NF-κB/RelA controlled A20 limits TRAIL-induced apoptosis in pancreatic cancer

Abstract

The emergence of resistance to systemic therapies in pancreatic ductal adenocarcinoma (PDAC) is still a major obstacle in clinical practice. Both, constitutive and inducible NF-κB activity are known as key players in this context. To identify differentially expressed and TRAIL resistance mediating NF-κB target genes, TRAIL sensitive and resistant PDAC cell lines were analyzed by transcriptome assays. In this context, A20 was identified as an NF-κB/RelA inducible target gene. Translational PDAC tissue analysis confirmed the correlation of elevated A20 protein expression with activated RelA expression in PDAC patients. In in vitro experiments, an elevated A20 expression is accompanied by a specific resistance toward TRAIL-mediated apoptosis but not to chemotherapeutic-induced cell death. This TRAIL resistance was attributed to A20´s E3-ligase activity-mediating Zink finger domain. Furthermore, the ubiquitin-binding scaffold protein p62 was identified as indispensable for the TRAIL-mediated apoptosis-inducing pathway affected by A20. The results of this study identify A20 as a possible therapeutic target to affect resistance to TRAIL-induced apoptosis in PDAC cells.

Fig. 1. A20 is a TRAIL inducible target gene in TRAIL-resistant PDAC cells.

A Cells were treated with TRAIL for 6 h and whole-cell lysates were analyzed by western blot with indicated antibodies. HSP90 was used as a loading control. n = 5. B GSEA of RNA-Seq expression data from human Panc1 cells treated with 10 ng/ml TRAIL for 5 h. Depicted is the HALLMARK signature TNFA SIGNALLING VIA NF-κB, including the q value. (C + D) PDAC cells were left untreated C or transfected with RelA-specific siRNA D for 48 h. Afterward, cells were treated as described in B and a genome-wide array was performed. Top 10 differentially TRAIL-induced genes of a genome-wide expression array in Panc1 and MiaPaca2 cells, ranked by fold change are depicted.

Fig. 2. RelA-dependent A20 expression correlates with chemoresistance of PDAC cells.

A Panc1, Patu8988t, PaTu8902, and MiaPaca2 cells were left untreated or treated with TRAIL for indicated periods. Isolated total RNA was submitted to reversed transcription and A20 mRNA was measured by real-time PCR. An external standard curve was applied. A20 mRNA expression was related to RPL13 gene expression. The mean values of 5 independent experiments performed in duplicates ± S.D. are depicted, *P-values <0.05. B Cells were treated with TRAIL for 6 h and whole-cell lysates were analyzed with indicated antibodies. HSP90 was used as a loading control, n = 5. (C + D) Resistant PDAC cells were treated with indicated siRNAs and left untreated or with TRAIL for 3 h or 5 h C or 6 h D. C Isolated total RNA was reversed transcribed and analyzed by real-time PCR for A20 mRNA expression and normalized to housekeeper gene expression. Depicted are the mean values of 5 independent experiments performed in duplicates ± SD *P-values < 0.05. D western blot analysis with total lysates was done with indicated antibodies and β-actin as a loading control, n = 5.

Fig. 3. RelA and A20 expression are elevated in PDAC tissue.

A Total RNA from 31 PDAC tissues and corresponding normal tissues were isolated, reversed transcribed, and analyzed by qPCR for A20 expression. For normalization RPL13 expression was analyzed. Displayed is the mRNA expression of tumor and normal tissue and the respective mean ± SD. *P-values <0.02. B Depicted is the expression score (ES = P*S) of A20 and phospho-RelA staining in 22 PDAC and 8 normal tissues ± SEM. *P-values < 0.05. C Representative images of immunohistochemical staining for A20, phospho-p65, and IgG-control (scale bar = 100 nm) of tissues from two PDACs and one normal pancreas are shown. D Depicted are the Pearson correlation coefficient and the linear regression of the A20 and phospho-RelA expression score (ES) of PDAC tissues (n = 22). P-value is indicated. E Results of GSEA hallmark analysis for A20 (TNFAIP3) high and low expressing PDAC of TCGA and ICGC Data.

Fig. 4. NF-κB/RelA binds to the A20 promoter.

A Sequence of the A20 5´flanking region containing two κB elements (highlighted in boxes; #1 NF-κB site: −45 to −54 (GGAATCCCC) and #2 NF-κB site: −57 to −66 (GGAAAGTCCC)). Underlined are the EMSA oligos in grey and the ChIP primers are marked in green. B Panc1 or PaTu8902 cells were treated with TRAIL for indicated times. Nuclear extracts were analyzed by EMSA assays with oligonucleotides spanning one putative κB binding site, n = 2. C For competitive EMSA assays, TRAIL-treated PaTu8902 nuclear extracts were incubated in addition to the indicated ³²P-labeled NF-κB probes with non-labeled #1/#2 NF-κB oligonucleotides, a consensus NF-κB probe, or two unrelated probes (Oct1 and AP1), n = 2. D For supershift experiments with Panc1 nuclear extracts indicated antibodies were used, n = 2. E Panc1 cells were treated with TRAIL for 3 h or left untreated and lysates were examined by ChIP assay using IgG, RelA, or Rpb1 antibody for precipitation. Primers spanning both κB sites and results were normalized to input. Mean ± SD of 4 independent experiments is shown. *p < 0.02.

Fig. 5. A20 mediates chemoresistance towards TRAIL in PDAC cells.

SiRNA-treated Panc1 cells (left panel) or Patu A20cc (right panel) were treated with therapeutic drugs (20 µg/ml etoposide, 10 µg/ml gemcitabine, or 100 ng/ml TRAIL) for indicated periods. A, B whole-cell lysates were conducted to western blot analysis with indicated antibodies and HSP90 and β-actin as loading controls, n = 5. C Panc1 cells (left panel) and Patu8988t (right panel) were analyzed for apoptotic cell death by subG1 assay. Shown are results from 5 independent experiments, *p < 0.05, n = 5.

Fig. 6. Znf4 domain of A20 accountable for chemoresistance mediation.

Patu A20cc cells were transfected with indicated A20 expression plasmids, treated with TRAIL for 24 h, and analyzed by subG1 assay. Plotted are mean ± S.D. from 5 independent experiments.

Fig. 7. p62 is needed for TRAIL-mediated apoptosis induction.

A, C Panc1 and B, D PaTu8902 cells were transfected with control, A20, p62, or A20 + p62 siRNA and treated with TRAIL for 0 h, 4 h, or 6 h. A, B caspase 3/7-assay was performed in duplicates and normalized to mg protein. Depicted are the mean ± SD from 5 independent experiments. C, D whole-cell lysates were analyzed by western blot experiments with indicated antibodies and HSP90 as a loading control, n = 5. E, F MiaPaca2 cells were transfected with control or p62 siRNA and subsequently treated with TRAIL for indicated periods. E caspase 3/7-activity was measured and normalized to mg protein. F cells were fixed with ethanol and analyzed by subG1 assay. Depicted are means ± SD from 5 independent experiments. G Survival of patients with high (>75^th percentile) and low (<25^th percentile) A20 mRNA expression were subdivided by their p62 mRNA expression (high (>75^th percentile), low (<25^th percentile)). Clinical data based on the curated ICGC data set (n = 81)).