The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression

Abstract

Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis whose expression suppresses tumor growth in vivo. Like many angiogenesis-related genes, TSP-1 expression is tightly controlled by various mechanisms, but there is little data regarding the contribution of post-transcriptional processing to this regulation. NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype and suppresses tumor growth, in part through upregulation of TSP-1. Here we demonstrate that NOL7 is an mRNA-binding protein that must localize to the nucleoplasm to exert its antiangiogenic and tumor suppressive effects. There, it associates with the RNA-processing machinery and specifically interacts with TSP-1 mRNA through its 3'UTR. Reintroduction of NOL7 into SiHa cells increases luciferase expression through interaction with the TSP-1 3'UTR at both the mRNA and protein levels. NOL7 also increases endogenous TSP-1 mRNA half-life. Further, NOL7 post-transcriptional stabilization is observed in a subset of angiogenesis-related mRNAs, suggesting that the stabilization of TSP-1 may be part of a larger novel mechanism. These data demonstrate that NOL7 significantly alters TSP-1 expression and may be a master regulator that coordinates the post-transcriptional expression of key signaling factors critical for the regulation of the angiogenic phenotype.

Figure 1.

NOL7 must reside in the nucleus to modulate TSP-1 expression, inhibit endothelial cell migration and suppress tumor growth. (a) Cells were transfected with GFP-tagged wild-type NOL7 or mutants that target NOL7 to the nucleoplasm (N23(−), clones 1 and 2) or the cytoplasm (N123(−), clones 1 and 2). Cell nuclei were counterstained with DAPI and imaged by fluorescence microscopy. (b) Conditioned media from transfected cells was analyzed by enzyme-linked immunosorbent assay and concentration calculated from a standard curve. Data are represented as mean±s.e.m. Significance was calculated using Student’s t-test from three independent experiments. *P<0.005; **P<0.002; ***P<0.001; n.s., not significant. (c) Serum-free conditioned media from parental SiHa, GFP and NOL7 wt- and mutant-transfected clones were functionally tested for their ability to stimulate endothelial cell migration. Data are represented as mean±s.e.m. Significance was calculated using Student’s t-test from four independent experiments. *P<1×10⁻⁵; **P<1×10⁻⁸; n.s., not significant. (d) SiHa parental, GFP and NOL7 wt- and mutant-transfected cells were subcutaneously injected into nude mice and monitored over a period of 30 (n=6 animals per group). Data are represented as mean±s.e.m. Significance was calculated using two-way analysis of variance. *P<0.001; **P<0.0001; n.s., not significant. (e) Tumor angiogenesis was assessed by CD31 staining, followed by microvessel density quantification. Data are represented as mean±s.e.m. Significance was calculated using Student’s t-test (n=6 animals per group). **P<2×10⁻⁶; ** *P<1×10⁻¹⁰; n.s., not significant.

Figure 2.

NOL7 interacts with a large RNP complex in an RNA-dependent manner. NOL7-transfected lysate was separated on a 10–30% gradient. Individual fractions were collected and plotted on the basis of their absorbance at 260 nm. Individual fractions were also subjected to western blotting for NOL7. The co-migration of NOL7 with a large RNP complex is marked with a box. (a) Total lysate was evaluated for association of NOL7 into RNP complexes. (b) Lysate was either mock-treated or digested with RNase A before separation.

Figure 3.

NOL7 interacts with 3’ end-processing proteins. (a) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. (b) GFP-V5 or NOL7-V5 lysate was mock-treated ( − ) or digested with RNase ( + ). Lysates were immunoprecipitated using α-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RT-PCR against the 18S rRNA was performed.

Figure 4.

NUL7 specifically interacts with mRNA. HtK293l cells were transfected with GFP control or NOL7. Proteins associated with poly(A) mRNA were pulled down using oligo(dT) cellulose. Input lanes represent 10% of total input. As controls, lysate was digested with RNase A before incubation or bound proteins were competed using five or ten times input RNA of polyA or polyC. Coprecipitation of GFP and NOL7 was evaluated by western blot. Data are represented as mean ± s.e.m. Significance was calculated using Student’s t-test relative to NOL7 input (a, b) and relative to the NOL7-bound (c, d) fractions. ^aP<0.002; ^bP<4 × 10⁻⁴; ^cP<0.001; ^dP< 0.0001. Statistical significance between groups was also calculated (bars). *P<0.001, **P<1 × 10⁻⁴. Data are represented as mean ± s.e.m. from three independent assays.

Figure 5.

NOL7 coimmunoprecipitates TSP-1 mRNA. (a) Lysate from cells expressing GFP, NOL7 or HuR were immunoprecipitated with α-V5 beads and RNA was extracted. RNA was then subjected to northern blotting using a probe against the 3^’UTR of TSP-1. Western blotting to confirm equivalent protein IP was performed. Densitometry scanning was performed on northern bands and normalized to protein IP lanes. Data are represented as mean ± s.e.m. from three independent experiments. *P<0.01. (b) The 3^’UTR of TSP-1 and a negative-control sequence R01 were in vitro transcribed and biotinylated. Increasing amounts of transcript were incubated with lysate expressing GFP, NOL7 or HuR. Transcripts were precipitated using streptavidin beads and coprecipitating proteins were analyzed by western blot.

Figure 6.

The 3’UTR of TSP-1 is sufficient for NOL7-mediated post-transcriptional upregulation. Two clones each from SiHa cells stably co-expressing GFP, NOL7 or HuR and luciferase-EMP, -R01 or –TSP-1 were assayed for luciferase expression at the mRNA (dark-gray bar) or protein (light-gray bar). To control for vector expression artifacts, values were normalized to luciferase-EMP and reported as a percentage of GFP expression for each set of constructs. Data are represented as mean±s.e.m. from four independent experiments. *P<3×10⁻⁵; **P<9×10⁻⁵; ***P<2×10⁻⁷.

Figure 7.

NOL7 post-transcriptionally stabilizes TSP-1 mRNA. SiHa cells stably expressing GFP, NOL7 or HuR were treated for 0, 2, 4 or 6 with 5 μg/ml α-amanitin. Endogenous TSP-1 levels were assayed by real-time PCR and calculated via ΔΔC_t method. Half-life calculations were calculated from the nonlinear regression of the exponential decay curve N₀ = N(t)e^−λt, where the TSP-1 mRNA half-life t_½ = − ln(2)/λ. Data are represented as mean±s.e.m. from four independent experiments. *P<0.03; **P< 0.001; ***P< 0.0001.

Figure 8.

NOL7 specifically regulates distinct subset of angiogenic transcripts. Total complementary DNA from GFP- or NOL7-expressing cells untreated or transcriptionally inhibited with a-amanitin were analyzed by real-time PCR on TaqMan angiogeniesis array cards. Data are represented as mean ± s.e.m. from three independent experiments. (a) Steady-state expression (untreated NOL7 normalized to untreated gFp). Dashed line indicates the GFP control normalization. ^aP<0.05; ^bP<0.01; ^cP< 0.001; ^dP< 0.0001. (b) Post-transcriptional expression (α-amanitin-treated GFP/NOL7 normalized to untreated controls). *P<0.05, **P<0.01. (c) Genes significantly upregulated (yellow), unchanged (white), or downregulated (blue) are represented schematically based on their functional annotations, ligand-receptor interactions, and signaling. Those genes that were post-transcriptionally stabilized by NOL7 are outlined in red.