Targeting the NRF-2/RHOA/ROCK signaling pathway ...

Cancer Letters 424 (2018) 97e108

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Original Articles

Targeting the NRF-2/RHOA/ROCK signaling pathway with a novel aziridonin, YD0514, to suppress breast cancer progression and lung metastasis Dengfeng Li a, b, 1, Hong Wang a, c, 1, Ye Ding d, 1, Ziwei Zhang a, e, Zhi Zheng a, f, Jiabin Dong a, g, Hyejin Kim a, Xiaojing Meng h, Qianjun Zhou i, Jia Zhou d, ***, Lin Fang b, **, Qiang Shen a, * a

Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 1013, Houston, TX, 77030, United States b Department of Thyroid and Breast, Division of General Surgery, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, 200072, PR China c Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, 200032, PR China d Chemical Biology Program, Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX, 77555, United States e Department of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, PR China f Department of Internal Medicine 5th Division, Jiangxi Provincial Key Laboratory of Translational Medicine and Oncology, Jiangxi Cancer Hospital, Jiangxi Cancer Center, Nanchang, 330029, PR China g Department of Maxillofacial and Otorhinolaryngological Oncology, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Cancer Prevention and Therapy, Tianjin Cancer Institute, National Clinical Research Center of Cancer, Tianjin, 300060, PR China h Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515, PR China i Shanghai Lung Cancer Center, Shanghai Chest Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200030, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 January 2018 Received in revised form 6 March 2018 Accepted 21 March 2018

Metastasis is a major cause of breast cancer-associated mortality. Natural products extracted from herbs provide rich bioactive compounds with anticancer efficacy but may have limited or moderate potency and considerable toxicity. We developed a novel aziridonin, YD0514, by aziridinating oridonin, a natural product of the medicinal herb Rabdosia rubescens. In this study, we found that YD0514 significantly inhibited proliferation, motility, and adhesion of metastatic breast cancer cell lines MDA-MB-231, GI101, GILM2, and GILM3. YD0514 also decreased the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9), focal adhesion kinase (FAK), and integrin family members. Importantly, YD0514 suppressed the growth of metastatic breast cancer xenograft tumors and significantly inhibited lung metastasis in vivo. Lastly, we showed that YD0514's anti-metastatic effect on highly aggressive breast cancer is mediated via regulating the NRF-2/RHOA/ROCK signaling pathway. These results demonstrate that YD0514, the first active analog based on an oridonin D-ring modification, has the potential to be developed as an anti-metastasis therapy for patients with metastatic cancers. © 2018 Elsevier B.V. All rights reserved.

Keywords: YD0514 Aziridonin Breast cancer Metastasis NRF-2/RHOA/ROCK pathway Oridonin

Abbreviations: BSA, bovine serum albumin; CFL, cofilin; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; FAK, focal adhesion kinase; FBS, fetal bovine serum; KEAP1, Kelch-like ECH-associated protein; LIMK, LIM domain kinase; MMP, matrix metalloproteinase; MTT, 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; NRF-2, nuclear factor erythoid 2-related factor 2; Rho, Ras homology; RHOA, Ras homolog family member A; ROCK, Rho-associated protein kinase; TNBC, triple-negative breast cancer. * Corresponding author. Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population Science, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 1013, Houston, TX, 77030, United States. ** Corresponding author. Department of Thyroid and Breast, Division of General Surgery, Shanghai Tenth People's Hospital, Tongji University School of Medicine, No. 301 Yanchang Middle Road, Shanghai, 200072, PR China. *** Corresponding author. Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX, 77555, United States. E-mail addresses: [email protected] (J. Zhou), [email protected] (L. Fang), [email protected] (Q. Shen). 1 Equal contribution. https://doi.org/10.1016/j.canlet.2018.03.029 0304-3835/© 2018 Elsevier B.V. All rights reserved.

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D. Li et al. / Cancer Letters 424 (2018) 97e108

