LUBAC enables tumor-promoting LTβ receptor signaling by activating canonical NF-κB

Abstract

Lymphotoxin β receptor (LTβR), a member of the TNF receptor superfamily (TNFR-SF), is essential for development and maturation of lymphoid organs. In addition, LTβR activation promotes carcinogenesis by inducing a proinflammatory secretome. Yet, we currently lack a detailed understanding of LTβR signaling. In this study we discovered the linear ubiquitin chain assembly complex (LUBAC) as a previously unrecognized and functionally crucial component of the native LTβR signaling complex (LTβR-SC). Mechanistically, LUBAC-generated linear ubiquitin chains enable recruitment of NEMO, OPTN and A20 to the LTβR-SC, where they act coordinately to regulate the balance between canonical and non-canonical NF-κB pathways. Thus, different from death receptor signaling, where LUBAC prevents inflammation through inhibition of cell death, in LTβR signaling LUBAC is required for inflammatory signaling by enabling canonical and interfering with non-canonical NF-κB activation. This results in a LUBAC-dependent LTβR-driven inflammatory, protumorigenic secretome. Intriguingly, in liver cancer patients with high LTβR expression, high expression of LUBAC correlates with poor prognosis, providing clinical relevance for LUBAC-mediated inflammatory LTβR signaling.

Fig. 1. Unbiased functional and proteomic analysis reveals LUBAC as a new component of the native LTβR-SC.

A The indicated four cell lines were stimulated with 500 ng/ml of TAP-LIGHT overnight or left untreated, and the correspondent supernatants were collected and subjected to a secretome analysis by using a human cytokine array. B The indicated cell lines were stimulated with increasing concentrations TAP-LIGHT for 24 h, and the concentration of IL-8 or CCL20 was determined by ELISA. Error bars represent the mean ± SEM of three independent experiments. Statistical significance was assessed by two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C Schematic representation of TAP-LIGHT. D Hep3B cells were stimulated with TAP-LIGHT (2000 ng/ml) followed by total protein extraction. The native LTβR-SC was purified via anti-FLAG M2 beads, and analyzed by mass spectrometry (n = 2). A STRING functional connectivity network was generated for the LTβR-SC. Relative protein abundances are represented by the node size and sequence coverage is depicted on the outer ring in red. E Analysis of native LTβR-signaling complex (SC) in four different cancer cell lines. Cells were stimulated with TAP-LIGHT (2000 ng/ml) for the indicated times, LTβR-SC was immunoprecipitated as in (D) and analyzed by Western blotting. Representative results of at least two independent replicates are shown.

Fig. 2. Secretion of proinflammatory cytokines by LTβR-induced canonical NF-κB is dependent on LUBAC.

A HOIP proficient or deficient cell lines were stimulated with TAP-LIGHT (200 ng/ml) for the indicated time points followed by total protein extraction. Cell lysates were subjected to Western blot analysis and probed for the indicated proteins. Representative results of at least three independent replicates are shown. HOIP proficient or deficient Hep3B cells were stimulated overnight with the indicated concentrations of LIGHT. Supernatants were collected and levels of IL-8 (B), CCL20 (C) and CCL2 (D) were quantified by ELISA. Error bars represent the mean ± SEM of three independent experiments. Statistical significance was assessed by two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. E A549 WT, HOIP KO or HOIP-C885S cell lines were stimulated with TAP-LIGHT (200 ng/ml) for the indicated time points followed by total protein extraction. Cell lysates were subjected to Western blot analysis and probed for the indicated proteins. Representative results of at least three independent replicates are shown. F A549 WT, HOIP KO or HOIP-C885S cell lines were stimulated overnight with TAP-LIGHT (200 ng/ml). Supernatants were collected and levels of IL-8 were quantified by ELISA. Error bars represent the mean ± SEM of three independent experiments. Statistical significance was assessed by two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3. LUBAC is required for the recruitment of NEMO, A20 and OPTN to the LTβR-SC.

