In the present study, we show a novel function of the vegf 5′UTR in tumor cell survival and growth. Both treatment of vegf-knockdown HCT116 cells with rhVEGF165 and treatment of VEGF165-overexpressing HCT 116 cells with a neutralizing anti-VEGF165 Ab suggest the presence of vegf mRNA-mediated anti-apoptotic action against anti-cancer drugs (5-FU, etoposide, and doxorubicin). We determined that the anti-apoptotic action resided in a 270-nt-long element between positions nt 475 and nt 745 of the 5′ UTR of vegf mRNA. The 5′ UTR could exert the anti-apoptotic action even in the presence of a protein synthesis inhibitor. These results suggest that the 5′UTR may function as a regulatory RNA.
To further clarify the potential role of vegf 5′UTR RNA, we established HCT116/vegf 5′ and HCT116/vegf 5′mut clones. HCT116/vegf 5′ cells, but not HCT116/vegf 5′mut cells, showed anchorage-independent growth in vitro and rapidly grew when implanted in athymic nude mice, indicating that vegf mRNA 5′UTR facilitates tumor progression. The rapid growth observed in the vegf 5′-tumors might be due to the acquisition of resistance to apoptosis (the process that eliminates defective cells), which contributes to tumor development and resistance to drug therapy. Microarray and quantitative RT-PCR analyses demonstrated that expression of pro-apoptotic genes (fas, pdcd1, nrg1, and bax) and an anti-apoptotic gene (mia) was up-regulated and markedly down-regulated, respectively, in the rapidly growing vegf 5′-tumors. In addition, microarray analysis also showed that the expression of cell growth-promoting genes (cxcl1, cxcl2, cxcr4, mia1, and fgf-19) was up-regulated in the rapidly growing vegf 5′-tumors.
The combination of both dysregulated cell proliferation and suppressed cell death are required for neoplastic progression [36
]. In addition, survival and growth under anchorage-independent conditions are required for the progression [37
]. This anchorage-independent property of tumor cells correlates with their in vivo oncogenic potential. Thus, we consider that the vegf
5′UTR may increase the ability not only to suppress apoptosis but also to survive and grow in an anchorage-independent manner, resulting in the acceleration of tumor formation.
The precise sequence and structure of the 5′UTR of vegf mRNA as well as the molecular target(s) that interact with the RNA remain to be elucidated. However, it seems likely that such a profound change in cell function induced by RNA would require a global change of gene expression in cells. Our results support this notion by showing that the vegf mRNA 5′UTR modulated expression of numerous genes. One of the striking findings is that many of the down-regulated genes belong to a set of IFN-inducible genes, including stat1.
Since it is reported that IFN down-regulates IRES-dependent translation of distinct mRNAs [38
], overexpression of the IRES-containing 5′UTR might attenuate the IFN signal, leading to the down-regulation of IFN-inducible genes. We therefore tested whether IFNα affected IRES activity of c-myc
, or bip
using a bicistronic luciferase assay system (Figure S7
A) in our experimental conditions, and found that IFNα did not modify at least these IRES activities (Figure S7
C). At the same time, we also confirmed that IFNα signaling, estimated using the ISRE-dependent transcription reporter assay, was not inhibited in the cells overexpressing bip
, or fgf-2
5′UTR, nor was it inhibited in the cells overexpressing the pdgf-b
5′UTR that have no IRES activity (Figure S7
D). Although these results suggest that the suppression of IFN signaling may be specific for the vegf
5′UTR, further studies are needed to clarify the molecular mechanism for the suppression.
The IFNs/STAT1 signal is one of the key pathways for tumor suppression. Mice with a targeted deletion of the STAT1 or IFNγ receptor develop chemically induced or spontaneous tumors more rapidly than wild-type mice [39
]. Similarly, mouse embryonic fibroblasts deficient in the IFNα receptor undergo spontaneous malignant transformation, and the IFNα receptor-deficient mice develop papillomas of the skin at a high rate when treated with a chemical carcinogen [40
]. In contrast, the reconstitution of STAT1 suppressed the tumorigenicity of STAT1-deficient tumor cells in vivo, demonstrating that STAT1 acts as an important tumor suppressor [41
]. Indeed, the majority of cancer cell lines and primary tumors are resistant to IFNs, often through inhibition of STAT1 expression [42
]. We propose here that the vegf
mRNA 5′UTR negatively regulates expression of STAT1, leading to suppression of STAT1-dependent transcriptional activities. It has been suggested that STAT1 directly regulates DNA damage-induced apoptosis by transcriptional activation of apoptosis modulating genes, such as TRAIL, Fas, and XAF-1 [33
], all of which were down-regulated in the vegf
5′-tumors (). Thus, the suppression of STAT1 may be crucial for malignant transformation of HCT116/vegf
5′ cells. In fact, the vegf
5′-tumors did not response to IFNα therapy ().
