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MTMR3 Is Upregulated in Patients with Breast Cancer and Regulates Proliferation, Cell Cycle Progression and Autophagy in Breast Cancer Cells

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MTMR3 Is Upregulated in Patients with Breast Cancer and Regulates Proliferation, Cell Cycle Progression and Autophagy in Breast Cancer Cells ONCOLOGY REPORTS 42: 1915-1923, 2019 MTMR3 is upregulated in patients with breast cancer and regulates proliferation, cell cycle progression and autophagy in breast cancer cells ZHAN WANG1*, MIN ZHANG1*, RONG SHAN1, YU-JIE WANG1, JUAN CHEN2, JUAN HUANG3, LUN-QUAN SUN4 and WEI-BING ZHOU1 1Department of Oncology, 2Department of Pharmacy, 3Hunan Province Clinic Meditech Research Center for Breast Cancer, and 4Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China Received April 9, 2019; Accepted July 23, 2019 DOI: 10.3892/or.2019.7292 Abstract. As a member of the myotubularin family, myotu- Introduction bularin related protein 3 (MTMR3) has been demonstrated to participate in tumor development, including oral and colon Breast cancer is the second leading cause of cancer-related cancer. However, little is known about its functional roles in deaths in women, and the incidence of breast is estimated breast cancer. In the present study, the expression of MTMR3 in to increase by ~0.5% annually (1,2). Despite enormous breast cancer was evaluated by immunohistochemical staining progress on breast cancer therapy, 20% of patients eventu- of tumor tissues from 172 patients. Online data was then ally die of their disease (3,4). Multiple studies have revealed used for survival analysis from the PROGgeneV2 database. that breast cancer is a heterogeneous and complex disease, In vitro, MTMR3 expression was silenced in MDA-MB-231 whose pathophysiology cannot be explained by one or several cells via lentiviral shRNA transduction. MTT, colony forma- mechanisms (5). A number of reliable biological markers have tion and flow cytometry assays were performed in the control been found to predict the risk of recurrence and metastasis and MTMR3-silenced cells to evaluate the cell growth, of breast cancer. Among these prognostic factors, estrogen proliferation and cell cycle phase distribution, respectively. receptor (ER), progesterone receptor (PR) and erb-b2 receptor Western blotting was used to evaluate the protein expression tyrosine kinase 2 (HER2) status are significantly associated levels of autophagy-related markers. The results demonstrated with overall clinical outcome (6). In addition, triple-negative that the expression of MTMR3 in breast cancer tissues was breast cancer (TNBC), which is characterized by absent or significantly increased compared with adjacent normal tissues. minimal expression of ER, PR and HER2, is more aggressive MTMR3 was highly expressed in triple-negative breast and has a worse prognosis compared with the other subtypes cancer and was associated with disease recurrence. MTMR3 of breast cancer (7). Recently, the expression profile of TNBC knockdown in MDA-MB-231 cells inhibited cell prolifera- was described in a homogeneous population from northeastern tion and induced cell cycle arrest and autophagy. The present Mexico and a novel gene signature related to metabolism results indicated that MTMR3 may have an important role in was proposed (8). Such expression profiles are valuable promoting the progression of breast cancer, and its inhibition for elucidating the mechanisms involved in tumorigenesis, may serve as a promising therapeutic target for breast cancer diagnosis, prognosis and potential therapies. treatment. Myotubularin related protein 3 (MTMR3) was first identified as an inositol lipid 3‑phosphatase and belongs to the myotubularin (MTM) family (9). It confers a unique substrate specificity to phosphatidylinositol(3)‑phosphate (PtdIns3P) and phosphatidylinositol(3,5)-biphosphate [PtdIns(3,5)P2], and the hydrolysis products, PtdIns and PtdIns5P, regulate cell activity Correspondence to: Professor Wei-Bing Zhou, Department of and tumor progression (9,10). MTMR3 contains a N-terminal Oncology, Xiangya Hospital, Central South University, 87 Xiangya phosphatidylinositol lipids binding domain, a pleckstrin Road, Changsha, Hunan 410008, P.R. China E-mail: [email protected] homology-GRAM (PHG) domain, and a C-terminal modula- tory domain, that is responsible for endosomal localization *Contributed equally and for binding to Ptdins3P (10). Inositol lipids are thought to be involved in various cellular functions, including prolifera- Key words: breast cancer, myotubularin related protein 3, proli- tion, invasion, survival, cell cycle progression, migration and feration, cell cycle, autophagy membrane trafficking (11). Several investigations into MTMR3 have underlined the key role of this gene in cancer develop- ment (12-14). Oppelt et al (14) reported that MTMR3 is widely expressed in most cancer cell lines and