A Schematic overview of the proteomic analysis of the native LTβR-SC performed in HOIP proficient and deficient Hep3B cells. B Heatmap represents a linear model-based of log2 protein abundances of five independent experiments. C, D HOIP proficient or deficient Hep3B cells were stimulated with TAP-LIGHT (2000 ng/ml) for the indicated times followed by total protein extraction. Native LTβR-SC was isolated by using M2-beads and analyzed by Western blotting. Representative results of at least three independent replicates are shown. E A549 WT, HOIP KO or HOIP-C885S were stimulated with TAP-LIGHT (2000 ng/ml) for the indicated times followed by total protein extraction. Native LTβR-SC was isolated by using M2-beads and analyzed by western blotting. Representative results of at least three independent replicates are shown. F Gene Set Enrichment Analysis (GSEA) comparing the results obtained by LC-MS/MS on the composition of the LTβR-SC obtained from WT vs. HOIP-deficient cells using the quantitative peptide number data obtained from five independent experiments. The thresholds for this analysis were established as follows: (1) an FDR of less than 0.25, and (2) an adjusted P-value of less than 0.01.

Fig. 4. NEMO, A20 and OPTN cooperatively and inversely regulate LTβR-mediated NF-κB activation.

A–C A549 WT or lacking expression of NEMO (A), OPTN (B) or A20 (C) were stimulated with TAP-LIGHT (200 ng/ml) for the indicated times followed by total protein extraction. Cell lysates were subjected to western blot analysis and probed for the indicated proteins. Representative results of at least three independent replicates are shown. D A549 WT or lacking expression of NEMO, OPTN or A20 were stimulated overnight with TAP-LIGHT (500 ng/ml). Supernatants were collected and levels of IL8 were quantified by ELISA. Error bars represent the mean ± SEM of three independent experiments. Statistical significance was assessed by two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5. Loss of NEMO, A20 and OPTN differentially affect composition of the LTβR-SC.

A–C A549 WT or lacking expression of NEMO (A), A20 (B) or OPTN (C) were stimulated with TAP-LIGHT (2000 ng/ml) for the indicated times followed by total protein extraction. Native LTβR-SC was isolated by using M2-beads and analyzed by Western blotting. Representative results of at least three independent replicates are shown. D Model of the regulation of LTβR signaling by LUBAC, NEMO and OPTN. In normal conditions, LTβR triggers a balanced activation of canonical and non-canonical NF-κB pathways. This balance is controlled by LUBAC mediated linear ubiquitination of several components within the LTβR-SC, which enables the recruitment of NEMO, OPTN and A20; NEMO positively regulates the canonical NF-κB pathway, whereas OPTN and A20 are negative regulators of this pathway and are involved in the late switch towards activation of the non-canonical NF-κB pathway. Absence of LUBAC causes an impaired recruitment of NEMO, A20 and OPTN, disrupting the signaling balance and causing an uncontrolled activation of the non-canonical NF-κB pathway.

Fig. 6. HOIP is a novel prognostic marker in liver cancer patients with high LTβR expression.

A RNA-seq expression data obtained from TCGA and GTEx databases were analyzed. All normalized expression data were downloaded from UCSC Xena platform (unit: log2(expected_count-deseq2 + 1)). Generally, the transcripts of LTβR in tumor tissues were retrieved from TCGA database with different cancer types. The normal tissues from different organs in this analysis were obtained from GTEx and TCGA matched normal samples. Statistical significance was calculated using a two tailed student’s t test, and the asterisks indicate the statistical significance (* means: p < 0.05, **** means: p < 0.001). B Primary samples obtained from 14 liver cancer patients were stratified according to their LTβR expression (quantified by IHC staining intensity), and their respective survival rates were plotted in a Kaplan-Meier survival graph. Representative IHC images of normal tissue (negative control), low LTβR expression and high LTβR expression are shown. Statistical significance was calculated using a log-rank test, and p value is shown in the graph. C GSEA of RNA-seq expression data from TCGA liver cancer database (LIHC), comparing patients with high expression of LTβR (n = 183) versus low expression of LTβR (n = 182). Correlation between expression levels of LTβR and gene signatures of either canonical NF-κB pathway (GO:0043123) or non-canonical NF-κB pathway (GO:0038061) are analyzed. The median value of normalized mRNA transcripts was set up as a cut-off value for separating high expression and low expression LTβR group. D RNA-seq expression data from TCGA LIHC was used to stratify patients in two groups according to their LTβR expression. The median value of normalized LTβR mRNA transcripts was set up as a cut-off value for separating high and low expression groups. Subsequently, in the high LTβR expression group, patients were stratified again into high- or low-HOIP expression groups based on the quartile value, and survival results were represented in Kaplan-Meier survival graphs. Statistical significance was calculated using a log-rank test, and p values are shown in each graph.