Background
Tongue squamous cell carcinoma (TSCC) is one of the most common and lethal oral cancer [
1,
2], which is characterized by its preferring of lymph node and distant metastasis [
3]. Clinical evidences indicate that metastasis is the most important poor prognostic factors for patient diagnosis with TSCC [
4]. Despite its significance and the enormous studies accumulated in the past decades on the molecular mechanisms of TSCC progression, little is known about the underlying molecular mechanisms regulating metastatic dissemination.
More and more studies demonstrated that epithelial mesenchymal transition (EMT) is a key process which has been shown to be of critical biological function and significance during embryogenesis and carcinogenesis [
5‐
7]. Increasing evidences have recognized that the epithelial to mesenchymal transition (EMT), a driver of invasion and metastasis of cancer, may play a pivotal role in multiple types of tumor cell metastatic dissemination by endowing cells with a more motile, invasive potential [
8‐
11].
High mobility group 2 (HMGA2) is a chromatin remodeling factor which can change the chromatin architecture to activate or impair the activity of transcriptional enhancers [
12]. HMGA2 is highly expressed in most malignant epithelial tumors, including breast cancer [
13,
14], colorectal cancer [
15], gastric cancer [
16], lung cancer [
17], melanoma [
18], myeloid [
19], oral cancer [
20], ovarian cancer [
21], pancreas cancer [
22], pituitary adenomas [
23,
24]. HMGA2 overexpression in transgenic mice causes tumorigenesis; however, HMGA2-knockout in mice can severely impair the mice growth and development, leading a nanous shape [
25].
Despite the fact that both the HMGA2 and EMT play a significant role in the development and progression of TSCC, the relationship between these factors has not yet been reported in TSCC. In the present study, we demonstrate that overexpression of HMGA2 is closely associated with progression and poorer overall survival in human TSCC, and provide evidence that the expression of HMGA2 can promote the progression of TSCC by upregulating Snail and inducing the EMT.
Methods
Patients and tissue samples
A total of 60 human TSCC tissues and 20 adjacent non-tumor tissue samples were examined in this study. The patients were histopathologically and clinically diagnosed at Sun Yat-sen Memorial Hospital, Sun Yat-sen University from 2008 to 2010; the pathological diagnosis was verified for each case. For each case, tumor samples with matched adjacent non-tumor tissue samples were collected during surgical resection and frozen in liquid nitrogen and stored at −80 °C. Sample collection was performed in accordance with the policies of the National Research Ethics Committee and informed consent was obtained from each patient. The clinicopathological features of the patients are summarized in Table
1.
Table 1
Clinicopathological parameters and HMGA2, Snail1 expression in 60 primary tongue carcinomas
Age | | | 0.863 | | 0.134 |
≤55 | 34 | 22 (64.7) | | 17 (50.0) | |
>55 | 26 | 15 (57.7) | | 8 (30.8) | |
Sex | | | 0.005 | | 0.394 |
Female | 23 | 9 (39.1) | | 8 (34.8) | |
Male | 37 | 28 (75.7) | | 17 (45.9) | |
T stage | | | 0.074 | | 0.895 |
T1 + T2 | 33 | 17 (63.6) | | 14 (45.5) | |
T3 + T4 | 27 | 20 (81.5) | | 11 (59.3) | |
Clinical stage | | | 0.001 | | 0.003 |
I + II | 23 | 8 (34.8) | | 4 (17.4) | |
III + IV | 37 | 29 (78.4) | | 21 (56.8) | |
N status | | | 0.000 | | 0.000 |
N−
| 35 | 14 (40.0) | | 6 (17.1) | |
N+
| 25 | 23 (92.0) | | 19 (76.0) | |
Histological differentiation | | | 0.002 | | 0.001 |
Well | 24 | 9 (37.5) | | 4 (16.7) | |
Moderate/poor | 36 | 28 (77.8) | | 21 (58.3) | |
Survival | | | 0.000 | | 0.001 |
Survival | 32 | 11 (34.4) | | 7 (21.9) | |
Die | 28 | 26 (92.9) | | 18 (64.3) | |
Cell lines and cell cultures
The human TSCC cell Cal27, SCC9, SCC15, SCC25 and UM1 were used in our study. Cal27, SCC9, SCC15 and SCC25 cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and UM1 was reserved by our lab. Cal27 cells were maintained in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) and other cells were cultured in RPMI-1640 medium supplemented with 10 % FBS. For all TSCC cell lines, 1 % penicillin/streptomycin was added to the culture medium and all TSCC cell lines were cultured at 37 °C in a humidified atmosphere containing 5 % CO2.
