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FGF1–FGFR1 axis promotes tongue squamous cell carcinoma (TSCC) metastasis through epithelial–mesenchymal transition (EMT)
Accepted Manuscript FGF1-FGFR1 axis promotes tongue squamous cell carcinoma (TSCC) metastasis through epithelial-mesenchymal transition (EMT) Jiuyang Jiao, Xiaopeng Zhao, Yancan Liang, Dongxiao Tang, Chaobin Pan PII:
S0006-291X(15)30538-6
DOI:
10.1016/j.bbrc.2015.09.021
Reference:
YBBRC 34521
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 27 August 2015 Accepted Date: 4 September 2015
Please cite this article as: J. Jiao, X. Zhao, Y. Liang, D. Tang, C. Pan, FGF1-FGFR1 axis promotes tongue squamous cell carcinoma (TSCC) metastasis through epithelial-mesenchymal transition (EMT), Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.09.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT FGF1-FGFR1 axis promotes tongue squamous cell carcinoma (TSCC) metastasis
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through epithelial-mesenchymal transition (EMT)
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Jiuyang Jiao1, 2, 3, Xiaopeng Zhao1, 2, 3, Yancan Liang1, 2, Dongxiao Tang1, 2, Chaobin
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Pan1, 2, *
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1. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene
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Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,
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China
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2. Department of Oral & Maxillofacial Surgery, Sun Yat-sen Memorial Hospital, Sun
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Yat-sen University, Guangzhou, China
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3. These authors contribute equally to this work
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* Corresponding author: Chaobin Pan，Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Increasing evidences suggest a close association between tumor metastasis and the
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inflammatory factors secreted by tumor microenvironment. It has been reported that
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epithelial mesenchymal-transition (EMT) plays a significant role during multiple
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types of tumor metastasis and progression induced by inflammatory factor from tumor
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microenvironment. Previous researches implied that fibroblast growth factor 1 (FGF1)
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can promote tumor progression and cause poor prognosis in several types of
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malignant tumors via interacting with its receptor fibroblast growth factor receptor 1
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(FGFR1). However, the effects of FGF1-FGFR1 on tongue squamous cell carcinoma
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(TSCC) are not yet completely understood. In the present study, we evaluated the
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effects and function of FGF1-FGFR1 axis on TSCC metastasis. In addition, we
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investigated whether the EMT pathway is involved in these effects, thus modulating
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the TSCC progression. The expression of FGFR1 was measured both in tongue cancer
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cell lines and tissues by qRT-PCR and western blot. We found that FGFR1 was
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up-regulated in TSCC tissues compared to non-neoplastic tongue tissues. Additionally,
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overexpression of FGFR1 is positively associated with poor differentiation and
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metastasis potential. Furthermore, the function of FGF1-FGFR1 was examined in
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TSCC cell line. The results implied that FGF1 can obviously promote Cal27 cells
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migration and invasion abilities through FGFR1, while the motile and invasive
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capabilities can be severely attenuated when knockdown the expression of FGFR1 by
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specific siRNAs. Further investigation results show that FGF1-FGFR1 axis promotes
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TSCC metastasis by modulating EMT pathway. However, this effect can be inhibited
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ACCEPTED MANUSCRIPT by blocking the FGF1-FGFR1 axis using FGFR1 specific siRNAs. In conclusion, our
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findings of the present study provide the evidences that FGF1-FGFR1 axis promotes
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the TSCC metastasis through the EMT pathway.
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Keywords: FGF1-FGFR1 axis; tongue squamous cell carcinoma; epithelial
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mesenchymal-transition; tumor metastasis
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ACCEPTED MANUSCRIPT Introduction
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Tongue squamous cell carcinoma (TSCC) is one of the leading causes of
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cancer-related mortality of oral cancer in the world.[1, 2] Most of patients with TSCC
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had bad prognosis and short survival time even underwent systematic therapy. It has
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been believed that tumor metastasis which disseminated tumor cells from the in situ to
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distant organs mainly accounted for these events.[3]
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Epithelial mesenchymal-transition is an important process which characterized by
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epithelial cells loosing polarity and cells to cells contact.[4] During EMT, cells
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acquire a spindle-shaped, fibroblastic-like phenotype, loss of epithelial cell adhesion.
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Meanwhile, cells undergoing EMT express mesenchymal markers and acquire motile
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features. Increasing evidences show that EMT plays a significant role during the
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tumor metastasis and development.[5] EMT, a driver of invasion and metastasis of
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cancer, even partial or transient, is one of the initial and major events in the tumor
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progression.[6]
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Recent studies on tumorigenesis demonstrated that tumor microenvironment has
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involved in the progression of tumor metastasis via inducing tumor EMT by secreting
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inflammatory factors through autocrine or paracrine loop pathway.[7] Numerous
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investigations have been proven that many inflammatory factors from tumor
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microenvironment can influence tumor progression. Transforming growth factor-β
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(TGF-β) and tumor necrosis factor (TNF-α) can directly induce multiple types of
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tumor cells EMT and promote cancer metastasis and progression;[8, 9] Chemokines, a
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family of chemotactic cytokines, can directly induce tumor cells chemotaxis, then
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ACCEPTED MANUSCRIPT promote tumor development, such as: Chemokine (C-C motif) ligand 18 (CCL18) and
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CCL2;[9-12] Interleukin family also can promote tumor progression by inducing
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tumor cells EMT, such as Interleukin 8 (IL-8);[13, 14] Fibroblast growth factor (FGF)
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family has also been related to the tumor metastasis in EMT.[14-16]
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In previous studies, fibroblast growth factor 1 (FGF1) has been associated with
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mitogenic, cell survival, morphogenesis, and modulating endothelial cell migration
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and proliferation, as well as an angiogenic factor.[17] However, recent studies implied
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that FGF1 may play a pivotal role in regulating a range of biological processes,
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including embryogenesis, differentiation, as well as increasing cellular motility and
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invasiveness in malignant tumor through autocrine and paracrine.[18] Through
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interacting with its cognate high-affinity receptors, FGF1-FGFR1 can activate
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downstream multiple signal pathways and have a crosstalk with other signal pathways
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to regulate tumor progression, including the MAPK/ERK, PI3K/AKT and
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WNT/β-catenin.[19]
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Despite the fact that FGF1and EMT is closely associated with multiple types of
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cancer development and progression, the relationship between these factors has not
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yet been well known in TSCC. The current study was designed to investigate and
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demonstrate the role of FGF1/FGFR1 and EMT in the progression and development
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of TSCC, as well as to elucidate the underlying molecular mechanisms.
