T-box 3 overexpression is associated with poor prognosis of non-small cell lung cancer

  • Authors:
    • Yueming Wu
    • Jiang Feng
    • Weiwei Hu
    • Yawei Zhang
  • View Affiliations

  • Published online on: March 13, 2017     https://doi.org/10.3892/ol.2017.5855
  • Pages: 3335-3341
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Abstract

T-box 3 (Tbx3), a member of the T-box transcription factor family, serves a crucial role in embryonic development and cancer progression. Previous studies have demonstrated the clinical significance of Tbx3 in numerous types of cancer. However, the expression level and pathological function of Tbx3 in non‑small cell lung cancer (NSCLC) are unknown. To the best of our knowledge, the present study provided the first evidence demonstrating the clinicopathological significance of Tbx3 in NSCLC. Tbx3 was revealed to be overexpressed in NSCLC cell lines and tissues obtained from patients with NSCLC by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Downregulation of Tbx3 by Tbx3‑specific short hairpin RNA decreased cell proliferation in NSCLC cell lines, but there was a slight increase in the cell population of the G1 phase. Furthermore, depletion of Tbx3 expression significantly decreased the cell migration distance. In addition, overexpression of Tbx3 was notably associated with tumor size, tobacco smoking status, tumor‑node‑metastasis stage and differentiation. These results demonstrated the importance of Tbx3 in the pathological progression of NSCLC and may serve as a potential therapeutic target for NSCLC.

Introduction

Lung cancer may be classified into two main groups, small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) (1). Epidemiological data revealed that tobacco smoke exposure is the leading risk factor of lung cancer and NSCLC accounts for 85% of lung cancer cases (2,3). Despite encouraging progress made in the early detection and therapeutic strategies, lung cancer remains one of the most common malignancies worldwide, with a poor prognosis of NSCLC patients and a relative low 5-year survival rate (3). Therefore, novel prognostic biomarkers should be identified and applied in combination with current staging systems to improve the prognostic evaluation and clinical management of patients with NSCLC.

T-box transcription factors are highly evolutionarily conserved and serve critical roles in a variety of cellular biological events (4). T-box 3 (Tbx3), a member of the T-box transcription factor family, is widely expressed in the nervous system, heart, lungs and pancreas (4). Accumulating evidence has demonstrated that Tbx3 is involved in the developmental process by binding to the T-box motif with a 20–24 nucleotide palindromic sequence, the T-site, or half of the sequence in the promoters of target genes (5). A mutation of Tbx3 was demonstrated to be closely associated with the pluripotency of embryonic stem cells and the invasiveness of cancer (6). In resectable pancreatic carcinoma (7), melanoma (8), bladder cancer (9) and colorectal cancer (10), Tbx3 was revealed to be overexpressed and associated with poor prognosis of cancer. Recently, Tbx3 was reported to serve a key role in melanoma migration and invasion by acting as a key substrate of protein kinase B and thus inducing the repression of E-cadherin (8). Additionally, Tbx3 is involved in the anti-proliferative event mediated by the transforming growth factor β1 signaling pathway by repressing the oncogenic Tbx2 in human breast epithelial MCF-12A cells (11).

The expression of Tbx3 in lung tissue under normal physiological conditions has previously been established (12). However, to the best of our knowledge, the expression status of Tbx3 in patients with NSCLC has not been investigated to date. In order to understand the role of Tbx3 in cancer and to improve the poor prognosis of NSCLC, further investigation into the expression status and clinical significance of Tbx3 in NSCLC is required. The present study evaluated Tbx3 expression at the messenger RNA (mRNA) and protein levels in NSCLC tissue samples, and analyzed the correlations between Tbx3 expression and various clinicopathological parameters. Finally, the present study aimed to explore the association between Tbx3 overexpression and the prognosis of NSCLC.

Materials and methods

Patients and sample collections

A total of 86 patients with NSCLC (46 males and 40 females) were enrolled in the present study. All patients underwent treatment at the People's Hospital of Dongyang City (Dongyang, China) between August 2007 and October 2010. The stage of cancer of patients was classified according to the tumor-node-metastasis (TNM) staging system. The tumor tissues and matched adjacent normal tissues were frozen in liquid nitrogen following surgical resection and preserved at −80°C prior to use. Clinicopathological data were collected, including age, gender, tumor size, tobacco smoking status, alcohol drinking status, TNM stage and differentiation. Patients characteristics are summarized in Table I. The present study was approved by the ethics committee of The People's Hospital of Dongyang City. Written informed consent was obtained from all patients prior to enrollment in the present study.