1. Introduction Approximately 1.7 million women worldwide and about 250,000 women in the United States are diagnosed each year with invasive breast cancer, with a yearly mortality of about 40,000 in the United States alone [1]. From 20% to 50% of breast cancer patients will develop metastatic disease [2], which is defined as aggressive cancer spreading to other organs of the body, such as the lung, bone, liver, and brain, with the lungs the most common site [3]. Although the 5-year survival rate for breast cancer patients without metastasis has reached 99%, patients with metastatic disease have a sharply lower 5-year survival rate of only 25% [4]. Therefore, new, more effective therapies for metastatic breast cancer are an urgent but unmet need. Medicinal herbs have long been used in Asia for their therapeutic efficacy and safety in the treatment of many diseases, including cancers, and have now become an important source of new bioactive compounds [5]. Not only are herbs used for treating invasive and metastatic breast cancer, but they also have potential value as cancer-preventing agents [6]. Oridonin, a natural product isolated from the medicinal herb Rabdosia rubescens, has been widely investigated for the treatment of many human cancers because it has unique anticancer activity [7,8]. Multiple studies demonstrated that oridonin is effective in inhibiting tumor growth, suppressing metastasis, and inducing apoptosis and autophagy in breast cancer [9e12]. Although oridonin shows promising safety and efficacy as a cancer therapy, several disadvantages, such as its moderate potency, poor aqueous solubility, and unfavorable bioavailability, limit its further preclinical development and clinical applications [13]. Therefore, researchers have sought to modify oridonin's structure to overcome these disadvantages and develop more potent oridonin-derivative compounds. A recent study reported that 2 oridonin-derived compounds with modifications of the A-ring of oridonin inhibited growth of MCF-7 breast cancer cells in vitro and tumor growth in vivo even more effectively than oridonin itself [14]. Our previous work also demonstrated that modifications of the Aring of oridonin could generate potent derivative compounds that significantly inhibit the growth of wild-type and chemoresistant breast cancer cells and xenograft tumors [15e19]. However, no research into whether modifying the D-ring of oridonin can yield more potent and safer anticancer agents has been reported. In the present study, we aimed to develop a candidate targeted therapy for metastatic breast cancer by modifying the D-ring of oridonin. Our newly developed aziridonin, YD0514, which was generated by oridonin D-ring aziridination, significantly inhibited the motility of multiple metastatic breast cancer cell lines but had less effect on normal breast epithelial cells. Interestingly, we found that YD0514's anticancer effect on metastatic breast cancer cells is primarily mediated by regulating multiple key molecules in the nuclear factor erythoid 2-related factor 2 (NRF-2)/Ras homolog family member A (RHOA)/Rho-associated protein kinase (ROCK) signaling pathway. Our results suggest that YD0514, the first active analog based on an oridonin D-ring modification, has the potential to be developed as an anti-metastasis therapy for treating metastatic breast cancer. 2. Materials and methods 2.1. Cell lines and reagents The triple-negative, highly aggressive metastatic breast cancer cell line MDA-MB-231 was purchased from ATCC (Manassas, VA). GI101, GILM2, and GILM3 breast cancer cell lines were provided by Dr. Janet Price at The University of Texas MD Anderson Cancer

Center; GILM2 and GILM3 cells are derived from GI101 cells and have significantly higher metastatic potential than their parental GI101 cell line, which itself is a highly metastatic breast cancer cell line [20,21]. MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning, Corning, NY) with 10% fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany) and 1% penicillin-streptomycin (Sigma-Aldrich). GI101, GILM2, and GILM3 cells were cultured in DMEM/F12 medium (50/50 mix; Corning) with 10% FBS and 1% penicillin-streptomycin. All cell lines were cultured in an incubator at 37 C in 5% CO2. The oridoninderived compound YD0514 was designed and synthesized in the laboratory of Dr. Jia Zhou at The University of Texas Medical Branch. Oridonin and sulforaphane were purchased from Sigma-Aldrich. All reagents were dissolved in dimethyl sulfoxide (DMSO; Corning). 2.2. Proliferation assay For the 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) proliferation assay, MDA-MB-231 (600/well), GI101 (2000/well), GILM2 (2000/well), or GILM3 (2000/well) cells were seeded in a 96-well plate and treated with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM). The cell proliferation rate was determined by measuring the absorbance with a spectrometer according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA) for 5 days. Proliferation was measured after incubating cells with MTT for 4 h. A microplate spectrophotometer (BioTek, Winooski, VT) was used to measure the absorbance values of each sample at 560 nm. Each individual compound was tested in quadruplicate wells for each concentration. 2.3. Colony-formation assay We prepared single-cell suspensions of each breast cancer cell line and plated cells in a 6-well plate at a density of 600 cells/well for MDA-MB-231 cells and 2000 cells/well for GI101, GILM2, and GILM3 cells. After 24 h, the cells were treated with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, and 20 mM, single treatment). The culture medium was changed every 3 days. After 10 days for MDA-MB-231 cells and 21 days for GI101, GILM2, and GILM3 cells, the surfaces of the 6-well plate were washed twice with phosphatebuffered saline (PBS) and then treated with 4% polyformaldehyde for 10 min, stained with 0.1% crystal violet for 10 min, and washed with double-distilled water 3 times. Photos of the culture surfaces on dried plates were taken for analysis. 2.4. Cellular motility assays We performed Transwell assays to evaluate cellular migration (without Matrigel) and invasion (with Matrigel). For MDA-MB231 cells, after 24 h of treatment with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM), 5 104 cells/well were seeded in the upper chambers of a 24-well Transwell plate (Corning) with 200 mL of DMEM supplemented with 0.1% bovine serum albumin (BSA). The lower chambers were filled with 600 mL of DMEM with 10% FBS. The cells were cultured for 8 h to assess migration and 16 h to assess invasion. GI101, GILM2, and GILM3 cells were treated with DMSO or YD0514 as were MDA-MB-231 cells. After treatment, 10 104 cells/ well were seeded in the upper chambers with 200 mL of DMEM/F12 supplemented with 0.1% BSA. The lower chambers were filled with 600 mL of DMEM/F12 with 10% FBS. The cells were cultured for 8 h for migration and 48 h for invasion assessments. After culturing, the outer surface of each insert was washed 3 times with PBS, and then the cells were fixed and stained for 10 min as described for the colony-formation assay. When the surfaces had dried, photographs of the bottoms of the inserts were captured for analysis. The images


were divided into 9 equal areas, and cells were counted in the central and four corner areas. The numbers of cells counted in each area were summed and averaged as shown in figures.