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Materials and methods
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Tissue specimens
ACCEPTED MANUSCRIPT In this study, all tongue cancer and adjacent non-neoplastic tissue samples were
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collected with informed consent under the institutional board-approved protocols
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from the Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Guangzhou, China).
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For each case, tumor samples and adjacent non-neoplastic tissue were collected and
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stored at –80ºC. This study was approved by the institutional research ethics
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committee of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University.
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Cell lines and cell cultures
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Four tongue cancer cell lines were used in this study, including Cal27, SCC9, SCC15
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and SCC25; all cell lines were obtained from American Type Culture Collection
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(ATCC; Manassas, VA, USA). Cal27 was cultured in Dulbecco’s modified Eagle’s
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medium (DMEM) supplemented with 10% FBS and other cell lines were maintained
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in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). For all cell
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lines, 1% penicillin/streptomycin was added to the culture medium.
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RNA extraction and quantitative real-time PCR
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For total RNA extraction, tumor specimens were homogenized and total RNA from
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fresh surgical tongue tissues and TSCC cells were isolated using the TRIzol reagent
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(Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.
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Complementary DNA (cDNA) was synthesized with the PrimeScript RT reagent Kit
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(TaKaRa, Dalian, China). Quantitative real-time PCR was performed with
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LightCycler Real Time PCR System (Roche Diagnostics, Switzerland).
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RNA interference
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Short interfering RNA (siRNA) against FGFR1 was synthesized and purchased from
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GenePharma Company (GenePharma, Shanghai, China) and the two specific siRNA
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against
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TGGTATTAACTCCAGCAGTCTTCAAGA;
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AAGTCGGACGCAACAGAGAAA. The cells were plated in 6-well plates and
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transfected with the siRNA using Lipofectamine 2000 (Invitrogen) following the
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manufacturer’s instructions.
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Western blotting
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Cells were harvested, lysed, and protein concentration was determined by the
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Bradford assay using a kit purchased from Bio-Rad Laboratories (Hercules, CA,
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USA). Equal quantities of protein were separated on 10% SDS-polyacrylamide gels
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and transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia
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Biotech). The membranes were probed with antibody against human FGFR1 (1:1000,
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Abcam), E-cadherin, Vimentin, N-cadherin (1:500, Santa Cruz), or GAPDH (1:3000,
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Proteintech), and then with peroxidase-conjugated secondary antibody (1:3000,
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Proteintech) and the signals were visualised by enhanced chemiluminescence kit (GE,
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Fairfield, CT, USA) according to the manufacturer’s instructions. GAPDH was used
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as a loading control.
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Boyden chamber assay
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The invasion and migration assay were done using a Transwell chamber consisting of
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8 mm membrane filter inserts (Corning Inc., USA) coated with or without Matrigel
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(BD Biosciences, Franklin Lakes, NJ, USA). Briefly, for migration assay, 1×105 cells
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were added to the upper chamber of a polycarbonate transwell filter chamber and
were
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FGFR1-siRNA1:
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FGFR1-siRNA2:
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ACCEPTED MANUSCRIPT incubated for 8 h. For invasion assay, the upper chamber was coated with Matrigel
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(BD Biosciences, USA) and incubated for 24 h. The non-invading cells were gently
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removed with a soft cotton swab, and the cells that invaded to the bottom chamber
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were fixed with 4% paraformaldehyde, stained with hematoxyli, photographed and
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counted.
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Immunohistochemistry
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Immunohistochemistry analysis was performed to investigate the expression of
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FGFR1 in different grades of human tongue cancer. Briefly, immunohistochemistry
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was performed on the paraffin-embedded human tongue cancer tissue sections using
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Rabbit polyclonal antibody against FGFR1 (1:100, Abcam). For the negative controls,
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isotype-matched antibodies were applied. The tissue sections were observed under a
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Zeiss AX10-Imager A1 microscope e (Carl Zeiss, Thornwood, NY) and all images
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were captured using AxioVision 4.7 microscopy software (Carl Zeiss, Thornwood,
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NY).
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Statistical analysis
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Statistical analysis was performed using a SPSS software package (SPSS Standard
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version 18.0, SPSS Inc). Differences between variables were assessed by the χ2 test or
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Fisher’s exact test. Two-tailed Student’s t-tests were used to determine statistical
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significance for all results. P<0.05 was considered to be statistically significant.
