Lysine N-methyltransferase SETD7 promotes bladder cancer progression and immune escape via STAT3/PD-L1 cascade

Abstract

Background: The immunotherapy sensitivity of patients with bladder cancer (BCa) remains low. As the role of protein methylation in tumorigenesis and development becomes clearer, the role of lysine N-methyltransferase SET domain containing 7 (SETD7) in the progression and immune escape of BCa is worth studying. Methods: The correlation between lysine methyltransferase family and prognosis or immunotheray sensitivity of BCa patients were analyzed, and SETD7 was screened out because of the significant correlation between its expression and survival data or immunotherapy sensitivity. The expression of SETD7 in BCa tissues and cell lines were explored. The functions of SETD7 were investigated by proliferation and migration assays. The role of SETD7 in BCa immune escape was validated by analyzing the correlation between SETD7 expression and tumor microenvironment (TME)-related indicators. The results were further confirmed by conducting BCa cell-CD8⁺ T cell co-culture assays and tumorigenesis experiment in human immune reconstitution NOG mice (HuNOG mice). Bioinformatic prediction, CO-IP, qRT-PCR, and western blot were used to validate the SETD7/STAT3/PD-L1 cascade. Results: SETD7 was highly expressed in BCa, and it was positively associated with high histological grade and worse prognosis. SETD7 promoted the proliferation and migration of BCa cells. The results of bioinformatics, in vitro co-culture, and in vivo tumorigenesis assays showed that SETD7 could inhibit the chemotoxis and cytotoxicity of CD8⁺ T cells in BCa TME. Mechanistically, bioinformatics analysis, CO-IP assay, qRT-PCR, and western blot results indicated that SETD7 could increase the expression of PD-L1 via binding and promoting STAT3. Conclusions: Taken together, SETD7 indicated poor prognosis and promoted the progression and immune escape of BCa cells. It has great potential to act as a new indicator for BCa diagnosis and treatment, especially immunotherapy.

Keywords: SETD7; bladder cancer; immune escape; migration; proliferation.

Figure 1

SETD7 acted as a high-risk factor for BCa progression and worse survival. SETD7 was an increased risk factor in BRCA, MESO, and BLCA. B. Gene enrichment analysis result showed that SETD7 was correlated with 'bladder cancer' pathway. C. GSEA analysis showed bladder cancer associated genes were influenced by SETD7. D. GSE48276 and GSE69795 data indicated that SETD7 was correlated with worse overall survival of BCa patients. E. TCGA-BLCA data indicated that SETD7 was highly regulated in high-grade BCa patients compared with low-grade patients.

Figure 2

SETD7 was highly regulated in BCa. The expression of SETD7 in 40 pairs of BCa tissues was validated by qRT-PCR (*P<0.05, Student's t-test). B. The expression of SETD7 in seven BCa cell lines and SV-HUC cell line was confirmed by qRT-PCR (**P<0.01, ***P<0.001, Student's t-test). C. The correlation between SETD7 and the overall survival of patients with BCa was validated by Kaplan-Meier analysis. D. The expression of SETD7 in BCa and normal tissues was validated using the Human Protein Atlas (HPA) database. E. The expression of SETD7 in our own BCa tissues and adjacent normal tissues was confirmed by IHC. Data are expressed as mean±SD, n=3.

Figure 3

SETD7 promoted the proliferation and migration of BCa cells. A. Efficiency of SETD7 overexpression in T24 and UMUC3 cells was confirmed by qRT-PCR (*P<0.05, Student's t-test). B. Efficiency of SETD7 overexpression in T24 and UMUC3 cells was confirmed by Western blot analysis. C. CCK8 assay showed that SETD7 could promote T24 and UMUC3 cell proliferation (***P<0.001, ****P<0.0001, Student's t-test). D. Colony formation assay indicated that SETD7 could promote T24 and UMUC3 colony formation (*P<0.05, ***P<0.001, Student's t-test). E. Scratch wound healing assays showed that SETD7 could promote the migration rate of T24 and UMUC3 cells (*P<0.05, Student's t-test). F. Transwell assays showed that SETD7 could promote the migration rate of T24 and UMUC3 cells (*P<0.05, Student's t-test). Data are mean±SD, n=3.