2.5. Cell adhesion assays Human lung microvascular endothelial cells (HMVEC-L) (Lonza, Walkersville, MD) were cultured in endothelial basal medium-2 supplemented with EGM-2MV SingleQuot Kit Supplement and Growth Factors (Lonza). The HMVEC-L cells were seeded in a 48well plate and allowed to form monolayers and reach a density of 100%. The 4 breast cancer cell lines were treated with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM). A CytoSelect TumorEndothelium Adhesion Assay Kit (Cell Biolabs, Inc., San Diego, CA) was used to perform cell adhesion assays according to the manufacturer's instructions. Photographs were taken under an inverted fluorescence microscope, and adherent cancer cells were counted. After counting, we aspirated the final wash and added 150 mL of 1 lysis buffer to each well containing cells, incubated the mixture for 5 min at room temperature with shaking, and transferred 100 mL of the mixture to a 96-well plate suitable for fluorescence measurement. Fluorescence was read with a fluorescence plate reader at 480 nm/520 nm. The assay was performed 3 times independently.

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2.6. Western blot assay The 4 breast cancer cell lines were treated with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM). After 48 h of treatment, cells were harvested and lysed. Total protein samples were extracted using radioimmunoprecipitation assay (RIPA) buffer. Protein concentrations of the samples were measured with a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Equal amounts of total cellular protein extract (40 mg) were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blotting membranes. After blocking with 5% nonfat milk, the membrane was incubated with the desired primary antibody overnight. The membrane was then incubated with the appropriate secondary antibody. After 3 washes with PBS with Tween 20, immunoreactive protein bands were detected using an Odyssey scanning system (LI-COR Biosciences, Lincoln, NE). The antibodies and concentrations used were: MMP9 (1:500; Calbiochem, Burlington, MA; Cat# IM37L), MMP2 (1:1000; Abcam, Cambridge, UK; Cat# ab37150), p-FAK (1:1000; Invitrogen, Carlsbad, CA; Cat# 700255), FAK (1:1000; Invitrogen; Cat# 39e6500), integrin family (1:1000; Cell Signaling Technology [CST], Danvers, MA; Cat# 4749), NRF-2 (1:1000; CST; Cat# 12721), RHOA (1:1000; CST; Cat# 2117), ROCK1 (1:1000; CST; Cat# 4035), ROCK2 (1:1000;

Fig. 1. YD0514 suppresses proliferation of metastatic breast cancer cells. A, Molecular structures of oridonin and aziridonin (YD0514). The D-ring modification of oridonin is highlighted in blue. B, YD0514 suppressed proliferation of MDA-MB-231, GI101, GILM2, and GILM3 metastatic breast cancer cells, assessed by MTT assays. Two-way ANOVA was used; P < 0.0001. C & D, YD0514 suppressed colony formation of metastatic breast cancer cells. One-way ANOVA was used; P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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CST; Cat# 9029), LIMK1(1:1000; CST; Cat# 3842), LIMK2 (1:1000; CST; Cat# 3845), p-CFL1 (1:1000; CST; Cat# 3313 P), CFL1 (1:1000; CST; Cat# 5175 P), b-actin (1:2000; Santa Cruz Biotechnology, Dallas, TX; Cat# 47778), and anti-rabbit or anti-mouse secondary antibody (1:2500; Thermo Fisher Scientific; Cat# 35521 [mouse], Cat# 35571 [rabbit]). 2.7. In vivo antitumor activity determination All procedures, including mouse and in vivo experiments, were approved by the Institutional Animal Care and Use Committee of MD Anderson Cancer Center. Twenty female nude mice (NCI Athymic NCr-nu/nu, strain code 553) obtained from Jackson Laboratory (Bar Harbor, ME) were used for orthotopic tumor studies at 4e6 weeks of age. For xenograft experiments, the mice were maintained in a barrier unit on a 12-h light-dark cycle. Freshly harvested MDA-MB-231 cells (1.0 106 cells per mouse, resuspended in 100 mL PBS) were injected into the third mammary fat pad of the mice, which were then randomly assigned into control or treatment groups. The mice were treated daily with 10 mg/kg of YD0514 or vehicle (DMSO) through intraperitoneal injection until the tumor volume was 80 mm3 or higher. Body weight and tumor volume were measured daily; tumor volume was calculated as we previously described [18,19]. For lung metastasis experiments, freshly harvested MDA-MB-231 cells (1.0 106 cells per mouse,

