Research Article: A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascularendothelial growth factor mRNA

Date Published: May 20, 2008

Publisher: Public Library of Science

Author(s): Kiyoshi Masuda, Shigetada Teshima-Kondo, Mina Mukaijo, Naoko Yamagishi, Yoshiko Nishikawa, Kensei Nishida, Tomoko Kawai, Kazuhito Rokutan, Pam Jones

Abstract: BackgroundVascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.Methods and FindingsKnockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.ConclusionsThese findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.

Partial Text: Vascular endothelial growth factor-A (VEGF) is one of the key regulators in tumor formation and progression [1–4]. The clinical significance of VEGF in tumors has been demonstrated by many studies showing that the VEGF expression level is correlated with tumor grade, depth of invasion, status of nodal and distant metastasis, and clinical stage [5–10]. In addition, high levels of circulating VEGF are associated with resistance to chemotherapy in patients with metastatic solid tumors, including colorectal cancer [11,12]. The stimulatory action of VEGF on tumor angiogenesis is believed to play a central role in promotion of tumor development. At the same time, VEGF is known to act as an autocrine survival and growth factor for tumor cells [13]. Based on the evidence outlined above, a number of strategies to target VEGF or VEGF receptors (VEGFR) have been developed and subjected to clinical evaluation [14]. In contrast to preclinical studies in animal models, clinical trials suggest that anti-VEGF monotherapy was less effective than standard chemotherapy [15], raising the possibility that VEGF or possibly vegf mRNA might possess unrecognized function(s).

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.



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