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Results
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Up-regulation of FGFR1 in TSCC cell lines and primary TSCC tissues
ACCEPTED MANUSCRIPT Western blotting analysis was performed to evaluate the ectopic expression phenotype
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of FGFR1 in TSCC cell lines Cal27, SCC9, SCC15 and SCC25, and the results
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showed that FGFR1 was overexpressed in TSCC cell lines (Fig 1A). To further
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investigate and confirm the FGFR1 expression, quantitative real-time PCR and
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western blotting analysis were carried out to quantify the expression level of FGFR1
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in tongue squamous primary cancer tissues. Consistent to the TSCC cell lines results,
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we found that FGFR1 protein expression was much higher in four human tongue
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squamous cancer tissues compared to the paired adjacent non-neoplastic tongue
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tissues (Fig 1B and C). These results indicate that FGFR1 protein expression is
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upregulated in tongue squamous cancer.
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Overexpression of FGFR1 was correlated with poor prognostic phenotype of
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TSCC
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To further investigate the relationship between FGFR1 expression and the
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clinicopathological features and prognostic significance in tongue squamous cancer
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patients, the expression levels of FGFR1 in TSCC tissues were examined by
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immunohistochemistry. Consistently, immunohistochemistry analysis indicated that
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FGFR1 was markedly upregulated in tongue squamous cancer samples. Meanwhile,
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the poorer differentiation of tongue squamous cancer has much higher expression of
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FGFR1, suggesting that there is a closely association between FGFR1 expression and
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tongue squamous cancer differentiation (Fig 2A).
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It has been known that metastasis is the predominant reason which causes tongue
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squamous cancer mortality, the relationship between FGFR1 expression and tongue
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ACCEPTED MANUSCRIPT squamous cancer metastasis has been evaluated in 20 paired primary tongue cancer
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tissues and adjacent non-neoplastic tongue tissues. The finding is consistent with the
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former results obtained in our immunohistochemical staining analysis, in primary
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TSCCs, most of cases had much higher FGFR1 expression levels, compared to
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adjacent non-neoplastic tongue tissues, indicating that the higher expression level of
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FGFR1 is positively associated with a metastatic phenotype (P<0.0001) (Fig 2B).
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Knockdown of FGFR1 expression attenuated tongue cancer cell migration and
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invasion in vitro
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As FGFR1 expression was positively related with tongue cancer metastasis, next we
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investigate the effect of FGFR1 on the migration and invasion of tongue cancer cell.
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At first, two specific siRNAs against to FGFR1 were used to determine the silence
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efficiency examined by western blotting in TSCC cell line Cal27. As shown in Figure
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3A, both the two siRNAs can efficiently knock down the endogenous FGFR1 protein
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expression in Cal27 cells. Next, we investigate the effect and function of FGFR1 on
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TSCC cell migration and invasion induced by FGF1. Using the transwell chamber
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assay, we found that knocking down FGFR1 expression severely attenuated TSCC
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cell Cal27 motile and invasiveness abilities induced by FGF1, compared to untreated
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control or vector transfected alone. Meanwhile, without the inducing of FGF1, the
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TSCC cell Cal27 exhibited weak migration and invasion ability, suggesting that the
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interaction between FGF1 and FGFR1 is significant to the TSCC cell migration and
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invasion (Fig 3B). These results indicated that the FGF1-FGFR1 interaction promotes
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the invasive and metastatic ability of tongue cancer cells.
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ACCEPTED MANUSCRIPT FGF1 promotes TSCC cell EMT via interacting with its receptor FGFR1
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All the results above demonstrate that up-regulation of FGFR1 is positively associated
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with poor prognosis in patients with tongue cancer; however, the underlying
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molecular mechanism which mediate this effect are little known. It has been
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well-known that FGF family can induce multiple types of cancer cells epithelial
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mesenchymal transition by up-regulating some transcriptional factors, such as Twist1,
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Zeb1 and Snail1 expression. Moreover, EMT is considered to contribute to cancer
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metastasis in several types of cancer. Therefore, Western blotting and quantitative
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real-time PCR were performed to investigate the expression of EMT markers in Cal27
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cells. We found that without the effect of FGF1, the epithelial marker of E-cadherin is
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highly expressed, and both the two mesenchumal marker of Vimentin and N-cadherin
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are lowly expressed in the epithelial type cell Cal27. However, when FGF1 was added,
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the epithelial marker of E-cadherin expression was severely impaired; meanwhile, the
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two mesenchymal markers of Vimentin and N-cadherin were up-regulated. To further
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demonstrate the effect and function of FGF1 and FGFR1 in EMT, two specific siRNA
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against FGFR1 was used. The results showed that silencing the expression of FGFR1
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can partially reverse the EMT progression induced by FGF1, including: E-cadherin
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expression is partially recovered and Vimentin and N-cadherin expression are
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inhibited (Fig 4A, B and C). Both the Western blotting and quantitative real-time PCR
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results imply that the FGF1 can mediate and promote TSCC metastasis by activating
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the EMT signaling pathway through interacting with its receptor FGFR1.
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Considering all the results above, our findings indicate that FGF1-FGFR axis may
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ACCEPTED MANUSCRIPT 218
activate TSCC EMT and promote TSCC metastasis.
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Discussion
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Tongue squamous cell carcinoma (TSCC) is one of the most common malignant
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cancers in oral cavity, which represented more than 90% of whole oral cancers.
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Meanwhile, TSCC is also a considerable threat to human health. Investigating and
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understanding the underlying molecular mechanisms which regulate tongue cancer
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cell proliferation and invasion is significant to interrupt tumor progression. However,
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by far, little is known about tongue cancer tumorigenesis and initiation.