Table I.

Correlation of Tbx3 expression level with the clinicopathological features of patients with non-small cell lung cancer.

Table I.

Correlation of Tbx3 expression level with the clinicopathological features of patients with non-small cell lung cancer.

Tbx3 expression level

VariableCases, nHighLowP-value
Gender
  Male4630160.116
  Female4031  9
Age, years
  ≥505133180.146
  <503528  7
Tumor size, cm
  ≥54532130.035
  <5412912
Tobacco smoking status
  Non-smoker3828100.036
  Smoker483315
Alcohol drinking status
  Non-drinker4933160.062
  Drinker3728  9
Differentiation
  Poor4632140.014
  Well/moderate402911
Tumor stage
  I–II4230120.002
  III–IV443113

[i] Tbx3, T-box 3.

Cell lines and cell culture

The A549 and NCI-H460 human NSCLC cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). For cell culture, Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 U/ml streptomycin were used, and a 100% humidified atmosphere was maintained (37°C, 5% CO2).

Tbx3 depletion in A549 and NCI-H460 cells

The sequences of Tbx3-specific short hairpin RNA (shRNA) and control shRNA were described in a previous study (13) and obtained from Invitrogen (Thermo Fisher, Scientific, Inc.). A549 and NCI-H460 cell lines stably expressing Tbx3 shRNA were synthesized by retroviral transduction of the pSUPER.Retro.Puro (Addgene, Inc., Cambridge, MA, USA) vector encoding shRNA against Tbx3, followed by selection in 1 µg/ml puromycin.

Cell proliferation assay

The MTT assay was performed to evaluate the level of cell proliferation. Following transfection, cells with Tbx3-specific shRNA or control shRNA were seeded in 96-well plates at a density of 4,000 cells/well and incubated under the aforementioned conditions (37°C, 5% CO2). Following 12, 24, 48 and 72 h culture, 20 µl of 5 mg/ml MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well. The plates were incubated for a further 4 h under the same conditions (37°C, 5% CO2), and subsequently 150 µl of dimethylsulfoxide was added until the purple formazan was completely dissolved. The absorbance of each well was determined using the Multiskan® Spectrum (Thermo Fisher Scientific, Inc.) at a wavelength of 490 nm. Each independent experiment was performed three times.

Total RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted from the transfected cells, tumor tissues and matched normal tissues using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol, and first-strand complementary DNA was synthesized from 5 ng total RNA using the QuantiTect Reverse Transcription kit (Qiagen GmbH).

The expression level of Tbx3 in transfected cells, tumor tissues and normal tissues was quantified using SYBR®-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using an ABI-7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and analyzed using ABI SDS software (version 2.0.6; Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used for RT-qPCR were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and listed as follows: Human Tbx3 sense 5′-CCCGAAGAAGACGTAGAAGATGAC-3′ and antisense 5′-CCCGAAGAAGAGGTGGAGGACGAC-3′; human β-actin (ACTB) sense 5′-CCTCCATCGTCCACCGCAAATG-3′ and antisense 5′-TGCTGTCACCTTCACCGTTCCA-3′. The following optimized cycling conditions were used: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 55°C for 15 sec. Each sample was analyzed in triplicate and the mean value was determined. The expression level of Tbx3 was normalized to the endogenous control, ACTB. The relative expression level of Tbx3 was evaluated using the 2−ΔΔCq method (14).

Western blot analysis

Western blotting was performed to assess the expression level of Tbx3 in transfected cells, tumor tissues and normal tissues. Protein concentration was determined using the standard Bradford assay kit (cat. no. P0006; Beyotime Institute of Biotechnology, Haimen, China). Equal amount of total protein was loaded onto a 10% SDS-PAGE gel and blotted onto a nitrocellulose membrane in a humid environment. The membrane was blocked with 5% milk in TBS containing 0.05% Tween 20 (TBST) and incubated with anti-Tbx3 antibody (dilution, 1:2,000; cat no. ab89220; Abcam, Cambridge, UK) at room temperature for 1 h. The anti-β-actin antibody (dilution, 1:2,000; cat. no. ab6276; Abcam) was used as the internal control and incubated at room temperature for 1 h. Subsequently, the membrane was washed three times with TBST and incubated with a secondary anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (dilution, 1:5,000; cat. no. SC-2054; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Blots were developed using the enhanced chemiluminescence method, according to the manufacturer's protocol (Pierce; Thermo Fisher Scientific, Inc.). Autoradiography signals were analyzed using an ImageQuant RT ECL gel imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The gray-scale ratio of Tbx3 to β-actin in each sample was considered as the relative protein expression level. Patients who exhibited Tbx3 expression levels in tumor tissues that was higher compared with adjacent noncancerous tissues were classified into the high Tbx3 expression group. Patients with a lower Tbx3 expression level in tumor tissues compared with the adjacent noncancerous tissues were classified into low Tbx3 expression group.

