Expression of Malic Enzyme 3 in Breast Cancer and Precancerous Lesions: a Promising Novel Biomarker for Carcinogenesis and Prognosis


 Purpose: While malic enzymes 1 (ME1) was correlated with breast cancer progression and prognosis, the association of ME3 (a homologue of ME1) with breast cancer is not known. The aim of this study is to explore the potential of ME3 as a biomarker in breast cancer carcinogenesis and prognosis.Methods: A total of 107 patients confirmed with breast cancer were enrolled. The ME3 expression was evaluated by IHC and correlated with clinicopathological indicators.Results: The ME3 positive immunostaining rate was higher in normal breast tissues and decreased stepwise from normal (97.60%) to usual ductal hyperplasia (91.1%), atypical ductal hyperplasia (64.2%), carcinoma in situ (62.5%) and invasive carcinoma (45.5%). Similarly, the decreasing tendency was observed for ME3 positive immunostaining rate from Tis (75.0%) through T1 (62.5%) and T2 (37.5%) to T3 (33.3%) and from stag 0 (75.0%) through I (72.0%), II (44.4%) to III (24.1%). ME3 expression was related with negative lymph node metastasis. Patients with positive expression of ME3 had better outcome. By incorporating ME3 into tumor TNM staging, the area under receiver operating characteristic curve for the 5-year survival was increased from 84.0% to 87.5%. Conclusions: ME3 may be a promising biomarker for better prognosis for breast cancer patients.


Introduction
Breast cancer remains the leading cause of cancer-related death among females worldwide. It accounts for 24% of all cancer cases and 15% of all cancer deaths among females 1 . With dramatic changes in lifestyles and economy, the incidence and mortality rates of breast cancer in Chinese women have a horrendous growth, whereas the overall 5-year survival rate for Chinese women with breast cancer is much lower than American women (82% vs. 91%) [2][3][4] . Breast cancer has been a major public health problem in China 5, 6 .
It has been well known that changes in cell metabolism can contribute to transformation and tumor progression 7,8 . Malic enzymes (MEs), including three isoforms of ME1, 2 and 3, catalyze the oxidative decarboxylation of malate to pyruvate, with a concomitant reduction of NAD(P) + to NAD(P)H. The different isoforms of MEs have different cofactor speci city and sub-cellular localizations in mammals: the cytosolic NADP + -dependent ME1, the mitochondrial NAD(P) + -dependent ME2, and the mitochondrial NADP + -dependent ME3 [9][10][11] . MEs are important for NADPH production: while ME1 is important for the NADPH production in the cytosol, ME3 takes a part in maintaining the redox homeostasis in mitochondria 12,13 .
Recently, a growing number of evidence has indicated that ME family plays an important role in different types of cancer. Jiang et al reported that up-regulation of MEs induced p53 inhibition 13 20 .
In this study, we sought to characterize ME3 protein expression in breast precancerous and cancerous lesions, aiming to explore the potential of ME3 as a biomarker for carcinogenesis and prognosis in breast cancer.

Results
The clinicopathological characteristics of the patients Table 1 summarized the clinicopathological characteristics of the 107 breast cancer patients. All the patients were con rmed with breast cancer, and were predominately at the invasive stage (92.5%), only 7.5% at CIS stage. Pathologically, there were 101 cases with ductal carcinoma, 3 cases with invasive lobular carcinoma, 1 case with medullary carcinoma, 1 case with mucinous carcinoma, and 1 case with mixed carcinoma. Based on the T staging, more than half of the patients were in T2 (59.8%), followed by T1 (29.9%), Tis (7.5%) and T3 (2.8%). About half of the patients (42.1%) had positive lymph node metastasis when they were diagnosed. 71.9% of the patients were in early stage based on TNM staging, including 7.5% patients in stage 0, 23.4% in stage I, 42.1% in stage II, and the rest (27.1%) in advanced stage III.  The Kaplan-Meier survival analysis showed that the patients with positive expression of ME3 had signi cant better outcome than those with negative expression (χ2 = 8.233, P = 0.004, Fig. 4).
ME3 is inversely associated with the progression of breast cancer

Receiver operating characteristic curve
Using multivariate logistic regression and receiver operating characteristic curve (ROC), we explored the potential of ME3 expression in the classi cation of 5-year survival outcome. Logistics regression analysis showed that TNM staging was the in uencing factor for 5-year survival outcome in the available clinical information in this study. Using TNM alone, the 5-year survival model based on the prediction probability showed that the area under ROC curve was 84.0% (Fig. 5A). Using TNM combined with ME3 expression, the area under ROC curve increased to 87.5% (Fig. 5B), suggesting a potential role of the ME3 in prediction of patients at risk of breast cancer death.