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Metastasis is an important feature and character of cancer, mainly accounted for the
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cancer-related lethality in humans. EMT is a key cellular morphological change that is
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always observed during cancer progression and enables cancer cells abilities to
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disseminate from the primary site to distant organs. It has been well-demonstrated
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before that cancer metastasis is closely associated with EMT, in which epithelial cells
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lose cell to cell adherence and contact, decrease E-cadherin expression and acquire
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mesenchymal properties, resulting in enhanced cell motility, invasiveness, increased
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Vimentin expression and resistance to apoptosis.[15] Current advances have unveiled
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the underlying molecular mechanisms and regulation networks which govern EMT in
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tumor progression. Inflammatory factors is a big families of small molecular
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compounds in tumor microenvironment, which included TGF-β, TNF-α, chemokine
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family members, interleukin family members. In tumor microenvironment,
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inflammatory factors can trigger and activate the transcription factors expression,
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ACCEPTED MANUSCRIPT containing Snail, Slug, Twist, ZEB1, ZEB2, which can subsequently promote
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EMT.[20-23] Therefore, EMT is a crucial process to promote cancer metastasis in
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tumor and revealing the mechanisms that regulate EMT is essential and necessary to
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design and develop novel therapeutic strategies to conquer cancer metastasis.
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In previous studies, FGF-FGFR axis has been reported for involving in multiple types
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of cancers progression, including breast cancer,[24] prostate cancer,[16] lung cancer,
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[25]skin cancer,[26] thyroid cancer,[27] hepatocellular carcinoma[28]and pancreas
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cancer.[29] Besides, many researches have revealed that several signal pathways
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downstream the FGF-FGFR axis may account for the progression of cancer, such as:
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activated FGFR can phosphorylate FGF receptor substrate 2 (FRS2) to recruit the
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GRB2 adaptor molecule; or FGF signals can trigger the RAS/MAPK or PI3K/AKT
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signaling cascades through FRS2 and GRB2, which promotes cancer cells
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proliferation.[19] FGF-FGFR signal pathway also can enhance cancer development
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by crosstalk with other signal pathway, including Notch, WNT, Hedgehog,
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TGFβ/BMP signaling cascades, since their correlation and involvement in
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carcinogenesis had been well established for a long run.[18]
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In present study, we found that the receptor of FGF1-FGFR1 was up-regulated in
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tongue cancer tissues compared to the paired non-tumor tissues. Further investigation
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showed that overexpression of FGFR1 was positively correlated with tongue cancer
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metastasis and differentiation. In vitro functional assay indicated that FGFR1mediated
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and promoted tongue cancer cell migration and invasion, under the induction of FGF1.
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At last, the underlying molecular mechanism was investigated and EMT pathway was
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ACCEPTED MANUSCRIPT found to take responsibility for the tongue cancer metastasis which governed by
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FGF1-FGFR1 axis.
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In summary, we have determined the ectopic expression phenotype of FGFR1 in
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TSCC and high expression of FGFR1 was associated with poor differentiation and
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metastasis. Moreover, the expression of FGFR1 enhanced the metastatic capability of
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TSCC cells, and the expression of FGFR1 prevented the EMT phenotype in TSCC
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cells in vitro.
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Competing interests
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The authors declare that they have no competing interests
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Author Contributions
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Chao-Bin Pan conceived and designed the experiments; Jiu Yang Jiao and Xiao Peng
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Zhao performed the experiments; Yan Can Liang analyzed the data; Dong-Xiao Tang
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contributed reagents/materials/analysis tools; Jiu Yang Jiao wrote the paper.
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Acknowledgements
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This study was supported by the Specialized Research Fund for the Doctoral Program
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of Higher Education (20130171110095) and Guangdong Provincial Department of
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Science and Technology (2012B031800252)
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ACCEPTED MANUSCRIPT Figure 1: Up-regulation of FGFR1 in human primary tongue cancer tissues and
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cell lines
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(A) Western blot analysis of FGFR1 protein in tongue squamous cell carcinoma
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(TSCC) cell lines. GAPDH was probed as loading control. (B) Quantitative real-time
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PCR analysis of FGFR1 mRNA from four pairs of tongue cancer and adjacent
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non-neoplastic tongue tissues. Error bars represent SEM calculated from three
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independent experiments. (C) Western blot analysis of FGFR1 protein from four
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human primary tongue cancer tissues (T) and paired adjacent non-neoplastic tongue
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tissues (N), as each pair tissues were taken from the same patient.
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Figure 2: Over-expression of FGFR1 is correlated with poor prognostic
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phenotypes of TSCC
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(A) Immunohistochemitry analysis detecting the expression of FGFR1 in normal
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tongue tissues and primary tongue tumor tissues with different histological
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differentiation. (B) Expression levels of FGFR1 in 20 paired TSCC and adjacent
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non-neoplastic tongue tissues. Alteration of expression is shown as box plot
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presentations and the mean level of FGFR1 expression in TSCCs were significantly
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higher than that in non-neoplastic tissues. (P<0.0001, independent t test).
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Figure 3: Knockdown of FGFR1 expression attenuated tongue cancer cell
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migration and invasion in vitro
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examined by Western blot in Cal27. (B) The migration and invasion abilities were
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analyzed in Cal27 by boyden chamber assay (scale bar: 200µm, *P < 0.05, **P < 0.01,
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***P < 0.001).
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Figure 4: Knockdown of FGFR1 expression can partially reverse EMT induced
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by FGF1
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(A) The expression of EMT markers including: E-Cadherin, Vimentin and
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N-Cadherin were measured by western blot. GAPDH was probed as the loading
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control. (B) and (C) Quantitative real-time PCR were performed to evaluate the
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expression of E-Cadherin and Vimentin (*P < 0.05, **P < 0.01, ***P < 0.001).
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ACCEPTED MANUSCRIPT Highlights overexpression of FGFR1 in TSCC was related to poor differentiation and metastasis. the expression of FGFR1 enhanced the metastatic capability of TSCC cells the expression of FGFR1 prevented the EMT phenotype in TSCC cells in vitro.
