Double-Stranded RNA-Binding Protein Regulates Vascular Endothelial Growth Factor mRNA Stability, Translation, and Breast Cancer Angiogenesis

Cellular adaptation to nutrient and metabolic stress is a critical mechanism for tumor cell survival and progression. Hypoxia induces cells to express genes that function to shift cellular metabolism from aerobic oxidative phosphorylation to anaerobic glycolytic pathways. One essential mechanism of this adaptive response is the transcription of target genes through activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Genes transcribed by HIF-1 include those in the glycolytic pathway, including glucose transporter 1 (glut1), hexokinase 1 (HK), phosphoglycerate kinase 1 (PGK1), pyruvate kinase (PK), glyceraldehyde phosphate dehydrogenase (GAPDH), and lactate dehydrogenase (LDH) (14, 35). In addition to the genes necessary for glycolysis, survival factors such as vascular endothelial growth factor (VEGF), which can induce local vascular permeability and angiogenesis, are also effectively transcribed by HIF-1 (8, 25, 41). VEGF expression under hypoxia also requires posttranscriptional mRNA stability and mRNA transport mechanisms. VEGF mRNA is highly labile under normal oxygen and nutrient conditions (3, 5, 17) and is mediated through AU-rich elements (AREs) in the 3′ untranslated region (3′-UTR) (5). A consensus destabilization motif (AUUUA) occurs eight times in the human VEGF 3′-UTR, which is 1.6 kb in length (3). ARE-binding proteins such as AUF1 and tristetraprolin (TTP) have all been shown to destabilize mRNAs in various mammalian cell types (2, 4, 11, 40, 44). In addition to their destabilization effects, ARE elements can contribute to mRNA stabilization through interactions with the ELAV family of RNA-binding proteins, which includes Hel-N1, HuC, HuD, and HuR (7, 19, 22, 30). Interestingly, poly(A)-binding protein has been predominantly a stabilizing factor for polyadenylated mRNAs; however, recent investigations suggest that it may also have destabilizing effects (2, 11, 26). Hypoxia-induced mRNA stability has been shown to be a mechanism that can facilitate VEGF expression in tumors even without HIF-1 transcription (32). The identification of 3′-UTR elements in VEGF which promote mRNA stability has determined that AU-rich regions also confer hypoxia-dependent mRNA stability (3, 10, 24). The RNA-binding proteins that interact with these 3′-UTR elements include HuR, hnRNP A1, hnRNP L, poly(A)-binding protein, PAIP2, and TI5IId, according to literature reports (23). HuR and hnRNP L are predominantly nuclear proteins that have the capacity to shuttle between nuclear and cytoplasmic compartments, especially under hypoxic conditions (6, 13, 18, 31). Previous identification of a predicted stem-loop structure in the 3′-UTR of VEGF mRNA showed that this element can provide hypoxia-induced stability to a heterologous mRNA (3). Cross-linking and affinity purification experiments identified both HuR and hnRNP L as RNA-binding proteins for this hypoxia stability region (HSR) element (36). An additional protein with an apparent molecular mass of 90/88 kDa was also found to cross-link to the HSR 126-bp 3′-UTR stem-loop RNA under hypoxic conditions but has not been identified to date (3). VEGF protein synthesis under hypoxic conditions also requires 5′-UTR mRNA elements to maximize expression. In the case of VEGF, the 5′-UTR contains predicted internal ribosome entry sites that facilitate mRNA loading onto ribosomes and efficient translation (15, 27, 39). These sequences are G/C-rich, have a predicted secondary structure that makes the translation start site accessible, and have been shown to confer increased expression of chloramphenicol acetyltransferase (43) reporter protein under hypoxia in HeLa cells (39). In a recent analysis of translational control mechanisms, eIF-4F initiation complexes were found to be disrupted under conditions of hypoxia (42). This would dictate that 5′-cap-dependent translation would be blocked and that mRNAs with internal ribosome entry site sequences would be preferentially translated under hypoxia. The increased association of carbonic anhydrase IX (CAIX) mRNA with polysomes was demonstrated in prolonged hypoxia up to 16 h, suggesting that mRNA shuttling and loading mechanisms are also important for hypoxia-dependent gene expression (20). In this study, a 90- to 88-kDa protein complex that binds to the VEGF HSR 3′-UTR A/U-rich stem-loop element that confers hypoxia-dependent mRNA stability was identified. Affinity purification and proteomic analysis revealed that the characteristics of this protein complex were consistent with those of the double-stranded RNA-binding protein-interleukin enhancer binding protein factor-3/nuclear factor family of alternatively spliced DRBPs. One of these alternatively spliced proteins, double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), was found to contribute to VEGF expression under hypoxia, and silencing its expression reduced VEGF mRNA and protein levels. The role for DRBP76/NF90 in VEGF mRNA and protein synthesis under hypoxic conditions and in tumor progression was evaluated, and DRBP76/NF90 was shown to significantly contribute to hypoxia-induced VEGF expression and tumorigenesis in vivo in a breast carcinoma model.