ESCC is the seventh most common cancer and the sixth leading cause of cancer death globally[1]. Despite advances in the diagnosis and surgical treatment of the disease during the past several decades, the prognosis for patients remains disappointing. Therefore, finding new molecular biological markers and effective therapeutic targets for ESCC are urgently required.
According to previous studies, All FOXA members play a key role in esophagus cancer, and the FOXA3’s effect on esophagus cancer seems to be inconsistent with the results of our preliminary experiments which the expression of FOXA3 mRNA is down-regulated in human ESCC tissues (n = 11) by using RT-PCR.
In this study, the expression level of FOXA3 in ESCC tissues (n = 93) were examined using immunohistochemistry. Subsequently, we investigated the correlation of FOXA3 with clinicopathologic characteristics of ESCC patients. Kaplan–Meier survival analysis indicated that ESCC patients with low FOXA3 expression in general had worse prognosis than those with high FOXA3 expression. Univariate and multivariate Cox regression analysis indicated that low FOXA3 expression was an independent risk factor for the prognosis of ESCC patients. The clinical data also showed that low level of FOXA3 was correlated with lymph node metastasis.
To further explore whether FOXA3 is involved in the development of ESCC as a tumor suppressor factor, we used CCK8 assay, colony formation assay and EdU assay to test the proliferation ability of ESCC, and the results all confirmed that FOXA3 overexpression could indeed inhibit the proliferation of ESCC cells effectively. Subsequently, the Eca-109 cells were injected subcutaneously into nude mice. And it was shown that the average weight of implanted tumor in FOXA3 overexpression group is significantly lighter than control group which proved that FOXA3 could also inhibit tumor growth in vivo.
Since the 93 cases we chosen were all post-operation patients, there was no distant metastasis before surgery, we still found that lower expression of FOXA3 was closely related to lymph node metastasis, thus we speculate that FOXA3 can not only inhibit tumor proliferation, but also affect tumor metastasis. Tumor metastasis itself is the biggest cause of death in cancer patients and is an extremely complex process involving multiple steps[22, 23]. Furthermore, the ability of cell invasion and migration is considered the most crucial to tumor metastasis[24]. The results of wound healing assay and Transwell migration/invasion assay indicated that the ability of invasion and migration in ESCC cell was either significantly reduced after overexpression of FOXA3 .
Taking these results together, FOXA3 may exert tumor suppressive roles in ESCC via the inhibition of cell proliferation and invasion activities. However, intriguingly, it is previously reported FOXA3 is highly expressed in esophageal cancer and is closely related to the metastasis of esophageal cancer[21]. To analyze that, it's worth noting that the esophageal cancer case data in the GSE6059, GSE13898, and Oncomine datasets chosen in the above study did not distinguish the specific pathological type of esophageal cancer, and 96 pairs of surgically removed tumor tissues also covered multiple pathological types involving not only ESCC, but also undifferentiated carcinoma, and Siewert type I gastroesophageal junction adenocarcinoma. Furthermore, the specific number of each pathological type was not mentioned. Since ESCC and EAC are obviously two different diseases according to their oncogenic characteristics and all tissue specimens we chose for our study are all ESCC, it is understandable that the two studies have inconsistent results.
The limitation of this study is that there was only subcutaneous tumorigenesis assay was done on nude mice and the effect of FOXA3 on tumor metastasis in vivo have not been studied yet. The lack of research on the mechanism of how FOXA3 is involved in the process of tumor proliferation and metastasis is another disadvantage which may also partly explain the inconsistency with the results of previous studies.
The specific role of FOXA3 maybe controversial, but there is no doubt that FOXA3 is quite crucial for the development and progression of ESCC, and FOXA3 is expected to become a molecular biomarker, a prognostic indicator and an effective therapeutic target for ESCC. Therefore, in the next step, animal models of tumor metastasis need to be constructed to clarify the role of FOXA3 on metastasis in vivo, and further research should be carried out to seek the signaling pathways and molecular networks involved in the function of FOXA3 in ESCC.