Background
Worldwide, cervical cancer is ranked as the second most common cancer in women and the third leading cause of death from cancer in women [
1,
2]. The incidence of cervical cancer is very high in developing countries [
3]. Until recently, therapeutic options for hysterectomy-resistant cervical cancers have been limited with treatments largely palliative [
4]. Therefore, detecting or preventing cervical cancers with progressions in early is critical, which could help to prolong patient survival. As we know TFF3 is a soluble peptide containing trefoil domain and C-terminal dimerization domain which is not only a novel prognostic marker but also a therapeutic target in various cancers, such as mammary carcinoma, gastric cancer and prostate carcinoma [
5‐
8]. Upregulation of TFF3 after rectal cancer chemo-radiotherapy is an adverse prognostic factor [
9]. Furthermore, in prostate carcinoma cells, TFF3 reduces the sensitivity to ionizing-radiation [
10].
TFF3, behaved as an oncogene, promotes proliferation and invasion, improves survival, and increases oncogenicity in cancer cells, such as mammary carcinoma, gastric cancer and prostate carcinoma [
5,
11]. TFF3 promoted epithelial tumorigenesis by inducing aberrant proliferation and inhibiting apoptosis [
7]. TFF3 also may contribute to cancer metastasis with epithelial-to-mesenchymal transition (EMT) potentially through the regulation of genes such as androgen receptor (AR), FOXA1 and human epidermal growth factor receptor-type 2 (HER2) [
12,
13]. Moreover, TFF3, a secreted protein, is a valuable predictive serum biomarker in patients with metastatic colorectal cancer [
9]. In cancer cells, TFF3 promotes cell migration, invasion and metastasis by reducing cell–cell and cell–matrix interactions and enhancing cell scattering in bronchiole or other epithelia cells [
14,
15]. Up-regulation of TFF3 in cancer cells was accompanied by activation of multiple pathways including PI3K, MAPK and JAK/STAT pathways which were associated with cellular proliferation, apoptosis, migration, invasion and clonogenic survival [
16]. Despite the evidence that TFF3 could influence various cancer cells function in vitro, the role of TFF3 in cervical cancer cells has not been examined.
In the present study, we found that TFF3 protein was overexpressed in cervical cancer cells and weakly expressed in human non-tumor keratinocytes. We detected up-regulated expression of TFF3 promoted growth, proliferation and invasion, and inhibited apoptosis in SiHa and Hela cells. These finding demonstrate that TFF3 may be a potential therapeutic target in invasive cervical cancers with multidrug resistance.
Methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO (Carlsbad, California, USA). Mouse anti-GPADH polyclonal antibody (Lot#ab37168), Rabbit anti-Trefoil Factor 3 monoclonal antibody (Lot#ab108599), Mouse anti-E Cadherin monoclonal antibody (Lot#ab1416), Mouse anti-Phospho-STAT3 monoclonal antibody (Lot#ab119672), Mouse anti-Total STAT3 monoclonal antibody (Lot#ab119672) were obtained from Abcam (Cambridge, UK). JSI-124 was obtained from Enzo Life Science (USA). Goat anti-Rabbit IgG IR Dye 800cw (Lot#C30626-03) and Goat anti-Mouse IgG IR Dye 800cw (Lot#C40528-02) were from Odyssey (Licor, USA). Click-iT Edu imaging kit and Live/Dead Bac Light Viability Kit for microscopy were from Invitrogen (Carlsbad, CA, USA).
Cell cultures and transfection
Human cervical cancer cell lines SiHa, CaSki, Hela, Me180 and human non-tumor keratinocyte line HaCaT were obtained from Nanjing KeyGen Biotech Co, Ltd (Nanjing,China). The cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Carlsbad, California, USA) containing 10% FBS in a humidified atmosphere of 5% CO
2 at 37 °C. Human TFF3 expression, TFF3 siRNA and CDH1 siRNA plasmid constructs have been previously described [
7,
17]. Luciferase assays were performed as previously described [
18]. Briefly, transfections were carried out in triplicate using 1 μg of the appropriate luciferase reporter construct and empty vector per transfection along with 0.1 μg of Renilla luciferase construct as control for transfection efficiency. Luciferase activities were assayed after 24 h of transfection using the Dual Luciferase Assay System (Promega Corp, Madison, WI, USA).
RT-PCR and semi-quantitative RT-PCR
Total RNA was isolated from cells using Trizol plus RNA Purification system as previously described [
19]. DNase I treatment, total RNA to complementary DNA, PCR, and qPCR assays were performed as previously described. Gene expression analysis was performed as previously described [
20] and the sequence of the primers were described in Additional file
1: Table S1.
