Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells

Abstract

Resistance of cancer cells to radiotherapy is a major clinical problem in cancer treatment. Therefore, understanding the molecular basis of cellular resistance to radiotherapy and identification of novel targets are essential for improving treatment efficacy for cancer patients. Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress. We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation. Methylation PCR array analysis identified that ERp29 expression increased promoter hypomethylation of the DNA repair gene, O(6)-methylguanine DNA-methyltransferase (MGMT), by downregulating DNA methyltransferase 1. Knockdown of MGMT in the ERp29-transfected cancer cells increased radiosensitivity, leading to a decreased post-irradiation survival. In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis. Hence, our studies prove a novel function of ERp29\MGMT in cancer cell survival against radiation. Targeting ERp29\MGMT axis may be useful for providing better treatment efficacy in combination with radiotherapy in breast cancer.

Figure 1. ERp29 regulates post-irradiation survival of MDA-MB-231 cells.

(a) ERp29 overexpression increased post-irradiation survival rate. ERp29 expressing construct was transfected into MB-231 cells and two stable clones (clone B and E) showing high expression of ERp29 were selected for radiation treatment. (b) Repression of exogenously expressed ERp29 by siRNA in the ERp29-transfected MDA-MB-231 cells (clone B) attenuated the post-irradiation survival rate. (c) ERp29 knockdown by siRNA in parent MB-231 cells reduced post-irradiation survival rate. ERp29-transfected or knockdown cells (48 hours of treatment with siRNA #1) were seeded on six-well plates and irradiated with the indicated dose of radiation. After 10 days incubation at 37 °C, colonies with >50 cells per colony were counted. The survival fraction of irradiated cells was normalized to the plating efficiency of non-irradiated control cells. The level of ER29 in ERp29-transfected cells (a) siRNA-treated, ERp29-overexpressed clone B cells (b) and siRNA-treated parental MDA-MB-231 cells (c) was examined by Western blot. Data represent the mean ± SD of three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, relative to controls at the indicated dose. The level of β-actin was used as a loading control.

Figure 2. ERp29 modulates the expression of tumour suppressors and pro-oncogenes by epigenetic regulation.

(a) ERp29 expression promoted/inhibited promoter demethylation of tumour suppressors/pro-oncogenes identified by Methylation PCR arrays. (b) Tumour suppressor genes CDH1 and MGMT were transcriptionally activated by ERp29. The mRNA and protein expressions were examined by RT-PCR and Western blot. (c) MS-PCR analysis for MGMT promoter methylation/demethylation. Note that the ratio of demethylation/methylation was highly increased in the ERp29-transfected cells (clone B and E). Cells treated with 5′-aza-dC was used as a positive control for demethylation. Genomic DNA was extracted and converted with sodium bisulfite. MS-PCR was performed as described in “Materials and Methods”. **p < 0.01 versus control.

Figure 3. ERp29 expression reduces DNMT1 to increase MGMT promoter demethylation in MDA-MB-231 cells.

(a) ERp29 expression decreased the level of DNMT1 whereas ERp29 knockdown upregulated the expression of DNMT1. The expression of DNMT3A or 3B was not markedly affected by ERp29. *p < 0.05, **p < 0.01, relative mock-transfected control or controL siRNA. (b) Reduction of DNMT1 by siRNA upregulated MGMT expression in MDA-MB-231 cells. MDA-MB-231 cells were transiently transfected with control siRNA or DNMT1 siRNA (#1) for 48hours and the expression of DNMT1 and MGMT was examined. (c) MGMT promoter methylation/demethylation. Genomic DNA was extracted from the MDA-MB-231 cells transfected with control siRNA or DNMT1 siRNA and the MS-PCR was done as described in “Materials and Methods”. **p < 0.01, ***p < 0.001, versus control.

Figure 4. MGMT is a downstream target of ERp29.

