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Up-regulated ADP-Ribosylation factor 3 promotes breast cancer cell proliferation through the participation of FOXO1
Journal Pre-proof Up-regulated ADP-Ribosylation factor 3 promotes breast cancer cell proliferation through the participation of FOXO1 Danping Huang, Yuanyuan Pei, Changping Dai, Yun Huang, Han Chen, Xuhong Chen, Xiaolan Zhang, Chun Lin, Hongying Wang, Rui Zhang, Xinhong Wan, Lan Wang PII:
S0014-4827(19)30486-0
DOI:
https://doi.org/10.1016/j.yexcr.2019.111624
Reference:
YEXCR 111624
To appear in:
Experimental Cell Research
Received Date: 8 April 2019 Revised Date:
12 September 2019
Accepted Date: 15 September 2019
Please cite this article as: D. Huang, Y. Pei, C. Dai, Y. Huang, H. Chen, X. Chen, X. Zhang, C. Lin, H. Wang, R. Zhang, X. Wan, L. Wang, Up-regulated ADP-Ribosylation factor 3 promotes breast cancer cell proliferation through the participation of FOXO1, Experimental Cell Research (2019), doi: https:// doi.org/10.1016/j.yexcr.2019.111624. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Up-regulated ADP-Ribosylation Factor 3 Promotes Breast Cancer Cell Proliferation Through the Participation of FOXO1 Running title: ARF3 Promotes the Proliferation of Breast Cancer Danping Huang1*, Yuanyuan Pei 2*, Changping Dai1, Yun Huang3, Han Chen4, Xuhong Chen4, Xiaolan Zhang4, Chun Lin4, Hongying Wang1, Rui Zhang1, Xinhong Wan2, Lan Wang3# 1
Department of Ultrasonography, Guangzhou Women and Children's Medical Center,
Guangzhou Medical University, Guangzhou 510623, China 2
Shenzhen Long-gang Maternal and Child Health Hospital Centralab, Shenzhen 518172,
China 3
Department of Pathogen Biology and Immunology, School of Basic Courses, Guangdong
Pharmaceutical University, Guangzhou 510006, China 4
Key Laboratory of Protein Modification and Degradation, Department of Pathophysiology,
School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China * These authors contributed equally to this work. #
Corresponding author
Correspondence to: Lan Wang, Department of Pathogen Biology and Immunology, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China. e-mail: [email protected]. Abbreviations: ARF=ADP-ribosylation factor; FOXO1=forkhead box O1; Ras=KRAS proto-oncogene, GTPase; ER=estrogen receptor; PR=progesterone receptor; HER2=human epidermal growth factor type 2 receptor; GSEA=Gene Set Enrichment Analysis; TNBC=triple negative breast cancer; TCGA=The Cancer Genome Atlas; ANT=adjacent normal tissues; RT-qPCR=Real time-quantitative polymerase chain reaction; NBEC=normal breast epithelial cells; AKT=AKT serine/threonine kinase 1; ERK=extracellular signal-regulated kinase.