resuspended in 100 mL PBS) were injected into the tail vein. The mice were treated with 5 mg/kg YD0514 through intraperitoneal injection on the same day. The mice then received intraperitoneal injections of YD0514 or vehicle (DMSO) every other day for 6 weeks. The mice were then killed, and the lungs were dissected and fixed with 4% polyformaldehyde for hematoxylin-and-eosin staining analysis. A microscopic metastatic focus was defined as having 40 or more cells in a 10-mm-thick section; lung metastatic foci were quantified using a previously described method [20,21]. 2.8. Statistical analysis Data were presented as the mean ± standard deviation or as the mean ± standard error. A two-way analysis of variance test or Student t-test were used for comparisons between groups. Differences were considered significant for p values of less than 0.05. GraphPad Prism software version 6.0 (GraphPad, San Diego, CA) was used to perform all statistical analyses. 3. Results 3.1. YD0514 suppresses the proliferation of metastatic breast cancer cells We performed MTT assays to evaluate the antiproliferation

Fig. 2. YD0514 suppresses migration and invasion of metastatic breast cancer cells. A & B, YD0514 suppressed migration of metastatic breast cancer cells at the indicated doses. C & D, YD0514 suppressed invasion of metastatic breast cancer cells at the indicated doses. One-way ANOVA and Student t-test were used to analyze the differences. ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.


effect of YD0514 in a panel of highly metastatic breast cancer cells including the triple-negative MDA-MB-231 and estrogen receptor (ER)-positive GI101 and its derivative GILM2 and GILM3 clones. As shown in Fig. 1A, YD0514 was designed and synthesized by oridonin D-ring aziridination (highlighted in blue); the detailed medicinal chemistry was described in our recent publication [22]. We treated the metastatic breast cancer cells with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM). YD0514 showed a significant antiproliferative effect in all metastatic breast cancer cells in a dosedependent manner (Fig. 1B). Colony-formation assays confirmed that YD0514 significantly inhibited proliferation of the 4 tested highly metastatic breast cancer cell lines (Fig. 1C and 1D). These results demonstrate that YD0514 inhibits the proliferation of metastatic breast cancer cells. 3.2. YD0514 suppresses the motility of metastatic breast cancer cells We next performed Transwell assays to explore whether oridonin-derived YD0514 inhibits the migration and invasion of metastatic breast cancer cells. We treated MDA-MB-231, GI101, GILM2, and GILM3 cells with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM). After 24 h of treatment, YD0514 significantly decreased the migration of these metastatic breast cancer cells in a

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dose-dependent manner (Fig. 2A & 2B). Consistently, the Transwell assays also showed that YD0514 significantly inhibited invasion of these metastatic breast cancer cells in a dose-dependent manner (Fig. 2C and 2D). In particular, YD0514 robustly inhibited invasion of the highly aggressive breast cancer cell lines GI101, GILM2, and GILM3 at high doses. These results suggest that YD0514 significantly inhibits the motility of metastatic breast cancer cells.

3.3. YD0514 suppresses the adhesion of metastatic breast cancer cells to lung-specific endothelial cells To further explore the effects of YD0514 on metastatic breast cancer cells, we performed adhesion assays. After treatment with DMSO or YD0514 (20 mM), cells were labeled with CytoTracker fluorescence marker and seeded on top of a monolayer of HMVEC-L cells, a lung-specific endothelial cell line. YD0514 treatment significantly inhibited the adhesion of the 4 tested metastatic breast cancer cell lines (Fig. 3). Most importantly, we found that YD0514 most strongly inhibited adhesion of GILM2 and GILM3 cells, which have high metastatic ability. These results demonstrate that YD0514 significantly inhibits the adhesion of metastatic breast cancer cells to the lung-specific endothelial cells.

