Overexpression of HMGA2 promotes tongue cancer metastasis through EMT pathway
- Xiao-Peng Zhao†1, 2,
- Hong Zhang†3,
- Jiu-Yang Jiao1, 2,
- Dong-Xiao Tang1, 2,
- Yu-ling Wu1, 2 and
- Chao-Bin Pan1, 2Email author
© Zhao et al. 2016
Received: 19 June 2015
Accepted: 8 January 2016
Published: 27 January 2016
Metastasis to long distance organs is the main reason leading to morality of tongue squamous cell carcinoma (TSCC); however, the molecular mechanisms are still unknown. High mobility group AT-hook 2 (HMGA2) is highly expressed in multiple metastatic carcinomas, in which it contributes to cancer progression, metastasis and poor prognosis by upregulating Snail expression and inducing epithelial mesenchymal transition (EMT). This study focuses on investigating the role and mechanism of regulation of HMGA2 in the metastasis of TSCC.
HMGA2 mRNA and protein expression were examined in TSCC specimens by quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry (IHC). Western blotting, IHC and immunofluorescence were also used to measure the expression and localization of EMT marker E-Cadherin and Vimentin both in TSCC cells and tissues. Knockdown assay was performed in vitro in TSCC cell lines using small interfering RNAs and the functional assay was carried out to determine the role of HMGA2 in TSCC cell migration and invasion.
TSCC mRNA and protein expression were significantly up-regulated in tumor tissues when compared to adjacent non-tumor tissues, and the overexpression of HMGA2 was closely correlated with lymph nodes metastasis. Clinicopathological analysis indicated that HMGA2 expression was associated with clinical stage (P = 0.001), lymph node metastasis (P = 0.000), histological differentiation (P = 0.002) and survival (P = 0.000). Silencing the HMGA2 expression in Cal27 and UM1 resulted in the inhibition of cell migration and invasion, meanwhile down-regulation of HMGA2 impaired the phenotype of EMT in TSCC cell lines and tissues. The Multivariate survival analysis indicates that HMGA2 can be an independent prognosis biomarker in TSCC.
Our findings demonstrate that HMGA2 promotes TSCC invasion and metastasis; additionally, HMGA2 is an independent prognostic factor which implied that HMGA2 can be a biomarker both for prognosis and therapeutic target of TSCC.
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 . Clinical evidences indicate that metastasis is the most important poor prognostic factors for patient diagnosis with TSCC . 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 . HMGA2 is highly expressed in most malignant epithelial tumors, including breast cancer [13, 14], colorectal cancer , gastric cancer , lung cancer , melanoma , myeloid , oral cancer , ovarian cancer , pancreas cancer , 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 .
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.
Patients and tissue samples
Clinicopathological parameters and HMGA2, Snail1 expression in 60 primary tongue carcinomas
T1 + T2
T3 + T4
I + II
III + IV
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.
RNA extraction, reverse transcription and quantitative real-time PCR (qRT-PCR)
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′.
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.
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.
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).
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 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.
HMGA2 expression is up-regulated in TSCC cells lines
HMGA2 is overexpressed in primary tongue cancer
Overexpression of HMGA2 was associated with a poor prognostic phenotype of TSCCs
To further investigate the clinicopathological and prognostic significance of HMGA2 levels in patients with TSCC, the levels of HMGA2 in a large cohort of 60 TSCC tissues were examined by qRT-PCR and then verified by IHC. Using qRT-PCR, the correlation between HMGA2 expression and metastatic status was analyzed in 60 TSCC samples. The result showed that, 39/60 (65 %) TSCC had much higher expression of HMGA2, which was significantly associated with a more aggressive tumor phenotype (P < 0.001, Fig. 2e). To further confirm the verified the results above, IHC was performed in all the 60 TSCC samples. The median value of all 60 TSCC samples was chosen as the cut-off point for separating tumors with negative expression of HMGA2 from positive expression HMGA2 tumors; thus 37/60 (61.7 %) TSCCs had positive expression of HMGA2, while 23/60 (38.3 %) TSCCs had negative expression of HMGA2 (Table 1). Furthermore, as shown in Table 1, HMGA2 expression strongly correlated with clinical stage (P = 0.001), lymph node status (P = 0.000), histological differentiation (P = 0.002) and survival (P = 0.000) in patients with tongue cancer; however, the analysis data indicated that HMGA2 expression was not correlated with age and tumor stage. Taken together, our analyses revealed that the expression of HMGA2 was upregulated during the clinical progression of tongue cancer, indicating that the expression of HMGA2 may promote the progression of tongue cancer.
Association between HMGA2 expression and patient survival
Univariate and multivariate analysis of factors associated with disease-free survival of patients with TSCC
95 % CI
95 % CI
T1 + T2
T3 + T4
I + II
III + IV
Down-regulation HMGA2 expression inhibited tongue cancer cell migration and invasion in vitro
HMGA2 promotes EMT phenotype in tongue cancer cells
Snail is potential involved in tongue cancer EMT activated by HMGA2
To further illustrate the relationship between HMGA2 and Snail, the correlation between HMGA2 and Snail were analyzed by Person analysis and the result indicated that there is a positive correlation between them (R 2 = 0.8876, P < 0.000, Fig. 6c). The clinicopathological and prognostic significance of Snail in tongue cancer is also analyzed by immunohistochemical staining. As shown in Table 1, Snail expression strongly correlated with clinical stage (P = 0.003), lymph node metastasis (P = 0.000), histological differentiation (P = 0.001) and survival (P = 0.001). However, multivariate survival analysis revealed that Snail expression was not an independent prognostic factor (P = 0.97), whereas HMGA2 was (P = 0.042) (Table 2). Collectively, these findings indicate that HMGA2 protein expression, but not Snail protein expression, correlates significantly with the prognosis of patients with tongue cancer.
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 .
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 . 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 . 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.
In sum, overexpression of HMGA2 promotes tongue cancer cell migration and invasion in vitro. In addition, up-regulation of HMGA2 was closely associated with poor prognosis in tongue cancer patients. HMGA2 enhances tongue cancer metastasis and progression via interaction with Snail through EMT signal pathway. Together, our findings suggest that HMGA2 participates in the progression of TSCC via Snail through EMT.
CP conceived and designed the experiments; XZ and HZ performed the experiments; JJ and DT analyzed the data; YW contributed reagents/materials/analysis tools; XZwrote the paper. All authors read and approved the final manuscript.
Our work is supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20130171110095) and Science and Technology Planning Project of Guangdong Province, China (2014A020212625).
The authors declare that they have no competing interests.
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