effect of SIX1 expression on DNA replication and cell cycle pathways (gene sets), Gene set enrichment analysis (GSEA) package was used as described previously (18). For this analysis, SIX1 expression was set as continuous label. Heat map was generated by GSEA.

BrdU incorporation

The BrdU colorimetric ELISA kit (Roche) was used to detect 5′-bromo-2′-deoxyuridine (BrdU) incorporated into newly synthesized DNA, according to the manufacturer’s protocol. Briefly, tumor cells were incubated with BrdU solution for 2–16 h at 37°C, then fixed and denatured and incubated with a peroxidase-conjugated antibody against BrdU for 1 h at room temperature. The cells were then incubated with the tetramethyl-benzidine (TMB) substrate solution and absorbance was read at 450 nm using a multiskan spectrum microplate reader (μQuant Bio-Tek Instruments, USA).

Flow cytometry analysis

Cells were harvested by trypsinization and fixed in 70% ice-cold ethanol overnight at −20°C, and then washed in PBS and incubated in RNase A (10 U/ml) and propidium iodide (20 mg/ml) for 30 min at room temperature. The cells in different phases were detected by flow cytometry on a FACS Calibur system (Becton-Dickinson) and analyzed using FlowJo software. Serum starvation was performed as described previously (19).

Tumor cell proliferation assay

Tumor cells were seeded at 2×103 per well into 96-well plates. Using cell counting kit-8 (CCK8, Boster, China), the proliferation of the cells at each time point was measured with multiskan spectrum microplate reader (μQuant Bio-Tek Instruments, USA) at 450 nm wavelength. Cell proliferation index was expressed as fold change related to day 0.

Soft agar assay

Cells were suspended in 0.35% agar in DMEM (20% FBS) and plated (1.5×103 per well) on a layer of 0.5% agar in culture medium in 6-well plates. After two-week culture, colonies were counted and photographed by phase-contrast microscopy (Nikon, Japan). The size of 20 randomly chosen colonies per well was measured. The average size of colony was calculated as (length + width) × 0.5. The colony formation rate was calculated as the number of colonies/1,500 ×100%.

Animal studies

Female NOD-SCID mice (4 weeks old) were purchased from Beijing HFK Bio-Technology Co. Ltd. (Beijing, China). The studies were approved by the Committee on Ethics of Animal Experiments of Tongji Medical College. The mice were maintained in the accredited animal facility of Tongji Medical College. Intramuscular tumors were initiated by injecting 1×106 tumor cells in 50 μl of media into the hind limb of NOD-SCID mice. The sizes of ectopic tumors were monitored by a caliper and calculated by the formula: volume = (width)2 × length/2. Orthotopic xenograft model of cervical cancer was performed as previously described (20). Briefly, intramuscular tumors were used as donor tumor fragments for orthotopic transplantation into the cervix of NOD-SCID mice. Tumor growth in cervix was dynamically monitored in living mice by optical imaging of luciferase activity using the IVIS Spectrum system (Caliper, Xenogen, USA). After anaesthetization with 3% pentobarbital sodium, mice were imaged 10 min after intraperitoneal injection of 100 mg/kg D-luciferin. Tumor size was measured by three-dimensional reconstruction of image using Living Image version 4.3.1 software.

Statistical analysis

Each experiment was repeated at least three times independently. SPSS (version 13.0) software package was used for statistical analysis. Results are expressed as mean value ± standard error of the mean (SEM), and interpreted by one-way ANOVA. Differences were considered to be statistically significant at P<0.05.