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Abstract ADP-ribosylation factor 3 (ARF3) is a member of the KRAS proto-oncogene, GTPase(Ras) super-family of guanine nucleotide-binding proteins that mediates Golgi-related mitosis, but its role in malignant cells is unclear. In the present study, we found that mRNA and protein expression of ARF3 is up-regulated in breast cancer cells. Immunohistochemical analysis of 167 paraffin-embedded archived breast cancer tissues showed that ARF3 expression was localized primarily in the cytoplasm and was significantly up-regulated in malignant specimens compared to benign specimens. There were strong associations between ARF3 expression and clinicopathological characteristics in breast cancer. We also found that overexpressing ARF3 promoted, while silencing endogenous ARF3 inhibited, the proliferation of breast cancer cells by regulating cell cycle G1-S transition. Moreover, the pro-proliferative effect of ARF3 on breast cancer cells was associated with inactivation of the forkhead box O1 (FOXO1) transcription factor. ARF3 promotes breast cancer cell proliferation through the participation of FOXO1 and represents as a novel prognostic marker and therapeutic target for breast cancer. Keywords: ARF3; FOXO1; Breast cancer; Proliferation; G1-S transition
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Introduction Breast cancer, the most common tumor in women, causes more than 400,000 deaths every year worldwide [1]. The best treatments are based largely on the growth rate and probability of recurrence in breast cancer, which was always first assessed by imaging [2, 3]. With positive expression of the estrogen receptor (ER) and progesterone receptor (PR), tumor tissue may grow more quickly and can be inhibited from growing by use of estrogen and progesterone blocking agents [4, 5]. Similarly, tumor tissue may grow more quickly and is more likely to spread to other parts of the body with high levels of human epidermal growth factor type 2 receptor (HER2) protein, which is targeted by Trastuzumab and Pertuzumab treatment [6-8]. However, numerous breast cancer patients classified as having triple negative breast cancer (TNBC), which is ER negative, PR negative and HER2 negative, have the highest recurrence rates and mortality rate due to lack of effective therapeutic targets[9]. Therefore, obtaining earlier diagnosis and identifying new prognostic indicators are critical for ameliorating poor survival outcomes in breast cancer. ADP-ribosylation factors (ARFs) include six related gene products, ARF1-ARF6, which are ubiquitously expressed in almost all human tissues, such as the breast, brain, bladder, etc [10-13]. As members of the Ras super-family of 20-kDa guanine nucleotide-binding proteins,
ARFs play important roles in the regulation of normal human cellular physiology [14]. Jackson CL clarified that ARF1 is involved in the progression of cell mitosis [15]. Transfection of cells with epitope-tagged ARFs revealed that ARF2 displays perinuclear Golgi localization [16]. ARF3 mediates cytidine phosphate-guanosine oligodeoxynucleotide-induced responses by regulating Toll-like receptor 9 trafficking [17]. ARF4 is associated with a variety of human pathogens, such as Chlamydia trachomatis and Shigella flexneri, invading human target cells [18]. ARF5 mediates cell adhesion by regulating integrin surface expression [19, 20]. ARF6 is locally activated around the ingressing cleavage furrow and recruited to the Flemming body during late phases of cytokinesis and is involved in faithful completion of cytokinesis. As malignant cells are characterized by enhanced and uncontrolled cell proliferation and migration [21], it follows that ARFs play important roles in the development of cancer, which has already been confirmed by several studies. In prostate cancer, ARF1 promotes cell proliferation by activating mitogen-activated protein kinases signaling pathways [22]. In ovarian cancer,
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overexpression of ARF1 is associated with cell proliferation and migration mediated by the phosphatidylinositol 3-kinase signaling pathway [23]. However, the role of ARF3 in breast cancer is unknown. Herein, we demonstrate that ARF3 mRNA and protein expression is up-regulated in human breast cancer cells and tissues. Expression of ARF3 is positively correlated with the proliferation of breast cancer cells. Moreover, the phosphorylation status, localization and activity of the forkhead box O1 (FOXO1) transcription factor are modified by ARF3 protein. These findings suggest that ARF3 plays an important role in the proliferation of human breast cancer cells and identifies a potentially valuable therapeutic target for breast cancer.
