Possible role of p53/Mieap‐regulated mitochondrial quality control as a tumor suppressor in human breast cancer

Mitochondria‐eating protein (Mieap), encoded by a p53‐target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase‐dependent apoptosis, with activation of both caspase‐3/7 and caspase‐9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap‐regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap‐regulated MQC pathway resulted in significantly shorter disease‐free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap‐regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.

driver of tumor development even in breast cancer. 9 p53 is clinically very important not only because of its high mutation rate but also because mutation is associated with more aggressive disease and worse overall survival. 10 p53 is a transcription factor that activates the expression of various downstream genes in response to DNA damage. 11 The central functions of this protein in tumor suppression are cell cycle arrest, apoptosis, DNA repair and anti-angiogenesis. [12][13][14][15][16] In particular, apoptosis is so important a function for p53-related tumor suppression that p53 activates target genes, including Bax, Noxa, Puma, Apaf-1 and p53AIP1, in response to DNA damage by radiation, UV and oxidative stress. 11,13,17 Although the mechanisms of apoptosis induced by DNA damage have been clarified, mitochondria are a pivotal organ for apoptosis, where these apoptosisrelated proteins localize and play an important role in mitochondria through caspase activation. 18 Recently, mitochondrial quality control has been revealed to be a novel function of p53. This function is regulated by a novel p53-inducible protein called mitochondriaeating protein (Mieap). 19 and NIX at the mitochondrial outer membrane may play a critical role in the translocation of lysosomal proteins from the cytoplasm to the mitochondrial matrix. 21 Although Mieap has been shown to be a key player in mitochondrial quality control, emerging evidence suggests that it also plays an important role in tumor suppression. In a mouse model, Mieap-deficient (adenomatous polyposis coli) ApcMin/+ mice had a much shorter lifespan and increased numbers and sizes of intestinal tumors compared to those in Mieap-wild type ApcMin/+ mice, suggesting that loss of Mieap increases ROS production in the intestinal mucosa and accelerates tumor progression. 22 Furthermore, Mieap-regulated mitochondrial quality control is frequently inactivated in human colorectal cancer by Mieap promoter methylation, BNIP3 promoter methylation or p53 mutation. 23 Considering that Mieap is a downstream target of p53, this novel mechanism for mitochondrial quality control is a new function of the p53 tumor suppressor. These results strongly prompted us to speculate that p53/Mieap-regulated mitochondrial quality control might be involved in tumor suppression in human breast cancer. In this study, we investigated this role.

| Fluorescence activated cell sorting analysis
We overexpressed Mieap using the constructed adenovirus in breast cancer cell lines. In addition, we treated these cells with etoposide (10 μmol/L). MCF-7, SK-BR-3 and MDA-MB-231 cells were infected with the adenovirus at a moi of 30 and 60, respectively; cells were then trypsinized and washed with PBS.
Subsequently, the cells were fixed in cold 70% ethanol at 4°C.
Fixed cells were washed and then incubated for 30 minutes at 37°C with PBS containing 0.2 mg/mL RNase, and then incubated for a further 30 minutes at approximately 22°C with PBS containing 20 μg/mL propidium iodide. Cells were analyzed using the EC800 Flow Cytometry Analyzer (Sony, Tokyo, Japan). For each analysis, 5000-20 000 cells were recorded, and the percentages of cells in different cell cycle phases (subG1, G1, S and G2/M) were determined using EC800 analysis software. The subG1 fraction was regarded as the apoptotic cell population. 25

| Caspase activity assay
Breast cancer cell lines were treated using the same conditions as described previously herein. Caspase activities were measured as described previously. 25 The cell pellets were resuspended in cell lysis buffer (50 mmol/L HEPES, 100 mmol/L NaCl, 0.1% CHAPS, 1 mmol/L EDTA, 10% glycerol, 0.1% NP40, pH 7.4) for 15-30 minutes on ice. Total protein (10-30 μg) was added to the reaction buffer (the same as the cell lysis buffer (except for the addition of 0.1% NP40) containing NAc-Asp-Glu-Val-Asp-pNA (Ac-DEVE-pNA, a substrate of caspase-3/7; Sigma-Aldrich), followed by incubation for 1-2 hours at 37°C. The free pNA cleaved from its precursor was quantified using a spectrometer at 405 nm (Bio-Rad, Hercules, CA, USA). The Caspase-Glo 9 Assay reagent (Promega, Madison, MI, USA) was used as the substrate for activated caspase-9. An equal volume of cell lysate and caspase-Glo 9 reagent was added to each well, mixed, and incubated in the dark for 1 hour at room temperature. The resultant luminescence, which is proportional to caspase-9 activity, was measured using a luminometer (Bio-Rad). Absorbance was determined using an Envision 2104 multilabel reader (Perkin-Elmer, Waltham, MA, USA). Data were analyzed using Student's t-test. A P-value <0.05 was considered statistically significant.

