B-Myb deficiency boosts bortezomib-induced immunogenic cell death in colorectal cancer

Abstract

B-Myb has received considerable attention for its critical tumorigenic function of supporting DNA repair. However, its modulatory effects on chemotherapy and immunotherapy have rarely been reported in colorectal cancer. Bortezomib (BTZ) is a novel compound with chemotherapeutic and immunotherapeutic effects, but it fails to work in colorectal cancer with high B-Myb expression. The present study was designed to investigate whether B-Myb deletion in colorectal cancer could potentiate the immune efficacy of BTZ against colorectal cancer and to clarify the underlying mechanism. Stable B-Myb knockdown was induced in colorectal cancer cells, which increased apoptosis of the cancer cells relative to the control group in vitro and in vivo. We found that BTZ exhibited more favourable efficacy in B-Myb-defective colorectal cancer cells and tumor-bearing mice. BTZ treatment led to differential expression of genes enriched in the p53 signaling pathway promoted more powerful downstream DNA damage, and arrested cell cycle in B-Myb-defective colorectal cancer. In contrast, recovery of B-Myb in B-Myb-defective colorectal cancer cells abated BTZ-related DNA damage, cell cycle arrest, and anticancer efficacy. Moreover, BTZ promoted DNA damage-associated enhancement of immunogenicity, as indicated by potentiated expression of HMGB1 and HSP90 in B-Myb-defective cells, thereby driving M1 polarization of macrophages. Collectively, B-Myb deletion in colorectal cancer facilitates the immunogenic death of cancer cells, thereby further promoting the immune efficacy of BTZ by amplifying DNA damage. The present work provides an effective molecular target for colorectal cancer immunotherapy with BTZ.

Keywords: B-Myb (MYBL2); Bioinformatics; Bortezomib (BTZ); DNA damage; Immunogenic death (ICD); Macrophages.

Figure 1

Stable knockdown of B-Myb led to increased apoptosis in colorectal cancer cells. (A–B) The B-Myb (MYBL2) expression in pan-cancer, COAD and READ was analyzed by bioinformatics. (C–D) The prognostic correlation between the expression of B-Myb was presented. The SW480 cells with or without B-Myb knockdown were cultured for 24 h. (E) The expression of apoptosis proteins Bax, Caspase-3 and B-Myb was detected by WB, which was also utilized to analyze the DNA damage-associated molecules p53 and γ-H2A.X expression. (F–H) The apoptosis of SW480 cells was measured through PI staining. The PI positive cells were detected with confocal microscopy (F) and flow cytometry (G–H). Geometric means were used to quantify the MFI. Values were means ± SD (n = 3, ****p < 0.0001).

Figure 2

Bortezomib further boosted apoptosis in colorectal cancer cells of B-Myb stable knockdown. The colorectal cancer cells (SW480 or SW620) were treated by BTZ for 24 h. (A) The chemical structure of bortezomib was presented. (B–C) CCK-8 assay indicated BTZ significantly suppressed the cell viability in SW480 cells (B) and SW620 cells of B-Myb (C) deletion when the concentration reached to 30 nM in response to different concentrations treatment (1, 5, 10, 30, 60, and 100 nM). Values were means ± SD (n = 3, ***p < 0.001, ****p < 0.0001). (D–E) Cellular apoptosis rate was assayed using PI staining. The PI positive cells were calculated by flow cytometry. Geometric means were applied to quantify the MFI. Values were means ± SD (n = 3, **** p < 0.0001). (F) The molecules of B-Myb, proliferation and apoptosis (PCNA, Bax) were measured by WB. (G) The PI positive cells were also observed by laser confocal microscopy.

Figure 3

Bortezomib showed excellent anti-cancer efficacy in B-Myb knockdown colorectal cancer cell-bearing mice. BTZ was administered every three days for a total of 5 administrations (i.p.). The tumor grafts were harvested upon the end of treatment. (A) The tumor grafts were photographed. (B) The tumor volume was monitored during the treatment. (C) The tumor tissues were weighed after the extraction of tumor grafts. (D–G) IHC and TUNEL staining were utilized to evaluate cancer cells’ apoptosis, including Bax and Caspase-3 expression (G), which was quantified (D–F). Values were means ± SD (n = 4, *p < 0.05,****p < 0.0001).

Figure 4

BTZ activated DNA damage in B-Myb defective colorectal cancer cells. The cancer cells were treated by BTZ for 24 h. (A–D) The DNA double-strand break of cells at different BTZ concentrations (10, 30, and 60 nM) was observed. (E–F) The DNA double-strand break was assayed by single-cell gel electrophoresis. The length of the comet tail was assayed. (G) The p53, γ-H2A.X molecules were detected using WB, suggesting prominent DNA damage in B-Myb defective SW480 cells treated by BTZ. (H–I) The expression of CDK4, CyclinD1 and CDC25A, which manipulate cell cycle progression, was investigated using WB. The mean gray was quantitatively assayed. (J–L) The cell cycle was assayed by PI staining and flow cytometry. The G1, S and G2/M phases were calculated. Geometric means were used to quantify the MFI. Values were means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Prominent DNA damage was observed in BTZ-treated shB-Myb SW480 cell-bearing mice. (A–B) IHC staining evidenced BTZ treatment induced significant DNA damage in tumor-bearing mice of B-Myb deletion, as characterized by enhanced expression of p53 and γ-H2A.X. (C–D) The proteins controlling cell cycle (CDK4 and CyclinD1) in tumor grafts were assayed by IHC. (E–H) The area of positive cells were calculated. Values were means ± SD (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6

Over-expression of B-Myb reduced the DNA damage, cell cycle arrest, and apoptosis induced by BTZ in shB-Myb SW480 cells. The shB-Myb SW480 cells were transfected with B-Myb plasmid, then treated by BTZ. (A–B) Comet experiments were applied to assay the DNA double-strand breakage. The length of the comet tail was analyzed. (C) The expression of B-Myb, p53 and γ-H2A.X was assayed using WB. (D–E) The cell cycle was analyzed by flow cytometry. (F) Apoptosis-associated molecules Casepase-3 and Bax were measured using WB. (G–I) The apoptosis of cells was measured by PI staining. The positive cells were observed by laser confocal microscopy (G) and flow cytometry (H–I). Geometric means were used to quantify the MFI. Values were means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 7

Type-I macrophages were accumulated in B-Myb defective colorectal cancer in the presence of BTZ. (A) The expression of GBP5 and iNOS, which are biomarkers of M1, was detected by WB. (B–E) Multiple immunofluorescence staining revealed the macrophages’ infiltration (expression of CD11b) and M1 polarization (iNOS and GBP5). Blue fluorescence came from the nucleus. Green fluorescence came from CD11b. Red fluorescence came from iNOS (D) or GBP5 (E). The CD11b/iNOS positive cells and CD11b/BGP5 positive cells were quantified (B–C). Values were means ± SD (n = 4, **p < 0.01, ***p < 0.001).

Figure 8

The schematic diagram of the current work. High expression of B-Myb leads to DNA repair and drug resistance in colorectal cancer. BTZ could promote cell apoptosis in B-Myb defective tumors, wherein pronounced DNA damage and cell cycle arrest were induced. The immunogenic cell death and macrophage M1 polarization were observed thereupon. Recovery of B-Myb attenuates BTZ-introduced DNA damage and blockage of the cell cycle, thereby attenuating the anti-cancer effect.