Results

SIX1 expression is increased by HPV oncoprotein in cervical cancer

To determine the role of SIX1 in cervical cancer, we analyzed the expression of SIX1 in each stage of cancer development. SIX1 expression was negligible in normal cervical epithelium, but was significantly increased in low-grade cervical intraepithelial neoplasia (CIN I, precancerous lesion) (Fig. 1A). Higher expression levels of SIX1 were observed in almost all cases of high-grade cervical intraepithelial neoplasia (CIN II-III). SIX1-expressing cells shared the same distribution area with the extent of intraepithelial neoplasia (Fig. 1A). In three cervical squamous cell carcinoma cell lines, the expression of SIX1 was very low in HPV-negative C33a cells, slightly elevated in Siha cells which contain 1–2 copies of HPV DNA, and much higher in Caski cells which contain 60–600 copies of HPV DNA (Fig. 1B), suggesting that HPV infection increased the expression of SIX1. We then further tested whether SIX1 was induced by E6 or E7 oncoprotein, the driving force of cervical cancer. Overexpression of E7, but not E6, in HPV-negative C33a cells induced SIX1 expression (Fig. 1C). Consistently, when E7 were knocked down in C33a-E7, Siha or Caski cells, the expression of SIX1 was significantly decreased (Fig. 1D). These results suggested that HPV-E7 is one of the factors that are required for inducing the expression of SIX1. Moreover, the correlation of SIX1 expression to the progression of cervical cancer suggested that SIX1 might play an important role in tumorigenesis and progression of cervical cancer.

SIX1 may modulate the expression of multiple genes to promote tumor cell proliferation

To identify the effect of SIX1 on the development of cervical cancer, bioinformatic analysis was performed to predict the effect of SIX1 on gene expression in cervical cancer. The RNA-sequencing data of 116 cervical cancer specimens was obtained from TCGA database. The specimens were divided into SIX1-low expression group (n=58) and SIX1-high expression group (n=58). Bioconductor edgeR package was used to identify the genes which were significantly up-regulated in SIX1-high expression group. Subsequent pathway analysis indicated five pathways which were significantly enriched in the genes potentially upregulated by SIX1 (Fig. 2A). Two of the pathways were correlated with cell proliferation: DNA replication and the cell cycle. Further analysis by using GSEA package confirmed that the enrichment of the upregulated genes related to DNA replication was significantly correlated with the increase of SIX1 gene expression (Fig. 2B, P<0.05, FDR<0.25). The enrichment of the upregulated genes related to cell cycle was correlated, although without statistical significance, with the increase of SIX1 gene expression (Fig. 2C). Importantly, the analysis with both edgeR-Onto and GSEA methods showed that the upregulated genes included many genes related to DNA replication and the cell cycle, and that these genes could be considered as candidate genes which might be up-regulated by SIX1 (Fig. 2D). These results implied that SIX1 may regulate the expression of the genes involved in DNA replication to promote tumor cell proliferation.

SIX1 regulates the expression of genes responsible for DNA replication and enhances DNA synthesis

Based on the above results, we overexpressed SIX1 in C33a cells (Fig. 3A) to investigate the regulatory effect of SIX1 on DNA replication genes (Fig. 3B). SIX1 expression was knocked down in Caski cells (Fig. 3C) to further confirm the effect of SIX1 (Fig. 3D). The results showed that SIX1 could upregulate the expression of several genes coding for the proteins in five complexes related to DNA replication, including MCM complex (MCM2, MCM3, MCM6), DNA polymerase α-primase complex (POLA1, PRIM1, PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA polymerase δ complex (POLD3) and DNA polymerase ɛ complex (POLE2). These five complexes play critical roles in three important steps of DNA replication initiation: DNA unwinding, synthesis of primer, and switching from DNA polymerase α to polymerase δ or polymerase ɛ (Fig. 4A). Consistently, BrdU incorporation assay indicated that DNA synthesis was more active in SIX1 highly expressed cervical cancer cells (Fig. 4B).

SIX1 modulates the cell cycle and promotes G1 to S phase progression

In accordance with the potential of SIX1 to promote DNA replication, overexpression of SIX1 in C33a cells resulted in a decrease of the percentage of cells in G0/G1 phase and a concomitant increase of the percentage of cells in S phase (Fig. 5A), indicating more active cell proliferation. After synchronization by serum starvation and following serum addition, C33a-SIX1 cells progressed into S phase faster than corresponding control cells (Fig. 5B), and knockdown of SIX1 in Caski cells resulted in the opposite effect (Fig. 5C and D). These results demonstrated that SIX1 modulated the cell cycle and promoted G1 to S phase progression.