Total RNA was isolated using Trizol plus RNA Purification Kit (Invitrogen, Carlsbad, CA) as previously described [
19]. Semi-quantitative RT-PCR was performed using a Super Script One Step RT-PCR kit (Invitrogen, Carlsbad, CA, USA). Sequences of the nucleotide primers for RT-PCR were: TFF3 5′-GCTGCCAGAGCGCTCTGCATG-3′ and 5′-AAGGTGCATTTCTGCTTCCTGCAG-3′ (35 cycles; wild-type cell lines); β-Actin, 5′-ATGATATCGCCGCGCTCG-3′ and 5′-CGCTCGGTGAGGATCTTCA-3′ (23 cycles). Amplified RT-PCR products were visualized on a 1.5% agarose gel.
In vitro invasion analysis
An in vitro invasion assay was carried out to examine the invasion of cervical cancer cells, as previously described [
21]. Briefly, 24-well Transwell units with 8 µm polycarbonate nucleopore filters (Corning, NY, USA) were coated with 0.1 mL 0.8 mg/mL Engelbreth Holm-Swarm sarcoma tumor extract (EHS Matrigel) at room temperature for 1 h to form a genuinely reconstituted basement membrane. Cervical cancer cells (5 × 10
4 cells) were placed in the upper compartment and 500 µL DMEM culture medium containing 10% fetal calf serum was added to the lower compartment. The Transwell plates were incubated at 37 °C for 36 h in a humidified atmosphere with 5% CO
2 and stained with 10% crystal violet. Invading cells were defined as cells that had degraded the Matrigel and moved into the lower surface of the membrane. The non-invading cells retained on the upper surface of the membrane were removed by a cotton swab.
Click-iT EdU test
We performed the Click-iT Edu test to analyze the cervical cancer cell proliferation according to the manufacturer’s instructions. Cervical cancer cells were incubated with EDU for 12 h and then images were obtained to determine percentages of EdU-labeled cervical cancer cells.
Western blot
Cervical cancer cells lysates were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and probed with primary antibodies, followed by Goat anti-rabbit or mouse IgG IR Dye 800 cw (1:15,000), respectively. Images were obtained with an Odyssey Imager (LI-COR, Lincoln, NE, USA).
Flow cytometry and analysis
Flow cytometry analysis was performed either on FACSCAN using Cell Quest software, or on MACS quant seven color analyzer. Data analysis was performed using Flow Jo software.
Total cell number
1 × 105 (SiHa and Hela) cells were seeded into 35 mm2 falcon tissue culture dish in monolayers in 10% serum media in quadruplicate. On indicated days, cells were trypsinised and the cell number was determined using a haemocytometer.
Graphs and statistical analysis
All graphs were generated using Prism 4 (GraphPad Software, Inc., California, USA). Statistical significance was assessed by using an unpaired two-tailed Student’s t test (P < 0.05 was considered as significant) using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Columns are the mean of triplicate experiments; bars ± SD. *P < 0.05, **P < 0.01.
Discussion
This study contributes to our understanding of the molecular mechanism by which overexpression of TFF3 in human cervical cancers promotes tumor progression. The present work first found that TFF3 was overexpressed in cervical cancer cells and weakly expressed in human non-tumor keratinocytes. Several studies demonstrated that TFF3 overexpression strongly correlated with poor prognosis in various tumors [
26,
27], which indicated that TFF3 could be a potentially superior diagnostic marker or therapeutic target for cervical cancer. In this study we demonstrated that TFF3 functionally promoted the malignant progression of cervical cancer cells. Forced expression of TFF3 promoted the proliferation and invasion, and inhibited the apoptosis in SiHa and Hela cells. Conversely, decreased expression of TFF3 inhibited the proliferation and invasion, and induced the apoptosis in the two cell lines. Our data suggested that TFF3 stimulated an invasive phenotype in cervical cancer cells through STAT3 mediated repression of CDH1. Furthermore, we found TFF3 decreased the sensitivity of cervical cancer cells to etoposide by increasing P-gp functional activity in the two cell lines. TFF3 silencing increased the sensitivity of the two cell lines to etoposide chemotherapy.
TFF3, behaved as an oncogene, promotes cancer cell proliferation, survival, oncogenicity and invasion in various cancers, such as mammary carcinoma, gastric cancer and prostate carcinoma [
7‐
9]. For the first time, we found that TFF3 was overexpressed in cervical cancer cells. Elevated expression level of TFF3 has also been reported in the molecular apocrine subtype of estrogen receptor-negative mammary carcinoma characterized by the expression of AR, FOXA1 and a high frequency of HER2 expression [
12,
13]. In SiHa and Hela cells, forced expression of TFF3 promoted cervical cancer cells growth, proliferation and invasion. Overexpression of TFF3 was caused changes in mRNA levels associated with the cellular proliferation, apoptosis, migration, invasion and clonogenic survival. Forced expression of TFF3 decreased mRNA expression of BAX, TIMP2, CDKN2A, SERPINB5 and CDH1, but increased mRNA levels of CDH2, VIM, TGFB1, TERT, SERPINE1, TWIST, KI67, SURVIVIN, MMP2 and MMP3 which closely correlated with increasing cell cycle progression, anti-apoptosis, proliferation, metastasis and invasion of cervical cancer cells [
9,
10,
28,
29].