MTMR3‑deficiency 1916 WANG et al: ROLE OF MTMR3 IN BREAST CANCER leads to reduced motility in rhabdomyosarcoma Rh30 cells and sectioned at 4 µm. Sections of tumors were dewaxed with and osteosarcoma U2OS cells. xylenes and dehydrated in gradient ethanol, followed by antigen Previously, a key role for MTMR3 was revealed in oral retrieval in citrate antigen retrieval solution (cat. no. P0081; cancer. Kuo et al (15) demonstrated that miR-99a exerts Beyotime Institute of Biotechnology). Endogenous peroxidase antimetastatic effects through decreasing MTMR3 levels, blocking buffer (100 µl; cat. no P0100A; Beyotime Institute of suggesting that MTMR3 may serve as a potential therapeutic Biotechnology) was added for 10 min to block the endogenous target for oral cancer. In breast cancer, although MTMR3 has peroxidase activity. Then, the sections were treated with 100 µl been shown to be associated with cell cycle regulation and blocking solution (cat. no. B10710; Invitrogen; Thermo Fisher apoptosis in the SK-BR-3 cell line (16), the clinical impacts Scientific, Inc.) and covered with parafilm. Subsequently, and functional role of MTMR3 remain unclear. Autophagy is the sections were incubated with primary anti-MTMR3 a critical intracellular pathway that is associated with the bulk antibody (1:100; cat. no. 12443; Cell Signaling Technology, degradation of cytoplasmic components (17). In addition to Inc.) overnight at 4˚C. After incubation with horseradish acting as a tumor inhibitor, autophagy can also enhance cell peroxidase (HRP)‑conjugated secondary antibody (1:5,000; survival to drive tumor growth and metastasis (18). Notably, cat. no. ab205718; Abcam) for 1 h at room temperature, the depletion of MTMR3 was demonstrated to trigger autopha- slides were stained with diaminobenzidine (cat. no. D3939; gosome formation, but overexpression of MTMR3 resulted Sigma-Aldrich; Merck KGaA) for 60 min at room temperature, in smaller nascent autophagosomes, subsequently blocking followed by counterstaining with hematoxylin (cat. no. C0107; autophagy (19). In breast cancer, MTMR3 has been reported Beyotime Institute of Biotechnology). The staining was visual- to be regulated by miR-100, which could mediate apoptosis ized using a light microscope (CKX41; Olympus Corporation) of breast cancer (16). However, the functions of MTMR3 in at x100 and x400 magnification. Positivity and intensity were breast cancer have not been elucidated to date. assessed by two independent pathologists in a blinded manner, The present study explored the prognostic role of MTMR3 according to a previous report (21). The staining intensity and in breast cancer, and the effects of MTMR3 silencing in proportion of immune reactive cells were scored, as previously MDA-MB-231 cells. The aim of the present study was to described (22). investigate the clinical implication of MTMR3 and its potential biological or functional mechanisms. Online overall survival analysis. Survival analysis for the MTMR3 gene in breast cancer was performed using the Materials and methods PROGgeneV2 prognostic database (http://genomics.jefferson. edu/proggene/) (18). This database includes the prognostic data Tissue specimen collection and follow‑up. A total of from The Cancer Genome Atlas (TCGA) and Gene Expression 172 patients were enrolled in the present study. All samples Omnibus (GEO) databases, while providing an easy operator were collected at Xiangya Hospital (Changsha, China) interface. The following parameters were selected in the first between January 2013 and December 2013. For 52 of them, interface: ‘MTMR3’ in Single/multiple user input genes; paired primary tumor tissues and adjacent normal tissues ‘breast cancer’ in cancer type; ‘death’ in survival measure; (>5 cm away from tumor area) were obtained. Formalin‑fixed and ‘median’ in bifurcate gene expression. Then, in the second paraffin‑embedded (FFPE) tumor tissues from 120 patients interface, all the filters were selected, and the plot was created. that underwent surgical removal were used to analyze MTMR3 According to the median expression level of MTMR3 in protein expression levels. These patients were divided into two breast cancer tissues, the patient samples were classified into groups: Relapse group and non-relapse group. Relapse was two groups, higher and lower expression levels. The survival defined as metastases or local recurrence occurring within patient information, including 3 and 5 year-survival rates, was 5 years; the terminal date for follow-up was January 2018. compared using Kaplan-Meier survival curves. The clinicopathological information was obtained from the patients' records: age, pathology subtypes, status of ER, PR Cell lines and culture. Human breast cancer cell lines MCF-7, and HER2 expression levels, and clinical stage. All cases met T47D, BT474, MDA-MB-231 and ZR-75-30, as well as human the following inclusion criteria: i) Histologically
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