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FGF1-FGFR1 axis promotes the TSCC metastasis through the EMT pathway.
S0006-291X(15)30538-6
DOI:
10.1016/j.bbrc.2015.09.021
Reference:
YBBRC 34521
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 27 August 2015 Accepted Date: 4 September 2015
Please cite this article as: J. Jiao, X. Zhao, Y. Liang, D. Tang, C. Pan, FGF1-FGFR1 axis promotes tongue squamous cell carcinoma (TSCC) metastasis through epithelial-mesenchymal transition (EMT), Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.09.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT FGF1-FGFR1 axis promotes tongue squamous cell carcinoma (TSCC) metastasis
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through epithelial-mesenchymal transition (EMT)
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Jiuyang Jiao1, 2, 3, Xiaopeng Zhao1, 2, 3, Yancan Liang1, 2, Dongxiao Tang1, 2, Chaobin
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Pan1, 2, *
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1. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene
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Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,
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China
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2. Department of Oral & Maxillofacial Surgery, Sun Yat-sen Memorial Hospital, Sun
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Yat-sen University, Guangzhou, China
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3. These authors contribute equally to this work
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* Corresponding author: Chaobin Pan，Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Increasing evidences suggest a close association between tumor metastasis and the
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inflammatory factors secreted by tumor microenvironment. It has been reported that
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epithelial mesenchymal-transition (EMT) plays a significant role during multiple
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types of tumor metastasis and progression induced by inflammatory factor from tumor
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microenvironment. Previous researches implied that fibroblast growth factor 1 (FGF1)
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can promote tumor progression and cause poor prognosis in several types of
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malignant tumors via interacting with its receptor fibroblast growth factor receptor 1
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(FGFR1). However, the effects of FGF1-FGFR1 on tongue squamous cell carcinoma
23
(TSCC) are not yet completely understood. In the present study, we evaluated the
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effects and function of FGF1-FGFR1 axis on TSCC metastasis. In addition, we
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investigated whether the EMT pathway is involved in these effects, thus modulating
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the TSCC progression. The expression of FGFR1 was measured both in tongue cancer
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cell lines and tissues by qRT-PCR and western blot. We found that FGFR1 was
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up-regulated in TSCC tissues compared to non-neoplastic tongue tissues. Additionally,
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overexpression of FGFR1 is positively associated with poor differentiation and
30
metastasis potential. Furthermore, the function of FGF1-FGFR1 was examined in
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TSCC cell line. The results implied that FGF1 can obviously promote Cal27 cells
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migration and invasion abilities through FGFR1, while the motile and invasive
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capabilities can be severely attenuated when knockdown the expression of FGFR1 by
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specific siRNAs. Further investigation results show that FGF1-FGFR1 axis promotes
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TSCC metastasis by modulating EMT pathway. However, this effect can be inhibited
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ACCEPTED MANUSCRIPT by blocking the FGF1-FGFR1 axis using FGFR1 specific siRNAs. In conclusion, our
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findings of the present study provide the evidences that FGF1-FGFR1 axis promotes
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the TSCC metastasis through the EMT pathway.
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Keywords: FGF1-FGFR1 axis; tongue squamous cell carcinoma; epithelial
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mesenchymal-transition; tumor metastasis
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ACCEPTED MANUSCRIPT Introduction
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Tongue squamous cell carcinoma (TSCC) is one of the leading causes of
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cancer-related mortality of oral cancer in the world.[1, 2] Most of patients with TSCC
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had bad prognosis and short survival time even underwent systematic therapy. It has
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been believed that tumor metastasis which disseminated tumor cells from the in situ to
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distant organs mainly accounted for these events.[3]
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Epithelial mesenchymal-transition is an important process which characterized by
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epithelial cells loosing polarity and cells to cells contact.[4] During EMT, cells
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acquire a spindle-shaped, fibroblastic-like phenotype, loss of epithelial cell adhesion.
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Meanwhile, cells undergoing EMT express mesenchymal markers and acquire motile
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features. Increasing evidences show that EMT plays a significant role during the
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tumor metastasis and development.[5] EMT, a driver of invasion and metastasis of
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cancer, even partial or transient, is one of the initial and major events in the tumor
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progression.[6]
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Recent studies on tumorigenesis demonstrated that tumor microenvironment has
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involved in the progression of tumor metastasis via inducing tumor EMT by secreting
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inflammatory factors through autocrine or paracrine loop pathway.[7] Numerous
59
investigations have been proven that many inflammatory factors from tumor
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microenvironment can influence tumor progression. Transforming growth factor-β
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(TGF-β) and tumor necrosis factor (TNF-α) can directly induce multiple types of
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tumor cells EMT and promote cancer metastasis and progression;[8, 9] Chemokines, a
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family of chemotactic cytokines, can directly induce tumor cells chemotaxis, then
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ACCEPTED MANUSCRIPT promote tumor development, such as: Chemokine (C-C motif) ligand 18 (CCL18) and
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CCL2;[9-12] Interleukin family also can promote tumor progression by inducing
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tumor cells EMT, such as Interleukin 8 (IL-8);[13, 14] Fibroblast growth factor (FGF)
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family has also been related to the tumor metastasis in EMT.[14-16]
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In previous studies, fibroblast growth factor 1 (FGF1) has been associated with
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mitogenic, cell survival, morphogenesis, and modulating endothelial cell migration
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and proliferation, as well as an angiogenic factor.[17] However, recent studies implied
71
that FGF1 may play a pivotal role in regulating a range of biological processes,
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including embryogenesis, differentiation, as well as increasing cellular motility and
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invasiveness in malignant tumor through autocrine and paracrine.[18] Through
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interacting with its cognate high-affinity receptors, FGF1-FGFR1 can activate
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downstream multiple signal pathways and have a crosstalk with other signal pathways
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to regulate tumor progression, including the MAPK/ERK, PI3K/AKT and
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WNT/β-catenin.[19]
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Despite the fact that FGF1and EMT is closely associated with multiple types of
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cancer development and progression, the relationship between these factors has not
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yet been well known in TSCC. The current study was designed to investigate and
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demonstrate the role of FGF1/FGFR1 and EMT in the progression and development
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of TSCC, as well as to elucidate the underlying molecular mechanisms.
