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

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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. Cancer Biology Oncology Breast cancer ME3 expression carcinogenesis prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 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 – 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 specificity and sub-cellular localizations in mammals: the cytosolic NADP + -dependent ME1, the mitochondrial NAD(P) + -dependent ME2, and the mitochondrial NADP + -dependent ME3 9–11 . 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 . Lu and Murai et al demonstrated that ME1 inhibited proliferation and invasion by regulating NADPH homeostasis in gastric cancer and colon cancer cell lines 14 , 15 . Ren et al showed that ME2 depletion increased reactive oxygen species (ROS) and NADP+/NADPH ratio, inhibited cell proliferation and induced cell death and differentiation in lung cancer cell 16 . Woo et al reported high-expression of ME2 was associated with poorer overall survival (OS) for patients with head and neck squamous cell carcinoma 17 . Dey et al found that ME3 depletion selectively killed ME2-null pancreatic cancer cells and Zhang Q et al discovered that ME3 promoted the proliferation, invasion and metastasis of pancreatic cancer cells 18 , 19 . Moreover, Liao et al reported that ME1 expression was positively correlated with large tumor size, high grade, poor survival and chemotherapy resistance, indicating that ME1 promoted breast cancer progression and was associated with poor prognosis 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 confirmed 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. Table 1 Clinicopathological characteristics of 107 breast cancer patients. Variables No. of patients examined N (%) Age < 50 years 64 (59.8) ≥ 50 years 43 (40.2) Gender Male 0 (0) Female 107 (100.0) T staging Tis 8 (7.5) T1 32 (29.9) T2 64 (59.8) T3 3 (2.8) Lymph node metastasis No 62 (57.9) Yes 45 (42.1) Pathological staging 0 8 (7.5) I 25 (23.4) II 45 (42.1) III 29 (27.1) Vascular cancer embolus No 87 (81.3) Yes 20 (18.7) ER status Negative 38 (35.5) Positive 69 (64.5) PR status Negative 42 (39.3) Positive 65 (60.7) Her-2 status Negative 45 (42.1) Positive 39 (36.4) Not available 23 (21.5) Ki67 index Low expression (≤ 14%) 27 (25.2) High expression (> 14%) 80 (74.8) ME3 positive expression is decreased with the progression from precancerous to cancerous lesions The immunohistochemical staining showed that the immunoreactivity for ME3 protein was located in the cytoplasm both in breast cancerous and precancerous tissue (Fig. 1 ). The ME3 positive expression is defined by semiquantitative scoring system as described in Materials and Methods. The ME3 positive immunostaining rate was higher in the normal breast tissue (97.60%, 82/84), and then the positive rate decreased from UHP (91.1%, 72/79) to AHP (64.2%, 43/67), CIS (62.5%, 40/64) and invasive carcinoma (45.5%, 45/99). The decreasing tendency was inversely correlated with the progression from precancerous to cancerous lesions (R 2 = 0.9414, P = 0.006, linear regression analysis, Table 2 , Fig. 2 ). Table 2 ME3 expression in the normal mammary tissue, precancerous and cancerous lesions. Pathological classification No. of lesions examined Expression of ME3 protein χ2 P Positive N (%) Negative N (%) Normal 84 82 (97.6) 2 (2.4) 195.89 0.000 Usual hyperplasia 79 72 (91.1) 7 (8.9) Atypical hyperplasia 67 43 (64.2) 24 (35.8) Carcinoma in situ 64 40 (62.5) 24 (37.5) Invasive carcinoma 99 45 (45.5) 54 (54.5) ME3 is associated with better prognosis in breast cancer patients The median follow-up of the entire cohort was 73.7 months (range 11.7–94.8 months) and 21 (19.6%) patients died because of the deteriorated breast cancer during the follow-up. The survival rates of 1-, 3- and 5-year were 98.1%, 87.9%, and 82.2%, respectively. It was noteworthy that the median survival time of the 21 dead patients was only 36.6 months, ranging from 11.7 to 67.2 months. Of the 21 died cases, one third (33.3%) was in early stage I and II, and the rest (66.7%) was in advanced stage III. In this study, the younger patients with ages ≤ 40 years accounted 16.8% (18/107) and about half of the patients (44.4%, 8/18) were in advanced stage III. The Kaplan–Meier survival analysis showed that the patients with positive expression of ME3 had significant better outcome than those with negative expression (χ2 = 8.233, P = 0.004, Fig. 4 ). In this study, lymph node metastasis (χ2 = 12.284, P = 0.000), TNM staging progression (χ2 = 18.232, P = 0.000) and high expression of Ki67 index (χ2 = 5.155, P = 0.024) were also negatively associated with survival (Supplementary Table S1). However, no influence was observed for age, T staging, vascular cancer embolus, ER status, PR status and Her-2 status on survival (Supplementary Table S1). ME3 is inversely associated with the progression of breast cancer Furthermore, linear regression analysis showed that ME3 expression decreased from Tis (75.0%, 6/8) to T1(62.5%, 20/32), T2 (37.5%, 24/64) and T3 (33.3%, 1/3), (R 2 = 0.9394, P = 0.031, Table 3 , Fig. 3 A). The analysis on TNM staging and ME3 expression showed ME3 positive immunostaining rate was much higher in stage 0 (75.0%, 6/8) and stage I (72.0%, 18/25) than in stage II (44.4%, 20/45) and stage III (24.1%, 7/29) (χ2 = 14.953, P = 0.002, Table 3 ). Similarly, the decreasing tendency was also significant from stage 0 to I, II and III (R 2 = 0.9283, P = 0.037, linear regression analysis, Fig. 3 B). ME3 positive expression in breast cancer tissue was related with negative lymph node metastasis (χ2 = 6.393, P = 0.011). No significant difference was observed for ME3 expression with age, vascular cancer embolus, ER status, PR status, Her-2 status, or Ki67 index (Table 3 ) Table 3 Relationship between ME3 expression and clinicopathological characteristics in breast cancer. Variables No. of patients examined Expression of ME3 protein χ2 P Positive(n = 51) N (%) Negative(n = 56) N (%) Age 1.914 0.166 < 50 years 64 27 (42.2) 37 (57.8) ≥ 50 years 43 24 (55.8) 19 (44.2) T staging 8.117 0.044 Tis 8 6 (75.0%) 2 (25.0%) T1 32 20 (62.5%) 12 (37.5%) T2 64 24 (37.5%) 40 (62.5%) T3 3 1 (33.3%) 2 (66.7%) Lymph node metastasis 6.393 0.011 No 62 36 (58.1) 26 (41.9) Yes 45 15 (33.3) 30 (66.7) Pathological staging 14.953 0.002 0 8 6 (75.0) 2 (25.0) I 25 18 (72.0) 7 (28.0) II 45 20 (44.4) 25 (55.6) III 29 7 (24.1) 22 (75.9) Vascular cancer embolus 0.070 0.791 No 87 42 (48.3) 45 (51.7) Yes 20 9 (45.0) 11 (55.0) ER status 0.730 0.393 Negative 38 16 (42.1) 22 (57.9) Positive 69 35 (50.7) 34 (49.3) PR status 2.537 0.111 Negative 42 16 (38.1) 26 (61.9) Positive 65 35 (53.8) 30 (46.2) Her-2 status 1.439 0.487 Negative 45 22 (48.9) 23 (51.1) Positive 39 16 (41.0) 23 (59.0) Not available 23 13 (56.5) 10 (43.5) Ki67 index 3.389 0.066 Low expression (≤ 14%) 27 17 (63.0) 10 (37.0) High expression (> 14%) 80 34 (42.5) 46 (57.5) Receiver operating characteristic curve Using multivariate logistic regression and receiver operating characteristic curve (ROC), we explored the potential of ME3 expression in the classification of 5-year survival outcome. Logistics regression analysis showed that TNM staging was the influencing 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. 