Meta-analysis identifies NF-κB as a therapeutic target in renal cancer

Abstract

Objective: To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes.

Methods: Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC.

Results: A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an 'interferon signature' may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis.

Conclusions: These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently-incurable malignancy.

Figure 1. An NF-κB signature in ccRCC .

(a) Immuno-histochemical staining showing prominent nuclear RelA signal in ccRCC samples, but not in normal kidney tissue. The arrow indicates cytoplasmic RelA staining in cells of the proximal tubular epithelium. Scale bar = 100 µM. (b) Up-regulated genes (X-axis) from the meta-analysis of the four ccRCC datasets were plotted against false-discovery rate (FDR, Y-axis). Up-regulated genes with FDR < 0.05, shown in red, were used to define NF-κB and IFN signatures. (c) Heatmaps showing expression of NF-κB signature genes in each of the four indicated studies. N = normal, T = tumor. Heat bar = expression levels (log₂ scale).

Figure 2. Box plots depicting individual mRNA expression levels of representative genes from the NF-κB signature.

(a) Cell-survival genes BCL2 and SOD2, (b) pro-inflammatory genes CCL5 and ICAM1, (c) NF-κB regulators NFKB1 and TNFAIP3 and (d) interferon regulatory factors IRF1 and IRF7. Data were normalized using RMA. Fold-changes for Tumor (T) versus Normal (N) comparison were obtained by LIMMA. Y-axis shows RMA-normalized expression level (on log₂ scale) of each mRNA. Blue boxes represent gene expression levels in normal tissue, and red depicts expression in ccRCC. The white line within each box is the median, and distance between box and whiskers indicate interquartile ranges. Each of the four graphs per gene represent expression profiles in arrays generated by (from left to right) the Cifola, Gumz, Lenburg, and Yusenko studies.

Figure 3. An IFN signature in ccRCC .

(a) Immuno-histochemical staining showing robust nuclear STAT1 signal in ccRCC samples, but not in normal kidney tissue. (b) Heatmap showing expression of IFN signature genes after IFN stimulation of murine embryo fibroblasts. Heat bar = fold-change (log₂ scale). Expression levels of untreated cells were arbitrarily set to 1 (beige). (c) Heatmaps showing expression of IFN signature genes in each of the four indicated studies. N = normal, T = tumor. Heat bar = expression levels (log₂ scale).

Figure 4. Molecular network of NF-κB and IFN signatures in ccRCC .

The network was constructed by Ingenuity Pathway Analysis software (Ingenuity® Systems, HUhttp://www.ingenuity.comUH) using all genes in the NF-κB and IFN signatures. Key clusters in NF-κB (brown) and IFN (blue) arms are identified, and regulatory molecules controlling gene-network nodes are shown as large circles.

Figure 5. VHL mutational status correlates with expression of NF-κB and IFN signatures.

Heatmaps showing fold-changes in expression levels of NF-κB signature genes (a) or IFN signature genes (b) between VHL+/- samples from cases of familial VHL disease compared to VHL +/+ normal renal epithelium (column 1) ; VHL-/- cases of familial ccRCC compared to normal renal tissue (column 2) ; or VHL-/- cases of sporadic ccRCC compared to normal renal tissue (column 3). Heat bar = fold-change (log₂ scale).

Figure 6. A subset of NF-κB regulators and target genes correlate with poor outcome in ccRCC .

Kaplan-Meier survival curves for genes in the NF-κB signature whose increased expression levels significantly correlate with poorer overall survival outcome are shown. Individual gene expression profiles were dichotomized by median split into ‘high’ (red) or ‘low’ (blue) expression groups. Gene names are indicated above each graph. Please see Table S5 for p-values.

Figure 7. Model linking pVHL loss to NF-κB and IFN gene signatures.

(a) Heatmap showing basal expression of the IFN gene signature in RelA+/+ and RelA-/- MEFs. Heat bar = fold-change (log₂ scale). Expression levels in RelA+/+ were arbitrarily set to 1 (beige). Note that ~80% of IFN signature genes are expressed at lower basal levels in cells lacking RelA than in controls, indicative of a role for NF-κB in controlling ISG expression. (b) Schematic depicting the model presented in this study. ccRCC cells display elevated levels of NF-κB activity, perhaps as a result of result of constitutive EGFR or CARD9 activity stemming from loss of pVHL, that drives expression of cell-survival, pro-inflammatory, NF-κB regulatory genes, as well as genes encoding key nodes of type I interferon signaling (IFN-β, IRFs). Type I IFNs produced in this manner then function in an autocrine fashion to induce the expression of an IFN gene signature.