Figure 4

SETD7 was positively associated with the immune escape ability of GSE48075. A-B. Infiltration level of different immune cells in high- and low-SETD7 groups on the basis of GSE48075 data. C. Expression of different immunomodulators in high- and low-SETD7 groups based on the GSE48075 data. D. Expression of immune cells effector genes in high- and low-SETD7 groups on the basis of GSE48075 data. E. Correlation analysis between SETD7 and inhibitory immune checkpoints on the basis of GSE48075 data.

Figure 5

SETD7 was positively associated with immune escape ability of BLCA. A-B. Infiltration level of different immune cells in high and low SETD7 groups on the basis of TCGA-BLCA data. C. Expression of different immunomodulators in high- and low-SETD7 groups on the basis of TCGA-BLCA data. D. The expression of immune cells effector genes in high- and low-SETD7 groups on the basis of TCGA-BLCA data. E. Correlation analysis between SETD7 and inhibitory immune checkpoints on the basis of TCGA-BLCA data.

Figure 6

SETD7 promoted the expression of PD-L1 in BCa. A. Correlation analysis of SETD7 and PD-L1 in TCGA-BLCA. B. Correlation analysis of SETD7 and CD8⁺ T cell infiltration in BCa based on TCGA-BLCA. C. Pearson's correlation analysis was applied to confirm the correlation of SETD7 and PD-L1. D. QRT-PCR results revealed that SETD7 promoted the mRNA level of PD-L1 in T24 and UMUC3 cells (**P<0.01, Student's t test). E. Western blot results revealed that SETD7 promoted the protein level of PD-L1 in T24 and UMUC3 cells. F. Genemania database was used to predict the potential binding proteins of SETD7. G. Hitpredict database was used to predict the potential binding proteins of SETD7. H. Correlation analysis of SETD7 and STAT3 in TCGA-BLCA. I. CO-IP assay was used to confirm the binding of SETD7 and STAT3. J. Western blot assay indicated that SETD7 promoted the expression of STAT3. Data are expressed as mean±SD, n=3.

Figure 7

SETD7 inhibited the chemotaxis and function of CD8⁺ T cells in vitro. A. Schematic diagram of the process of the sorting and activatation of CD8⁺ T cells. B. Schematic diagram of CD8⁺ T cell chemotaxis assay. C. Chemotaxis assays results showed the migration ability of CD8⁺ T cells was decreased when co-culturing with SETD7 overexpression T24 or UMUC3 cells (*P<0.05, **P<0.01, Student's t-test). D. Schematic diagram of CD8⁺ T cell-mediated BCa cell killing assay. E. CD8⁺ T cell-mediated BCa killing assay results show that the killing ability of CD8⁺ T cell was decreased when co-culturing with SETD7 overexpression T24 or UMUC3 cells. F. ELISA assay results show that CD8⁺ T cells produced less IFN-γ and granzyme B when co-culturing with SETD7 overexpression T24 or UMUC3 cells (*P<0.05, **P<0.01, Student's t-test). Data are expressed as mean±SD, n=3.

Figure 8

SETD7 promoted the tumor proliferation and inhibited function of CD8⁺ T cells in vivo. A. Schematic diagram of the HuNOG mice model construction. B. Representative picture of the tumor samples of HuNOG mice injected with SETD7 overexpression or control T24 cells (n=3). C. Weights of the tumor samples dissected from HuNOG mice (*P<0.05, Student's t-test). D. Tumor volumes of two groups of HuNOG mice were measured every 3 days (*P<0.05, Student's t-test). E. IHC staining results indicated SETD7 inhibited CD8 positive rate in tumor samples dissected from HuNOG mice (**P<0.01, Student's t-test). Data are expressed as mean±SD, n=3.

Figure 9

Mechanism network of SETD7 in bladder cancer immune escape.