value of ≤0.05 was considered to indicate a statistically significant difference (*p<0.05,
**p<0.01 and ***p<0.001).
五、結果與討論
1. The ProT expression pattern in clinical lung cancer specimen
We collected lung cancer specimens at different cancer stages and measured the expression patterns of ProT by immunostaining. As shown in Figure 1, the expression of ProT was significantly higher in tumor at early stage (stage I-II) and also the heavy staining of ProT was shown in nucleus. However, with the process of disease progression, ProT expression was decreased and its nuclear expression was even lost.
Next, we assayed the ProT expression patterns in clinical lung cancer specimen using GEPIA database. GEPIA(Gene Expression Profiling Interactive Analysis) is an online tool for gene expression analysis between normal and tumor data from The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx). As shown in Figure 2A, the expression of ProT in lung adenocarcinoma(LUAD) and lung squamous cell carcinoma(LUSC) was elevated compared with adjacent lung tissues. However, in clinical stage analysis, ProT expression was not higher in patients with clinical Stage II, Stage III or Stage IV than that in Stage I(Figure 2B and 2C). To further elucidate whether ProT contributed to the overall survival(Os) in lung cancer patients, we analyzed the OS for the ProT by GEPIA. The result (Figure 2D) showed that high level of ProT mRNA was associated with worse OS in lung adenocarcinoma patients.
Those results(Figure 1 and Figure 2) were consistent with previous study that loss of nuclear ProT expression was detected in more malignant or metastatic cancer.
Therefore, we demonstrate that nuclear ProT may play a pivotal role in tumor progression and act to slow or prevent EMT, the mechanisms that promote tumor metastasis.
Figure 1. Immunohistochemical staining for prothymosin α (ProT) expression of lung
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adenocarcinoma tissues. Clinical specimens were collected from patients with lung adenocarcinoma at the Thoracic Division, Department of Surgery, National Cheng Kung University (NCKU) Hospital. Informed consent was obtained from all subjects, and the experimental protocol was approved by the Human Experiment and Ethics Committee, NCKU Hospital. The overexpression of nuclear ProT of stage I and II (a-d) were higher than those in stage III, IV (e-g) and metastatic stage (h).
Figure 2 The ProT expression patterns in clinical lung cancer specimen. Analysis of ProT expression in clinical lung cancer specimen by GEPIA. (A) The level of ProT in lung carcinoma(LUAD) and
To evaluate the effect of nuclear ProT on suppressing tumor metastasis in vivo, we established stable clones of human lung adenocarcinoma A549 cells, overexpressing ProT or GFP which were subcutaneously inoculated into the back of immunodeficient NOD/SCID mice. Tumors were measured externally every 3 or 4 days using vernier calipers and the growth rate of tumor showed no significant difference between two groups which were injected with GFP-overexpressing and ProT-overexpressing A549 cells respectively. By 3 months post injection, the mice were sacrificed to examine for the lung metastasis and tumor were removed for analyzed. Metastatic nodules on lung were detected by H&E staining and it showed that five of the seven mice which inoculated with GFP-overexpressing A549 cells, exhibited lung metastasis. However, only one of the seven mice which inoculated with ProT-overexpressing A549 cells, had a few small nodules on lung and the other six mice did not demonstrate any lung
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metastasis (Figure 3). The results indicate that ProT overexpression in cancer cells markedly inhibits metastasis in vivo.
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Figure 3. ProT-overexpressing A549 cells exhibit lower metastatic potential in vivo.
A549 cells transducing with lentiviral vectors encoding ProT or GFP (as a control) were subcutaneously inoculated into the backs of NOD/SCID mice (n=7 in each group, 4x106 cells per mouse). Mice were sacrificed after 3 months post injection. (A) Tumor volume was measured every 3 to 4 days starting at day 25 after inoculated, which showed no significant difference between two groups. (B,C) Histological analyses of lung in the in vivo tumor metastasis assay. The analyses were carried out using H&E staining and lung images showed the lung nodules. Arrowheads indicate the nodules.
(B) Inoculated with GFP-overexpressing A549 cells, five of the seven mice showed lung metastasis. (C) Inoculated with ProT-overexpressing A549 cells, only one of the seven mice showed lung metastasis.
3. High expression of ProT inhibits EMT-associated transcription factors expression by interrupting TGF-β signaling
To assess the inhibitory effect of ProT on repressing invasion and migration ability in lung cancer cells, we generated A549 cells with constitutive overexpression of ProT by lentivirus vector. To investigate that ProT could inhibit TGF-β-induced EMT, western blotting showed that TGF-β1 treatment caused a decrease in epithelial marker (E-cadherin) and increase in mesenchymal markers (N-cadherin, Snail and Twist) in A549 cells.