5 A). Using TNM combined with ME3 expression, the area under ROC curve increased to 87.5% (Fig. 5 B), 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 first 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 – 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 The State Key Laboratory of Esophageal Cancer Prevention & Treatment and Henan Key Laboratory of Esophageal Cancer Research in The First Affiliated Hospital of Zhengzhou University. All the patients had been underwent mastectomy from August 2012 to December 2014 without any radiation therapy or chemotherapy prior to surgery before operation. All patients were females with a mean age of 48.41±11.18 years and a median age of 46 years (range from 26 to 82 years). Staging for breast cancer were based on American Joint Committee on Cancer (AJCC) TNM staging system in 2017 30 . The follow-up was performed with hospital medical records, either through telephone or through home interview to each patient yearly until death. The last follow-up was May 2020. Overall survival time was calculated from the day of mastectomy to death or to the last follow-up date. The study protocol was approved by the Medical Ethics Committee of Zhengzhou University. Tissue collection and processing A total of 107 breast cancer tissues and 87 matched adjacent non-cancerous tissues were collected from surgically resected specimens from the patients with primary breast cancer. The tissues were routinely 10% neutral formalin fixed, paraffin-embedded and then sectioned. Serial 4 µm sections were prepared for histopathological analysis (hematoxylin and eosin stain) and immunohistochemical staining. Histopathological analysis Histopathological diagnoses and staging for breast cancer and adjacent non-cancerous lesions were made based on WHO criteria in 2012 31 . The epithelial proliferative lesions in mammary gland acini and duct were categorized as “normal”, usual hyperplasia (UHP), atypical hyperplasia (AHP) and carcinoma in situ (CIS). Histopathologically, UHP was usually found adjacent to the tumor tissue; AHP and CIS were frequently observed as isolated lesions in the surgically resected breast specimens. Pathologically, of the 87 cases with adjacent cancer tissue, there were 84 cases identified with normal tissue (96.6%, 84/87), 79 with UHP (90.8%, 79/87), 67 with AHP (77.0%, 67/87) and 64 with CIS (73.6%, 64/87). Histological examination was carried out by two pathologists independently. Immunohistochemical staining Follow the manufacturer’s instructions, tissue sections were deparaffinized, 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. According to immunohistochemical scoring system previously with some modification 32,33 , ME3 immunostaining score (S) was determined semiquantitatively by multiplying the percentage of positive cells (P) with intensity (I), according to the formula: S= P×I. The range score for percentage of positive cells is 0 to 4 (0, ＜1%; 1, 1%-10%; 2, 11%-50%; 3, 51%-75%; 4, 76%-100%). The range score for intensity is 0 to 3 (0, no staining; 1, weak; 2, moderate; 3, strong.). The index score was obtained a range from 0 to 12. The final immunostaining results were denoted as negative expression of ME3 (0～1 scores) and positive expression of ME3 (≥2 scores). Statistical analysis All statistical analyses were conducted using IBM SPSS version 21.0. Data was represented as the mean ± standard deviation for continuous variables and frequency (number-percent) for categorical data. Relationships of ME3 expression and clinicopathological features were evaluated by the chi-square (χ2-value) and linear regression analysis (R 2 -value). The Kaplan–Meier survival analysis was used to estimate the association between eligible variables and survival. Cox proportional hazards regression models were used for multivariate survival analysis in stepwise regression manner. Receiver operating characteristic curve was based on the prediction probability of 5-year survival outcome from the logistic regression model, and the area under ROC curve was used to assess the classification performance of the model. Any result with a P-value of less than 0.05 was considered as statistically significant. Declarations Acknowledgments This work has been supported in part by the China National 973 project (2013CB911303), China Natural Sciences Foundation projects (81470126), a key project (2018C03009) funded by Zhejiang Provincial Department of Sciences and Technologies, and the Fundamental Research Funds for the Central Universities, National Ministry of Education, China, to XH. Authors' contributions Conception and design of the study: XH and DY; Sample acquisition and analysis: DD, and DY; Histopathological diagnosis: XML and DD; Manuscript preparation: All authors; Approval of the final version: All authors. Competing interests The authors declare that they have no conflicts of interest with the contents of this article. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics declarations Informed consent was obtained from patients in this study. The human study protocol was in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans were approved by the Medical Ethics Committee of Zhengzhou University. Consent to participate Not applicable. References Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68 , 394-424, doi:10.3322/caac.21492 (2018). Li, T., Mello-Thoms, C. & Brennan, P. C. Descriptive epidemiology of breast cancer in China: incidence, mortality, survival and prevalence. Breast Cancer Res Treat 159 , 395-406, doi:10.1007/s10549-016-3947-0 (2016). Zeng, H. et al. Changing cancer survival in China during 2003–15: a pooled analysis of 17 population-based cancer registries. The Lancet Global Health 6 , e555-e567, doi:10.1016/s2214-109x(18)30127-x (2018). Miller, K. D. et al. Cancer treatment and survivorship statistics, 2019. CA Cancer J Clin 69 , 363-385, doi:10.3322/caac.21565 (2019). Chen, W. et al. Cancer statistics in China, 2015. CA Cancer J Clin 66 , 115-132, doi:10.3322/caac.21338 (2016). Fan, L. et al. Breast cancer in China. The Lancet Oncology 15 , e279-e289, doi:10.1016/s1470-2045(13)70567-9 (2014). Pavlova, N. N. & Thompson, C. B. The Emerging Hallmarks of Cancer Metabolism. Cell Metab 23 , 27-47, doi:10.1016/j.cmet.2015.12.006 (2016). Vander Heiden, M. G. & DeBerardinis, R. J. Understanding the Intersections between Metabolism and Cancer Biology. Cell 168 , 657-669, doi:10.1016/j.cell.2016.12.039 (2017). Chang, G.-G. & Tong, L. Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 42 , 12721-12733 (2003). Xu, Y., Bhargava, G., Wu, H., Loeber, G. & Tong, L. Crystal structure of human mitochondrial NAD (P)+-dependent malic enzyme: a new class of oxidative decarboxylases. Structure 7 , 877-889 (1999). Yao, P. et al. Evidence for a direct cross-talk between malic enzyme and the pentose phosphate pathway via structural interactions. J Biol Chem 292 , 17113-17120, doi:10.1074/jbc.M117.810309 (2017). Hasan, N. M., Longacre, M. J., Stoker, S. W., Kendrick, M. A. & MacDonald, M. J. Mitochondrial malic enzyme 3 is important for insulin secretion in pancreatic beta-cells. Mol Endocrinol 29 , 396-410, doi:10.1210/me.2014-1249 (2015). Jiang, P., Du, W., Mancuso, A., Wellen, K. E. & Yang, X. Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence. Nature 493 , 689-693, doi:10.1038/nature11776 (2013). Lu, Y. X. et al. ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth and Metastasis. Cancer Res 78 , 1972-1985, doi:10.1158/0008-5472.CAN-17-3155 (2018). Murai, S. et al. Inhibition of malic enzyme 1 disrupts cellular metabolism and leads to vulnerability in cancer cells in glucose-restricted conditions. Oncogenesis 6 , e329, doi:10.1038/oncsis.2017.34 (2017). Ren, J. G. et al. Knockdown of malic enzyme 2 suppresses lung tumor growth, induces differentiation and impacts PI3K/AKT signaling. Sci Rep 4 , 5414, doi:10.1038/srep05414 (2014). Woo, S. H. et al. Down-regulation of malic enzyme 1 and 2: Sensitizing head and neck squamous cell carcinoma cells to therapy-induced senescence. Head Neck 38 Suppl 1 , E934-940, doi:10.1002/hed.24129 (2016). Dey, P. et al. Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer. Nature 542 , 119-123, doi:10.1038/nature21052 (2017). Zhang, Q., Li, J., Tan, X. & Zhao, Q. Effects of ME3 on the proliferation, invasion and metastasis of pancreatic cancer cells through epithelial-mesenchymal transition. Neoplasma 66 , 896-907 (2019). Liao, R. et al. ME1 promotes basal-like breast cancer progression and associates with poor prognosis. Sci Rep 8 , 16743, doi:10.1038/s41598-018-35106-y (2018). Society, A. C. Breast cancer facts & figures 2019–2020. Am. Cancer Soc , 1-44 (2019). Fan, J. et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510 , 298-302, doi:10.1038/nature13236 (2014). Lewis, C. A. et al. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol Cell 55 , 253-263, doi:10.1016/j.molcel.2014.05.008 (2014). Sabharwal, S. S. & Schumacker, P. T. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel? Nat Rev Cancer 14 , 709-721, doi:10.1038/nrc3803 (2014). DeBerardinis, R. J. & Chandel, N. S. Fundamentals of cancer metabolism. Science advances 2 , e1600200 (2016). Liu, C. et al. Malic enzyme 1 indicates worse prognosis in breast cancer and promotes metastasis by manipulating reactive oxygen species. OncoTargets and therapy 13 , 8735 (2020). Birch‐Machin, M. The role of mitochondria in ageing and carcinogenesis. Clinical and Experimental Dermatology: Experimental dermatology 31 , 548-552 (2006). Ježek, J., Cooper, K. F. & Strich, R. Reactive oxygen species and mitochondrial dynamics: the yin and yang of mitochondrial dysfunction and cancer progression. Antioxidants 7 , 13 (2018). Yang, Y. et al. Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy. Journal of cellular physiology 231 , 2570-2581 (2016). Amin, M. B. et al. The Eighth Edition AJCC Cancer Staging Manual: Continuing to build a bridge from a population-based to a more "personalized" approach to cancer staging. CA Cancer J Clin 67 , 93-99, doi:10.3322/caac.21388 (2017). Frank, G. A., Danilova, N. V., Andreeva, I. & Nefedova, N. A. [WHO classification of tumors of the breast, 2012]. Arkhiv patologii 75 , 53-63 (2013). Fedchenko, N. & Reifenrath, J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. Diagn Pathol 9 , 221, doi:10.1186/s13000-014-0221-9 (2014). Fitzgibbons, P. L. et al. Template for reporting results of biomarker testing of specimens from patients with carcinoma of the breast. Arch Pathol Lab Med 138 , 595-601, doi:10.5858/arpa.2013-0566-CP (2014). Supplementary Files SupplementaryTableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-144916","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":8133260,"identity":"a96e8bd4-64ee-445b-a714-6a72a79dfd55","order_by":0,"name":"Duo You","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Duo","middleName":"","lastName":"You","suffix":""},{"id":8133261,"identity":"bf87d900-c660-412b-a8a9-03b3879ce1a2","order_by":1,"name":"Danfeng Du","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Danfeng","middleName":"","lastName":"Du","suffix":""},{"id":8133262,"identity":"abb14ccb-b99d-4cc2-a8b3-944104467817","order_by":2,"name":"Xinmin Li","email":"","orcid":"","institution":"Zhengzhou maternal and child health hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinmin","middleName":"","lastName":"Li","suffix":""},{"id":8133263,"identity":"66cabcc3-6f70-495e-bfde-36f2be9996f6","order_by":3,"name":"Xun Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACAzBZwcAMoiRI0HKGZC2MbRAOcVrMJXLMpAvn1bEbHGA+eJuHwS6PoBbLGWlp0jO3sTEbHGBLtuZhSC4m7LAbycekebfxALXwmEnzMBxIbCCsJbFNmneOBFAL/zditYBsaTAA2cJGpJYzz4BeOJbALHmYzdhyjkEyEVqO5xje5qmpS+Y73vzwxpsKO8JaYCAZEpkGxKoHAjsS1I6CUTAKRsFIAwBimjHjrre6BAAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xun","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2021-01-11 08:59:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-144916/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-144916/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5003851,"identity":"2b3ba217-6873-42c1-afc9-a66836d8874a","added_by":"auto","created_at":"2021-01-15 18:44:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2755142,"visible":true,"origin":"","legend":"A-E are the microscopic representatives of hematoxylin and eosin staining for normal tissue (A), usual hyperplasia (B), atypical hyperplasia (C), carcinoma in situ (D) and invasive carcinoma (E). A’–E’ are the matched tissues of ME3 immunostaining. Scale bar = 150 μm.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/f967337b1a485fc97425c369.jpg"},{"id":5003849,"identity":"64257b7c-82ac-42ae-9289-bda76daf056a","added_by":"auto","created_at":"2021-01-15 18:44:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199073,"visible":true,"origin":"","legend":"The ME3 positive immunostaining rate decreased from normal to usual hyperplasia (UHP), atypical hyperplasia (AHP), carcinoma in situ (CIS) and invasive carcinoma (IC) (R2=0.9414, P=0.006, linear regression analysis). Data are from table 2.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/9801fffbf01245e10fb3f89a.jpg"},{"id":5003652,"identity":"0218b527-921e-48b4-8f40-9c0b8758e165","added_by":"auto","created_at":"2021-01-15 18:41:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287556,"visible":true,"origin":"","legend":"Linear regression of ME3 positive immunostaining rate in T staging and TNM staging. (A) ME3 positive immunostaining rate in Tis, T1, T2 and T3, (R2=0.9394, P=0.031); (B) ME3 positive immunostaining rate in stage 0 to I, II and III (R2=0.9283, P=0.037). Data are from table 3.