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Material and methods Immunohistochemistry Immunohistochemistry and scoring of ARF3 expression were performed as previously described [24]. Normal breast tissues and 167 paraffin-embedded breast cancer samples were obtained from patients first diagnosis by ultrasonic examination, with final resection of the tumor performed at Guangzhou Women and Children’s Medical Center. Clinicopathological classification and staging were determined according to criteria of the American Joint Committee on Cancer (AJCC). This study was approved by the Ethics Committee of Southern Medical University and meets the standards of the Declaration of Helsinki. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay The 2,000 cells/well was seeded in seven 96-well plates, with each well in triplicate. Every 24 hours, a plate was taken out randomly and every well of this plate was incubated with 100 µl of 0.5 mg/ml sterile MTT (Sigma) for 4 h at 37 ºC. One hundred fifty microliters DMSO (Sigma, St. Louis, MO, USA) was added to dissolve the residue after pipetting of MTT solution gently into every well. Absorbance values were measured by a spectrophotometer according to the instructions provided. Anchorage-independent growth assay Two milliliters serum-free medium with 1% agar (Sigma) was smoothly plated into each well of a six-well plate to form a transparent solidified layer. Five hundred single cells were mixed with 2 ml complete media containing 0.3% agar and were placed on the top of this layer. When magnified 200 times under a microscope, colonies that were larger than 0.1 mm or that were greater than 50 cells were counted[25]. BrdU labeling and immunofluorescence BrdU (Abcam, Cambridge, MA) was diluted to be a final concentration of 10 µM and was incubated with cells grown on coverslips (Fisher, Pittsburgh, PA) for 1 h followed by staining with an anti-BrdU antibody as previously described [24]. Colony formation assay Five hundred single cells were evenly plated into each well of 6-well plates. Seven days later, adherent cells were fixed with 10% formaldehyde for 5 min and stained with 1% crystal violet for 30 s. After natural drying, plates were placed on the scanner to obtain images and
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count the number of clones. Xenograft tumor model NOD/SCID mice (female, 4-5 weeks old, 18-20 g) were purchased from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, Hunan, China). Each mouse was injected in the mammary pads with vector-transfected cells (5,000,000 cells) on the left side with ARF3-RNAi2 cells (5,000,000 cells) on the right side. Every three days, the length (L) and width (W) of tumors were measured using calipers, and volumes were calculated using the equation (L×W2)/2. On day 18, animals were euthanized, and tumors were excised and weighed. All animal experiments should comply with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
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Results ARF3 is up-regulated in breast cancer cells and tissues Analysis of mRNA expression of various genes from TCGA indicates that ARF3 exhibits expression differences between normal and malignant breast tissues. After grouping and comparison, ARF3 was up-regulated in 1,092 malignant breast tissues (Tumor) compared to 111 paracancerous normal breast tissue samples (Normal, P < 0.001, Fig. 1A). To eliminate the impact of individual differences, we further compared expression of ARF3 in the 111 paracancerous tissues and their corresponding cancerous tissues. As shown in Fig. 1B, ARF3 was significantly up-regulated in malignant tissues compared to their corresponding paracancerous tissues (P < 0.001). To verify the above expression differences, normal breast epithelial cells (NBEC1 and NBEC2) and six breast cancer cell lines were experimentally examined. Real time-quantitative polymerase chain reaction (RT-qPCR) analysis and western blot demonstrated that mRNA and the protein