Tumor cell growth and survival may require both expression of VEGF and down-regulation of the IFN pathway. Consistent with this notion, it is known that IFNα transcriptionally suppresses VEGF expression [44
]. In contrast, we suggest here that vegf
mRNA may negatively regulate the IFN signaling pathway by repressing STAT1. This reciprocal regulation of VEGF and STAT1 is entirely feasible as these two molecules exert opposing biological functions: VEGF promotes proliferation, metastasis, and angiogenesis [1
], and inhibits apoptosis [1
]; in contrast, STAT1 negatively regulates proliferation, metastasis, and angiogenesis [41
], and promotes apoptosis [33
]. Tumor cells abundantly expressing vegf
] may use this negative regulatory mechanism to gain advantages of growth and survival to escape from the IFN-mediated anti-tumor machinery. Indeed, we found here that stable knockdown of endogenous vegf
mRNA by siRNA increased STAT1 expression.
At present, the precise mechanisms for the vegf
5′UTR-mediated anti-apoptotic action are still unknown. However, our preliminary experiments showed that the vegf
5′UTR RNA might interact with double-stranded RNA-activated protein kinase (PKR) protein in the cells expressing the full-length vegf
mRNA or the vegf
5′UTR RNA, which were examined by the pull-down assay using an antisense oligonucleotide probe specific for the vegf
5′UTR sequence (unpublished data). It is reported that PKR binds several structured UTRs of mRNA, such as the 5′UTR of ifn-
γ mRNA [50
], the 3′UTR of α-tropomyosin
], and the 3′UTR of tnf
-α mRNA [52
]. PKR associates with tumor suppressor, such as IRF-1 and p53 [53
] and induces apoptosis. Our preliminary data suggest that the vegf
5′UTR might interact with PKR and lead to suppression of PKR-mediated apoptotic pathway. However, this is still an unproven hypothesis, and further experiments are being carried out in our laboratory.
Recent studies have revealed that a large number of non-coding RNAs play a critical role in tumorigenesis [17
]. Our findings support the concept originally put forward by Blau and colleagues [18
] that non-coding regions of mRNAs can act as RNA regulators for tumor malignancy. There is increasing evidence that the UTR of certain mRNAs significantly suppresses the tumorigenic properties of cancer cells both in vitro and in vivo. The 3′UTR of α-tropomyosin
mRNA suppresses the proliferation, invasion, and destruction of muscle tissues characteristic of rhabdomyosarcoma cells [18
]. The 3′UTR of ribonucleotide reductase
, a key rate-limiting enzyme in DNA synthesis, and the 3′UTR of prohibitin
, an inhibitor of cell proliferation, significantly suppress the tumorigenic properties and metastatic phenotype of transformed fibroblasts and MCF7 cells [19
]. In addition, the 5′UTR of the human c-myc
P0 transcript suppresses the malignant phenotype of human breast cancer cells with decreased anchorage-independent proliferation, enhanced susceptibility to programmed cell death, and complete loss of the ability to form tumors in the intact animal [21
]. To the best of our knowledge, the 5′UTR vegf
mRNA is the first example of tumor-promoting UTR RNA.
VEGF protein is considered to be an important therapeutic target for cancer treatment, and anti-VEGF strategies are undergoing clinical evaluation [14
]. A number of preclinical studies have demonstrated that anti-VEGF therapy alone can suppress the growth of established tumors [55
]. Unlike these preclinical studies, anti-VEGF-specific Ab (bevacizumab) alone has not been shown to increase survival in lung and colorectal cancer patients [14
]. The combined use of bevacizumab with standard chemotherapy increased overall survival in metastatic colorectal cancer [57
], but did not improve the clinical outcome in metastatic breast cancers in previously treated patients [58
]. Furthermore, the combination of the VEGF receptor tyrosine kinase inhibitor, vatalanib, with chemotherapy did not show an increased survival rate in metastatic colorectal cancer patients [59
]. Our finding that the novel intrinsic tumor-promoting activity presents in the vegf
mRNA might explain, at least in part, the inconsistencies of outcome associated with VEGF-VEGFR strategies. The present study suggests that both vegf
mRNA and VEGF protein may synergistically promote the malignancy of tumor cells. Thus anti-vegf
transcript therapy, such as siRNA-based gene silencing, in combination with anti-VEGF therapy might provide optimal anti-tumor effects, including inhibition of angiogenesis, blockade of tumor cell survival, and enhanced sensitivity to radiation and drug therapies.