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/1dee6f9df50b72504e4da2d0.jpg"},{"id":5003850,"identity":"ed24009d-f4dd-446b-99f9-b54811237f72","added_by":"auto","created_at":"2021-01-15 18:44:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33706,"visible":true,"origin":"","legend":"The Kaplan–Meier survival analysis showed that the patients with positive expression of ME3 had significant better outcome than those with negative expression (χ2=8.233, P=0.004).","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/3d62fc5480410a959202f5de.png"},{"id":5003650,"identity":"44cdd0e3-daa5-4568-9173-c04d7bf2cb88","added_by":"auto","created_at":"2021-01-15 18:41:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":362047,"visible":true,"origin":"","legend":"Receiver operating characteristic curves for prediction of breast cancer 5-year survival outcome. (A) 5-year overall survival rate based on tumor TNM staging (the area under ROC curve =84.0%); (B) 5-year overall survival rate based on tumor TNM staging combined with ME3 expression (the area under ROC curve =87.5%).","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/459f417de8fa6828ea188a22.jpg"},{"id":13646874,"identity":"d2104f5b-25d9-48ff-97a4-1533ab9f1f50","added_by":"auto","created_at":"2021-09-17 09:25:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":972808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/d2e910e1-d1dd-41ef-b234-cb2123d46754.pdf"},{"id":5003655,"identity":"4219c283-1727-4027-9ca0-b1bdce7296cd","added_by":"auto","created_at":"2021-01-15 18:41:20","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":28859,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-144916/v1/0855729ad901003361d3e1b6.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExpression of Malic Enzyme 3 in Breast Cancer and Precancerous Lesions: a Promising Novel Biomarker for Carcinogenesis and Prognosis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eBreast 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 \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. 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%) \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Breast cancer has been a major public health problem in China \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt has been well known that changes in cell metabolism can contribute to transformation and tumor progression \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. 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)\u003csup\u003e+\u003c/sup\u003e to NAD(P)H. The different isoforms of MEs have different cofactor specificity and sub-cellular localizations in mammals: the cytosolic NADP\u003csup\u003e+\u003c/sup\u003e-dependent ME1, the mitochondrial NAD(P)\u003csup\u003e+\u003c/sup\u003e-dependent ME2, and the mitochondrial NADP\u003csup\u003e+\u003c/sup\u003e-dependent ME3 \u003csup\u003e9\u0026ndash;11\u003c/sup\u003e. 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 \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, 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 \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Lu and Murai et al demonstrated that ME1 inhibited proliferation and invasion by regulating NADPH homeostasis in gastric cancer and colon cancer cell lines \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Ren et al showed that ME2 depletion increased reactive oxygen species (ROS) and NADP+/NADPH ratio, inhibited cell proliferation and induced cell death and differentiation in lung cancer cell \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Woo et al reported high-expression of ME2 was associated with poorer overall survival (OS) for patients with head and neck squamous cell carcinoma \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Dey et al found that ME3 depletion selectively killed ME2-null pancreatic cancer cells and Zhang Q et al discovered that ME3 promoted the proliferation, invasion and metastasis of pancreatic cancer cells \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Moreover, Liao et al reported that ME1 expression was positively correlated with large tumor size, high grade, poor survival and chemotherapy resistance, indicating that ME1 promoted breast cancer progression and was associated with poor prognosis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe clinicopathological characteristics of the patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e summarized the clinicopathological characteristics of the 107 breast cancer patients. All the patients were confirmed 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.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eClinicopathological characteristics of 107 breast cancer patients.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVariables\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNo. of patients examined\u003c/p\u003e\n\u003cp\u003eN (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 50\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64 (59.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026ge; 50\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e43 (40.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGender\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0 (0)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e107 (100.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT staging\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (7.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32 (29.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64 (59.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (2.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLymph node metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62 (57.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45 (42.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePathological staging\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (7.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25 (23.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45 (42.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29 (27.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVascular cancer embolus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e87 (81.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20 (18.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eER status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38 (35.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69 (64.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePR status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42 (39.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65 (60.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHer-2 status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45 (42.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39 (36.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNot available\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23 (21.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKi67 index\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow expression (\u0026le;\u0026thinsp;14%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27 (25.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh expression (\u0026gt; 14%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80 (74.