Vascular Endothelial Growth Factor Receptor-1 Is Synthetic Lethal to Aberrant {beta}-Catenin Activation in Colon Cancer

Abstract

PURPOSE: The Wnt/beta-catenin (beta-cat) signaling cascade is a key regulator of development, and dysregulation of Wnt/beta-cat contributes to selected cancers, such as colorectal, breast, and hepatocellular carcinoma, through abnormal activation of Wnt target genes. To identify novel modulators of the Wnt/beta-cat pathway that may emerge as therapeutic targets, we did an unbiased high-throughput RNA interference screen. EXPERIMENTAL DESIGN: A synthetic oligonucleotide small interfering RNA library targeting 691 known and predicted human kinases was screened in Wnt3a-stimulated human cells in a live cell luciferase assay for modulation of Wnt/beta-cat-dependent transcription. Follow-up studies of a selected high-confidence "hit" were conducted. RESULTS: A robust quartile-based statistical analysis and secondary screen yielded several kinases worthy of further investigation, including Cdc2L1, Lmtk3, Pank2, ErbB3, and, of note, vascular endothelial growth factor receptor (VEGFR)1/Flt1, a receptor tyrosine kinase (TK) with putative weak kinase activity conventionally believed to be a negative regulator of angiogenesis. A series of loss-of-function, genetic null, and VEGFR TK inhibitor assays further revealed that VEGFR1 is a positive regulator of Wnt signaling that functions in a glycogen synthase kinase-3beta (GSK3beta)-independent manner as a potential synthetic lethal target in Wnt/beta-cat-addicted colon carcinoma cells. CONCLUSIONS: This unanticipated non-endothelial link between VEGFR1 TK activity and Wnt/beta-cat signaling may refine our understanding of aberrant Wnt signaling in colon carcinoma and points to new combinatorial therapeutics targeted to the tumor cell compartment, rather than angiogenesis, in the context of colon cancer. (Clin Cancer Res 2009;15(24):7529-37).

Figure 1. High-throughput RNA interference screen for kinase modulators of Wnt signaling

Screen results including “hits” at various significance thresholds. Log[normalized photon flux] represents bioluminescence reporter signal intensity normalized for cell viability and a control non-targeting sequence placed on each plate to facilitate experiment-wide analysis. Values are plotted for siRNA against all 691 kinase targets. Log[normalized photon flux] values of < −0.37 and > 0.38 score as significant by high stringency criteria (α ≤ 0.0027) and are indicated in red. Values that lie between −0.37 and −0.25 and between 0.38 and 0.26 score as significant by lower stringency criteria (α ≤ 0.046) and are indicated in orange. “Hits” that scored at α ≤ 0.0027 in both an experiment-wide and plate-by-plate analysis are indicated as red triangles (see Supplementary Table 1), and VEGFR1/Flt1 by a blue triangle. The cluster of red circles between targets 1-150 represents “hits” that score as significant by high stringency criteria in an experiment-wide analysis only, but not by a plate-by-plate analysis (listed in Supplementary Table 3). For details on the quartile-based statistical analysis see Materials and Methods; siRNA sequences targeting genes identified as high stringency “hits” in the primary screen are listed in Supplementary Table 4.

Figure 2. Validation of Wnt/β-cat signaling changes following shRNA knock-down of “hits”

Box and whisker plot of one representative experiment demonstrating luciferase reporter signal changes in STF293 cells 48 hrs after transfection with shRNA plasmids. Four different shRNA sequences targeting non-overlapping segments of the coding regions of the indicated genes were tested in quadruplicate (x-axis). Photon flux in each well was normalized for cell viability (Abs). Lines represent medians, boxes represent 25^th to 75^th percentile interquartile ranges, and whiskers represent the highest and lowest values for each replicate within a given gene. The dashed horizontal line represents the median value of a negative control. Genes reproducible across at least two of three independent experiments are indicated by an underline.

Figure 3. Validation of negative regulation of Wnt/β-cat signaling upon loss of VEGFR1 using pooled and individual siRNA duplexes targeting VEGFR1

(a) Gel electrophoresis following semi-quantitative RT-PCR from STF293 cells mock transfected (mock) or transfected with individual duplexes (D1-D4) targeting VEGFR1, a non-targeting siCONTROL sequence (siC), or a set of 4 pooled ON-TARGET plus sequences against VEGFR1 (pool). Gene-specific primers were used to amplify VEGFR1 or control GAPDH. Effect of (b) a set of 4 pooled siRNA sequences against VEGFR1, or (c) individual sequences (D1-D4) targeting VEGFR1, along with non-targeting siC as well as a Firefly luciferase (PGL3)-targeting siRNA on STF293 cells stimulated with Wnt3a (gray) or control media (black). Data are represented as mean photon flux +/− S.E.M (n=3 per condition). (d) Knock-down of VEGFR1 had no effect on control 293Luc cells expressing a non-Wnt-dependent reporter, both in the absence (shown) or presence of Wnt3a.

