SULT1E1 Inhibits Cell Proliferation and Invasion by Activating Pparγ in Breast Cancer

Journal of Cancer 2018, Vol. 9 1078 Ivyspring International Publisher Journal of Cancer 2018; 9(6): 1078-1087. doi: 10.7150/jca.23596 Research Paper SULT1E1 inhibits cell proliferation and invasion by activating PPARγ in breast cancer Yali Xu1, Xiaoyan Lin1, Jiawen Xu1, Haiyan Jing1, Yejun Qin1, Yintao Li2 1. Department of Pathology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, P.R. China 2. Department of Medical Oncology, Shandong Cancer Hospital and Institute, Jinan, Shandong, P.R. China Corresponding author: Yejun Qin, Department of Pathology, Shandong Provincial Hospital Affiliated to Shandong University, No. 324 Jingwu Road, Jinan, Shandong, 250021, China. Tel: +86-531-68776430. E-mail: [email protected] and Yintao Li, Department of Medical Oncology, Shandong Cancer Hospital and Institute, Shandong University, No. 440 Jiefang Road, Jinan, Shandong, 250117, China. Tel: +86-531-87984777. E-mail: [email protected] © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2017.10.31; Accepted: 2018.01.29; Published: 2018.02.28 Abstract Sulfotransferase family 1E member 1 (SULT1E1) is known to catalyze sulfoconjugation and play a crucial role in the deactivation of estrogen homeostasis, which is involved in tumorigenesis and the progression of breast and endometrial cancers. Our previous study has shown that the protein levels of SULT1E1 were decreased in breast cancer; however, the underlying mechanism is still poorly understood. In this study, we explored the functional and molecular mechanisms by which SULT1E1 influenced breast cancer. Here, we identified that overexpression of SULT1E1 inhibited breast cancer cell growth through inducing apoptosis and arresting cell cycle progression. Furthermore, enforced expression of SULT1E1 suppressed tumor cell migration and invasion. Moreover, we found that the activation of PPARγ was required for SULT1E1-mediated downregulation of C-myc, Cyclin D1, MMP-2 and MMP-9 as well as for cell apoptosis, migration and invasion. In addition, the overexpression of SULT1E1 significantly inhibited tumor growth in vivo. Taken together, our findings indicated that SULT1E1 performed its tumor suppressor characteristics by activating PPARγ, which provided a novel target for patients with breast cancer. Key words: breast cancer, SULT1E1, PPARγ, proliferation, invasion Introduction Breast cancer is the most universal of malignant that catalyzes the sulfation of estrogen and estradiol, tumors in females and accounts for 23% of all leading to their inactivation by inhibiting their ability diagnosed cancers[1]. Although current anti-tumor to bind to the estrogen receptor [8-10]. Previous strategies, including surgery, radiotherapy, hormonal studies have shown that SULT1E1 negatively therapy, or a combination thereof, have greatly regulated adipogenesis by inactivating ERK1/2 improved in recent years, recurrence and distant MAPK and attenuating insulin signaling in mice [11]. metastasis are the leading causes of mortality in breast Furthermore, SULT1E1 promoted human cancer patients[1-3]. Although significant adipogenesis by deactivating estrogen in humans [12]. improvement in the molecular mechanisms of breast SULT1E1 was found to be correlated with breast and cancer development and progression have been endometrial cancer, and the inhibition of SULT1E1 made[4-7], the mechanisms underlying breast cancer might lead to increased estrogen levels that promote remain largely unknown. Therefore, gaining a better carcinogenesis [13, 14]. High SULT1E1 levels have understanding of the molecular mechanisms of breast been found in the tumor tissues of patients with breast cancer and identifying a novel therapeutic target can cancer and have been associated with a poor lead to better treatments. prognosis for breast cancer in women [15-17]. Our Sulfotransferase family 1E member 1 (SULT1E1), previous study also found that SULT1E1 levels were known as estrogen sulfotransferase, is a key enzyme higher in breast cancer tissues than in normal breast http://www.jcancer.org Journal of Cancer 2018, Vol. 9 1079 tissues[18]. However, the overexpression of SULT1E1 Cell viability and colony formation assay inhibits proliferation in vitro and tumorigenesis in Cell proliferation ability was measured using the vivo[18]. SULT1E1 also significantly suppresses cell Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, proliferation and induces cell apoptosis. However, the Japan). Cells were seeded in 96-well plates at a density potential roles and molecular mechanism of SULT1E1 3 × 104 cells/mL in a volume of 100 µL/well. Ten remain poorly understood. microliters of CCK-8 was added