Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBK1 and the IRF3 pathway

Abstract

A genome-wide phenotype screen was used to identify factors and pathways that induce proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC proliferation is a recognized marker for factors that modulate vascularization. Screening "hits" included known proangiogenic factors, such as VEGF, FGF1, and FGF2 and additional factors for which a direct association with angiogenesis was not previously described. These include the kinase TBK1 as well as Toll-like receptor adaptor molecule and IFN regulatory factor 3. All three proteins belong to one signaling pathway that mediates induction of gene expression, including a mixture of secreted factors, which, in concert, mediate proliferative activity toward endothelial cells. TBK1 as the "trigger" of this pathway is induced under hypoxic conditions and expressed at significant levels in many solid tumors. This pattern of expression and the decreased expression of angiogenic factors in cultured cells upon RNA-interference-mediated ablation suggests that TBK1 is important for vascularization and subsequent tumor growth and a target for cancer therapy.

Fig. 1.

Factors that specifically induce HUVEC proliferation. (A) TBK1 induces proliferation of HUVEC. Proliferative activities were detected by incubating HUVEC for 5 days with supernatants of transfected HEK293 producer cells [four independent experiments (different shades) with multiple samples each (individual bars)]. Increased signals of Alamar blue assays indicate proliferation. Supernatants of HEK293 cells producing VEGF (positive controls) confirm that the processing machinery releases secreted/shed factors in sufficient concentrations. For normalization of results, the mean value of the controls was set to 100% for each experiment; individual values for each experiment were calculated relative to that. Indicated are also the mean values ± SD of each group, and the significances of the signal increases (Student’s t test). (B–D) Representative single experiments. (B) Proliferative activities were detected as described in A. Increased signals of Alamar blue assays (fold increase relative to vector control = 1) indicate proliferation. Proliferation was also mediated by intracellular proteins through induction of signaling pathways. One pathway is defined by activities of TBK1, TRIF, and IRF3, which stimulate cells to release proliferative activities. (C) Endothelial cell proliferation accompanied by DNA replication: TIME cells were analyzed, applying a [³H]thymidine-incorporation assay. Increased signals (fold increase relative to control = 1) indicate DNA synthesis and cell proliferation (shown with SD). (D) Specificity. Supernatants were applied in parallel to endothelial cells (HUVEC, gray) and human fibroblasts (NHDF, black). TBK1, VEGF, and FGF2 cause HUVEC proliferation. Only FGF2 mediates proliferation of NHDF, confirming the specificity for endothelial cells of TBK1-induced activity. (E) HUVEC proliferation induced by supernatants of transiently transfected cancer cell lines.

Fig. 2.

TBK1 induces expression of secreted factors. The induction of RANTES and IL8 in recombinant TBK1-expressing cells was analyzed by qPCR using as reference the housekeeper G6PDH, which was tested in parallel in those experiments. (A) RNA of TBK1-transfected HEK293 cells was extracted at different time points after transfection, and mRNA levels were determined by qPCR. The relative signals (normalized to housekeeper expression) of the PCR reactions of RANTES and IL8 are different (RANTES, left axis; IL8, right axis), but both factors are induced with similar kinetics. (B) RANTES and IL8 induction by supernatants of transiently transfected cells is also observed in the MCF7 breast cancer cell line. Induction of the cytokines was detected as described in A.

Fig. 3.

NF-κB is not sufficient for induction of proliferative activity. TBK1 and IKKα were analyzed for NF-κB activation and induction of HUVEC proliferation. NF-κB activation (left axis): HEK293 were cotransfected with a NF-κB-promoter luciferase reporter construct and plasmids for expression of TBK1, IKKα, or empty vector. The relative signal (light units, fold above vector control) reflects NF-κB-dependent transcription. HUVEC proliferation (right axis): HEK293 cells were transfected (TBK1, IKKα, or vector control) and proliferative activities analyzed as described above. TBK1 and IKKα induce NF-κB-dependent transcription, but only supernatants of cells expressing TBK1 mediate HUVEC proliferation.

Fig. 4.

TBK1 levels increase under hypoxic conditions and correlate with expression of VEGF. (A) Induction of TBK1 under hypoxic conditions. HEK293 cells were exposed to 50 mM CoCl₂ for 24 h (chemical induction of hypoxia). Expression of VEGF and TBK1 was analyzed by qPCR (references were G6PDH or β2 microglobulin, relative values, control expression set to 1′). The housekeepers showed no evidence of nonspecific effects of RNAi; the presented data are all shown relative to the housekeeper. VEGF and TBK1 are induced by CoCl₂ treatment. (B) RNAi-mediated reduction of TBK1. The effects of RNAi targeting TBK1 were analyzed by qPCR in CoCl₂-treated cells. TBK1-RNAi reduces the expression of TBK1 and VEGF in CoCl₂-treated cells. (C) Effects of RNAi against TBK1 on VEGF levels analyzed by ELISA. Hypoxia-induced HEK293 cells were exposed to TBK1-RNAi. VEGF levels were detected by ELISA (OD 492 nm) in two independent experiments (left axis, signal of experiment 1; right axis, experiment 2). Under our experimental conditions, VEGF expression is barely detectable without CoCl₂ induction, and VEGF signals rise above background in CoCl₂-treated cells and are reduced in TBK1-RNAi-treated cells.

Fig. 5.

Elevated expression levels of TBK1 in solid tumors. (A) RT-PCR (qPCR) for detection of TBK1 transcripts. RNAs from tumor and normal colon and breast were subjected to TBK1-specific qPCR. Each bar represents the qPCR-signal from one individual (y axis signal represents mRNA amount). The first (far left) colon tumor sample showed a very high expression (value, 21.6). The hatched horizontal lines represent the mean + SD of the expression of the nontumor samples. Thus, everything above these thresholds is increased expression. The mean values of expression were 0.43 and 0.12 for all normal colon or normal breast, respectively, and 1.52 or 0.40 for all colon or breast cancer samples, respectively. Preparations from tissues from control individuals display low signals, whereas many tumors show increased levels of the mRNA of TBK1. (B) Detection of TBK1 protein by immunohistochemistry. Exemplar shown is the analysis of tumor and adjacent normal tissue from colon and breast cancer. Both tumors show increased expression of TBK1 in malignant cells. IHC, immunohistochemistry.