Targ Oncol DOI 10.1007/s11523-014-0316-y

ORIGINAL RESEARCH

Study on mouse model of triple-negative breast cancer: association between higher parity and triple-negative breast cancer Chun Huang & Xuan Wang & Baocun Sun & Man Li & Xiulan Zhao & Yanjun Gu & Yanfen Cui & Yan Li

Received: 16 February 2014 / Accepted: 15 April 2014 # Springer International Publishing Switzerland 2014

Abstract To investigate the association between high parity and triple-negative breast cancer (TNBC), and explore the etiologic mechanisms of TNBC in Tientsin Albinao 2 (TA2) mice model. After the TA2 mice model with high parity and TNBC had been established, the cell proliferation and apoptosis were detected by immunohistochemical and TUNEL staining in mammary epithelia from different conditions, which included non-pregnancy, low and high gravidity in pregnancy, and carcinogenesis. Apoptotic signaling was Chun Huang and Xuan Wang contributed equally. C. Huang Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), Department of Medical Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Tianjin 300060, People’s Republic of China X. Wang (*) Department of Nutrition and Food Science, School of Public Health, Tianjin Medical University, Qi Xiang Tai Road 22#, Heping District, Tianjin 300070, People’s Republic of China e-mail: [email protected] X. Wang : B. Sun (*) : Y. Cui : Y. Li Department of Pathology, Tianjin Cancer Institute and Hospital, Ti Yuan Bei, Huan Hu Xi Road, He Xi District, Tianjin 300060, People’s Republic of China e-mail: [email protected] B. Sun : X. Zhao Department of Pathology, Tianjin Medical University, Tianjin, People’s Republic of China M. Li Department of Digestive, The Second Hospital of Tianjin Medical University, Tianjin, People’s Republic of China Y. Gu Department of Pathology, Medical College of the Chinese People’s Armed Police Forces, Tianjin, People’s Republic of China

analyzed by measuring bcl-2, bax, caspase-3, and caspase-9 expression using immunohistochemistry (IHC), western blot, and real-time PCR technique. Estrogen receptor α (ERα) and progesterone receptor (PR) were determined by immunohistochemical staining and real-time PCR. Both proliferation and apoptosis in mammary epithelia changed with the increasing of parity. Immunohistochemistry revealed increased cell proliferation and apoptosis were related with upregulation of ERα, PR, bcl-2, bax, caspase-3, and caspase-9 expression, especially during the fourth pregnancy. Mammary gland in the fourth pregnancy stage was the closest to precancerous. In precancerous mammary gland, cell proliferation rate was much higher than apoptosis rate. High parity could increase the ovarian hormones level and alter ovarian hormone receptor levels in TA2 mice, and their sensitivity to ovarian hormones result in the imbalance between cell proliferation and apoptosis in precancerous mammary epithelial cells. Keyword Spontaneous breast cancer . Triple-negative phenotype of breast cancer . TA2 mice . Pregnancy

Introduction Breast cancer is one of the most common malignant tumor in females, while the mechanism of carcinogenesis is not yet been clarified definitively. Most study indicated that null parity or low parity is risk factors for breast cancer, and high parity may have a protective effect [1, 2]. The parity exacts its protective effects through inducing the differentiation which accompanies pregnancy and lactation [3]. However, recent research has shown that pregnancy does not result in an immediate protective effect [4, 5]; sometimes it becomes a risk factor for breast cancer. Researchers indicated that parity has dual association with breast cancer, parity is associated with an increased risk among women