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eME3 positive expression is decreased with the progression from precancerous to cancerous lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical staining showed that the immunoreactivity for ME3 protein was located in the cytoplasm both in breast cancerous and precancerous tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The ME3 positive expression is defined by semiquantitative scoring system as described in Materials and Methods. The ME3 positive immunostaining rate was higher in the normal breast tissue (97.60%, 82/84), and then the positive rate decreased from UHP (91.1%, 72/79) to AHP (64.2%, 43/67), CIS (62.5%, 40/64) and invasive carcinoma (45.5%, 45/99). The decreasing tendency was inversely correlated with the progression from precancerous to cancerous lesions (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9414, P\u0026thinsp;=\u0026thinsp;0.006, linear regression analysis, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eME3 expression in the normal mammary tissue, precancerous and cancerous lesions.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePathological classification\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNo. of lesions\u003c/p\u003e\n\u003cp\u003eexamined\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eExpression of ME3 protein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u0026chi;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003cp\u003eN (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003cp\u003eN (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNormal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82 (97.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 (2.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e195.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.000\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUsual hyperplasia\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72 (91.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (8.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAtypical hyperplasia\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e43 (64.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24 (35.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCarcinoma in situ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40 (62.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24 (37.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInvasive carcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e99\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45 (45.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e54 (54.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eME3 is associated with better prognosis in breast cancer patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe median follow-up of the entire cohort was 73.7\u0026nbsp;months (range 11.7\u0026ndash;94.8\u0026nbsp;months) and 21 (19.6%) patients died because of the deteriorated breast cancer during the follow-up. The survival rates of 1-, 3- and 5-year were 98.1%, 87.9%, and 82.2%, respectively. It was noteworthy that the median survival time of the 21 dead patients was only 36.6 months, ranging from 11.7 to 67.2\u0026nbsp;months. Of the 21 died cases, one third (33.3%) was in early stage I and II, and the rest (66.7%) was in advanced stage III. In this study, the younger patients with ages\u0026thinsp;\u0026le;\u0026thinsp;40\u0026nbsp;years accounted 16.8% (18/107) and about half of the patients (44.4%, 8/18) were in advanced stage III.\u003c/p\u003e\n\u003cp\u003eThe Kaplan\u0026ndash;Meier survival analysis showed that the patients with positive expression of ME3 had significant better outcome than those with negative expression (\u0026chi;2\u0026thinsp;=\u0026thinsp;8.233, P\u0026thinsp;=\u0026thinsp;0.004, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn this study, lymph node metastasis (\u0026chi;2\u0026thinsp;=\u0026thinsp;12.284, P\u0026thinsp;=\u0026thinsp;0.000), TNM staging progression (\u0026chi;2\u0026thinsp;=\u0026thinsp;18.232, P\u0026thinsp;=\u0026thinsp;0.000) and high expression of Ki67 index (\u0026chi;2\u0026thinsp;=\u0026thinsp;5.155, P\u0026thinsp;=\u0026thinsp;0.024) were also negatively associated with survival (Supplementary Table S1). However, no influence was observed for age, T staging, vascular cancer embolus, ER status, PR status and Her-2 status on survival (Supplementary Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eME3 is inversely associated with the progression of breast cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, linear regression analysis showed that ME3 expression decreased from Tis (75.0%, 6/8) to T1(62.5%, 20/32), T2 (37.5%, 24/64) and T3 (33.3%, 1/3), (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9394, P\u0026thinsp;=\u0026thinsp;0.031, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The analysis on TNM staging and ME3 expression showed ME3 positive immunostaining rate was much higher in stage 0 (75.0%, 6/8) and stage I (72.0%, 18/25) than in stage II (44.4%, 20/45) and stage III (24.1%, 7/29) (\u0026chi;2\u0026thinsp;=\u0026thinsp;14.953, P\u0026thinsp;=\u0026thinsp;0.002, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, the decreasing tendency was also significant from stage 0 to I, II and III (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9283, P\u0026thinsp;=\u0026thinsp;0.037, linear regression analysis, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). ME3 positive expression in breast cancer tissue was related with negative lymph node metastasis (\u0026chi;2\u0026thinsp;=\u0026thinsp;6.393, P\u0026thinsp;=\u0026thinsp;0.011). No significant difference was observed for ME3 expression with age, vascular cancer embolus, ER status, PR status, Her-2 status, or Ki67 index (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eRelationship between ME3 expression and clinicopathological characteristics in breast cancer.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVariables\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNo. of patients\u003c/p\u003e\n\u003cp\u003eexamined\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eExpression of ME3 protein\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u0026chi;2\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePositive(n\u0026thinsp;=\u0026thinsp;51)\u003c/p\u003e\n\u003cp\u003eN (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNegative(n\u0026thinsp;=\u0026thinsp;56)\u003c/p\u003e\n\u003cp\u003eN (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.914\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.166\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;50\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27 (42.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37 (57.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026ge;\u0026thinsp;50\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24 (55.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19 (44.