levels of ARF3 were indeed up-regulated in cultured breast cancer cell lines compared to NBEC1 (Fig. 1C and D). Primary human tissues removed directly from patients reflect the clinical trend of ARF3 expression more accurately. Therefore, we assessed expression levels of ARF3 in five paired malignant tissues (T) and matched adjacent normal tissues (ANT) by RT-qPCR and Western blot. As shown in Fig. 1E and F, ARF3 was markedly overexpressed in malignant tissues compared to adjacent normal tissues. ARF3 is related to clinicopathological characteristics in breast cancer patients To investigate the frequency of ARF3 up-regulation in breast cancer, we examined ARF3 expression by immunohistochemistry staining of 167 paraffin-embedded, archived human breast cancer tissues. Tissue sections were stained using an ARF3 antibody, followed by scoring and grouping. As shown in Fig. 2A, ARF3 protein was localized primarily in the cytoplasm and up-regulated in 92.8% (155 cases) of malignant specimens compared to benign specimens. Dividing specimens into high-group and low-group ARF3 expression revealed that all 167 malignant specimens were positively associated with clinical stage (P < 0.001, Fig. 2B), representing ARF3’s potential for use in clinical diagnosis. Summarized analyses between ARF3 expression and other clinical characteristics are listed in Supplemental Table 1. Briefly, there were strong associations between ARF3 expression and T classification (P < 0.001), N classification (P < 0.001), M classification (P = 0.013) and
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histologic grade (P < 0.001). In contrast, expression of ARF3 did not correlate with ER (P = 0.55), PR (P = 0.178) or HER2 (P = 0.398) status. Also shown in Fig. 2C, Kaplan-Meier survival curves demonstrate that the overall survival of patients with high expression of ARF3 was significantly shorter than those with low ARF3 expression (P < 0.001). In vitro assays reveal the role of ARF3 in promoting proliferation To investigate the biological role of ARF3 in the malignant progression of breast cancer, MCF-7 and SKBR-3 cell lines were transduced to stably overexpress exogenous ARF3 or silence endogenous ARF3 (Fig. 3A and B). MTT assays revealed that growth levels in ARF3-transfected cells were significantly increased, while ARF3-silenced cells (Ri1 and Ri2) exhibited significantly decreased levels of growth compared to negative-control-transduced cells (Vector or Ri-vector, Fig. 3C). Overexpression of ARF3 also increased mean colony number, while silencing of ARF3 decreased mean colony number in colony formation assay (Fig. 3D and E) compared to negative-control-transfected cells. Moreover, overexpressing ARF3 increased anchorage-independent growth ability, while silencing endogenous ARF3 reduce this ability (Fig. 3F and G). In vivo assay reveals ARF3 promotes proliferation To validate this pro-proliferative effect of ARF3 in vivo, we created a xenograft model in NOD/SCID nude mice. As shown in Fig. 4A, ARF3-tranfected cells showed a pro-proliferative tendency in nude mice (M1, mouse 1; M2, mouse 2, etc.). ARF3-silenced SKBR-3 cells showed an anti-proliferative tendency in nude mice. As tumor volume measurements revealed, the growth rate of ARF3-tranfected cells was faster than that of vector-transfected cells, while the growth rate of ARF3-silenced cells was slower than that of vector-transfected cells (Fig. 4B). Final xenograft tumor weight also reflected a positive correlation between the growth rate of transplanted tumor and expression of ARF3 (Fig. 4C). ARF3 promotes cell-cycle G1-S transition The percentage of cells being in a certain portion of the cell cycle generally reflects the proliferation rate of a cell population. In terms of biological function, overexpression of ARF3 significantly reduced the percentage of cells in G0/G1 phase, i.e. MCF-7 cells decreased from 63.07% to 43.60%, and SKBR-3 cells decreased from 58.37% to 47.12%. At the same time, the proportion of cells in S phase increased significantly when ARF3 overexpressed, i.e. MCF-7 cells increased from 23.15% to 42.43%; SKBR-3 cells increased