Targ Oncol

younger than 45 years, on the other hand, parity is associated with a decreased risk among women 45 years and older [6, 7]. Triple-negative breast cancer (TNBC), a subtype of invasive breast cancer with estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER’s-2-negative phenotype, is an important subtype of breast cancer due to its relationship with basal-like breast cancer. TNBC is more likely to affect younger women and has a more advanced and aggressive stage. Regardless of stage at diagnosis, women with TNBC had poorer survival than those with other breast cancers. Recent epidemiologic studies showed high parity and lack of breast feeding are associated with TNBC [8–11]. However, the mechanism of its carcinogenesis is still unclear. Hence, a specific animal model may conducive to further elucidation of the mechanism of TNBC. Mice are frequently used in genetic studies because the genetic background of many particular strains of mice has been clarified. The Tientsin Albinao 2 (TA2) mouse strain has a high incidence of spontaneous breast cancer (SBC) without any induction by chemical stimulus [12]. Pathologic analysis showed that breast cancer cells in TA2 mice are triple negative [13–15]. Our previous studies showed that the mammary carcinogenesis might be closely related to high parity and genetic background [16, 17]. Therefore, TA2 mice which have multiple pregnancy-induced changes in the mammary gland may serve as a good model to investigate the association between high parity and TNBC. Cumulative exposure to estrogen and progesterone lead to high risk of developing breast cancer. Estrogen, progesterone, and other hormones during pregnancy increased significantly than non-pregnant female. It is presumed that cellular proliferation promoted by these two hormones increases the likelihood of stochastically acquired mutations, and leads to development of tumor [18]. Besides regulate cell proliferation, sex hormones and their receptors control cell apoptosis [19]. Although TNBC is an estrogen-independent phenotype of breast cancer, sex hormone might be indirectly associated with the tumor genesis of TNBC in high parity. Several regulation proteins, such as B cell leukemia/lymphoma-2 (bcl-2) show hormone-dependent expression patterns. We hypothesized that the mechanism of TNBC could attribute to the disrupted cell balance which is caused by high parity. In order to confirm the effect of high parity on the genesis of TNBC, we took TA2 mice as a model and observed the changes of cell proliferation and apoptosis during different stages of pregnancy and puerperium, and analyzed the tumorigenesis of mammary gland in TA2 mice.

Materials and methods Animals The TA2 mice (weighing 18–22 g, 6–8 weeks) were obtained from the Experimental Animal Center at Tianjin Medical

University, China. All animal protocol followed the Guide of Chinese Academy of Medical Sciences for the Care and Use of Laboratory Animals. All mice were fed ad libitum with standard rodent food and water in a climate-controlled environment with a light:dark photoperiod of 12:12. Three to four female mice were housed together with male TA2 mouse; female mice were inspected for the presence of vaginal plugs every morning. The day a vaginal plug was first observed was defined as the first day of pregnancy. Non-pregnant adult mice (6–8 weeks) served as normal control (NC group); other animals were grouped by the duration of pregnancy (5, 10, 20 days: P5, P10, P20 groups) and puerperium (1, 7, 14, 21, 42 days: PP1, PP7, PP14, PP21, PP42 groups). The mammary glands of mice were removed for histological analysis. The tissues were collected on days 5, 10, and 20 of pregnancy and days 1, 7, 14, 21, and 42 of puerperium in the first pregnancy. And P10, P20, PP42 in four gravidities were chosen as the base of the first gravidity for comparison (Table 1). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay using the in situ cell death detection kit (Roche Applied Science, Indianapolis, IN, USA) conjugated with horseradish peroxidase (POD) according to the manufacturer’s instructions. Briefly, prepared sections were washed, permeabilized, and then incubated with 50 μL of TUNEL reaction mixture for 60 min at 37 °C in a humidified atmosphere in the dark. Slides were rinsed with phosphate-buffered saline (PBS), incubated with 50 μL of converter-POD for 30 min at 37 °C, rinsed with PBS again, and then incubated with 50 μL of 3, 3′-diaminobenzidine (DAB) substrate solution for 10 min at 25 °C. The slides were then rinsed with PBS, mounted under glass coverslips. Immunohistochemical staining Sections (4 μm thick) were mounted on polylysine-coated slides and deparaffinized in xylene. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide in 50 % methanol for 10 min at room temperature (RT). Sections were rehydrated in alcohol, washed with PBS, and then pretreated with citrate buffer [0.01 mol/L citric acid (pH 6.0)] for 20 min at 95 °C in a microwave oven. After nonspecific binding sites were blocked by exposing them to 10 % normal goat serum in PBS for 20 min at 37 °C, sections were incubated overnight at 4 °C with rabbit polyclonal anti-PCNA (Boster Biological Technology, Hubei, CHN; dilution 1:100), rabbit polyclonal