Fig. 3. YD0514 suppresses adhesion of metastatic breast cancer cells to lung-specific endothelial cells. A, YD0514 suppressed the adhesion of MDA-MB-231 cells. B, YD0514 suppressed the adhesion of GI101 metastatic breast cancer cells. C, YD0514 suppressed the adhesion of GILM2 metastatic breast cancer cells. D, YD0514 suppressed the adhesion of GILM3 metastatic breast cancer cells. Quantitative data was obtained by naked-eye counting and fluorescence detection. Student t-test was used to analyze the differences. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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3.4. YD0514 suppressed the growth of metastatic triple-negative breast cancer xenograft tumors and the development of lung metastasis in vivo Because YD0514 showed significant inhibition of the proliferation, motility, and lung-specific endothelial adhesion of metastatic breast cancer cells in vitro, we further evaluated its activity in suppressing tumor growth and lung metastasis in nude mice with highly metastatic triple-negative MDA-MB-231 tumors. We injected MDA-MB-231 cells into the mammary fat pads of mice to develop xenograft tumors. As shown in Fig. 4A & 4B, xenografted mice that were intraperitoneally injected with 10 mg/kg of YD0514 showed significantly less tumor growth (Student t-test p < 0.05). We then established an experimental lung metastasis model by injecting highly aggressive and metastatic triple-negative breast cancer (TNBC) MDA-MB-231 cells into the tail vein of nude mice to develop metastatic lung lesions. The lungs of the mice were examined 6 weeks after cell injection, and there were no distinguishable macrometastatic foci on the lung surfaces in either vehicle- or YD0514-treated mice. We then examined microscopic metastatic foci in thick lung sections. As shown in Fig. 4C and 4D, hematoxylin and eosin staining of lung tissue revealed that the number of microscopic metastatic foci was significantly lower in the lungs of mice treated with 5 mg/kg YD0514 than in vehicletreated mice. These results demonstrate that YD0514 significantly reduces the development of metastatic lesions in the lungs, suggesting that YD0514 is a promising agent for treating metastatic breast cancer. 3.5. YD0514 suppresses the expression of metastasis-related proteins in metastatic breast cancer cells It was reported that oridonin inhibits the migration and invasion of MDA-MB-231 breast cancer cell via suppression of matrix metalloproteinases (MMPs) and the integrin b1/focal adhesion kinase (FAK) pathway [11]. We speculated that YD0514 may have

retained this property from oridonin. We therefore performed Western blot assays to demonstrate whether YD0514 inhibits MMPs and the FAK/integrin pathway. We treated MDA-MB-231, GI101, and GILM3 cells with DMSO or YD0514 (1 mM, 5 mM, 10 mM, 15 mM, or 20 mM). As shown in Fig. 5, we found that YD0514 decreased the expression of MMP2 and MMP9, particularly at a dose of 20 mM. Meanwhile, YD0514 also significantly inhibited expression of FAK/p-FAK and integrin family proteins, in addition to integrin b1. At a concentration of 20 mM, expression of several proteins was decreased. In fact, in immortalized mammary epithelial cell lines such as MCF-10 A, YD0514 was less toxic than oridonin at concentrations of up to 100 mM (Supplementary Fig. 1). Our results suggest that YD0514 suppresses the expression of MMPs and FAK/integrin family members in metastatic breast cancer cells, consistent with the mechanism by which oridonin acts on MDA-MB-231 cells while sparing normal epithelial cells. 3.6. YD0514 suppresses the NRF-2/RHOA/ROCK signaling pathway in metastatic breast cancer cells To identify the pathway targeted by YD0514, we analyzed our reverse phase protein array data obtained from the cells treating with oridonin and YD0514, and found that NRF-2 may be a target of YD0514. It was reported that NRF-2 can regulate the RHOA/ROCK signaling pathway to promote proliferation and metastasis in breast cancer [23], and we therefore speculated that YD0514 could target NRF-2 to regulate the RHOA/ROCK pathway and suppress the proliferation and motility of metastatic breast cancer cells. To test this hypothesis, we searched the GeneCards database of human genes (http://www.genecards.org) and found that NRF-2 can bind to the promoter of RHOA (Fig. 6A), suggesting that NRF-2 may induce the expression of RHOA. Sulforaphane has been reported to be an activator of NRF-2, releasing NRF-2 from the Kelch-like ECHassociated protein 1 (KEAP1)/NRF-2 complex [24,25]. As shown in Fig. 6B, we found that YD0514 inhibited the expression of NRF-2 in metastatic breast cancer cells, especially after activation of

Fig. 4. YD0514 suppresses the growth of triple-negative breast cancer xenograft tumors and the development of lung metastases in vivo. A & B, YD0514 suppressed the growth of triple-negative breast cancer xenograft tumors in vivo. Student t-test was used to analyze differences; *, P < 0.05. C & D, YD0514 suppressed the development of microscopic metastatic foci in the lungs of mice. Student t-test was used to analyze differences; ***, P < 0.001.