per well at 24, 48 and In this study, we analyzed the effects of 72 h and incubated for 1 h. The absorbance of each SULT1E1 on proliferation, angiogenesis, invasion and sample was measured at a wavelength of 450 nm by metastasis of breast cancer. In addition, the using a SoftMaxPro5 Microplate Reader (Molecular mechanisms of SULT1E1 regulation in breast cancer Devices, California, USA). cell apoptosis and invasion were investigated. Our To complete the colony formation assay, 1x103 results indicate that SULT1E1 inhibits cell invasion by cells per well were plated in 6-well plates and upregulating the expression of PPARγ, supporting the cultured for an additional 14 days in complete growth notion that SULT1E1 may offer new possibilities for media. The medium was removed, and colonies (a the treatment of breast cancer. colony was defined as >50 cells) were stained with Materials and Methods 0.5% crystal violet for 15 min, photographed and counted. Cell lines and cultures Flow cytometric analysis The human breast cancer cell lines MCF-7 and For the cell apoptosis assay, cell apoptosis was T47D were purchased from the Cell Bank of the tested using FITC Apoptosis Detection Kit I (BD Chinese Academy of Sciences (Shanghai, China). All Pharmingen). Cells were harvested and resuspended cells were maintained in RPMI 1640 (Invitrogen, in 1× binding buffer. Then, cells were incubated with Carlsbad, CA, USA) supplemented with 10% fetal calf 5 μl FITC Annexin V and 5 μl propidium iodide for 15 serum (Invitrogen) and 1% penicillin/streptomycin min in a dark place at room temperature. Cell (Thermo Fisher Scientific, Waltham, MA). Cells were apoptosis was measured by flow cytometry cultured in a humidified incubator at 37°C with 5% (FACScan®; BD Biosciences, San Jose, CA), and data CO2. were analyzed using FlowJo software. Lentivirus vectors construction and For cell cycle analysis, cells were synchronized transfection by serum starvation for 24 h. Then, the cells were harvested and fixed in 70% ice cold ethanol. RNase A To upregulate SULT1E1 expression, the was added and incubated at 37°C for 15 min. Finally, PCR-amplified human SULT1E1 coding sequence was cells were stained with propidium iodide (50 μg/ml) inserted into the EcoR I/Xho I site of the lentivirus for 30 min at 4°C. Cellular DNA content was analyzed expression vector pLV-CMV-hSULT1E1-EGFP-Puro. using FlowJo software. Lentiviruses were produced by HEK293T cells with vector or SULT1E1 using the Lipofectamine® 2000 Migration and invasion assay Transfection Reagent (Thermo Fisher Scientific, Cell migration and invasion assay was Waltham, MA) according to the manufacturer’s performed using 8-μm pore size membranes (Corning instructions. Lentiviral particles were harvested 48 h Costar Corp, Cambridge, MA, USA). Cells (8 × 105 in after transfection and filtered using a 0.45-mm filter 600 µl serum-free media) were placed into the upper (Sartius, Gottingen, Germany). For infection, cells well of the Transwell chamber for migration assays were transduced with 1 × 109 TU lentivirus and then (without Martrigel, BD Biosciences, Franklin Lakes, cultured in medium with a concentration of 2 mg/ml NJ, USA) and invasion assays (with Martrigel). Next, puromycin (Sigma, USA) for 72 h prior to selection. 800 μl of media containing 10% FBS was added to the The small interfering RNA (siRNA) targeting lower chamber as a chemoattractant. After a 36-h PPARγ was obtained from GenePharma (Shanghai, incubation, the cells that migrated or invaded to the China). SiRNA sequences targeting human PPARγ are lower compartment were fixed with methanol and 5ʹ-GATAAGCTTCAATCTGATT-3ʹ. MCF-7 and T47D stained with 0.5% crystal violet. Cells on the cells were transfected with 50 nM negative control undersides of the filters were imaged with an inverted small interfering RNA (NC) or PPARγ siRNA using IX51 microscope (OLYMPUS, Japan) and counted Lipofectamine® 2000 Transfection Reagent. using ImageJ software. Forty-eight hours after transfection, cells were harvested for Western blot analysis. Tube formation assay Stable SULT1E1 overexpressed breast cancer http://www.jcancer.org Journal of Cancer 2018, Vol. 9 1080 cells were cultured with serum-free media. Twelve upregulated in both breast cancer cells (Figure 1A). In hours later, the supernatant was collected and stored vitro experiments showed that overexpression of at -80°C until use. For the tube formation assay, 6×104 SULT1E1 significantly decreased cell proliferation HUVECs per well were seeded in a 24-well plate (Figure 1B) and impaired colony formation ability coated with Matrigel containing medium with the (Figure 1C) in MCF-7 and T47D cells.

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