Discussion
The present work has led to identification of an E1A-downregulated miRNA (miR-520h) that inhibits synthesis of the PP2A/C protein and enhances the expression of Twist. Importantly, overexpression of this miRNA in cells stably expressing E1A stimulates invasion and migration in vitro, and exacerbates metastatic potential in vivo. Conversely, silencing of miR-520h inhibits invasive and metastatic behavior in cancer cells in vitro and in vivo. Therefore, miR-520h plays a major role in cancer metastasis and E1A can suppress cancer metastasis through downregulation of this oncogenic miRNA. In addition, miR-520c (which is located within the same gene cluster as the miR-520h gene) has recently been reported to target the cell surface receptor CD44 (Huang, 2008). CD44 acts as a metastasis suppressor in cancers of the breast, prostate, and colon (Choi, 2000; Jaeger, 2001; Lopez, 2005; Pereira, 2001), indicating that miR-520c functions like miR-520h as a metastasis promoter in breast cancer. In addition to suppress metastasis, E1A gene therapy has been shown to increase sensitivity to different categories of anticancer drug in multiple clinical trials. Recent studies have shown an association of miR-27a with drug-resistance in cancer cells (Zhu, 2008). Moreover, our data show that miR-27a is downregulated by E1A. Whether other miRNA family members are involved in E1A-mediated chemosensitization remains to be investigated. On the other hand, E1A regulates the expression of several miRNAs (Table S1) and its involvement in the transcription control of miRNA expression has been suggested. Future studies should be conducted to clarify the mechanism of E1A-mediated miRNA regulation.
Increasing data suggest that miRNAs might affect metastasis through regulation of EMT, which is a genetic developmental program regulated by a number of transcription factors. These transcription factors, such as ZEB1 and ZEB2, are expressed at the invasive front of tumors and correlate with distant metastases and poor prognosis (Maeda, 2005; Spoelstra, 2006). Recently, it has been confirmed that miR-200 family members target ZEB1 and ZEB2 directly for repression (Burk, 2008; Gregory, 2008;
Korpal, 2008) and mediate inhibition of ZEB1 and ZEB2 at the crossroads of EMT where they are likely to have considerable impact on metastatic progression. Twist plays a key role in EMT-mediated
intravasation of tumor cells (i.e., entry into the circulation to seed metastases) (Yang, 2004). Furthermore, Twist is required to bypass oncogene-induced cellular senescence (Ansieau, 2008). Recent research shows that tumor cells undergoing EMT resemble cancer stem cells (Mani, 2008). While investigating E1A-mediated mesenchymal-epithelial transition (MET), we discovered that Twist is markedly reduced by ectopic expression of E1A in various cancer cells. However, overexpression of miR-520h in 231/E1A and ip1/E1A cells leads to restoration of Twist expression. On the other hand, administration of miR-520h inhibitor lowers Twist level in 231/VC cells, and transfection of Twist siRNA in cells overexpressing miR-520h inhibits cell mobility. Therefore, our data suggest that Twist is a downstream target of E1A and imply that E1A gene therapy (through its ability to downregulate Twist) can have efficacy against cancer cells as well as cancer stem cells. On the other hand, due to its short half-life, we do not clearly detect the change of Snai1 protein expression in different miR-520h-expressing cells. This will need to be clarified by further study.
Bioinformatics analyses predict that conserved vertebrate miRNAs target more than 100 to 400 regulatory genes. The seed sequence within PP2A/C 3’UTR for miR-520h is poorly conserved in different species. However, in our study, PP2A/C was shown to be a relevant target of miRNA-520h. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that suppresses tumor growth. The report that PP2A might be a tumor suppressor is based on the observation that okadaic acid, a strong inhibitor of PP2A, is a potent tumor promoter (Cohen, 1989). Mutation of PP2A was found in human cancers including breast, colon, and lung cancers and melanoma (Schonthal, 2001). The participation of PP2A in E1A-mediated sensitization to anticancer drugs involves activation through up-regulation of PP2A/C expression, which results in activation of p38 and repression of Akt (Liao, 2004;
Liao, 2003). In the current study, PP2A/C was identified as a target of miR-520h, which inhibits PP2A/C protein translation by binding to PP2A/C 3’UTR. Consequently, we suggest that E1A induces PP2A/C expression through an miRNA regulatory mechanism. As reported in this article, overexpression of PP2A/C in various cancer cells inhibits their motility, suggesting a putative role for PP2A/C in the suppression of metastasis. Furthermore, Twist has been identified as the conserved target of NF-κB
(Kanegae, 1998; Pham, 2007; Sosic, 2003), and PP2A/C may exert its tumor suppression activity by inhibiting IKK activity. Our findings imply that E1A might participate in the regulation of Twist by inducing PP2A/C expression and thereby suppressing cancer cell metastasis.
We have identified Twist and PP2A/C as downstream targets of E1A, suggesting that E1A may act through different mechanisms to inhibit the expression of putative oncogenic miRNAs (miR-520h) and oncogenes. Likewise, numerous genetic studies have allowed the identification of miRNA abnormalities in human cancer by dissecting their transcriptional regulators (Chang, 2008; He, 2007). For instance, p53 promotes the transcription of all members of the miR-34 family (He, 2007) and MYC can both positively and negatively regulate transcription of different miRNAs to promote tumorigenesis (Chang, 2008;
O'Donnell, 2005). Our miRNA microarray data indicate that E1A can influence the expression of different miRNAs. Possibly, E1A can be used to regulate miRNA expression through interacting with several transcription factors or coactivators, such as c-Jun, p300, and YY-1. However, the mechanism of this regulation remains to be seen. A future challenge will be to identify the entire complement of miRNAs and their mRNA targets to elucidate more fully the contributions of these miRNAs to high-grade malignancy. In this study, we provide a new insight into the regulation of miRNA expression by E1A and its possible use to suppress metastasis.