SETD6 regulates NF-κB signaling in urothelial cell survival: Implications for bladder cancer

Abstract

Non-muscle invasive bladder cancer has a high recurrence rate of 45-70%, progressing to muscle invasive disease in about 15% of those patients over a 5-year period. Administration of the mycobacterium, Bacillus Calmette-Guerin (BCG) that induces local inflammation resulting in tumor remission in responsive patients is frequently used for treatment. BCG-treated patients with NF-κB del/del genotype have an increased risk of recurrence suggesting an important role of NF-κB in bladder cancer. Since protein methyltransferases play critical roles in modulating chromatin structure and gene expression, we screened a focused array of epigenetic modification genes to identify differential expression between normal urothelial and bladder cancer cells. We found and validated high expression of the SET-domain-containing protein methyltransferase, SETD6. SETD6 monomethylates NF-κB-p65 at lysine 310. Our results show that primary urothelial cells and normal bladder tissue have nearly undetectable message and protein level of SETD6 that increases in transformed urothelial cells and is further increased in bladder cancer cells and tissues. Overexpression of SETD6 in transformed urothelial cells increased cell survival and colony formation while knockdown in cancer cells decreased both parameters. Luciferase reporter assays showed that SETD6 induced the canonical NF-κB signaling pathway. Further, the use of catalytic SETD6 and IκBα mutant shows that SETD6 positively regulates survival by affecting p65 message, protein level and its function as determined by increased expression of NF-κB target genes. Our findings suggest that SETD6 plays an important role in NF-κB regulation and may have an important role in NF-κB-mediated local inflammatory response following BCG treatment.

Keywords: NF-κB; SETD6; bladder cancer; methyltransferase; signaling.

Figure 1. Upregulation of SETD6 in bladder cancer

(A) Validation of relative SETD6 expression in different bladder cells was carried out using qPCR. p ≤ 0.05 were considered to be statistically significant (*). The samples were measured in triplicates and the experiment was repeated 3 times. (B) cDNA array was performed with bladder cancer tissues (n = 24) representing different stages and SETD6 expression was analyzed by qPCR. (C) Basal level of SETD6 protein was detected using anti-SETD6 antibody by western blotting. (D) Immunocytochemistry in different bladder cancer cell lines cells showing cytoplasmic and nuclear SETD6. (E) SETD6 protein was detected using anti-SETD6 antibody by western blot in bladder cancer tissues (n = 9) compared to non-cancerous bladder tissues.

Figure 2. SETD6 positively regulates survival

Wild type (wt) SETD6 was overexpressed in the SVHUC1 cell line. EV: pCAG; pSD6: pCAG-Flag SETD6. (A) Trypan blue and (B) crystal violet staining were used to determine cell survival. The samples were counted in duplicates and the experiments were repeated 2 times. p ≤ 0.05 were considered to be statistically significant (*). (C) MTT assay was used to measure growth. The samples were measured in triplicates and the experiments were repeated 3 times. p ≤ 0.05 were considered to be statistically significant (*). Mutant (mut) SETD6 Y285A was overexpressed in the SVHUC1cell line. EV: pCAG; pmSD6: pCAG-Flag mutant SETD6 (Y285A). (D) Trypan blue and (E) crystal violet staining were used to determine cell survival. The samples were counted in duplicates and the experiments were repeated 2 times. p ≤ 0.05 were considered to be statistically significant (*). (F) MTT assay was used to measure growth. The samples were measured in triplicates and the experiments were repeated 2 times. p ≤ 0.05 were considered to be statistically significant (*). SETD6 was knocked down in bladder cancer cell lines (G) in UMUC3 and (H) in RT4 and trypan blue assay was used to determine cell survival. NTC: non-targeting control siRNA; si-SD6: SETD6 siRNA. Samples were counted in duplicate and the experiments were repeated twice. p ≤ 0.05 were considered to be statistically significant (*).

Figure 3. SETD6 induces canonical NF-κB signaling

Effect of SETD6 overexpression on (A) p65 mRNA and on (B) IκBα mRNA (18 h post SETD6 overexpression) measured by RT-PCR. Samples were measured in triplicates and experiments were repeated 3 times. p ≤ 0.05 were considered to be statistically significant (*). EV: (empty vector) pCAG; pSD6: pCAG-Flag SETD6. (C) Immunoblots to show the effect of wild type (wt) SETD6 overexpression on p65 using whole cell and nuclear extracts. EV (empty vector): pCAG; pSD6: pCAG-Flag SETD6. Other members of the NF-κB family were tested using whole cell extracts. (D) Effect of mutant (mut) SETD6 overexpression on p65 and other members of the NF-κB family as in panel C. EV (empty vector): pCAG; pmSD6: pCAG-Flag mutant SETD6 (Y285A). Effect of SETD6 knockdown on p65 in (E) UMUC3 and (F) RT4. p ≤ 0.05 were considered to be statistically significant (*). Experiments were repeated twice. NTC: non-targeting control siRNA; si-SD6: SETD6 siRNA.

Figure 4. SETD6 mediated p65 level increases cell survival

(A) Consequence of wild type (wt) SETD6 and mutant (mut) IκB overexpression on survival of SVHUC1 cells determined by trypan blue assay. The samples were measured in duplicates and experiments were repeated twice. p ≤ 0.05 was considered to be statistically significant (*). Lane 1 EV (empty vector): pCAG+ pcDNA; Lane 2 EV (empty vector): pCAG; pSD6: pCAG-Flag SETD6; mIκBα: pcDNA-mutant IκBα (S32/36A). Effect of wt SETD6 overexpression on (B) nuclear level of p65 determined by western blotting and the effect on (C) transcription reporter activity of NF-κB measured by luciferase activity. The samples were measured in triplicates and experiments were repeated twice. p ≤ 0.05 was considered to be statistically significant (*). (D) Effect of mut SETD6 overexpression on transcriptional activity of NF-κB measured by luciferase activity. Samples were measured in triplicates and experiments were repeated twice. p ≤ 0.05 was considered to be statistically significant (*). (E) Effect of SETD6 overexpression on NF-κB target gene expression (24 h post SETD6 overexpression) was measured by RT-PCR. The samples were measured in triplicates and experiments were repeated twice. p ≤ 0.05 was considered to be statistically significant (*). EV (empty vector): pCAG; pSD6: pCAG-Flag SETD6. (F) Model of the role of SETD6 in bladder cancer: SETD6 induces NF-κB signaling by increasing p65 level and its nuclear translocation resulting in increased survival of transformed bladder cells.