Flow cytometry for cell cycle assessment

Following transfection of Tbx3-specific shRNA or control shRNA, the A549 and NCI-H460 cells were collected by trypsinization (Sigma-Aldrich; Merck KGaA), washed with PBS and fixed with 70% cold ethanol overnight at −20°C. The ethanol-suspended cells were then collected, washed and stained with propidium iodide (PI) staining buffer (5 µg/ml PI and 0.25 µg/ml RNase) for 30 min in the dark at room temperature. Finally, cell cycle analysis was performed by evaluating the DNA content of control shRNA and Tbx3-specific shRNA-transfected NSCLC cell lines using a FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. The results were analyzed using the ModFit LT software (version 3.2; Verity Software House, Inc., Topsham, ME, USA).

Cell migration assay

Cell migration in culture was determined using a two-dimensional in vitro scratch motility assay, as previously described (15). The A549 and NCI-H460 cell lines transfected with control shRNA or Tbx3-specific shRNA were seeded at 2×106 cells/well in 6-well plates. A linear wound was made by scratching cells with the tip of a p1000 pipette (Axygen; Corning Life Sciences, Tewksbury, MA, USA). To prevent cell proliferation, mitomycin C (Sigma-Aldrich; Merck KGaA) was added at a final concentration of 10 µg/ml. Cells were incubated at 37°C in 5% CO2. The wound widths were measured at the time of the scratching (0 h) and at various time points (2, 4 and 6 h). Images were captured using a BX53 microscope (Olympus Corporation, Tokyo, Japan), and migration distances were measured using AxioVision software version 4.8 (Zeiss GmbH, Jena, Germany). The difference in width represents the distance migrated in µm.

Statistical analysis

All the data collected in the present study were analyzed using SPSS software (version 20.0; IBM SPSS, Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Differences between groups were determined by the χ2 test. The Kaplan-Meier log-rank test was used for survival analysis, whereas prognostic factors were examined by univariate and multivariate analyses (Cox's regression test). P<0.05 was considered to indicate a statistically significant difference.

Results

Tbx3 is highly expressed in NSCLC tissues

In order to evaluate the expression level of Tbx3 in NCLC tissues, RT-qPCR and western blot analysis were performed to quantify the Tbx3 expression levels. As presented in Fig. 1A and B, the mRNA expression level of Tbx3 in normal or tumor tissues was determined. The present study demonstrated that 61/86 patients with NSCLC (70.9%) displayed a significantly higher Tbx3 gene expression level in tumor tissues compared with that in adjacent normal tissues (overexpressed group); thus, in the majority of cases, Tbx3 was overexpressed in patients with NSCLC. The remaining 25 cases displayed lower Tbx3 gene expression level in tumor tissues compared with those in normal tissues (underexpressed group). Furthermore, the results of the western blot analysis verified the conclusions drawn from RT-qPCR. The protein level of Tbx3 in the overexpressed group was significantly higher in tumor tissues compared with that in normal tissues (P<0.001, Fig. 1C and E). Additionally, the expression level of Tbx3 was lower in tumor tissues compared with that in the normal tissues of the underexpressed group (P<0.05; Fig. 1D and F).

Tbx3 overexpression promotes cell proliferation

To evaluate the role of Tbx3 in the process of tumorigenesis, Tbx3-specific shRNA was introduced to the A549 and NCI-H460 NSCLC cell lines with the aim of downregulating the expression level of Tbx3. The gene expression level of Tbx3 in transfected cells was determined by RT-qPCR. As demonstrated in Fig. 2A, the mRNA expression level of Tbx3 in A549 and NCI-H460 cells transfected with Tbx3-specific shRNA was significantly decreased (P<0.05) compared with that in the control shRNA-transfected cells. Western blot analysis confirmed that the level of Tbx3 was significantly downregulated with the transfection of Tbx3-specific shRNA (P<0.05; Fig. 2B). These results indicated that the knockdown of Tbx3 significantly decreased the mRNA and protein expression levels of Tbx3, which suggested the highly regulated control of Tbx3-specific shRNA (P<0.05).