Discussion
To the best of our knowledge, this is the rst report to investigate the potential of ME3 as a biomarker in breast cancer carcinogenesis and progression. The present study demonstrated a stepwise decrement for ME3 protein expression at the different stages of breast malignant progression, indicating that ME3 aberrant loss of expression may be involved in breast carcinogenesis.
Furthermore, ME3 positive immunostaining rate was decreasing from Tis through T1, T2 to T3 and from stag 0 through I, II to III. In addition, ME3 expression was strongly related with lymph node status. The close association between ME3 expression and pathological staging suggests that ME3 may involve in the progression of in-situ invasion and lymph node metastasis of breast cancer. Meanwhile, patients with ME3 positive expression have a better survival than the negative expression of ME3.
Breast cancer is the most frequent cancer among female and TNM staging at diagnosis is still one of the most important factors affecting prognosis so far. Breast cancer has a heterogeneous prognosis of the 5- year survival rate varies substantially among patients, range from 27% for distant metastasis to 86% for regional breast cancer and 99% for localized breast cancer in the report from the American Cancer Society 21 . In this study, addition of ME3 expression into TNM staging has a stronger predictive value of 5-year survival outcome than TNM staging alone. Hence, ME3 could be a potential biomarker for better outcome of breast cancer patients.
It has not been well characterized for the ME3 and the associated metabolic pathway in carcinogenesis, especially in breast cancer. The mechanism of ME3 aberrant expression in breast cancer is largely unknown. Biologically, ME3 catalyzes a reaction by oxidizing malate to pyruvate and concomitantly reducing NADP + to NADPH and this reaction is crucial for maintaining the NADPH pool in mitochondria 22,23 . As NAPDH is the essential molecule for antagonizing mitochondrial ROS (mROS), ME3 downregulation may enhance the accumulation of mROS. It has been well recognized that mROS overproduction would promote carcinogenesis and tumor progression by inducing genetic instability, modifying gene expressions, and activating diverse signaling 24,25 . Therefore, ME3 downregulation may take a part in breast carcinogenesis and breast cancer progression via perturbing the homeostasis of NADPH/NADP + in mitochondria, leading to accumulation of mROS. Further investigation is required to validate the hypothesis.
It is interesting to note that ME3 and ME1 showed opposite effect on diagnosis on the survival of breast cancer patients. While it has been extensively studied that higher ME1 expression is associated with worse prognosis of breast cancer patients than the lower ME1 expression 26 , the higher ME3 expression was associated with a better prognosis of breast cancer patients than the lower ME3 expression. Because ME1 and ME3 share the same catalytic function catalyzing the oxidative decarboxylation of malate, the different effect of the enzymes on prognosis may emanate from their different locations. ME1 is located in cytoplasm, whereas ME3 is located in mitochondria. Because it is well recognized that the vicious cycle of ROS in mitochondria is important for carcinogenesis and cancer progression [27][28][29] , it is conceivable that this vicious cycle is to some extent associated with ME3 activity. The lower ME3 expression may enhance the vicious cycle and hence may be correlated with the progression from normal to precancerous tissue and to cancerous tissue as well as the progression from early to later stage of breast cancer.
In conclusion, our data supported ME3 as a potential biomarker for breast carcinogenesis as well as for the progression of breast cancer patients. ME3 positive expression may be a promising biomarker for better outcome for breast cancer patients.

Materials And Methods
Patients 107 breast cancer patients were retrieved in this study from a database, which has been established by

Immunohistochemical staining
Follow the manufacturer's instructions, tissue sections were depara nized, rehydrated, subjected to antigen retrieval. Polyclonal rabbit anti-human ME3 antibody was purchased from Sigma (HPA038473, USA). The samples were incubated with primary antibody (ME3, 1:400) overnight at 4 °C. The secondary antibody was conjugated to horseradish peroxidase. The immunoreactivty for ME3 was visualized through adding diaminobenzidine and subsequently counterstained with hematoxylin. The sections were evaluated under microscope by two pathologists independently who were blinded to the patients' clinical information.