Members of the Prenylated Rab acceptor domain family (PRAF) are essential for the regulation of many cellular processes [22, 23]. Here we have demonstrated that PRAF3 could induce apoptosis and inhibit migration and invasion of ESCC cells and hence may serve as a tumor suppressor in ESCC.
Although the role of PRAF3 has been studied in several other tumors using tumor cell lines [7, 9, 10, 24], there is a lack of investigation into the relationship between PRAF3 expression and the clinical features of ESCCs. In the present study, we found that the expression level of PRAF3 mRNA and protein in ESCC tissues was significantly lower than that in the matched normal tissues. In addition, we found that down-regulation of PRAF3 expression was significantly correlated with poorly differentiated grading, advanced tumor stage and lymph node metastasis of ESCC. These results would imply that PRAF3 may play an important role in regulating the progression and metastasis of ESCCs, although further in vivo studies are needed to substantiate this conclusion. We noticed that the expression of PRAF3 in normal squamous tissue is not homogeneous and appeared as a decreasing gradient from the differentiating squamous compared to the transit amplifying and stem cell compartment. However, there is a lack of information regarding the role of PRAF3 in the regulation of differentiation of normal squamous cells. In this sense, it would be interesting to see whether down-regulation of PRAF3 expression would result in retarded differentiation of normal squamous cells.
Apoptosis is a form of cellular suicide that closely related to the progression and metastasis of tumor cell . Previous studies showed that PRAF3 serves as an important regulator in cell apoptosis [8, 9, 24]. For example, Zhou et al. demonstrated that the expression of PRAF3 is up-regulated and required for the arsenic trioxide induced tumor cell apoptosis in Hela and MCF-7 . Wang et al showed that PRAF3 is a key mediator in C/EBP-α induced cell apoptosis of HEK293 and NIH3T3 . However, whether changes in PRAF3 expression alone affect tumor cell apoptosis has not been reported. In the present study, we have examined the effect of PRAF3 on the cell apoptosis through adenovirus mediated PRAF3 gene transfer. We demonstrate that overexpression of PRAF3 induces cell apoptosis in human ESCC.
Cell apoptosis mainly involves two signaling pathways: the death receptor pathway and the death receptor-independent (or mitochondrial) pathway . Caspase-8 and caspase-9 have been identified as the key signal molecules of the two pathways respectively [26, 27]. In the present study, we showed that overexpression of PRAF3 could significantly increase the activity of both caspase-8 and caspase-9 in ESCC cells, suggesting that PRAF3 could induce ESCC cell apoptosis through both caspase-8 and caspase-9 dependent pathways. Notably, we found that the overexpression of PRAF3 induced greater changes in caspase-9 than caspase-8 expression, suggesting that PRAF3 may induce cell apoptosis mainly through the death receptor-independent pathway in human ESCC.
Tumor metastasis is the leading cause of low survival rate of ESCC patients. Previous studies showed that PRAF3 acts as a down-regulatory factor for cell migration and invasion in melanoma, osteosarcoma, cervical cancer and breast cancer [7, 10]. In the present study, we investigated the role of PRAF3 in ESCC metastasis by clinical investigation and cellular experiment. As mentioned above, our clinical investigation suggests that the expression of PRAF3 in ESCC is negatively related to the tumor metastasis. Moreover, with ESCC cell lines infected with Ad.PRAF3, we further demonstrate that overexpression of PRAF3 significantly inhibits the migration and invasion of ESCC cells. Our results indicate that down regulation of PRAF3 may contribute to the metastasis of ESCC.
It is well known that matrix metalloproteinases (MMPs), a family of zinc- dependent endopeptidases, are involved in tumor metastasis in many aspects such as tumor-induced angiogenesis, tumor invasion, and establishment of metastatic foci at the secondary site, etc [28–30]. Among the many MMPs, MMP-2 and MMP-9 are reported to be closely related to the tumor metastasis in ESCC . However, whether PRAF3 regulates the activity of MMP-2 and MMP-9 in ESCC has not been documented. In the present study, we found that overexpression of PRAF3 significantly suppressed the activity of both MMP-2 and MMP-9, suggesting that PRAF3 could inhibit ESCC metastasis partially through the repression of MMP-2 and MMP-9 dependent pathway.
Previous study showed that PRAF3 could suppress the activity of MMP-2 by modulating the integrin avb3 signaling in melanoma . Here we show that overexpression of PRAF3 did not alter the expression of MMP-2 but significantly repressed the expression of integrin av and b3, suggesting that PRAF3 may inhibit the activity of MMP-2 probably by the inhibition of integrin avb3 signaling in ESCC cells.
On the other hand, we found that the expression of MMP-9 was significantly lower in Ad.PRAF3 treated cells than the controls. Since it was reported that the expression of MMP-9 was regulated by CCL5/CCR5 axis  and PRAF3 contains a CCR5 binding motif , we further studied the affection of PRAF3 on the CCR5. Our data show that overexpression of PRAF3 did not alter the level of CCR5 in the total cell lysate but significantly decreased the level of CCR5 at the plasma membrane. Considering that PRAF3 is a transmembrane protein located at the endoplasmic reticulum , we propose that PRAF3 might suppress the traffic of CCR5 from endoplasmic reticulum to plasma membrane, and thereby inhibits the expression and in turn the activity of MMP-9.