| Western blotting
Caspase plays a central role in apoptosis, and the cleavage of poly ADP-ribose polymerase (PARP) facilitates cellular disassembly.
Cleaved PARP is a marker of cells undergoing apoptosis. 26 Breast cancer cell lines were treated using the same conditions as those described previously herein. Cells were resuspended in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) with protease inhibitors for 15-30 minutes on ice. Protein (8 μg) was subjected to SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked for 1 hour and then incubated with respective primary antibodies overnight at 4°C. The anti-Mieap antibody (rabbit, 1:1000) was prepared as described previously. 19 Anti-

| Immunohistochemistry
We examined the expression of Mieap in vivo using surgical speci- were incubated with EnVision reagents for 1 hour at room temperature. 22 IHC evaluation was performed by experienced pathologists.
When we started IHC for human cancer tissues in vivo, we repeated preliminary experiments for appropriate conditions using preimmune serum. For subsequent runs, we did not use this due to its shortage. However, as we found endothelial cells and smooth muscle of blood vessels to be good positive controls, we show Mieapexpressing endothelial cells in Figure S1.
Informed consent was obtained from all patients who agreed to provide a surgical specimen. This study was approved by the central ethics committee of Gifu University.

| Methylation-specific PCR and p53 mutation search
Methylation-specific PCR was performed as described previously. 19 The p53 mutation search was performed as described previously. 23 Genomic PCR was performed, followed by DNA sequence analysis.

| Statistical analysis
Immunohistochemistry for Mieap was analyzed using the χ 2 -test.
Disease-free survival (DFS) was estimated using the Kaplan-Meier method, and comparisons between groups were performed using a 2-sided log-rank test. Results were considered significant at P < 0.05.  Figure 1D).

| Mieap-induced cell death is apoptosis
As a preliminary experiment, we treated 3 cell lines with etoposide.
We found cleaved caspase-3/7/9 with activation of mitochondrial apoptotic factors particularly in SK-BR-3 and MDA-MB-231 ( Figure   S3). 17,27 To investigate the association between Mieap-induced cell death and caspases, we determined the expression levels and activation of caspase-3/7 and caspase-9 by western blotting and caspase activity assays. We found that SK-BR-3 was the most sensitive cell line to Mieap-induced cell death ( Figure 1D). Forty-eight hours after Ad-Mieap infection (30 moi), not only caspase-3/7 but also caspase-9 was significantly elevated in SK-BR-3 cells (caspase-

| Mieap expression is downregulated in tumors from breast cancer patients with an aggressive and malignant phenotype
We next performed IHC using surgical specimens obtained from 120  (Table 1).

| The p53/Mieap-regulated mitochondrial quality control pathway is genetically and epigenetically inactivated in aggressive and malignant breast cancer
To investigate inactivation of the p53/Mieap-regulated MQC pathway, we performed MSP for Mieap, NIX and BNIP3 promoters, as well as a p53 mutation search, because NIX and BNIP3 have been shown to be co-factors for MQC. 21 We investigated 46 IDC, which had been  Table 2). Of 12 cases, 6 with methylated Mieap promoter were luminal B and the other 6 with mutant p53 were triple negative as follows: 4, HER2-rich; 1, luminal B; 1, whose subtypes were clinically more aggressive and malignant phenotypes in breast cancer (Table 3). 30,31 Interestingly, the group with inactivation of the p53/Mieapregulated MQC pathway showed shorter DFS than that without these genetic alterations (P = 0.021, Figure 5). Four of six recurrent cases showed inactivation in the p53/Mieap-regulated MQC pathway. Of 46 cases, we separately showed DFS curves for individuals with or without Mieap promoter methylation or those with or without p53 alterations ( Figure S4). As shown in Figure S4A, there was no significant difference between those with Mieap promoter methylation (n = 6) and those without (n = 40). Six cases with a methylated Mieap promoter showed no expression of Mieap by IHC.
However, as shown in Figure S4B, there was a significant difference between those with p53 mutations (n = 6) and those without (n = 40;

| D ISCUSS I ON
According to a previous report, we demonstrated the physiological functions related to MQC, which were classified mainly into 2 mechanisms.
One is referred to as MALM, whereas the other is MIV, which occurs when salvage of the damaged mitochondria appears to be impossible for recovery and survival. 20   TA B L E 3 (Continued)

ACK N OWLED G M ENTS
We thank Shizu K for technical support and Enya K, Hirata M, Iwata A, Mori K and Takano K for administrative assistance.

D I S C LO S U R E S TAT E M E N T
KY has received grants and personal fees from Taiho