Figure 4. Inhibition of VEGFR tyrosine kinase activity is synthetic lethal in “β-cat-addicted” colon cancer cells, acting through downregulation of Wnt/β-cat-dependent transcriptional activation

(a) Inhibition of VEGFR-TK activity selectively diminishes viability of SW480 (solid circle) and KM12L4a (solid triangle) colon cancer cells, but not Wnt3a-stimulated STF293 cells (open square), control STF293 cells (open diamond) or non-colon cancer HeLa cells (open circle). Each cell type was treated with increasing concentrations of Inh II for 72 hrs before cell viability analysis using an MTS assay. Data are represented as mean +/− S.E.M. of fold-untreated absorbance values from two independent experiments (n=3 each) for each concentration for each cell line. (b) Reporter concentration-response in STF293 cells stimulated with Wnt3a following pre-treatment with increasing concentrations of Inh II. Bioluminescence photon flux was normalized to cell viability using a resazurin dye-based fluorescence assay and plotted as fold-untreated normalized photon flux +/− propagated S.E.M. (n=3 per concentration). (Inset) In HEK293T cells transfected with pTOPFLASH, Inh II strongly attenuated β-cat-dependent transcription induced by Wnt3a (white bars), but showed a statistically significant lower effect on basal transcriptional activity in the absence of Wnt3a (black bars). Bioluminescence photon flux was normalized to cell viability using a resazurin dye-based fluorescence assay and plotted as fold-initial, fold-untreated normalized photon flux +/− propagated S.E.M. (n=3 per concentration). Statistical significance by Student's t-test is indicated as * (p <0.05). A two-way analysis of variance also found statistically significant effects of both Wnt treatment (p <0.05) and drug concentration (p <0.001). (c) Inhibitory effect of a 48-hour exposure to increasing concentrations of Inh II on constitutively active β-cat-dependent signaling in SW480 (gray bars) and KM12L4a (black bars) colon carcinoma cells transfected with pTOPFLASH. Data are represented as mean fold-initial, fold-untreated photon flux +/− propagated S.E.M. (n= 3 per concentration). Statistical significance by Student's t-test is indicated as * (p <0.05) and ** (p <0.01).

Figure 5. VEGFR1 acts in a GSK3β-independent manner and downstream of β-cat protein stabilization

(a) Effects of individual duplexes (D1-D4) targeting VEGFR1, a non-targeting siCONTROL sequence (siC) and a Firefly luciferase-targeting (PGL3) siRNA on STF293 cells treated with the GSK3β inhibitor SB216763 (10 μM). Bioluminescence photon flux is represented as mean photon flux +/− S.E.M. (n=4). (b) Western Blot analysis of STF293 cells transfected with ON-TARGET plus SMARTpool siRNA targeting VEGFR1 (Lanes 1 & 2) or off-target PGL3-targeting siRNA (Lanes 3 & 4), and stimulated with Wnt3a (Lanes 1 & 3) or control media (Lanes 2 & 4), or (c) treated with vehicle (Lanes 1 & 2) or 1 μM VEGFR-TK Inh II (Lane 3) for 18 hrs together with control media (Lane 1) or Wnt3a (Lanes 2 & 3).

Figure 6. Modulation of Wnt/β-cat signaling by VEGFR1 is independent of β-cat stabilization and nuclear translocation, but correlates with β-cat tyrosine phosphorylation

(a, b) Confocal immunofluorescence microscopy images showing β-cat subcellular localization (green) in STF293 cells transfected with either siRNA targeting VEGFR1 or a non-targeting control duplex for 48 hrs (a) or treated with Inh II (1 μM) or vehicle for 18 hrs (b). Cells were stimulated for two hours with either control media (top) or Wnt3a (bottom). Nuclei (blue) were stained with DAPI (magnification 40X). (c) Western blot analysis of immunoprecipitated β-cat from KM12L4a and SW480 colon carcinoma cells treated with Inh II (1 μM) for 48 hrs. Samples were first probed with anti-phospho tyrosine (pTyr) antibody, stripped and reprobed with anti-β-cat antibody.