2.1. Expression of TBX4 in PDAC Tissues and Normal Adjacent Pancreas
Immunohistochemical stain for TBX4 showed cytoplasm and nuclear staining patterns in both malignant and normal pancreatic ductal epithelial cells, but not in acinar cells, islet cells or stromal fibroblasts. Fifteen PDAC tumors showed no TBX4 staining while the other 62 PDAC cases showed TBX4 expression with different staining density. The mean percentage of TBX4 labeling cancer cells to total cancer cells (labeling ratio) in 77 PDAC samples was 38.8% ± 16.4%. None of the normal pancreatic ducts showed a TBX4 labeling ratio of more than 25%. In this study, 48 PDAC cases with a labeling ratio ≥25% were classified as high level of TBX4 expression, wherease those showing a labeling ratio <25% were classified as low level of TBX4 expression. Representative micrographs of PDAC tumors with high and low level of TBX4 expression are shown in .
Representative micrographs showing pancreatic ductal adenocarcinoma (PDAC) with high TBX4 expression with cytoplasmic and nuclear staining (A) and low TBX4 expression (B). Original magnification, ×400.
2.2. Correlation of Clinicopathological Features with TBX4 Expression
shows the relationship between TBX4 expression level and other clinicopathological factors of the study population. TBX4 expression levels showed an inverse correlation with higher grading (P = 0.015). Better differentiated PDAC tumors (Grade 1 and 2) more frequently expressed high level of TBX4 expression (45/48, 93.8%) than their poorly differentiated (Grade 3) counterparts (3/48, 6.2%). Liver metastasis recurrence was more frequently seen in the TBX4-low expressing group than the TBX4-high group (51.7% vs. 20.8%, P = 0.005). No significant association was identified between TBX4 expression and patient’s age, gender, tumor size, margin status, lymphnode metastasis and angiolymphatic invasion.
Clinicopathological features and correlation of TBX4 expression in stage II PDACs.
2.3. Correlation of TBX4 Expression Level and Overall Survival
To further investigate the prognostic significance of TBX4, we first performed univariate analysis to identify factors which might affect the survival of PDAC patients. After surgical resection, the median follow-up time was 25 months. As seen in , overall survival significantly correlated with margin status, lymph node status, liver metastasis recurrence, venous invasion and TBX4 expression levels, but not with age, gender, tumor size, histopathological grading, lymphatic invasion (). Kaplan-Meier analysis revealed that low TBX4 level in PDAC tissues significantly correlated with markedly reduced overall survival (P = 0.010) (). Additionally, we performed multivariate analysis to exclude the confounder effect. Cox proportional hazard model analysis confirmed that low TBX4 expression was an independent predictor for reduced overall survival of PDAC patients ().
Univariate analysis of overall survival in patients with stage II PDACs.
Kaplan–Meier survival curves show that TBX4-low expression PDAC patients had a shorter survival than those with TBX4-high expression. The difference is statistically significant based on the log-rank test (P = 0.010).
2.4. Methylation of Promoter Region CpG Island of TBX4 in PDAC and Normal Pancreas
The promoter region CpG island of the TBX4 gene were analyzed in 7 PDAC tumors (including both high and low expression) and 7 normal adjacent pancreas by bisulfite sequencing. As seen in , the CpG island regions of TBX4 promoter were hypermethylated in both PDAC and normal pancreas tissues, thus indicating that expression of TBX4 in pancreas is less likely to be regulated by DNA methylation.
Figure 3. Methylation status of 18 CpG sites in the TBX4 promoter region (−1842 to −1626) were analyzed in 7 PDAC tissues (T1-7) (3 cases were defined as low level of TBX4 expression, and the other 4 cases were defined as high level of TBX4 expression) (more ...)