For total RNA isolation, tumor specimens were finely minced with scissors and homogenized, then, the total RNA from fresh surgical tongue tissues and TSCC cells were extracted using the TRIzol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized with the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) primed with random hexamers. For amplification of HMGA2, reverse transcription PCR was programmed as follows: 95 °C for 2 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 45 s, 72 °C for 10 min, hold at 4 °C. The primer was as followed: HMGA2 forwared: 5′-AAGTTGTTCAGAAGAAGCCTGCTCA-3′; HMGA2 reverse: 5′-TGGAAAGACCATGGCAATACAGAAT-3′. RT-PCR products were analyzed via 2.0 % agarose gel electrophoresis and stained with ethidium bromide for visualization using ultraviolet light. Real-time PCR was performed with LightCycler Real Time PCR System (Roche Diagnostics, Switzerland) and the primer sequences for HMGA2 was used as followed: (F) 5′-AAAGCAGCTCAAAAGAAA GCA-3′; (R) 5′-TGTTGTGGCCATTTCCTAGGT-3′.
RNA interference
Short interfering RNA (siRNA) against HMGA2 and corresponding GFP siRNA (GFP-si) were synthesized and purchased from GenePharma Company (GenePharma, Shanghai, China). The two siRNAs specific against HMGA2 sequences were as followed: HMGA2-siRNA1: CACAACAAGUCGUUCAGAA; and HMGA2-siRNA2: AGAGGCAGACCUAGGAAAU. Transfection was performed in 6-well plates using Lipofectamine 2000 (inviztrogen) following the manufacturer’s instructions. The gene silencing efficiency was detected by western blotting after transfection.
Western blotting
Equal amounts of protein extracts were separated using 10 % polyacrylamide SDS gels (SDS–PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia Biotech) and the membranes were probed with antibody against human HMGA2 (1:1000, Cell Signal Technology, Danvers, MA, USA), E-cadherin, vimentin, snail (1:500, Santa Cruz, Santa Cruz, CA, USA), or GAPDH (1:3000, Proteintech, Chicago, IL, USA), and then with peroxidase-conjugated secondary antibody (1:3000, Proteintech) and the signals were visualised by enhanced chemiluminescence kit (GE, Fairfield, CT, USA) according to the manufacturer’s instructions. Anti-GAPDH antibody (Proteintech) was used as a loading control.
Modified boyden chamber assay
A total of 1 × 105 cells were plated into the upper chamber of a polycarbonate transwell filter chamber (Corning, New York, NY, USA) and incubated for 10 h. For invasion assay, the upper chamber was coated with Matrigel (R&D, Minneapolis, MN, USA) and incubated for 24 h. The non-invading cells were gently removed with a soft cotton swab, and the cells that had invaded to the bottom chamber were fixed, stained, photographed and counted.
Immunofluorescence analysis
Cells were seeded on glass coverslips, cultured, fixed and subjected to immunofluorescent analysis by incubation overnight at 4 °C with antibodies against E-cadherin or vimentin (1:100, Santa Cruz, Santa Cruz, CA, USA). After washing several times, the cells were incubated with Alexa Fluor 594-conjugated secondary antibodies (1:500, Invitrogen, USA) for 1 h at room temperature, then the cells were counterstained with DAPI and imaged by confocal laser-scanning microscopy (LSM710, Carl Zeiss, Thornwood, NY).
Immunohistochemistry
Immunohistochemical analysis was performed to investigate the expression of HMGA2, Snail, E-Cadherin and Vimentin in different grades of human tongue cancer. Briefly, immunohistochemistry (IHC) was performed on the paraffin-embedded human tongue cancer tissue sections. Antigen retrieval was performed in a pressure cooker in citrate solution, pH 6.0, for 15 min, followed by treatment with 3 % hydrogen peroxide for 5 min. Specimens were incubated with antibodies as followed: goat monoclonal antibodies against HMGA2 (1:100, CST), E-cadherin, vimentin, snail (1:100, Santa Cruz, Santa Cruz, CA, USA). For the negative controls, isotype-matched antibodies were applied. The tissue sections were observed under a Zeiss AX10-Imager A1 microscope (Carl Zeiss, Thornwood, NY) and all images were captured using AxioVision 4.7 microscopy software (Carl Zeiss, Thornwood, NY).