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT staging\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.117\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.044\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6 (75.0%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2 (25.0%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20 (62.5%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12 (37.5%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24 (37.5%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40 (62.5%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1 (33.3%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2 (66.7%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLymph node metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.393\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.011\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36 (58.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26 (41.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15 (33.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30 (66.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePathological staging\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.953\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6 (75.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2 (25.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18 (72.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7 (28.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20 (44.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25 (55.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7 (24.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22 (75.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVascular cancer embolus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.070\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.791\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e42 (48.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45 (51.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9 (45.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11 (55.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eER status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.730\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.393\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16 (42.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22 (57.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35 (50.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34 (49.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePR status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.537\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.111\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16 (38.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26 (61.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35 (53.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30 (46.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHer-2 status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.439\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.487\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22 (48.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23 (51.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16 (41.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23 (59.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNot available\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13 (56.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10 (43.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKi67 index\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.066\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow expression (\u0026le;\u0026thinsp;14%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17 (63.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10 (37.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh expression (\u0026gt; 14%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34 (42.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46 (57.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReceiver operating characteristic curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing multivariate logistic regression and receiver operating characteristic curve (ROC), we explored the potential of ME3 expression in the classification of 5-year survival outcome. Logistics regression analysis showed that TNM staging was the influencing 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Using TNM combined with ME3 expression, the area under ROC curve increased to 87.5% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting a potential role of the ME3 in prediction of patients at risk of breast cancer death.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, this is the first 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.\u003c/p\u003e\n\u003cp\u003eFurthermore, 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.\u003c/p\u003e\n\u003cp\u003eBreast 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 \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e\n\u003cp\u003eIt 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\u003csup\u003e+\u003c/sup\u003e to NADPH and this reaction is crucial for maintaining the NADPH pool in mitochondria \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. 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 \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, ME3 downregulation may take a part in breast carcinogenesis and breast cancer progression via perturbing the homeostasis of NADPH/NADP\u003csup\u003e+\u003c/sup\u003e in mitochondria, leading to accumulation of mROS. Further investigation is required to validate the hypothesis.\u003c/p\u003e\n\u003cp\u003eIt 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 \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, 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 \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, 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.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e107 breast cancer patients were retrieved in this study from a database, which has been established by The State Key Laboratory of Esophageal Cancer Prevention \u0026amp; Treatment and Henan Key Laboratory of Esophageal Cancer Research in The First Affiliated Hospital of Zhengzhou University. All the patients had been underwent mastectomy from August 2012 to December 2014 without any radiation therapy or chemotherapy prior to surgery before operation. All patients were females with a mean age of 48.41\u0026plusmn;11.18 years and a median age of 46 years (range from 26 to 82 years). Staging for breast cancer were based on American Joint Committee on Cancer (AJCC) TNM staging system in 2017 \u003csup\u003e30\u003c/sup\u003e. The follow-up was performed with hospital medical records, either through telephone or through home interview to each patient yearly until death. The last follow-up was May 2020. Overall survival time was calculated from the day of mastectomy to death or to the last follow-up date. The study protocol was approved by the Medical Ethics Committee of Zhengzhou University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue collection and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 107 breast cancer tissues and 87 matched adjacent non-cancerous tissues