SIX1 promotes tumor cell proliferation in vitro

We next investigated whether SIX1 could promote tumor cell proliferation. Overexpression of SIX1 in C33a cells significantly, although slightly, promoted anchorage-dependent cell growth, while knockdown of SIX1 in Caski cells decreased anchorage-dependent cell growth (Fig. 6A). Intriguingly, in colony-forming assays in soft agar, the tumor cells with highly expressed SIX1 showed remarkably larger size colonies (Fig. 6B) and much higher colony formation rate (Fig. 6C), indicating that SIX1 could more efficiently promote anchorage-independent cell growth.

SIX1 promotes tumor growth in vivo

Expression of the proliferation marker Ki-67, was significantly higher in SIX1 highly expressed cervical cancer specimens (Fig. 7A). Comparing with corresponding control groups, the percentage of Ki67-positive tumor cells was increased in C33a-SIX1 tumors and decreased in Caski-shSIX1 tumors (Fig. 7B), indicating more active cell proliferation in SIX1 highly expressed tumors. In line with this, in both ectopic (Fig. 7C) and orthotopic (Fig. 7D) xenograft models of cervical cancer, overexpression of SIX1 in C33a cells significantly promoted tumor growth, while knockdown of SIX1 in Caski cells decreased tumor growth. Taken together, these results demonstrated that SIX1 could promote not only tumor cell proliferation in vitro, but also tumor growth in vivo.

Discussion

Malignant proliferation is the fundamental trait of tumor cells. The initiation of DNA replication represents a key process for cell proliferation, and has a marked impact on tumorigenesis and progression (19,21). The factors that promote DNA replication are organized in multiprotein complexes that coordinately help to recognize regions of origin, unwind DNA and promote replisome formation (21). The data in this study showed that SIX1 could modulate the expression of several genes related to DNA replication, thus accelerating G1 to S phase progression, promoting the proliferation of cervical cancer cells in vitro, and promoting the growth of cervical cancer in vivo.

The expression of SIX1 was closely correlated with the progression of cervical cancer as shown by our data. High levels of SIX1 expression were observed in almost all regions that developed intraepithelial neoplasia, suggesting that SIX1 might play an important role in tumorigenesis and progression of cervical cancer. Our data showed that the expression of SIX1 was positively correlated with the copy number of HPV DNA in cervical squamous cell carcinoma cells. These results were also supported by a report that SIX1 was significantly increased in HPV-immortalized normal human keratinocytes (4). Moreover, we demonstrated that SIX1 expression in cervical cancer cells could be modulated by HPV oncoprotein. HPV-E7, but not E6, was required for sustaining the expression of SIX1 gene in cervical cancer cells, including C33a-E7, Siha and Caski cells. These results also implied that HPV-E7 oncoprotein might continuously promote the progression of cervical cancer after the formation of tumor by upregulating the expression of SIX1.

The general process of DNA replication initiation involves several key steps: loading of helicase at replication origins, unwinding of DNA by helicase, the synthesis of RNA primer by primase, elongation of primer by DNA polymerase α, and then the replacement of DNA polymerase α with polymerase δ or polymerase ɛ for extending DNA strands (22,23). SIX1 could modulate the expression of the genes that mainly affect the initiation of DNA replication as shown by out data. The upregulation of multiple genes by SIX1 might increase the efficiency of DNA unwinding, synthesis of primer, and switching from DNA polymerase α to polymerase δ or polymerase ɛ.

MCM complex serves as the primary helicase for unwinding DNA during replication (21). Members of MCM family are proposed as diagnostic or prognostic markers in various cancers due to their increased proliferative potential (24). Studies have demonstrated that CDC6 is necessary for the loading of MCM complex at replication origins (25). Our data in this study showed that SIX1 upregulated the expression of MCM2, MCM3 and MCM6. Bioinformatic analysis also suggested that SIX1 could upregulate the expression of CDC6. Therefore, SIX1 might accelerate the initiation of DNA replication by upregulating the expression of these genes.