Furthermore, the role of Tbx3 in cell proliferation was assessed by MTT assay. The results demonstrated that the optical densities of A549 or NCI-H460 cells transfected with Tbx3-specific shRNA were significantly lower compared with those of the A549 or NCI-H460 cells with the control shRNA (P<0.05; Fig. 2C), which indicated that Tbx3 overexpression may promote cell proliferation.

Downregulation of Tbx3 affects the distribution of cell cycle proportion

It has been well established that Tbx3 participates in the regulation of the cell cycle (16). In order to investigate the role of Tbx3 in regulating the cell cycle, flow cytometry was performed to determine whether Tbx3 overexpression affects the cell cycle of A549 and NCI-H460 cells. The results (Fig. 2D and E) indicated that the Tbx3-specific shRNA-transfected cells displayed a slight increase in cell population in the G1 growth phase, whereas the proliferation of cells in the S and G2/M phases were decreased compared with those of the control cells. The aforementioned results suggested that the expression level of Tbx3 may affect the cell cycle distribution and lead to the change in cell proliferation.

Tbx3 overexpression promotes cell migration

Investigating the clinicopathological function of Tbx3 is crucial for understanding tumorigenesis. The effect of Tbx3 overexpression on cell migration required further study. Therefore, the present study analyzed the mobility of cells transfected with Tbx3-specific shRNA or control shRNA by performing a two-dimensional scratch assay. As presented in Fig. 2F, the migration distance of cells transfected with Tbx3-specific shRNA was significantly lower (P<0.05) than that of the control shRNA-transfected group at all the selected time points (2, 4 and 6 h). The results presented (Fig. 2F) confirmed that Tbx3 overexpression promoted cell migration in A549 and NCI-H460 cells.

Clinical significance of Tbx3 overexpression in NSCLC

The clinical significance of Tbx3 overexpression in colorectal cancer (10) and pancreatic carcinoma (7) has been revealed in previous studies. Therefore, the clinical significance of Tbx3 expression in NSCLC patients was analyzed in the present study by evaluating the correlation of Tbx3 expression with clinicopathological characteristics. It was demonstrated that Tbx3 expression was significantly associated with tumor size (P=0.035), tobacco smoking status (P=0.036), TNM stage (P=0.002) and differentiation (P=0.014) (Table I). In addition, the 5-year overall survival rates were 13.14 and 49.56% in the overexpressed and underexpressed groups, respectively. Notably, the survival rates of these two groups were significantly different (P=0.046); thus, the patients with high Tbx3 expression levels had decreased 5-year survival rates compared with those with low Tbx3 expression (Fig. 3).

Tbx3 overexpression is associated with the poor prognosis of NSCLC

The association between Tbx3 expression and prognosis in patients with NSCLC was evaluated by univariate and multivariate analyses. Multivariate analysis using Cox's proportional hazards model was performed to analyze whether one factor can be considered as an independent factor for the prognosis of NSCLC patients, focusing on the factors that significantly affect the survival rate of patients with NSCLC obtained from univariate analysis. From the univariate and multivariate analysis results, the same conclusions could be drawn: Tumor differentiation, tumor size, tobacco smoking status, tumor stage and Tbx3 expression level were identified as independent prognosis factors for patients with NSCLC (Table II).

Table II.

Univariate and multivariate analyses of overall survival rate.

Table II.

Univariate and multivariate analyses of overall survival rate.

Univariate analysis Multivariate analysis


VariablesHR95% CIP-valueHR95% CIP-value
Tbx32.1311.022–4.0220.0182.2521.052–4.0520.026
Age0.9080.418–1.4180.5070.9130.421–1.4210.514
Gender1.0030.456–2.4560.3910.9970.453–2.4530.373
Tumor size2.2421.039–4.0390.0301.9170.931–3.9310.034
Tobacco smoking status2.2601.057–4.0570.0292.2291.026–4.0260.031
Alcohol drinking status1.5100.741–3.7410.1641.5880.761–3.7610.108
Differentiation2.3111.094–4.0940.0262.0151.066–4.0660.028
Tumor stage2.5531.177–5.1770.0142.3671.135–4.1350.016

[i] HR, hazard ratio; CI, confidence interval; Tbx3, T-box 3.