In the cervix cells, TFF3 expression was detected significantly higher level in cervical cancer cells than in human non-tumor keratinocytes. The results presented here clearly demonstrated that TFF3 overexpression accelerated cell cycle progression and a decrease in TFF3 levels slowed the progression of cells. In addition, TFF3 levels correlated with the proliferative potential of cervical cancer cells as revealed by correlation between TFF3 and Ki67 levels in vivo. As an oncogene, TFF3 is qualified with various functions that could impinge on normal cell proliferation. It is known that TFF3 induces the expressions of AR, FOXA1, HER2 and basic fibroblast growth factor (bFGF) in vitro in various cancers such as breast cancer and melanoma cells [
12,
13,
30]. Our study revealed that overexpression of TFF3 had no effect on apoptosis in SiHa and Hela cells. But siRNA-mediated depletion of TFF3 induced the apoptosis of cervical cancer cells by decreasing anti-apoptotic protein, Bcl-2 and increasing pro-apoptotic protein, Bax. The ratio of anti-apoptotic and pro-apoptotic members within the Bcl-2 family plays an important role to determine cell fate [
31‐
33].
It is well known that TFF3 is associated with invasion and metastasis which plays very important roles in progression of tumors. It was first discovered TFF3 might regulate migration via a Twist-dependent pathway in gastric cells [
6], which was an indispensable step in the process of cell invasion. TFF3 participated in cancer invasion metastasis in breast cancer through repression of CDH1 mediated by STAT3 [
7]. CDH1, as a tumor suppressor glyco-protein, is one of the major constituents of cell adhesion complexes and mediates calcium-dependent cellular interactions in epithelial cells, which plays a key role in the establishment of adherent type junctions [
34,
35]. Loss of CDH1 expression, or CDH1 dysfunction, contributes to the loss of cell–cell interaction which stimulates cancer cells to gain an invasive cell phenotype and metastasis [
36‐
38]. By two-dimensional Matrigel Transwell analysis we found TFF3 was critical for cervical cancers to involve invasion. Moreover, we observed that TFF3 repressed the expression of CDH1 to promote cell invasion in cervical cancer cells. Western blot analysis showed that siRNA against TFF3 increased the expression of CDH1 and decreased phosphorylation of STAT3 which up-regulated the expression of CDH1. Furthermore, up-regulated CDH1 via overexpression of TFF3 was significantly down-regulated by virtue of inhibitor of p-STAT3, JSI-124, which was similar to those reported by Pandey [
7]. Our results suggested that TFF3 stimulates invasion of cervical cancer cells probably by, at least partially, activating the STAT3/CDH1 signaling pathway. CDH1 is a downstream protein of TFF3 and may be a key modulator of TFF3-mediated cervical cancer invasion.
Surgery combined with chemotherapy or radiotherapy is still the optimal treatment for cervical cancer while MDR causes the cervical cancer cells to be resistant to chemotherapeutic drugs resulting in chemotherapy failure [
39‐
41]. Previously study showed that up-regulation of TFF3 after rectal cancer chemo-radiotherapy is an adverse prognostic factor. The physiological role of TFF3 in restoring the mucosa during neo-adjuvant chemotherapy could be interfering with treatment efficacy by increase neo-adjuvant chemotherapy resistance [
9]. In this study, we showed that TFF3 decreased the sensitivity of cervical cancer cells to etoposide by increasing P-gp functional activity and had no effect on the expression of P-gp. Drug resistance to chemotherapy is mediated mainly by the overexpression of P-gp which is a phosphorylated transmembrane glycoprotein pump with ATP enzyme activity encoded by mdr-1 gene [
42,
43]. P-gp exerts an impact on drug distribution by pumping a substantial amount of compounds from intracellular to extracellular sites, especially hydrated cation compounds. In addition, overexpression of TFF3 decreased the sensitivity of cancer cells to chemotherapy by mediating Bcl-2 [
44,
45].
Authors’ contributions
ZHY, DDC and YMW designed and performed experiments and analyzed data. DDC, XJC and HKY performed experiments and analyzed data. ZHY, XJC and CSC reviewed all data, prepared the figures, and wrote the manuscript. All authors read and approved the final manuscript.