were collected from surgically resected specimens from the patients with primary breast cancer. The tissues were routinely 10% neutral formalin fixed, paraffin-embedded and then sectioned. Serial 4 \u0026micro;m sections were prepared for histopathological analysis (hematoxylin and eosin stain) and immunohistochemical staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistopathological diagnoses and staging for breast cancer and adjacent non-cancerous lesions were made based on WHO criteria in 2012 \u003csup\u003e31\u003c/sup\u003e. The epithelial proliferative lesions in mammary gland acini and duct were categorized as \u0026ldquo;normal\u0026rdquo;, usual hyperplasia (UHP), atypical hyperplasia (AHP) and carcinoma in situ (CIS). Histopathologically, UHP was usually found adjacent to the tumor tissue; AHP and CIS were frequently observed as isolated lesions in the surgically resected breast specimens. Pathologically, of the 87 cases with adjacent cancer tissue, there were 84 cases identified with normal tissue (96.6%, 84/87), 79 with UHP (90.8%, 79/87), 67 with AHP (77.0%, 67/87) and 64 with CIS (73.6%, 64/87). Histological examination was carried out by two pathologists independently.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollow the manufacturer\u0026rsquo;s instructions, tissue sections were deparaffinized, 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 \u0026deg;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\u0026rsquo; clinical information.\u003c/p\u003e\n\u003cp\u003eAccording to immunohistochemical scoring system previously with some modification \u003csup\u003e32,33\u003c/sup\u003e, ME3 immunostaining score (S) was determined semiquantitatively by multiplying the percentage of positive cells (P) with intensity (I), according to the formula: S= P\u0026times;I. The range score for percentage of positive cells is 0 to 4 (0, ＜1%; 1, 1%-10%; 2, 11%-50%; 3, 51%-75%; 4, 76%-100%). The range score for intensity is 0 to 3 (0, no staining; 1, weak; 2, moderate; 3, strong.). The index score was obtained a range from 0 to 12. The final immunostaining results were denoted as negative expression of ME3 (0～1 scores) and positive expression of ME3 (\u0026ge;2 scores).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using IBM SPSS version 21.0. Data was represented as the mean \u0026plusmn; standard deviation for continuous variables and frequency (number-percent) for categorical data. Relationships of ME3 expression and clinicopathological features were evaluated by the chi-square (\u0026chi;2-value) and linear regression analysis (R\u003csup\u003e2\u003c/sup\u003e-value). The Kaplan\u0026ndash;Meier survival analysis was used to estimate the association between eligible variables and survival. Cox proportional hazards regression models were used for multivariate survival analysis in stepwise regression manner. Receiver operating characteristic curve was based on the prediction probability of 5-year survival outcome from the logistic regression model, and the area under ROC curve was used to assess the classification performance of the model. Any result with a P-value of less than 0.05 was considered as statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been supported in part by the China National 973 project (2013CB911303), China Natural Sciences Foundation projects (81470126), a key project (2018C03009) funded by Zhejiang Provincial Department of Sciences and Technologies, and the Fundamental Research Funds for the Central Universities, National Ministry of Education, China, to XH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design of the study: XH and DY; Sample acquisition and analysis: DD, and DY; Histopathological diagnosis: XML and DD; Manuscript preparation: All authors; Approval of the final version: All authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from patients in this study. The human study protocol was in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans were approved by the Medical Ethics Committee of Zhengzhou University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray, F.\u003cem\u003e et al.\u003c/em\u003e Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 394-424, doi:10.3322/caac.21492 (2018).\u003c/li\u003e\n\u003cli\u003eLi, T., Mello-Thoms, C. \u0026amp; Brennan, P. C. Descriptive epidemiology of breast cancer in China: incidence, mortality, survival and prevalence. \u003cem\u003eBreast Cancer Res Treat\u003c/em\u003e \u003cstrong\u003e159\u003c/strong\u003e, 395-406, doi:10.1007/s10549-016-3947-0 (2016).\u003c/li\u003e\n\u003cli\u003eZeng, H.\u003cem\u003e et al.\u003c/em\u003e Changing cancer survival in China during 2003\u0026ndash;15: a pooled analysis of 17 population-based cancer registries. \u003cem\u003eThe Lancet Global Health\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e555-e567, doi:10.1016/s2214-109x(18)30127-x (2018).\u003c/li\u003e\n\u003cli\u003eMiller, K. D.\u003cem\u003e et al.\u003c/em\u003e Cancer treatment and survivorship statistics, 2019. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 363-385, doi:10.3322/caac.21565 (2019).\u003c/li\u003e\n\u003cli\u003eChen, W.\u003cem\u003e et al.\u003c/em\u003e Cancer statistics in China, 2015. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 115-132, doi:10.3322/caac.21338 (2016).\u003c/li\u003e\n\u003cli\u003eFan, L.\u003cem\u003e et al.\u003c/em\u003e Breast cancer in China. \u003cem\u003eThe Lancet Oncology\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e279-e289, doi:10.1016/s1470-2045(13)70567-9 (2014).\u003c/li\u003e\n\u003cli\u003ePavlova, N. N. \u0026amp; Thompson, C. B. The Emerging Hallmarks of Cancer Metabolism. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 27-47, doi:10.1016/j.cmet.2015.12.006 (2016).\u003c/li\u003e\n\u003cli\u003eVander Heiden, M. G. \u0026amp; DeBerardinis, R. J. Understanding the Intersections between Metabolism and Cancer Biology. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e168\u003c/strong\u003e, 657-669, doi:10.1016/j.cell.2016.12.039 (2017).\u003c/li\u003e\n\u003cli\u003eChang, G.-G. \u0026amp; Tong, L. Structure and function of malic enzymes, a new class of oxidative decarboxylases. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 12721-12733 (2003).\u003c/li\u003e\n\u003cli\u003eXu, Y., Bhargava, G., Wu, H., Loeber, G. \u0026amp; Tong, L. Crystal structure of human mitochondrial NAD (P)+-dependent malic enzyme: a new class of oxidative decarboxylases. \u003cem\u003eStructure\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 877-889 (1999).\u003c/li\u003e\n\u003cli\u003eYao, P.\u003cem\u003e et al.\u003c/em\u003e Evidence for a direct cross-talk between malic enzyme and the pentose phosphate pathway via structural interactions. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e292\u003c/strong\u003e, 17113-17120, doi:10.1074/jbc.M117.810309 (2017).\u003c/li\u003e\n\u003cli\u003eHasan, N. M., Longacre, M. J., Stoker, S. W., Kendrick, M. A. \u0026amp; MacDonald, M. J. Mitochondrial malic enzyme 3 is important for insulin secretion in pancreatic beta-cells. \u003cem\u003eMol Endocrinol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 396-410, doi:10.1210/me.2014-1249 (2015).