Discussion

Each year, ~1.2 million novel NSCLC cases are diagnosed worldwide (2). Despite progress being made in the development of therapeutic strategies for NSCLC (1719), the 5-year overall survival rate remains low (10–15%) (3). One reason for this is the high frequency of tumor metastasis in patients with NSCLC (20). Metastasis is widely accepted as one of the most decisive factors influencing the prognosis of patients with cancer (21). Therefore, it is imperative to identify effective predictive factors for the diagnosis and treatment of NSCLC. As a member of the Tbx2 subfamily, Tbx3 has been confirmed to be implicated in a number of human diseases, particularly cancer (68,10). The overexpression of Tbx3 is present in several cancer subtypes, including colorectal cancer and pancreatic carcinoma (7,10). More importantly, the poor prognosis of cancer is also associated with the overexpression of Tbx3 (7,10). Previous studies have established that Tbx3 serves an important role in the regulation of cell proliferation, cell cycle, cell apoptosis and cell migration in tumor cells (7,8,10).

The expression of Tbx3 in lung tissues has previously been demonstrated (12); however, the role of Tbx3 in the pathogenesis of NSCLC requires further investigation. To the best of our knowledge, the present study evaluated the expression level of Tbx3 in NSCLC and its association with the clinicopathological characteristics of patients with NSCLC for the first time. Tbx3 was revealed to be overexpressed in NSCLC tissues compared with that in adjacent normal tissues by RT-qPCR and western blot analysis. Furthermore, the overexpression of Tbx3 is closely associated with TNM stage, differentiation and recurrence of cancer in patients with NSCLC. As a result, the present study demonstrated that patients with Tbx3 overexpression had a shorter 5-year survival rate compared with that of patients with underexpression of Tbx3. Additionally, the present study identified that Tbx3 overexpression was an independent prognostic biomarker for the prediction of prognosis for patients with NSCLC. Based on these findings, the present study further investigated the mechanisms underlying Tbx3 in tumorigenesis. Firstly, the shRNA transfection strategy was used to downregulate the expression level of Tbx3. RT-qPCR was performed to estimate the relative transcriptional level of Tbx3, and this indicated that the mRNA level of Tbx3 was significantly decreased following the Tbx3-specific shRNA transfection. Furthermore, the effect of Tbx3 expression on cell proliferation was analyzed by comparing the Tbx3-specific shRNA-transfected and control shRNA-transfected cells. The results demonstrated that Tbx3 overexpression promoted cell proliferation. In order to determine whether cell proliferation is affected by cell cycle progression, flow cytometry was performed to analyze the cell population in various phases of the cell cycle. It was demonstrated that the G1 phase was upregulated, whereas the S and G2/M phases were downregulated in the Tbx3-specific shRNA-transfected group compared with the findings in the control shRNA-transfected group. These results indicated that Tbx3 overexpression may promote cell proliferation. Furthermore, the cell migration distance of Tbx3-specific shRNA-transfected cells and control shRNA-transfected cells was evaluated, and the results suggested that Tbx3 overexpression may promote cell migration. This indicated that Tbx3 overexpression serves an important role in tumorigenesis via cell proliferation and cell migration promotion.

In conclusion, the present study demonstrated for the first time that Tbx3 is associated with the progression of NSCLC and may act as an independent prognostic biomarker for patients with NSCLC. Furthermore, the preliminary mechanism of Tbx3 in tumorigenesis was also discussed. Taken together, the results of the present study and previous findings (7,8,10) associated with the role Tbx3 in cancer suggested that Tbx3 may potentially be an effective therapeutic target and prognostic predictor for patients with NSCLC.

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Wu Y, Feng J, Hu W and Zhang Y: T-box 3 overexpression is associated with poor prognosis of non-small cell lung cancer. Oncol Lett 13: 3335-3341, 2017
APA
Wu, Y., Feng, J., Hu, W., & Zhang, Y. (2017). T-box 3 overexpression is associated with poor prognosis of non-small cell lung cancer. Oncology Letters, 13, 3335-3341. https://doi.org/10.3892/ol.2017.5855
MLA
Wu, Y., Feng, J., Hu, W., Zhang, Y."T-box 3 overexpression is associated with poor prognosis of non-small cell lung cancer". Oncology Letters 13.5 (2017): 3335-3341.
Chicago
Wu, Y., Feng, J., Hu, W., Zhang, Y."T-box 3 overexpression is associated with poor prognosis of non-small cell lung cancer". Oncology Letters 13, no. 5 (2017): 3335-3341. https://doi.org/10.3892/ol.2017.5855