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from 25.47% to 41.67%. Conversely, silencing ARF3 significantly increased the percentage of cells in the G0/G1 phase, but the cells in the S phase decreased dramatically (Fig. 5A). BrdU can be integrated into the DNA in the process of replication, so the amount of BrdU incorporation can be detected using an anti-BrdU antibody to detect the proliferative activity of the cells. As shown in Fig. 5B and 5C, the percentage of cells in or over the S phase in cells overexpressing exogenous ARF3 increased by at least 15%, but decreased by approximately 65% in endogenous ARF3 silenced cells. Compared with vector-transfected cells, protein and mRNA expression of p21Cip1 and p27Kip1, 2 G1-S transition inhibitor, was decreased in ARF3-transfected cells but increased in ARF3-silenced cells (Fig. 5D and E). FOXO1 activity is modified by ARF3 FOXO1 is the most well known cell cycle inhibitor associated with p21Cip1 and p27Kip1. As shown in Fig. 6A, luciferase reporter assay demonstrated that transcriptional activity of FOXO1 was decreased in ARF3-transfected cells but increased in ARF3-silenced cells, suggesting that FOXO1 may be involved in the regulation of ARF3. Transcriptional activity of FOXO1 is dependent on its phosphorylation status and subsequent protein localization. Phosphorylated FOXO1 is transported from the nucleus to the cytoplasm, and its tumor suppressor activity is also downregulated. As shown in Fig. 6B, phosphorylation of FOXO1 was increased in ARF3-transfected cells but decreased in ARF3-silenced cells (Fig. 6B). Cell immunofluorescence assay showed that FOXO1 protein was very rarely found in the nucleus of ARF3-transfected cells. Conversely, FOXO1 protein was almost entirely in the nucleus of ARF3-silenced cells (Fig. 6C). To confirm that ARF3 functions through FOXO1, overexpression of FOXO1 and an empty control plasmid (mock) were used to treat ARF3 overexpressing cells. Both FOXO1 protein levels and phosphorylation were up-regulated in FOXO1-treated cells (Fig. 6D). MTT analysis showed that overexpression of FOXO1 significantly inhibited ARF3-induced cell growth (Fig. 6E). At the same time, in the BrdU assay, overexpression of FOXO1 abolished the effect of ARF3 on the proportion of S phase cells (Fig. 6F). These results suggest that ARF3 may phosphorylate FOXO1 and promote translocation of FOXO1 protein from the nucleus to the cytoplasm.
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Discussion Each intracellular compartment involved in the biosynthetic/secretory pathway of eukaryotic cells bears at least one small GTP-binding protein [26, 27]. As important parts of small GTP-binding proteins, ARFs (ARF1-ARF6) play multiple roles in the regulation of cellular functions and are regarded as providing overlapping functions [11]. Studies have found that ARF1 binds to the microtubule minus-end motor protein dynein and control Golgi reorganization [28]. Activation of ARF1 triggers plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling [29]. ARF6 expression is enriched in human podocytes, and it modulates podocyte cytoskeletal dynamics [30]. As to the malignant process, more and more studies have shown the important role of
ARFs in the malignant progression of tumor cells. ARF1 promotes prostate tumorigenesis via targeting oncogenic MAPK signaling[31]. ARF1 also regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion [32]. Knockdown of ARF6 increases drug sensitivity in gastric cancer cells[33]. Expression of ARF6 is enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues [34]. It is well known that the growth rate of malignant cells is extremely critical for the prognosis of tumor patients [35]. In ovarian cancer, immunoreactivity of ARF1 is positively correlated with Ki-67