TBX4 belongs to the TBX gene family, which encode a group of transcription factors characterized by a highly conserved DNA-binding motif (T-box) and play a key role during organogenesis and pattern formation in both vertebrate and invertebrate embryos. Recent studies suggest that several T-box family members are implicated in the progression of multiple cancers. For example, TBX2 and TBX3 are overexpressed in several cancers including pancreatic cancer, and can contribute to cancer development and progression due to their ability to suppress cellular senescence and E-cadherin expression [10
]. However, different from TBX2 and TBX3, in a recent study, Yu et al
] recently found that loss of TBX5 gene expression in colon cancer tissues and cell lines was associated with promoter methylation. Furthermore, TBX5 methylation was detected in 68% (71/105) of primary colon tumors, and served as a potential biomarker for the prognosis of colon cancer. In a previous proteomic study, it was shown that TBX4 was overexpressed in a proportion of patients, however, the study included only a small sample size [7
]. Our data also confirm previous associations between higher TBX4 expression and better differentiated type of PDAC, and extend previous findings indicating that a low level of TBX4 expression in stage II PDACs was associated with poor overall survival and was an independent prognostic factor for patients with stage II PDACs. These findings suggested that TBX4 may play a role in the progression of pancreatic cancer.
In this study, although a nuclear pattern could be observed, the immunostaining of TBX4 protein was also located in the cytoplasm of cancer cells, which seems inconsistent with its function as a transcription factor. The cytoplasmic expression of TBX factors in cancer has been also identified in several other studies. For example, cytoplasmic overexpression of TBX2 had been identified in breast cancer tissues [12
]. Qi et al
] also identified cytoplasmic TBX4 staining in pancreatic cancer tissues using another TBX4 antibody. Recently, Krcmery et al
. characterized a functional nuclear export signal (NES) with an N-terminal DNA binding domain of all T-box proteins, by which TBX factors could shuttle between nuclear and cytoplasmic cell compartments, suggesting both nuclear and cytoplasmic roles for TBX family members [13
]. Therefore, the above findings suggest that the cytoplasmic expression of TBX4 in pancreatic cancer could be associated with NES-directed protein shuttling; however, its functional roles in carcinogenesis remain unclear.
Among the T-box family members, TBX4 and TBX5 are highly related in their genomic organization and gene function [15
]. Considering the possible role of TBX5 as a tumor suppressor gene in colon cancer, we speculated that loss of the Tbx4 gene might be also epigenetically controlled. However, in this study, in the CpG islands known to regulate TBX4 expression, we found that these regions were hypermethylated in both PDAC and matched normal pancreas controls through bisulfate sequencing, thus indicating that different from TBX5 expression in colon cancer, DNA methylation is less likely to regulate TBX4 expression in pancreas. The discrepancy may lie in the different patterns of gene regulation of TBX factors in different organs. Similarly, Peres et al
. also suggested that TBX2 and TBX3, although highly homologous, have distinct roles in the transformation process of cancers where they are both overexpressed [17
Currently available evidence indicates that TBX5 might exert its inhibitory action on cancer cells by inducing cancer cell apoptosis by targeting the extrinsic pathway, apoptotic Bax and Granzyme A signaling cascades, up-regulating the expression of a critical cell cycle inhibitor CDKN2A and an anti-metastatic gene MTSS1, or inhibiting the expression of oncogene SBCG and pro-metastatic gene MTA2 [9
]. However, until now the function of TBX4 is still not well understood. Although Tbx4 might share similar molecular mechanisms with TBX5, further works need to be done to clarify its definite function in cancer development.
Pancreatic cancer represents a devastating malignancy characterized by an extremely poor prognosis, rapid progression and resistance to chemo-radiotherapy. The majority of PDAC patients with surgically resectable cancer are at stage II disease, and identifying novel tumor biomarkers may lead to better prognosis prediction and more effective treatment options for this group of patients. In this study, we have reported for the first time a significant difference in the clinical outcome among stage II patents with different TBX4 expression levels. Our data suggests that TBX4 expression level might be a useful clinical predictive factor in PDAC patients undergoing surgically resection.
This present cohort study is limited by its moderate sample size and retrospective design; therefore, further larger, multi-center and prospective clinical trials are needed to validate the clinicopathological and prognostic values of TBX4 in PDAC in the future. Moreover, the exact mechanism of TBX4 in pancreatic carcinogenesis also deserves further investigation.