The synthesis of RNA primer by primase and the following elongation of the primer by DNA polymerase α are required for DNA replication in eukaryotes (23). The primase consists of two subunits, p49 (PRIM1) and p58 (PRIM2), both of which were upregulated by SIX1. Moreover, SIX1 could upregulate the expression of polymerase α1 (POLA1). The increased expression of these genes might make the initiation of DNA replication more efficient by improving the efficiency of primer synthesis. After the synthesis and elongation of primer, the synthesis of new DNA strands was catalyzed by DNA polymerase δ and polymerase ɛ. The switch from polymerase α to polymerase δ and polymerase ɛ is mediated by replication factor C (RFC) (26,27). DNA polymerases and RFC are important not only for DNA replication, but also for cell cycle control (26). RFC3 and RFC4 were reported to promote tumor cell proliferation (28,29). High RFC3 expression was associated with poor prognosis in a variety of cancers (28). Importantly, SIX1 not only increased the expression of DNA polymerase δ3 (POLD3) and polymerase ɛ2 (POLE2), but also upregulated the expression of RFC (RFC3, RFC4, RFC5). Therefore, SIX1 might not only promote the initiation of DNA replication by increasing the expression of primase and DNA polymerase α, but also promote the further synthesis of DNA strands by upregulating the expression of DNA polymerase δ3, polymerase ɛ2 and RFC.

SIX1 could significantly promote tumor cell proliferation in vitro. Correspondingly, ectopic and orthotopic xenograft models of cervical cancer further demonstrated that SIX1 could promote tumor growth in vivo. Intriguingly, colony formation assays in soft agar showed that SIX1 remarkably promoted anchorage-independent cell growth, suggesting that the promoting effect of SIX1 on tumor cell proliferation might promote not only the development of primary tumor but also the proliferation of disseminated tumor at metastatic sites.

In summary, this study revealed the important role of SIX1 in modulating DNA replication. SIX1 upregulated the expression of multiple key genes involved in the initiation of DNA replication. These genes coordinate to accelerate G1 to S phase progression, promote proliferation of cervical cancer cells and the growth of cervical cancer. These results implied that SIX1 might play a crucial role in the tumorigenesis and progression of cervical cancer, and also suggested that targeting SIX1 might have significant therapeutic value in cervical cancer treatment.

Acknowledgements

We thank Dr Qi-Lin Ao and Dr Shuang Guo for reviewing the histology data. This study was supported by National Science Foundation of China (nos. 81072135, 81372801, 81172466, 81072132 and 81372781).

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September 2014
Volume 45 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

2016 Impact Factor: 3.079
Ranked #33/217 Oncology
(total number of cites)

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APA
Liu, D., Zhang, X., Xi, B., Wan, D., Li, L., Zhou, J. ... Gao, Q. (2014). Sine oculis homeobox homolog 1 promotes DNA replication and cell proliferation in cervical cancer. International Journal of Oncology, 45, 1232-1240. https://doi.org/10.3892/ijo.2014.2510
MLA
Liu, D., Zhang, X., Xi, B., Wan, D., Li, L., Zhou, J., Wang, W., Ma, D., Wang, H., Gao, Q."Sine oculis homeobox homolog 1 promotes DNA replication and cell proliferation in cervical cancer". International Journal of Oncology 45.3 (2014): 1232-1240.
Chicago
Liu, D., Zhang, X., Xi, B., Wan, D., Li, L., Zhou, J., Wang, W., Ma, D., Wang, H., Gao, Q."Sine oculis homeobox homolog 1 promotes DNA replication and cell proliferation in cervical cancer". International Journal of Oncology 45, no. 3 (2014): 1232-1240. https://doi.org/10.3892/ijo.2014.2510