ERp29-transfected cells (cone B) (a) or MCF7 cells (b) were treated with ERp29 siRNA (#1) or MGMT siRNA (#3) or control siRNA and the expression of ERp29 and MGMT was analysed. ERp29 knockdown decreased the expression of MGMT whereas MGMT knockdown was unable to decrease the level of ERp29 in both clone B cells (a) and MCF-7 cells (b). **p < 0.01, ***p < 0.001, relative to control.

Figure 5. MGMT mediates ERp29-induced post-irradiation survival rate. and facilitates DNA damage in ERp29-transfected MDA-MB-231 cells.

(a) Repression of MGMT by siRNA (#3) reduced the ERp29-enhanced post-irradiation survival rate. Cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. Expression of MGMT was efficiently repressed by siRNA in the ERp29-overexpressed clone B cells. (b) Depletion of endogenous MGMT by siRNA in MCF-7 cells sensitized to radiation treatment. MCF-7 cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. The expression of MGMT in MCF-7 cells was efficiently reduced by siRNA. (c) Re-expression of MGMT in the MB-231/ERp29 siRNA cells restores radioresistance. Cells were transfected with pcDNA-MGMT or pcDNA for 24 hours and exposed to radiation treatment. MGMT expressed was examined by immunoblot. Post-radiation survival rate was assessed by clonogenic assay as described in Fig. 1

Figure 6. MGMT repression facilitates DNA damage in ERp29-transfected MDA-MB-231 cells and MCF-7 cells.

(a) Reduction of MGMT in ERp29-overexpressed cells increased irradiation-induced expression of γ–H2AX. Note that irradiation induces significant increase of γ–H2AX in the mock-transfected MDA-MB-231 cells (column 2 vs. 1). Depletion of MGMT alone in the ERp29-overexpressed clone B cells only slightly increased the expression of H2AX (column 5 vs. 1). However, combination treatment (column 4) of irradiation/MGMT siRNA in these cells led to a significant induction of γ–H2AX relative to the cells treated with MGMT siRNA (column 5) or with the irradiation/control siRNA (column 3). The level of γ–H2AX was examined after 12 hours of post-irradiation. (b) Irradiation treatment significantly increased the expression of γ–H2AX in MGMT-knockdown MCF-7 cells. MCF-7 cells were transfected with MGMT siRNA or control siRNA for 48 hours and then irradiated with 4 Gy. The expression of γ–H2AX was assayed after 12 hours of post-irradiation. (c) MGMT depletion induced high expression of γ–H2AX and reduced double strand DNA breaks repair after irradiation. MB-231/ERp29 (clone B) cells were treated with MGMT siRNA or control siRNA for 48 hours, irradiated with 4 Gy and incubated for 2, 6, 12 and 24 hours. Phosphorylation of H2AX was examined by Western blot. **p < 0.01, ***p < 0.001, relative to control at the indicated doses/time.

Figure 7. MGMT depletion by siRNA stimulates radiation-induced cell apoptosis in ERp29-transfected MDA-MB-231 cells.

(a) Reduction of MGMT in ERp29-overexpressed clone B cells increased irradiation-induced expression of cleaved caspase 3 compared to the control siRNA-treated cells. Expression of cleaved caspase 3 was highly induced by irradiation in the vector-transfected MDA-MB-231 cells (column 2 vs. 1). In the ERp29-overespressed clone B cells, depletion of MGMT alone did not activate expression of cleaved caspase 3 (column 3 vs. 1). However, these cells treated with MGMT siRNA (#3) markedly increased the expression of cleaved caspase 3 after irradiation (column 5 vs. 4). (b) Cell viability. Irradiation treatment caused significant reduction of cell viability in vector-transfected MDA-MB-231 cells (column 2 vs. 1). Combinatory treatment of irradiation/MGMT siRNA (column 5) in the ERp29-overexpressed clone B cells significantly reduced cell viability compared to the cells treated with MGMT siRNA alone (column 3) or the irradiation/control siRNA (column 4). **p < 0.01, ***p < 0.001, relative to control.