However, ProT-overexpressing A549 cells rescused E-cadherin expression and also reversed N-cadherin, Snail and Twist expression in the presence of TGF-β1 (Fig. 4).
Taken together, overexpression of ProT attenuates the effect of TGF-β-induced EMT in A549 cells, implying that ProT may function as an antagonist of TGF-β signaling.
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4. Overexpression of ProT inhibits migration and invasion abilities
To assess the inhibitory effect of ProT on repressing invasion and migration ability in lung cancer cells, we generated A549 cells with constitutive overexpression of ProT by lentivirus vector and performed transwell assay (Fig. 5A) and wound healing assay (Fig.
5B). Cells were treated with TGF-β1, which promoted the migratory ability of A549 cells.
We therefore assessed the capability for migration of A549 cells with TGF-β1 pre-treatment for 24 hours and performed migration assays. Both transwell assay and wound healing assay showed that ProT overexpression in A549 cells were significantly decreased migration and invasion capacity compared to control A549 cells in the present of TGF-β1.
Figure 5. Overexpression of ProT inhibits migration and invasion abilities. After treating with TGF-β1 (10 ng/ml) for 24 hours, the effect of ProT on TGF-β-regulated cell invasion and migration were measured by transwell assay (A) and Wound healing analysis (B). Cell invasion and migration were decreased in ProT-overexpressing A549 cells compared with parental and control-LV-GFP.
Figure 4. Immunoblot assay for EMT markers expression in A549 cells.
Western blotting analysis for EMT-associated markers expression from the cell lysates isolated from parental A549 or A549 cells those transduced with lentiviral vectors expressing ProT or GFP alone treated with or without TGF-β1 (10 ng/ml) for 24 h. ProT overexpression downregulated N-cadherin, Snail and Twist expression and upregulated E-cadherin expression, regardless of TGF-β1 treatment.
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5. Correlation of nuclear ProT, Smad7 and Snail expression in human lung patients
We initially identified that the decrease in nuclear ProT expression was associated with disease progression, particularly seen in metastatic lung tumors (Fig. 1). The expression of Sma7 and Snail in these patients was further evaluated by immunostaining. Snail expression was gradually increased from stage I to stage IV tumors with high expression levels at metastatic-stage tumors (Fig.6A). On the other hand, the expression of Smad7 varied during stage I but significantly decreased in stage II and III. Due to the low sample numbers and inevitable variation in metastatic specimens, significant reduction of Smad7 was not observed (Fig. 6B). However, in correlation analysis, a significantly negative correlation was found between the expression of Snail and ProT as well as Snail and Smad7 (Fig. 6C). In summary, our findings demonstrated that the expression of nuclear ProT was associated with EMT-related transcription factor suppression via the regulation of Smad7 protein, which was further corresponded to clinical lung cancer progression.
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Fig. 6. Expression of Snail and Smad7 in lung cancer patients. Immunostaining for (A) Snail and (B) Smad7 expression in tumor specimens from 34 lung cancer patients (stage I=13, stage II=3, stage III=14, stage IV/metastasis(M)=4). Intensity of Snail and Smad7 was analyzed by at least 20 randomly selected fields within the same specimen and quantified. Quantified result is presented as the relative intensity of immunoreactivity comparing with stage I specimens. (C) Quantitative analysis of correlation between Snail and nuclear ProT expression, and also Snail and Smad7 expression. Negative correlations are seen between Snail and ProT (P < 0.0001), and Snail and Smad7 (P = 0.0469).
Our study reveals an interesting insight to the biological function and molecular mechanism of ProT in tumor progression. The loss of nuclear ProT expression might serve as an indicator for tumor metastasis. Increased ProT expression was associated with tumor development but also accompanied with TGF-β signal interruption and EMT suppression. Lung cancer patients presented with a lack of nuclear ProT expression might have a higher risk of developing metastasis.
Targeting ProT as a therapeutic approach still requires more in depth understanding; nevertheless, ProT has shown the potential of serving as a biomarker for cancer diagnosis, prognosis, and monitoring tumor progression.
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106年度專題研究計畫成果彙整表
Our study reveals an interesting insight to the biological function and molecular mechanism of ProT in tumor progression. The loss of nuclear ProT expression might serve as an indicator for tumor metastasis. Increased ProT expression was associated with tumor development but also accompanied with TGF-β signal interruption and EMT suppression. Lung cancer patients presented with a lack of nuclear ProT expression might have a higher risk of developing metastasis. Targeting ProT as a therapeutic approach still requires more in depth understanding; nevertheless, ProT has shown the potential of serving as a biomarker for
cancer diagnosis, prognosis, and monitoring tumor progression.