A polymorphism in JMJD2C alters the cleavage by caspase-3 and the prognosis of human breast cancer

Abstract

JMJD2C is a candidate oncogene that encodes a histone lysine demethylase with the ability to demethylate the lysine 9 residue of histone H3 (H3K9). The expression levels of JMJD2C are associated with tumor development and clinical outcome. Here we identify JMJD2C as a new substrate for caspase-3. JMJD2C is cleaved by caspase-3 at DEVD396G motif and then loses its demethylase activity. Additionally, we uncover D396N polymorphism (rs2296067) in the cleavage site of JMJD2C and establish its influence on the resistant to the cleavage by caspase-3. Importantly, we determined that D396N polymorphism is significantly associated with the prognosis of human breast cancer. We further found that the basal levels of DSB (double strand DNA break) repair proteins γ-H2AX (gamma-H2AX) increased when cells were treated with tumor necrosis factor-α (TNF-α) which activates caspase-3 activity. We also show that knockdown of JMJD2C expression results in up-regulation of basal γ-H2AX. We propose that D396N polymorphism of JMJD2C affects the prognosis of human breast cancer via altering the cleavage by caspase-3 and the ability of DSB repair which may contribute to therapy resistance.

Figure 1. JMJD2C is cleaved by caspase-3-like protease

(A) Expression of JMJD2C protein in 8 breast-tumor samples was analyzed by western blotting with anti-JMJD2C (A300-885A) antibody. (B) Schematic representation of the structure of JMJD2C. (C) Schematic representation of A300-885A antibody mapping. Cell lysates of HeLa and MDA-MB-231 were analyzed by western blotting with A300-885A antibody. Where indicated, cells were treated with 20 mM z-DEVD-fmk, for 2 h before treatment. HeLa and MDA-MB-231 cells were treated with TNF-α (25 ng/ml) or CHX (10 μg/ml) for 6 h or with the combination for 6 h.

Figure 2. The JMJD2C D396N polymorphism in potential caspase-3-like protease cleavage site

(A) SNP rs2296067 in 8 breast-tumor samples (B) 293T cells were transiently transfected with empty pcDNA3.1 vector and pcDNA3.1-JMJD2C carrying G allele of the SNP rs2296067. After the transfection for 24 hours, the pcDNA3.1-JMJD2C transfected cells were treated with TNF-α (25 ng/ml) plus CHX (10 μg/ml) for 2 h. Cell lysates were analyzed by western blotting with anti-JMJD2C (A300-885A) antibody.

Figure 3. Caspase-3-like protease cleavage removes a C-terminal fragment from JMJD2C

(A) HeLa cells were stably transfected with full length JMJD2C (D396) tagged with FLAG at the C-terminus. The top schematic representation shows the C-terminal FLAG epitope tag of JMJD2C. Where indicated, HeLa cells were treated with 20 mM z-DEVD-fmk for 2 h before treatment and then were treated with TNF-α (25 ng/ml) plus CHX (10 μg/ml) for 6 h. Cell lysates of HeLa were analyzed by western blotting with FLAG antibody. Endogenous levels of total full-length PARP-1 and the large fragment produced by caspase-3 family protease cleavage at Asp214 were detected by PARP antibody. (B) HeLa cells were stably transfected with full length JMJD2C (D396) tagged with HA at the N-terminus. The top schematic representation shows the N-terminal HA epitope tag of JMJD2C. Treated with TNF-α (25 ng/ml) plus CHX (10 μg/ml) for 6 h, cell lysates of HeLa were analyzed by western blotting with HA antibody. The untransfected and untreated cells were set as the control. Full-length and the cleavage fragment of PARP-1 were also detected.

Figure 4. JMJD2C is cleaved at DEVD396G motif by caspase-3

(A) MCF-7 cells were stably transfected with full length JMJD2C (D396) with C-terminal FLAG epitope tag. Treated with TNF-α (25 ng/ml) plus CHX (10 μg/ml) for 2, 4, 6 and 8 h, the cell lysates of MCF-7 were analyzed by western blotting with FLAG antibody. (B) HeLa cells were stably transfected with full length JMJD2C (N396) with C-terminal FLAG epitope tag. The transfected HeLa cells were treated with TNF-α (25 ng/ml) plus CHX (10 μg/ml) for 2, 4, 6 and 8 h. Cell lysates were analyzed by western blotting with FLAG antibody.

Figure 5. Cleavage of JMJD2C by caspase-3 inactivates its demethylase activity

(A) HeLa and MCF-7 cells were treated with TNF-α (25 ng/ml) plus CHX (10 μg/ml) for 2, 4 and 6 h. The treated cells were lysed and then were subjected to western blotting analysis with H3K9me3 antibody and histone H3 antibody. Levels of histone H3 were taken as loading control. (B) MCF-7 cells were transiently transfected with the empty vector or full-length JMJD2C with N-terminal FLAG epitope tag or N-terminal cleavage JMJD2C (fragment 1-396 amino acid) with N-terminal FLAG epitope tag. The expression of JMJD2C (full length and fragment 1-396 amino acid), H3K9me3 and histone H3 were analyzed by western blotting with corresponding antibodies as indicated. Levels of histone H3 were taken as loading control. (C) Western blotting analysis demonstrated that the levels of γ-H2AX was significantly elevated during the treatment with TNF-α/CHX in HeLa cells but not in MCF-7 cells (left panel); HeLa cells were stably transfected with shRNA control (scramble) or with shRNA targeting JMJD2C (shJMJD2C). Western blotting analysis showing expression JMJD2C, H3K9me3, H2AX, γ-H2AX and histone H3 were analyzed by western blotting with corresponding antibodies as indicated. Levels of histone H3 were taken as loading control (right panel).

Figure 6. Association of SNP rs2296067 with breast cancer patient survival

Kaplan–Meier analysis of the overall survival of breast cancer patients grouped according to their genotype for SNP rs2296067.