expression [36]. Knockdown of ARF1 expression notably inhibited cell proliferation of ovarian cancer cells [36], and knockdown of ARF6 inhibits proliferation in gastric cancer cells[33]. In the present study, we identified that ARF3 is up-regulated in breast cancer cells. Statistical analysis of 167 clinical cases showed that there were positive associations between ARF3 expression and tumor growth in breast cancer. Cell function experiments also verified the proto oncogenic properties of ARF3 in promoting tumor growth. As a central link of many signal transduction pathways, GTP-binding proteins, including ARFs, have become target sites for many drugs and toxin attacks [37]. Investigation of ARF3 protein will aid in the effort to discover new tumor therapeutic targets. FOXO1 changes in response to cellular stimulation and maintains tissue homeostasis during cell proliferation, apoptosis, stem cell division, oxidative stress and metabolic dysregulation in many diseases [38]. It primarily remains in the inactive phosphorylated state in the cytosol, thus maintaining normal cellular proliferation and survival [39]. Translocation of non-phosphorylated FOXO1 into the nucleus promotes inhibition of the transcription of genes such as p27Kip1, thus inhibiting cell growth [40]. Phosphorylated FOXO1 translocates 10
into the cytoplasm and is inactivated. Low expression of FOXO1 is crucial for the malignant process of many tumors, such as tumorsphere formation capacity of gastric cancer, cell invasion of prostate cancer and the proliferation of hepatocellular carcinoma [41-43]. In the present study, we found that the pro-proliferative effect of ARF3 on breast cancer cells was associated with inactivation of FOXO1. Through construction of reporter plasmids and quantitative detection, we found that transcriptional activity of FOXO1 was enhanced in ARF3-silenced breast cancer cells, suggesting a relationship between ARF3 and FOXO1. Subsequently, we demonstrated by ELISA assay that phosphorylation levels of FOXO1 were up-regulated in response to silencing of ARF3. More accurately, cell in situ staining by immunofluorescent assay showed the distribution and localization of FOXO1 protein. However, the mechanism by which ARF3 regulates FOXO1 transcriptional activity is unclear. A large number of studies have confirmed that AKT1 is a direct upstream regulator of FOXO1 activity[44]. Our current research indicates a correlation between ARF3 and AKT. Jing-Yiing Wu et al found that ARF3 did not affect the expression of AKT protein, but positively correlated with the phosphorylation of AKT1 in mouse macrophages and human embryonic kidney cells[45]. However, it is unclear how non-enzymatic ARF3 acts on AKT1 remains unclear. The potential similarity between ARF3 and ARF1 functions, based on similar sequences and structures, suggests a meaningful research direction. Co-immunoprecipitation indicated that ARF1 interacts with PI3K in ovarian cancer cells. Furthermore, the loss of ARF1 significantly inhibited the activation of the PI3K pathway by AKT1 phosphorylation[46]. We speculate and will test in future studies that ARF3 may alter the phosphorylation of AKT1 by binding to PI3K protein, ultimately regulating FOXO1 activity. In conclusion, ARF3 status upon diagnosis and treatment is even more important, as it serves as an upstream control gene of the potential therapeutic target gene FOXO1. Conclusion we demonstrate that the frequently up-regulated ARF3 promotes the proliferation of breast cancer cells by inhibiting the activity of FOXO1.
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Funding: This work was supported by Natural science Foundation of Guangdong Province, China (grant numbers 2017A030313549, 2017) , Guangzhou Institute of Pediatrics/ Guangzhou Women and Children’s Medical Center (grant numbers YIP-2019-049); Shenzhen Science and Technology Project (grant numbers JCYJ20140414124506130, 2016; grant numbers, JCYJ20170307144612471, 2017). Disclosure of Potential Conflicts of Interest Conflicts of interest: none.