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Materials and methods
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Tissue specimens
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collected with informed consent under the institutional board-approved protocols
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from the Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Guangzhou, China).
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For each case, tumor samples and adjacent non-neoplastic tissue were collected and
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stored at –80ºC. This study was approved by the institutional research ethics
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committee of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University.
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Cell lines and cell cultures
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Four tongue cancer cell lines were used in this study, including Cal27, SCC9, SCC15
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and SCC25; all cell lines were obtained from American Type Culture Collection
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(ATCC; Manassas, VA, USA). Cal27 was cultured in Dulbecco’s modified Eagle’s
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medium (DMEM) supplemented with 10% FBS and other cell lines were maintained
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in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). For all cell
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lines, 1% penicillin/streptomycin was added to the culture medium.
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RNA extraction and quantitative real-time PCR
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For total RNA extraction, tumor specimens were homogenized and total RNA from
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fresh surgical tongue tissues and TSCC cells were isolated using the TRIzol reagent
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(Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.
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Complementary DNA (cDNA) was synthesized with the PrimeScript RT reagent Kit
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(TaKaRa, Dalian, China). Quantitative real-time PCR was performed with
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LightCycler Real Time PCR System (Roche Diagnostics, Switzerland).
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RNA interference
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Short interfering RNA (siRNA) against FGFR1 was synthesized and purchased from
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GenePharma Company (GenePharma, Shanghai, China) and the two specific siRNA
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against
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TGGTATTAACTCCAGCAGTCTTCAAGA;
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AAGTCGGACGCAACAGAGAAA. The cells were plated in 6-well plates and
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transfected with the siRNA using Lipofectamine 2000 (Invitrogen) following the
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manufacturer’s instructions.
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Western blotting
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Cells were harvested, lysed, and protein concentration was determined by the
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Bradford assay using a kit purchased from Bio-Rad Laboratories (Hercules, CA,
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USA). Equal quantities of protein were separated on 10% SDS-polyacrylamide gels
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and transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia
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Biotech). The membranes were probed with antibody against human FGFR1 (1:1000,
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Abcam), E-cadherin, Vimentin, N-cadherin (1:500, Santa Cruz), or GAPDH (1:3000,
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Proteintech), and then with peroxidase-conjugated secondary antibody (1:3000,
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Proteintech) and the signals were visualised by enhanced chemiluminescence kit (GE,
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Fairfield, CT, USA) according to the manufacturer’s instructions. GAPDH was used
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as a loading control.
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Boyden chamber assay
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The invasion and migration assay were done using a Transwell chamber consisting of
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8 mm membrane filter inserts (Corning Inc., USA) coated with or without Matrigel
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(BD Biosciences, Franklin Lakes, NJ, USA). Briefly, for migration assay, 1×105 cells
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were added to the upper chamber of a polycarbonate transwell filter chamber and
were
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FGFR1-siRNA1:
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FGFR1-siRNA2:
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ACCEPTED MANUSCRIPT incubated for 8 h. For invasion assay, the upper chamber was coated with Matrigel
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(BD Biosciences, USA) and incubated for 24 h. The non-invading cells were gently
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removed with a soft cotton swab, and the cells that invaded to the bottom chamber
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were fixed with 4% paraformaldehyde, stained with hematoxyli, photographed and
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counted.
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Immunohistochemistry
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Immunohistochemistry analysis was performed to investigate the expression of
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FGFR1 in different grades of human tongue cancer. Briefly, immunohistochemistry
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was performed on the paraffin-embedded human tongue cancer tissue sections using
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Rabbit polyclonal antibody against FGFR1 (1:100, Abcam). For the negative controls,
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isotype-matched antibodies were applied. The tissue sections were observed under a
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Zeiss AX10-Imager A1 microscope e (Carl Zeiss, Thornwood, NY) and all images
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were captured using AxioVision 4.7 microscopy software (Carl Zeiss, Thornwood,
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NY).
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Statistical analysis
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Statistical analysis was performed using a SPSS software package (SPSS Standard
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version 18.0, SPSS Inc). Differences between variables were assessed by the χ2 test or
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Fisher’s exact test. Two-tailed Student’s t-tests were used to determine statistical
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significance for all results. P<0.05 was considered to be statistically significant.
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Results
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Up-regulation of FGFR1 in TSCC cell lines and primary TSCC tissues
ACCEPTED MANUSCRIPT Western blotting analysis was performed to evaluate the ectopic expression phenotype
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of FGFR1 in TSCC cell lines Cal27, SCC9, SCC15 and SCC25, and the results
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showed that FGFR1 was overexpressed in TSCC cell lines (Fig 1A). To further
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investigate and confirm the FGFR1 expression, quantitative real-time PCR and
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western blotting analysis were carried out to quantify the expression level of FGFR1
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in tongue squamous primary cancer tissues. Consistent to the TSCC cell lines results,
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we found that FGFR1 protein expression was much higher in four human tongue
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squamous cancer tissues compared to the paired adjacent non-neoplastic tongue
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tissues (Fig 1B and C). These results indicate that FGFR1 protein expression is
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upregulated in tongue squamous cancer.