sulforaphane. Furthermore, we found more profound inhibition of NRF-2 in GI101 and GILM3 cells than in MDA-MB-231 cells, consistent with the metastatic potential of GI101 and its derivative cells. We also found that treatment with YD0514 inhibited the RHOA/ROCK signaling pathway (Fig. 6C). These results demonstrate that YD0514 suppresses the growth and progression of metastatic breast cancer at least partially via the NRF-2/RHOA/ROCK signaling pathway. 4. Discussion In the present study, we examined YD0514, the first compound derived from aziridinating the D-ring of oridonin, testing its in vitro and in vivo efficacy in blocking the growth and progression of metastatic breast cancer. We demonstrated that YD0514 significantly inhibits the growth and motility of highly metastatic breast cancer cells with ER-negative and ER-positive phenotypes. We also showed that YD0514 moderately suppresses the growth of metastatic breast cancer xenograft tumors and robustly inhibits the development of an experimental lung metastasis model derived from the metastatic TNBC cell line MDA-MB-231. We further demonstrated that the anti-metastasis efficacy of YD0514 is mediated by its modulation of the NRF-2/RHOA/ROCK signaling pathway both in vitro and in vivo. Our results provide a rationale for developing YD0514 as a new anti-metastasis therapy. Most breast cancer patients are diagnosed with stages 0 to III disease, without metastasis, but nearly 5% of breast cancer patients initially present with stage IV disease, meaning metastatic tumors have already spread to other organs at the time of diagnosis [26]. Many clinical trials have evaluated chemotherapy, radiotherapy, endocrine therapy, and combination therapies for metastatic breast cancer [27]. However, accumulating evidence has shown that after 3e5 years of post-adjuvant treatment, managing metastatic breast cancer becomes a major challenge [28]; approximately 90% of breast cancer deaths are a consequence of metastasis [29]. Surgery for metastatic breast cancer is difficult and carries a high risk of postoperative complications and 30-day mortality [26]. Therefore, developing new effective therapies for patients with metastatic breast cancer is an urgent task. Studies have reported that oridonin exerts its profound antitumor effects through several different mechanisms. Oridonin induced G2/M cell cycle arrest and apoptosis in human oral squamous cell carcinoma [30] and reversed drug resistance in cisplatinresistant human gastric cancer cells by decreasing expression of Pglycoprotein and multidrug resistance-associated protein [31]. Oridonin induced apoptosis and increased intracellular reactive oxygen species levels in the TNBC cell line MDA-MB-231 [12,32]. In addition, oridonin induced autophagy to enhance apoptosis of breast cancer cells [10]. Although oridonin has moderate safety and efficacy profiles for cancer treatment, its disadvantages, including moderate potency and limited aqueous solubility and oral bioavailability, hinder its further preclinical development and clinical applications [13]. Therefore, we developed a number of oridonin derivatives with oridonin A-ring modifications. These derivatives were designed to have enhanced anticancer activity and improved drug properties [13]. CYD0628, an oridonin-derivative compound we developed, strongly suppressed the growth of TNBC cells and xenografts [17]. However, the oridonin D-ring modification has been less thoroughly investigated; to date, no D-ring-modified derivatives of oridonin have been reported to be pharmacologically active, likely because of the importance of the enone pharmacophore. Aziridination of the parental natural product (e.g., the Food and Drug Administration-approved chemotherapeutic agent mutamycin) may enrich the nitrogen atom in the chemical scaffold, improving

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the drug properties such as aqueous solubility, cell permeability, and bioavailability [33,34]. YD0514 (aziridonin) is the first D-ringmodified analog of oridonin produced via aziridination to significantly inhibit proliferation of MDA-MB-231, GI101, GILM2, and GILM3 metastatic breast cancer cells (Fig. 1) and moderately but significantly inhibit the growth of TNBC xenografts in vivo (Fig. 4A). More importantly, YD0514 significantly inhibited the motility of metastatic breast cancer cells (Fig. 2). Furthermore, after treatment with YD0514, the adhesive ability of metastatic breast cancer cells was significantly suppressed (Fig. 3). It was previously reported that oridonin suppressed lung metastasis from pancreatic cancer at a relatively high dose of 10 mg/kg and suppressed mouse 4T1 cancer cell metastasis at a dose of 7.5 mg/kg [35,36]. Few studies have evaluated whether oridonin suppresses human TNBC metastasis in vivo. Our experiments demonstrate that YD0514 suppresses the growth of TNBC xenograft tumors (Fig. 4A and 4B) and significantly suppresses lung metastasis in an experimental model derived from a highly metastatic human TNBC cell line at a relatively low dose (Fig. 4C and 4D). The lung is one of the most common sites of metastasis in patients with breast cancer [3], and lung metastases are associated with a poor prognosis [37,38]. Although breast cancer can spread to distinct sites such as the lung, brain, bone, or other organs [3], metastasis to these sites may be regulated by a number of commonly shared signaling pathways, such as the MAPK pathway [39e41], AKT pathway [42e44], and epithelial-mesenchymal transition (EMT) [45e47]. In fact, organ-tropic MDA-MB-231 sublines that spread to the lung, brain, or bone have been developed [48e50]. These cells show activation of common signaling pathways including transforming growth factor b [49] and EMT [51]. Our study showed that YD0514 dramatically reduces the development of lung lesions from metastatic TNBC via inhibiting EMT and related molecules and signaling pathways. We speculate that YD0514 and other compounds in this series may also be useful to block the metastatic progression of breast cancer to other organ sites. We are currently exploring the in vitro and in vivo efficacy of YD0514 and these compounds in other metastasis models. Taken together, when combined with the aforementioned studies, our findings strongly suggest that YD0514 is a novel promising antimetastasis drug candidate for patients with metastatic cancer. Oridonin was reported to inhibit migration and invasion of highly metastatic breast cancer cells through suppressing protein expression of MMPs, integrin b1, and FAK [11]. MMP overexpression, especially of MMP-9 and MMP-2 in the serum, may indicate a worse prognosis for patients with breast cancer [52]. MMP2 is reported to be one of the signature genes that are only expressed in the most aggressive, lung-specific breast cancer metastases [53]. MMP-9 belongs to a “70-gene classifier” that is used to predict distant metastasis in lymph node-negative breast cancer cases [54]. Therefore, MMP-2 and MMP-9 are valuable clinical biomarkers for diagnosing and predicting the prognosis of metastatic breast cancer [55]. In our study, when metastatic breast cancer cells were treated with YD0514, the protein expression of MMP-2 and MMP-9 was decreased (Fig. 5A), demonstrating the anti-metastasis potential of YD0514. FAK is a key regulator of the focal adhesion complex, which is involved in several intracellular processes, including cell growth, motility, and survival, via regulating extracellular matrix and membrane signals [56]. When activated, FAK mediates the dissolution of intercellular junctions, upregulates mesenchymal markers such as MMP-2 and MMP-9, and downregulates membrane-bound E-cadherin, which is associated with EMT and metastasis in cancer [57]. Integrins are a family of cell adhesion receptors; they interact with extracellular matrix proteins and are primarily involved in cell adhesion [58]. Integrin-mediated signals are essential for breast