Statistical analysis
Statistical analysis was performed using a SPSS software package (SPSS Standard version 18.0, SPSS Inc). (SPSS, Chicago, IL, USA) Differences between variables were assessed by the χ2 test according to Pearson or Fisher’s exact test. For survival analysis, we analysed all patients with TSCC by Kaplane–Meier analysis. A log rank test was used to compare different survival curves. Multivariate survival analysis was performed on all parameters that were found to be significant in univariate analysis using the Cox regression model. Two-tailed Student’s t tests were used to determine statistical significance for all results. P < 0.05 was considered to be statistically significant in all cases.
Discussion
TSCC is a common and considerable threat to human health in the worldwide. Many researchers have explored the underlying mechanisms which may regulate cancer cell progression in TSCC. It is believed that metastasis is an essential feature of cancer and contributes to the majority of cancer-related deaths in humans and several signal pathways are involved in this procession, including EMT [
27,
32,
33]. Epithelial–mesenchymal transition (EMT) is a process whereby tumor cells lose the epithelial features to acquire a mesenchymal phenotype and become motile and invasive, which is closely associated with metastasis [
27,
34].
It has been reported that tumor cells can dedifferentiate to obtain the capability to migrate and invade, endowing cancer cells to disseminate from the primary tumor to distant organs, via triggering specific genes expression which associated with EMT signal pathway. Meanwhile, EMT is closely regulated by several signal pathways and involves regulation networks of transcription factors, such as Snail, ZEB and Twist family which regulate expression of E-cadherin, which is a major suppressor of tumor invasiveness and transcriptionally repressed during the EMT [
35‐
37].
HMGA2 is one of the members of the high-mobility group A (HMGA) family which binds to DNA sequences to orchestrate transcription activity by modulating chromatin structure. Besides, HMGA2 is frequently highly expressed in undifferentiated cells during embryogenesis, but silenced in most of the normal adult tissues [
21,
38]. So, HMGA2 rarely can be detected in normal adult tissues but is usually reactivated in a variety of benign and malignant tumors. Furthermore, highly expression of HMGA2 has been correlated with cancer proliferation, increased metastasis and poor prognosis in multiple types of cancer [
4].
It has been described that up-regulation of HMGA2 can activate the Snail, Twist and ZEB families expression and induce EMT process, which leads to tumor metastasis in various cancers [
14]. In this study, our results are consistent with numerous prior studies that HMGA2 is up-regulated both in TSCC cell lines and tissues; the high level expression of HMGA2 can activate the EMT process by repressing E-cadherin expression and the up-regulating of HMGA2 is closely associated with metastasis and poor prognosis in tongue squamous cell carcinoma. Meanwhile, previous researches have implied that Smad, TGF-β canonical pathway and NF-κB signal pathway also contribute to EMT procession through associating with HMGA2 [
26,
39,
40]. We show that the overexpression of HMGA2 can up-regulate Snail expression level and activate EMT, leading to poor clinical stage (
P = 0.001), lymph node status (
P = 0.000), poor histological differentiation (
P = 0.002) and short survival (
P = 0.000) in patients with tongue cancer. Interestingly, although Snail play a pivotal role in the regulating of EMT, multivariate survival analysis shown that Snail expression was not an independent prognostic factor (
P = 0.97), whereas HMGA2 was (
P = 0.042), implying that HMGA2 may be an independent prognosis biomarker in the tongue squamous cell carcinoma.
MicroRNA can regulate gene expression by binding to the 3′-untranslational region (3′-UTR) to degrade the target genes expression. In previous studies, HMGA2 was identified as the target gene of several microRNAs, such as
Let-
7, which is considered to be a tumor suppressor gene in multiple types of cancer [
41‐
44]. Several researches have revealed a new function and mechanism of HMGA2 as a competing endogenous to promote lung cancer progression [
17,
45].
Lymph node metastasis is the predominant invasive site of TSCC and predicted a poor prognosis [
31]. Our results show that overexpression of HMGA2 is closely associated with lymph node metastasis and immunohistochemical staining indicate that both HMGA2 and Snail are upregulated and co-localized in the nuclear. Correlation analysis also confirms that there is a positive correlation between them, implying the promoting cooperation during the TSCC progression.
In summary, our study demonstrated that HMGA2 was upregulated and positively associated with the overall survival, clinical stage, T classification and N classification. Moreover, HMGA2 expression is positively correlated with Snail expression in TSCC patients, implying the interaction between each other. In addition, knockdown of HMGA2 expression can severely impair tongue cancer cells migration, invasion and EMT process. This study suggests that HMGA2 may play a pivotal role in tumor metastasis and can be a novel diagnostic marker and potential therapeutic target in TSCC.