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Overexpression of FGFR1 was correlated with poor prognostic phenotype of
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TSCC
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To further investigate the relationship between FGFR1 expression and the
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clinicopathological features and prognostic significance in tongue squamous cancer
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patients, the expression levels of FGFR1 in TSCC tissues were examined by
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immunohistochemistry. Consistently, immunohistochemistry analysis indicated that
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FGFR1 was markedly upregulated in tongue squamous cancer samples. Meanwhile,
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the poorer differentiation of tongue squamous cancer has much higher expression of
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FGFR1, suggesting that there is a closely association between FGFR1 expression and
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tongue squamous cancer differentiation (Fig 2A).
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It has been known that metastasis is the predominant reason which causes tongue
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squamous cancer mortality, the relationship between FGFR1 expression and tongue
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ACCEPTED MANUSCRIPT squamous cancer metastasis has been evaluated in 20 paired primary tongue cancer
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tissues and adjacent non-neoplastic tongue tissues. The finding is consistent with the
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former results obtained in our immunohistochemical staining analysis, in primary
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TSCCs, most of cases had much higher FGFR1 expression levels, compared to
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adjacent non-neoplastic tongue tissues, indicating that the higher expression level of
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FGFR1 is positively associated with a metastatic phenotype (P<0.0001) (Fig 2B).
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Knockdown of FGFR1 expression attenuated tongue cancer cell migration and
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invasion in vitro
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As FGFR1 expression was positively related with tongue cancer metastasis, next we
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investigate the effect of FGFR1 on the migration and invasion of tongue cancer cell.
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At first, two specific siRNAs against to FGFR1 were used to determine the silence
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efficiency examined by western blotting in TSCC cell line Cal27. As shown in Figure
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3A, both the two siRNAs can efficiently knock down the endogenous FGFR1 protein
187
expression in Cal27 cells. Next, we investigate the effect and function of FGFR1 on
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TSCC cell migration and invasion induced by FGF1. Using the transwell chamber
189
assay, we found that knocking down FGFR1 expression severely attenuated TSCC
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cell Cal27 motile and invasiveness abilities induced by FGF1, compared to untreated
191
control or vector transfected alone. Meanwhile, without the inducing of FGF1, the
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TSCC cell Cal27 exhibited weak migration and invasion ability, suggesting that the
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interaction between FGF1 and FGFR1 is significant to the TSCC cell migration and
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invasion (Fig 3B). These results indicated that the FGF1-FGFR1 interaction promotes
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the invasive and metastatic ability of tongue cancer cells.
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ACCEPTED MANUSCRIPT FGF1 promotes TSCC cell EMT via interacting with its receptor FGFR1
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All the results above demonstrate that up-regulation of FGFR1 is positively associated
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with poor prognosis in patients with tongue cancer; however, the underlying
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molecular mechanism which mediate this effect are little known. It has been
200
well-known that FGF family can induce multiple types of cancer cells epithelial
201
mesenchymal transition by up-regulating some transcriptional factors, such as Twist1,
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Zeb1 and Snail1 expression. Moreover, EMT is considered to contribute to cancer
203
metastasis in several types of cancer. Therefore, Western blotting and quantitative
204
real-time PCR were performed to investigate the expression of EMT markers in Cal27
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cells. We found that without the effect of FGF1, the epithelial marker of E-cadherin is
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highly expressed, and both the two mesenchumal marker of Vimentin and N-cadherin
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are lowly expressed in the epithelial type cell Cal27. However, when FGF1 was added,
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the epithelial marker of E-cadherin expression was severely impaired; meanwhile, the
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two mesenchymal markers of Vimentin and N-cadherin were up-regulated. To further
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demonstrate the effect and function of FGF1 and FGFR1 in EMT, two specific siRNA
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against FGFR1 was used. The results showed that silencing the expression of FGFR1
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can partially reverse the EMT progression induced by FGF1, including: E-cadherin
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expression is partially recovered and Vimentin and N-cadherin expression are
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inhibited (Fig 4A, B and C). Both the Western blotting and quantitative real-time PCR
215
results imply that the FGF1 can mediate and promote TSCC metastasis by activating
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the EMT signaling pathway through interacting with its receptor FGFR1.
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Considering all the results above, our findings indicate that FGF1-FGFR axis may
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ACCEPTED MANUSCRIPT 218
activate TSCC EMT and promote TSCC metastasis.
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Discussion
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Tongue squamous cell carcinoma (TSCC) is one of the most common malignant
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cancers in oral cavity, which represented more than 90% of whole oral cancers.
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Meanwhile, TSCC is also a considerable threat to human health. Investigating and
224
understanding the underlying molecular mechanisms which regulate tongue cancer
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cell proliferation and invasion is significant to interrupt tumor progression. However,
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by far, little is known about tongue cancer tumorigenesis and initiation.
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Metastasis is an important feature and character of cancer, mainly accounted for the
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cancer-related lethality in humans. EMT is a key cellular morphological change that is
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always observed during cancer progression and enables cancer cells abilities to
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disseminate from the primary site to distant organs. It has been well-demonstrated
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before that cancer metastasis is closely associated with EMT, in which epithelial cells
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lose cell to cell adherence and contact, decrease E-cadherin expression and acquire
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mesenchymal properties, resulting in enhanced cell motility, invasiveness, increased
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Vimentin expression and resistance to apoptosis.[15] Current advances have unveiled
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the underlying molecular mechanisms and regulation networks which govern EMT in
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tumor progression. Inflammatory factors is a big families of small molecular
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compounds in tumor microenvironment, which included TGF-β, TNF-α, chemokine
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family members, interleukin family members. In tumor microenvironment,
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inflammatory factors can trigger and activate the transcription factors expression,
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ACCEPTED MANUSCRIPT containing Snail, Slug, Twist, ZEB1, ZEB2, which can subsequently promote
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EMT.[20-23] Therefore, EMT is a crucial process to promote cancer metastasis in
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tumor and revealing the mechanisms that regulate EMT is essential and necessary to
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design and develop novel therapeutic strategies to conquer cancer metastasis.