Targ Oncol Table 1 The groups of animals in different gravidities (n=5) Groups

8-month-old virgin

Day 10 of pregnancy

Day 20 of pregnancy

Day 42 of puerperium

Spontaneous breast cancer

Control 1st pregnancy 2nd pregnancy 3rd pregnancy 4th pregnancy

8MVa – – – –

– 1P10 2P10 3P10 4P10

– 1P20 2P20 3P20 4P20

– 1PP42 2PP42 3PP42 4PP42

SBCb – – – –

a b

Non-pregnant and non-tumor adult 8months old virgin mice were taken as negative control Mice with spontaneous breast cancer were taken as positive control

anti-Bcl2 (Santa Cruz, CA, USA; dilution 1:800), rabbit polyclonal anti-bax (Santa Cruz Biotechnology; dilution 1:800), rabbit polyclonal anti-caspase-3 (Newmarker, NY, USA; dilution 1:200), rabbit polyclonal anti-caspase-9 (Newmarker; dilution 1:200), rabbit polyclonal anti-ER (Santa Cruz; dilution 1:100), rabbit polyclonal anti-PR (Santa Cruz; dilution 1:100), and rabbit polyclonal anti-cyclinD1 (Santa Cruz; dilution 1:100). Then, the sections were rinsed with PBS and incubated with biotinylated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 37 °C. The slides were then incubated with 3,3′-DAB chromogen for 5 to 10 min at RT and washed with distilled water. Finally, sections were slightly counterstained with hematoxylin for 1 min followed by dehydration and coverslip mounting. PBS was used as primary antibodies for negative control. The staining systems used in this study were PicTure PV6000 (Zhongshan Chemical Co., Beijing, China) and Elivision Plus (Zhongshan Chemical Co., Beijing, China).

glycerol, 30 mM DTT, 6 % SDS) was added to yield a final 1× concentration and lysates were boiled at 95 °C for 5 min. Samples were subjected to SDS polyacrylamide gel electrophoresis on a 10 or 15 % gel (acrylamide: bis-acrylamide ratio of 29:1) and electro-blotted to Immobilin PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5 % defatting milk for 1 h at 37 °C, and then incubated with anti-Bcl2, anti-bax (Santa Cruz Biotechnology; both dilution 1:100), anti-caspase-3 and anti-caspase-9 (Newmarker; both dilution 1:1,000) at 4 °C overnight. Blots were washed by 0.5 % PBS-T (PBS with Tween-20) 3 times and then incubated with a 1:5,000 dilution of anti-rabbit conjugated HRP (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 1 h at 37 °C. The signals were detected by using the Western blotting chemiluminescent kit (Pierce Biotechnology, Rockford, Illinois, USA), quantified by densitometry and analyzed using Quantity One image analysis system (BioRad, Hercules, CA, USA). The β-actin (1:5,000, Santa Cruz, USA) was used as the inner control.

RNA isolation and quantitative RT-PCR analysis After mammary glands and tumor were isolated, the lymph nodes were removed and frozen in liquid nitrogen. Total tissue RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcript (TaKaRa, Otsu/Shiga, Japan), and real-time PCR. Real-time PCR was carried out using SYBR® Premix Ex Taq™ (Perfect Real Time) kit (TaKaRa, Japan) according to the manufacturer’s instructions. The copies of target cDNA were normalized by β-actin expression. Primers for Bcl2, bax, caspase-3, caspase-9, ER, PR and βactin were listed in Table 2. Western blots The frozen samples were grinded into fine powder in liquid nitrogen and homogenized in lysis buffer (pH 8.0 20 mM Tris, 137 mM NaCl, 10 % glycerol, 1 % NP-40, 0.1 % SDS, 0.5 % deoxycholate, 0.2 mM PMSF and 1× general protease inhibitor). Protein concentration in lysates was determined by Bradford assay prior to gel loading to ensure equal protein loading. A 6× sample buffer (300 mM Tris–HCl, pH 6.8, 60 %

Counting and statistical methods Protein expression levels were quantified according to the method described by Sun et al. [13]; both the intensity and the percentage of positive cells were evaluated. More than 10 microscopic fields in one section were observed with ×400 magnifications. For stain intensity, 0 was denoted for no staining; 1 for weak positive with faint yellow; 2 for moderate positive; and 3 for strong positive with brown staining. Positive cells were counted in 100 tumor cells per field with 10 fields in each section. The number of positive cells was visually evaluated and cell expression was stratified as follows: 0 (negative) for