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Fig. 6. YD0514 suppresses the NRF-2/RHOA/ROCK signaling pathway in metastatic breast cancer cells. A, Binding sites of NRF-2 and RHOA in the promoter region of NRF-2. B, YD0514 suppressed constitutive and induced expression of NRF-2 in metastatic breast cancer cells. C, YD0514 suppressed the expression of key molecules in the RHOA/ROCK signaling pathway in metastatic breast cancer cells. D & E, Relative quantification of MMPs and FAK/integrin family proteins in MDA-MB-231, GI101, and GILM2 metastatic breast cancer cells. One-way ANOVA and Student t tests were used to analyze the differences.

cancer invasion, metastasis, and radioresistance [59]. Indeed, FAK is the first activated downstream component when integrins interact with the extracellular matrix [60]. We found that YD0514 not only suppresses the expression and phosphorylation of FAK (particularly in highly metastatic GILM3 cells) (Fig. 5B) but also suppresses the expression of integrin family proteins (Fig. 6B). Our results strongly suggest that YD0514 possesses the essential properties to warrant its development as a potent anti-metastasis agent for treating metastatic cancers, including metastatic breast cancer. NRF-2 is a key transcription factor that regulates a wide array of genes coding for antioxidant and detoxification enzymes [61]. On one hand, NRF-2 acts as a tumor suppressor by modifying the tumor microenvironment; on the other hand, NRF-2 activity is enhanced in many solid cancers, and it contributes to proliferation, apoptosis evasion, and therapeutic resistance in cancer cells, including breast cancer cells [62]. When cancer cells are treated with NRF-2 inhibitors, apoptosis is induced and proliferation and migration are inhibited [63]. However, it was recently reported that NRF-2 promotes breast cancer cell proliferation and metastasis by upregulating the RHOA/ROCK signal transduction pathway [23]. Our unpublished reverse phase protein array data suggest that NRF-2 may be a target of YD0514. We examined the GeneCards database and found that NRF-2 can bind to the promoter region of RHOA (Fig. 6A), which is in accordance with a previous study [23]. Therefore, we hypothesized that YD0514 suppresses metastatic breast cancer via the NRF-2/RHOA/ROCK signaling pathway. NRF-2 is constantly synthesized but maintained at low levels; KEAP1 targets NRF-2 for ubiquitination and proteasome-mediated degradation [64]. With this in mind, we used sulforaphane to activate expression of NRF-2 so that it could be easily detected. After treatment with YD0514, the constitutive and induced expression of

NRF-2 decreased when compared with control cells (Fig. 6B). In addition, YD0514 decreased the expression of RHOA/ROCK and their downstream proteins, the LIM domain kinases (LIMKs) and cofilin (CLF1) (Fig. 6C). We further mined the publically available database Oncomine (http://www.oncomine.com) and examined the mRNA expression levels of the components of this pathway. The results are summarized in Supplementary Fig. 2. RHOA is a representative protein of the Ras homology (Rho) subfamily of GTPases, which are known as regulators of the actin cytoskeleton that participate in cell proliferation, differentiation, migration, and polarity. Recent research demonstrated that Rho family members are frequently and highly expressed and/or activated in cancers, playing a role in tumor progression [65]. ROCK, one of the downstream protein kinases of Rho proteins, is an appealing target for a number of small-molecule inhibitors for cancer therapy [66]. The ROCK pathway is involved in regulating cytoskeleton dynamics via actin filament stabilization and actin-myosin contractility. When RHOA/ROCK signaling is activated, cell-cell junctions decrease and cells' motility is promoted [67]. When RHOA-GTP expression is inhibited, for example by overexpressing diaphanous-related formin-3, the migration and invasion ability of TNBC cells is significantly decreased [68]. One study showed that in TNBC, co-inhibition of epidermal growth factor receptor and ROCK had a profound inhibitory impact on tumor growth and progression [69]. LIMK family members are key regulators of the actin cytoskeleton and are involved in cell motility and invasion [70]. In breast cancer cells, LIMK regulated tumor-cell invasion and matrix degradation [71], and LIMK1, a member of the LIMK family, enhanced MDA-MB-435 breast cancer cell invasion [72]. It is clear that LIMKs are downstream targets of ROCK [73] and that activation of LIMKs leads to phosphorylation of the actin-