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In previous studies, FGF-FGFR axis has been reported for involving in multiple types
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of cancers progression, including breast cancer,[24] prostate cancer,[16] lung cancer,
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[25]skin cancer,[26] thyroid cancer,[27] hepatocellular carcinoma[28]and pancreas
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cancer.[29] Besides, many researches have revealed that several signal pathways
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downstream the FGF-FGFR axis may account for the progression of cancer, such as:
249
activated FGFR can phosphorylate FGF receptor substrate 2 (FRS2) to recruit the
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GRB2 adaptor molecule; or FGF signals can trigger the RAS/MAPK or PI3K/AKT
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signaling cascades through FRS2 and GRB2, which promotes cancer cells
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proliferation.[19] FGF-FGFR signal pathway also can enhance cancer development
253
by crosstalk with other signal pathway, including Notch, WNT, Hedgehog,
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TGFβ/BMP signaling cascades, since their correlation and involvement in
255
carcinogenesis had been well established for a long run.[18]
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In present study, we found that the receptor of FGF1-FGFR1 was up-regulated in
257
tongue cancer tissues compared to the paired non-tumor tissues. Further investigation
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showed that overexpression of FGFR1 was positively correlated with tongue cancer
259
metastasis and differentiation. In vitro functional assay indicated that FGFR1mediated
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and promoted tongue cancer cell migration and invasion, under the induction of FGF1.
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At last, the underlying molecular mechanism was investigated and EMT pathway was
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ACCEPTED MANUSCRIPT found to take responsibility for the tongue cancer metastasis which governed by
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FGF1-FGFR1 axis.
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In summary, we have determined the ectopic expression phenotype of FGFR1 in
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TSCC and high expression of FGFR1 was associated with poor differentiation and
266
metastasis. Moreover, the expression of FGFR1 enhanced the metastatic capability of
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TSCC cells, and the expression of FGFR1 prevented the EMT phenotype in TSCC
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cells in vitro.
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Competing interests
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The authors declare that they have no competing interests
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Author Contributions
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Chao-Bin Pan conceived and designed the experiments; Jiu Yang Jiao and Xiao Peng
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Zhao performed the experiments; Yan Can Liang analyzed the data; Dong-Xiao Tang
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contributed reagents/materials/analysis tools; Jiu Yang Jiao wrote the paper.
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Acknowledgements
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This study was supported by the Specialized Research Fund for the Doctoral Program
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of Higher Education (20130171110095) and Guangdong Provincial Department of
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Science and Technology (2012B031800252)
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effects on tumor cells,
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ACCEPTED MANUSCRIPT Figure 1: Up-regulation of FGFR1 in human primary tongue cancer tissues and
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cell lines
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(A) Western blot analysis of FGFR1 protein in tongue squamous cell carcinoma
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(TSCC) cell lines. GAPDH was probed as loading control. (B) Quantitative real-time
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PCR analysis of FGFR1 mRNA from four pairs of tongue cancer and adjacent
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non-neoplastic tongue tissues. Error bars represent SEM calculated from three
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independent experiments. (C) Western blot analysis of FGFR1 protein from four
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human primary tongue cancer tissues (T) and paired adjacent non-neoplastic tongue
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tissues (N), as each pair tissues were taken from the same patient.
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Figure 2: Over-expression of FGFR1 is correlated with poor prognostic
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phenotypes of TSCC
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(A) Immunohistochemitry analysis detecting the expression of FGFR1 in normal
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tongue tissues and primary tongue tumor tissues with different histological
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differentiation. (B) Expression levels of FGFR1 in 20 paired TSCC and adjacent
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non-neoplastic tongue tissues. Alteration of expression is shown as box plot
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presentations and the mean level of FGFR1 expression in TSCCs were significantly
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higher than that in non-neoplastic tissues. (P<0.0001, independent t test).
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Figure 3: Knockdown of FGFR1 expression attenuated tongue cancer cell
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migration and invasion in vitro
ACCEPTED MANUSCRIPT (A) The knockdown efficiency of two specific siRNA against to FGFR1 was
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examined by Western blot in Cal27. (B) The migration and invasion abilities were
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analyzed in Cal27 by boyden chamber assay (scale bar: 200µm, *P < 0.05, **P < 0.01,
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***P < 0.001).
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Figure 4: Knockdown of FGFR1 expression can partially reverse EMT induced
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by FGF1
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(A) The expression of EMT markers including: E-Cadherin, Vimentin and
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N-Cadherin were measured by western blot. GAPDH was probed as the loading
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control. (B) and (C) Quantitative real-time PCR were performed to evaluate the
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expression of E-Cadherin and Vimentin (*P < 0.05, **P < 0.01, ***P < 0.001).
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ACCEPTED MANUSCRIPT Highlights overexpression of FGFR1 in TSCC was related to poor differentiation and metastasis. the expression of FGFR1 enhanced the metastatic capability of TSCC cells the expression of FGFR1 prevented the EMT phenotype in TSCC cells in vitro.
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FGF1-FGFR1 axis promotes the TSCC metastasis through the EMT pathway.