\u003c/li\u003e\n\u003cli\u003eJiang, P., Du, W., Mancuso, A., Wellen, K. E. \u0026amp; Yang, X. Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e493\u003c/strong\u003e, 689-693, doi:10.1038/nature11776 (2013).\u003c/li\u003e\n\u003cli\u003eLu, Y. X.\u003cem\u003e et al.\u003c/em\u003e ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth and Metastasis. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 1972-1985, doi:10.1158/0008-5472.CAN-17-3155 (2018).\u003c/li\u003e\n\u003cli\u003eMurai, S.\u003cem\u003e et al.\u003c/em\u003e Inhibition of malic enzyme 1 disrupts cellular metabolism and leads to vulnerability in cancer cells in glucose-restricted conditions. \u003cem\u003eOncogenesis\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e329, doi:10.1038/oncsis.2017.34 (2017).\u003c/li\u003e\n\u003cli\u003eRen, J. G.\u003cem\u003e et al.\u003c/em\u003e Knockdown of malic enzyme 2 suppresses lung tumor growth, induces differentiation and impacts PI3K/AKT signaling. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 5414, doi:10.1038/srep05414 (2014).\u003c/li\u003e\n\u003cli\u003eWoo, S. H.\u003cem\u003e et al.\u003c/em\u003e Down-regulation of malic enzyme 1 and 2: Sensitizing head and neck squamous cell carcinoma cells to therapy-induced senescence. \u003cem\u003eHead Neck\u003c/em\u003e \u003cstrong\u003e38 Suppl 1\u003c/strong\u003e, E934-940, doi:10.1002/hed.24129 (2016).\u003c/li\u003e\n\u003cli\u003eDey, P.\u003cem\u003e et al.\u003c/em\u003e Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e542\u003c/strong\u003e, 119-123, doi:10.1038/nature21052 (2017).\u003c/li\u003e\n\u003cli\u003eZhang, Q., Li, J., Tan, X. \u0026amp; Zhao, Q. Effects of ME3 on the proliferation, invasion and metastasis of pancreatic cancer cells through epithelial-mesenchymal transition. \u003cem\u003eNeoplasma\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 896-907 (2019).\u003c/li\u003e\n\u003cli\u003eLiao, R.\u003cem\u003e et al.\u003c/em\u003e ME1 promotes basal-like breast cancer progression and associates with poor prognosis. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 16743, doi:10.1038/s41598-018-35106-y (2018).\u003c/li\u003e\n\u003cli\u003eSociety, A. C. Breast cancer facts \u0026amp; figures 2019\u0026ndash;2020. \u003cem\u003eAm. Cancer Soc\u003c/em\u003e, 1-44 (2019).\u003c/li\u003e\n\u003cli\u003eFan, J.\u003cem\u003e et al.\u003c/em\u003e Quantitative flux analysis reveals folate-dependent NADPH production. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e510\u003c/strong\u003e, 298-302, doi:10.1038/nature13236 (2014).\u003c/li\u003e\n\u003cli\u003eLewis, C. A.\u003cem\u003e et al.\u003c/em\u003e Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 253-263, doi:10.1016/j.molcel.2014.05.008 (2014).\u003c/li\u003e\n\u003cli\u003eSabharwal, S. S. \u0026amp; Schumacker, P. T. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel? \u003cem\u003eNat Rev Cancer\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 709-721, doi:10.1038/nrc3803 (2014).\u003c/li\u003e\n\u003cli\u003eDeBerardinis, R. J. \u0026amp; Chandel, N. S. Fundamentals of cancer metabolism. \u003cem\u003eScience advances\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, e1600200 (2016).\u003c/li\u003e\n\u003cli\u003eLiu, C.\u003cem\u003e et al.\u003c/em\u003e Malic enzyme 1 indicates worse prognosis in breast cancer and promotes metastasis by manipulating reactive oxygen species. \u003cem\u003eOncoTargets and therapy\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 8735 (2020).\u003c/li\u003e\n\u003cli\u003eBirch‐Machin, M. The role of mitochondria in ageing and carcinogenesis. \u003cem\u003eClinical and Experimental Dermatology: Experimental dermatology\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 548-552 (2006).\u003c/li\u003e\n\u003cli\u003eJežek, J., Cooper, K. F. \u0026amp; Strich, R. Reactive oxygen species and mitochondrial dynamics: the yin and yang of mitochondrial dysfunction and cancer progression. \u003cem\u003eAntioxidants\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 13 (2018).\u003c/li\u003e\n\u003cli\u003eYang, Y.\u003cem\u003e et al.\u003c/em\u003e Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy. \u003cem\u003eJournal of cellular physiology\u003c/em\u003e \u003cstrong\u003e231\u003c/strong\u003e, 2570-2581 (2016).\u003c/li\u003e\n\u003cli\u003eAmin, M. B.\u003cem\u003e et al.\u003c/em\u003e The Eighth Edition AJCC Cancer Staging Manual: Continuing to build a bridge from a population-based to a more \"personalized\" approach to cancer staging. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 93-99, doi:10.3322/caac.21388 (2017).\u003c/li\u003e\n\u003cli\u003eFrank, G. A., Danilova, N. V., Andreeva, I. \u0026amp; Nefedova, N. A. [WHO classification of tumors of the breast, 2012]. \u003cem\u003eArkhiv patologii\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 53-63 (2013).\u003c/li\u003e\n\u003cli\u003eFedchenko, N. \u0026amp; Reifenrath, J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. \u003cem\u003eDiagn Pathol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 221, doi:10.1186/s13000-014-0221-9 (2014).\u003c/li\u003e\n\u003cli\u003eFitzgibbons, P. L.\u003cem\u003e et al.\u003c/em\u003e Template for reporting results of biomarker testing of specimens from patients with carcinoma of the breast. \u003cem\u003eArch Pathol Lab Med\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 595-601, doi:10.5858/arpa.2013-0566-CP (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, ME3 expression, carcinogenesis, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-144916/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-144916/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eWhile 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.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA total of 107 patients confirmed with breast cancer were enrolled. The ME3 expression was evaluated by IHC and correlated with clinicopathological indicators.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe 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%. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eME3 may be a promising biomarker for better prognosis for breast cancer patients.\u003c/p\u003e","manuscriptTitle":"Expression of Malic Enzyme 3 in Breast Cancer and Precancerous Lesions: a Promising Novel Biomarker for Carcinogenesis and Prognosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-15 18:41:18","doi":"10.21203/rs.3.rs-144916/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19de953d-5048-43ab-b379-668cd1b13f6e","owner":[],"postedDate":"January 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1905245,"name":"Cancer Biology"},{"id":1905246,"name":"Oncology"}],"tags":[],"updatedAt":"2021-06-30T05:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-15 18:41:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-144916","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-144916","identity":"rs-144916","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}