Fig. 5. YD0514 suppresses protein expression of FAK, p-FAK, and the integrin family. A & B, YD0514 suppressed the expression of matrix metalloproteinases (MMPs) and FAK/ integrin family proteins in MDA-MB-231, GI101, and GILM3 metastatic breast cancer cells, assessed by Western blotting. C & D, Relative quantification of MMPs and FAK/integrin family proteins in MDA-MB-231, GI101, and GILM3 metastatic breast cancer cells. One-way ANOVA was used to analyze the differences.

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Fig. 7. The signaling pathway network by which YD0514 exerts anticancer efficacy as assessed with Ingenuity Pathway Analysis. A, YD0514 inhibits the integrin family signaling pathway via potential targets. B, YD0514 inhibits the NRF-2/RHOA/ROCK signaling pathway via potential targets. Pathway Builder Tool 2.0 software was used to draw the picture.

depolymerizing protein cofilin (CFL) [74]. CFL plays an important role in actin filament dynamics and reorganization via stimulating the depolymerization and severance of actin filaments [75]. It has been reported that Rho proteins modulate actin CFL via ROCK and LIMK signal transduction pathways in many cell types in the presence of extracellular stimuli [76]. One study showed that CD74 interacts with CD44 to promote tumorigenesis and metastasis via RHOA-mediated CFL phosphorylation in human breast cancer cells [77]. RHOA/ROCKs were also found to crosstalk with the integrin family via modulating the actin cytoskeleton through LIMKs/CFL1 [78]. RHOA/ROCKs play an important role in metastasis [79], and NRF-2 is an important regulator to suppress the RHOA/ROCK signaling pathway [80,81]. As shown in Supplementary Fig. 2, enhanced expression of NRF2, RHOA, ROCK1, ROCK2, LIMK1, LIMK2, and CFL1 was significantly associated with higher tumor grade, advanced disease stage, local lymph node metastasis, metastatic events, and remote metastasis in patients with breast cancer. Taken together, our results fill in the map of the RHOA/ROCK/LIMK/CLF1 pathway in metastatic breast cancer. These results further support our findings that YD0514 regulates the NRF-2/RHOA/ROCK/LIMK/CFL1 pathway to block breast cancer metastasis and that YD0514 has promise as an antimetastasis therapy for patients with progressive and metastatic disease.

In summary, our study demonstrated that the aziridonin YD0514, a novel aziridinated oridonin compound, suppresses metastatic breast cancer cell proliferation, motility and adhesion, xenograft tumor growth, and lung metastasis in an animal model. The inhibitory effect and efficacy of YD0514 appears to be the consequence of suppressed expression of MMPs, integrins, and FAK, at least partially via the NRF-2/RHOA/ROCK/LIMK/CFL pathway, and may be mediated by yet-to-be-defined direct target(s) of YD0514 (Fig. 7), a question presently under vigorous investigation. Additional research, which is currently ongoing, is still needed to explore the potential targets of YD0514. With its potential as an anticancer drug candidate against metastatic breast cancer, YD0514 deserves further preclinical and clinical development.

Disclosure of potential conflicts of interest The authors have no potential conflicts of interest to disclose.

Conflicts of interest The authors declare that they have no conflicts of interest.


Acknowledgements This work was supported by a China Scholarship Council fellowship (No. 201606260219 to D.L.), Startup funds from The University of Texas MD Anderson Cancer Center (to Q.S.), the Breast Cancer Research Program Breakthrough Award from the U.S. Department of Defense (BC160038 to J.Z., BC160038P1 to Q.S.), a Holden Family Research Grant in Breast Cancer Prevention from the Prevent Cancer Foundation (to Q.S.), the Duncan Family Institute Seed Funding Research Program (Fund 0051934 to Q.S.), and a Cancer Prevention and Research Institute of Texas award (DP150074 to J.Z.). We also thank Amy Ninetto, PhD, ELS, Department of Scientific Publications, The University of Texas MD Anderson Cancer Center for her editing of the manuscript.

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Appendix A. Supplementary data [25]

Supplementary data related to this article can be found at https://doi.org/10.1016/j.canlet.2018.03.029.

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