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Overexpression of ARF1 is associated with cell proliferation and migration through PI3K signal pathway in ovarian cancer, ONCOL REP 37 (2017) 1511-1520. [37] X. Chang, H. Li, Y. Li, Q. He, J. Yao, T. Duan, K. Wang, RhoA/MLC signaling pathway is involved in Delta(9)-tetrahydrocannabinol-impaired placental angiogenesis, TOXICOL LETT 285 (2018) 148-155. [38] Y.Q. Xing, A. Li, Y. Yang, X.X. Li, L.N. Zhang, H.C. Guo, The regulation of FOXO1 and its role in disease progression, LIFE SCI 193 (2018) 124-131. [39] F. Shi, T. Li, Z. Liu, K. Qu, C. Shi, Y. Li, Q. Qin, L. Cheng, X. Jin, T. Yu, Di W, J. Que, H. Xia, J. She, FOXO1: Another avenue for treating digestive malignancy? SEMIN CANCER BIOL (2017). [40] C. Charvet, A.J. Canonigo, S. Becart, U. Maurer, A.V. Miletic, W. Swat, M. Deckert, A. Altman, Vav1 promotes T cell cycle progression by linking TCR/CD28 costimulation to FOXO1 and p27kip1 expression, J IMMUNOL 177 (2006) 5024-5031. [41] Y. Choi, J. Park, Y.S. Ko, Y. Kim, J.S. Pyo, B.G. Jang, M.A. Kim, J.S. Lee, M.S. Chang, B.L. Lee, FOXO1 reduces tumorsphere formation capacity and has crosstalk with LGR5 signaling in gastric cancer cells, Biochem Biophys Res Commun 493 (2017) 1349-1355. [42] Y. Yang, A.M. Blee, D. Wang, J. An, Y. Pan, Y. Yan, T. Ma, Y. He, J. Dugdale, X. Hou, J. Zhang, S.J. Weroha, W.G. Zhu, Y.A. Wang, R.A. DePinho, W. Xu, H. Huang, Loss of FOXO1 Cooperates with TMPRSS2-ERG Overexpression to Promote Prostate Tumorigenesis and Cell Invasion, CANCER RES 77 (2017) 6524-6537. [43] F. Yamaguchi, Y. Hirata, H. Akram, K. Kamitori, Y. Dong, L. Sui, M. Tokuda, FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801, BMC CANCER 13 (2013) 468. [44] B. Li, J. Lei, L. Yang, C. Gao, E. Dang, T. Cao, K. Xue, Y. Zhuang, S. Shao, D. Zhi, J. Hao, L. Jin, P. Qiao, W. Ouyang, G. Wang, Dysregulation of Akt-FOXO1 Pathway Leads to Dysfunction of Regulatory T Cells in Patients with Psoriasis, J INVEST DERMATOL (2019). [45] J.Y.
Wu,
C.C.
Kuo,
ADP-Ribosylation
Factor
3
Mediates
Cytidine-Phosphate-Guanosine
Oligodeoxynucleotide-Induced Responses by Regulating Toll-Like Receptor 9 Trafficking, J INNATE IMMUN 7 (2015) 623-636. [46] G. Gu, Y. Chen, C. Duan, L. Zhou, C. Chen, J. Chen, J. Cheng, N. Shi, Y. Jin, Q. Xi, J. Zhong, Overexpression of ARF1 is associated with cell proliferation and migration through PI3K signal pathway in ovarian cancer, ONCOL REP 37 (2017) 1511-1520.
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Figure legends Fig. 1. ARF3 is up-regulated in breast cancer. (A) ARF3 analyses in 1,092 breast cancer tissues (Tumor) compared with 111 normal breast tissue samples (Normal) in TCGA profile. ** P < 0.01 versus Normal. (B) ARF3 mRNA analyses in 111 paired breast tumor tissues (Tumor) and their adjacent normal tissues (Normal) in TCGA profile. ** P < 0.01 versus Normal. (C) RT-qPCR analysis of ARF3 expression in normal breast epithelial cells (NBEC1, NBEC2) and breast cancer cell lines (T47D, MCF-7, SKBR-3, ZR-75-30, MDA-MB-231, BT549). GAPDH gene was used as a normalized gene. Values are expressed as the means ± SD; n= 3. * p < 0.05 versus NBEC 1. (D) Western blot of ARF3 expression in normal breast epithelial cells (NBEC1, NBEC2) and breast cancer cell lines. β-actin was used as a loading control. (E) RT-qPCR analysis of ARF3 in five paired breast cancer tissues (T) and their adjacent normal tissues (ANT). P1 stand for Patient 1, and so on. GAPDH gene was used as a normalized gene. Values are expressed as the means±SD; * P < 0.05 versus ANT, respectively. (F) Western blot of ARF3 in five paired breast cancer tissues (T) and their adjacent normal tissues (ANT). β-actin was used as a loading control. Fig. 2. High ARF3 expression correlates with clinical characteristics and poor patient survival in breast cancer patients. (A) Representative images of ARF3 expression in normal breast tissues and breast cancer at different clinical stages. Magnification of the original images is 200×. (B) Quantification