Palmitoyl transferases act as potential regulators of tumor-infiltrating immune cells and glioma progression

Abstract

High immune-cell infiltration in glioblastomas (GBMs) leads to immunotherapy resistance. Emerging evidence has shown that zinc finger Asp-His-His-Cyc-type (ZDHHC) palmitoyl transferases participate in regulating tumor progression and the immune microenvironment. In the present study, a large cohort of patients with gliomas from The Cancer Genome Atlas (TCGA) and Rembrandt databases was included to perform omics analysis of ZDHHCs in gliomas. CCK-8, flow cytometry, quantitative real-time PCR, western blotting, and transwell assays were performed to determine the effects of ZDHHC inhibition on glioma cells and microglia. We found that five (ZDHHC11, ZDHHC12, ZDHHC15, ZDHHC22, and ZDHHC23) out of 23 ZDHHCs were aberrantly expressed in gliomas and might play their roles through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Further results indicated that inhibition of ZDHHCs with 2-bromopalmitate (2-BP) suppressed glioma-cell viability and autophagy, as well as promoted apoptosis. Targeting ZDHHCs also promoted the sensitivity of glioma cells to temozolomide (TMZ) chemotherapy. In addition, the inhibition of ZDHHCs weakened the migratory ability of microglia induced by glioma cells in vitro and in vivo. Taken together, our findings suggest that the inhibition of ZDHHCs suppresses glioma-cell viability and microglial infiltration. Targeting ZDHHCs may be promising for glioma treatments.

Keywords: MT: Bioinformatics; ZDHHCs; gliomas; immune-cell infiltration; post-translational modification; signaling pathway.

Graphical abstract

Figure 1

(A–H) Relative mRNA levels of the dysregulated ZDHHCs in glioma tissues and normal brain tissues. ∗p < 0.05 ; ∗∗p < 0.01

Figure 2

Relative protein levels of seven ZDHHCs in normal brain tissues and glioma tissues (A–G) Histograms of ZDHHC expression levels in glioma samples from the Protein Atlas. In total, 11 to 12 samples were analyzed for ZDHHCs. Immunohistochemical (IHC) staining was evaluated as high, medium, or low staining or not detected. No information could be retrieved for ZDHHC22. (H–N) Representative IHC staining for ZDHHCs in normal brain tissues and glioma tissues is shown.

Figure 3

ZDHHC-related risk assessment model for prognostic prediction in gliomas (A and B) Kaplan-Meier analysis and time-dependent ROC analysis of the ZDHHC-gene signature in gliomas (including IDH wild type and mutant). (C and D) Kaplan-Meier analysis and time-dependent ROC analysis of the ZDHHC-gene signature in IDH wild-type gliomas are shown. (E and F) Kaplan-Meier analysis and time-dependent ROC analysis of the ZDHHC-gene signature in IDH mutant gliomas are shown.

Figure 4

GO enrichment of ZDHHC12, ZDHHC15, ZDHHC22, and ZDHHC23 in gliomas (A) ZDHHC12 is associated with extracellular structure organization, extracellular structure organization, and leukocyte migration. (B) ZDHHC15 is correlated with cell cycle phase transition, mitotic nuclear division, and neurotransmitter transport. (C) ZDHHC22 is related to extracellular structure organization, extracellular matrix organization, blood vessel morphogenesis, and leukocyte migration. (D) ZDHHC23 is involved in the regulation of neuron differentiation, signal release, and cell development.

Figure 5

Immune-cell infiltration analysis of ZDHHC12 and ZDHHC22 in tumors (A) Pan-cancer analysis of the correlation between ZDHHC12 and immune-cell infiltration. (B) The differences of immune-cell infiltration between ZDHHC12 high- and low-expression group is shown. (C) Immune-cell infiltration analysis of ZDHHC22 in pan-cancer is shown. (D) The differences of immune-cell infiltration between ZDHHC22 high- and low-expression group is shown.

Figure 6

Impact of 2-BP on glioma-cell viability and apoptosis (A) Gl261 cells were treated with 50 μM 2-BP for indicated times, and cell viability was measured (∗∗p < 0.01; not significant [ns], p > 0.05 compared with the control group). (B) Gl261 cells were treated with 50 μM 2-BP for indicated times, and effects of 2-BP on GL261 apoptotic rate (Q2+Q3) at 0 h (6.10%), 24 h (10.06%), and 48 h (19.54%) were evaluated. (C) C6 cells were treated with 50 μM 2-BP for indicated times, and cell viability was measured (ns; p > 0.05 compared with the control group). (D) C6 cells were treated with 50 μM 2-BP for indicated times, and effects of 2-BP on C6 apoptotic rate (Q2+Q3) at 0 h (3.93%), 24 h (3.40%), and 48 h (5.12%) were evaluated.

Figure 7

2-BP inhibits glioma-cell autophagy (A) Autophagy-related key proteins of GL261 and C6 cells were detected by Western blotting assay. (B–D) Relative Beclin-1,P62, LC3B protein levels between control and 2-BP treated groups in GL261 and C6 cells. ∗p < 0.05; ∗∗p < 0.01; ns; p > 0.05 compared with the control group

Figure 8

2-BP promotes the sensitivity of glioma cells to TMZ chemotherapy (A) Gl261 cells were treated with 50 μM 2-BP and 200 μM TMZ for 48 h, and cell viability was measured (∗∗p < 0.01, compared with the control group; ^##p < 0.01, compared with TMZ). (B) Gl261 cells were treated with 50 μM 2-BP and 200 μM TMZ for 48 h, and effects of 2-BP and TMZ on GL261 apoptotic rate (Q2+Q3) at 48 h were evaluated: control group (5.82%), TMZ (12.01%), and 2-BP + TMZ (38.90%). (C) C6 cells were treated with 50 μM 2-BP and 200 μM TMZ for 48 h, and cell viability was measured (ns; p > 0.05, compared with the control group; ^#p < 0.05, compared with TMZ). (D) C6 cells were treated with 50 μM 2-BP and 200 μM TMZ for 48 h, and effects of 2-BP and TMZ on C6 apoptotic rate (Q2+Q3) at 48 h were evaluated: control group (3.99%), TMZ (7.50%), and 2-BP + TMZ (6.70%).

Figure 9

Effects of 2-BP on glioma-induced microglia migration (A) Glioma cells were pretreated with 2-BP for 48 h and then co-cultured with BV2 cells. Effects of 2-BP-treated GL261 and C6 cells on BV2 cell migration were evaluated. (B and C) Glioma cells were treated with 2-BP for 48 h, and CCL2 and CXCL16 expression was detected by qRT-PCR (∗p < 0.05; ns; p > 0.05; compared with the control group). (D) Effects of 2-BP on microglia migration in vivo are shown.