of the average mean optical density (MOD) for ARF3 in tissues at different stages. Values are expressed as the means±SD; * P < 0.05 versus Normal. (C) Kaplan–Meier overall survival curves for patients with breast cancer stratified by ARF3-high-expressing (93) and ARF3-low-expressing (74) cases. ** P < 0.01 versus ARF3-low. Fig. 3. ARF3 regulates the proliferation of breast cancer cells in vitro. (A) RT-qPCR analysis of ARF3 mRNA in indicated stable cell lines. Values are expressed as the means ± SD; n= 3. * P < 0.05 versus Vector & Ri-vector. (B) Western blot of ARF3 protein in indicated stable cell lines. β-actin was used as a loading control. (C) Cell growth curve by MTT assays. (D) Representative image of colony formation assay. (E) Mean count of colony numbers in colony formation assay. *P < 0.05. (F) Representative micrographs in anchorage-independent growth assay. (G) Average colony (>0.1 mm/colony) numbers of three independent experiments in the anchorage-independent growth assay. Each bar represents the mean of 16
three independent experiments. Values are expressed as the means±SD; * P < 0.05 versus Vector & Ri-vector, respetively. Fig.4. ARF3 promotes cell growth rate in vivo. (A) Images of excised tumors from five NOD/SCID mice 18 days after injection with indicated cells. M1 stand for mouse 1, and so on. (B) Measured tumor volumes at corresponding time points. (C) Average weight of excised tumors. *P < 0.05. Each bar represents the mean of three independent experiments. Values are expressed as the means±SD; * P < 0.05 versus Vector & Ri-vector, respetively. Fig. 5. ARF3 promotes cell-cycle G1-S transition. (A) Flow cytometric analysis of indicated cells, Ri represents RNAi, 2N represents amphiploid, 4N represents fortetraploid. Values are expressed as the means±SD; n= 3. * P < 0.05 versus Vector & Ri-vector, respetively. (B) Representative micrographs of BrdU incorporation in indicated cells. (C) Average quantification of BrdU incorporation in indicated cells from three independent experiments. Each bar represents the mean of three independent experiments. Values are expressed as the means±SD; * P < 0.05 versus Vector & Ri-vector. (D) Western blot analysis G1-S-transition-associated genes in indicated cells. β-actin was used as a loading control. (E) RT-qPCR analysis of key negative regulator p21Cip1and p27Kip1 in indicated cells. Each bar represents the mean of three independent experiments. Values are expressed as the means±SD; n= 3. * P < 0.05 versus Vector & Ri-vector. Fig. 6. FOXO1 activity is modified by ARF3. (A) Relative FOXO1 reporter activity in the indicated cells. Values are expressed as the means±SD; n= 3. * P < 0.05 versus Vector & Ri-vector. (B) Western blot analysis of phospho-FOXO1 and total-FOXO1. β-actin was used as a loading control. (C) Represented images of immunofluorescence staining. (D) Western blot analysis of phospho-FOXO1 and total-FOXO1. (E) Cell growth curve by MTT assays. (F) Average quantification of BrdU incorporation in indicated cells from three independent experiments. Each bar represents the mean of three independent experiments. * P < 0.05.
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Declaration of interests √ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
1
S0014-4827(19)30486-0
DOI:
https://doi.org/10.1016/j.yexcr.2019.111624
Reference:
YEXCR 111624
To appear in:
Experimental Cell Research
Received Date: 8 April 2019 Revised Date:
12 September 2019
Accepted Date: 15 September 2019
Please cite this article as: D. Huang, Y. Pei, C. Dai, Y. Huang, H. Chen, X. Chen, X. Zhang, C. Lin, H. Wang, R. Zhang, X. Wan, L. Wang, Up-regulated ADP-Ribosylation factor 3 promotes breast cancer cell proliferation through the participation of FOXO1, Experimental Cell Research (2019), doi: https:// doi.org/10.1016/j.yexcr.2019.111624. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Up-regulated ADP-Ribosylation Factor 3 Promotes Breast Cancer Cell Proliferation Through the Participation of FOXO1 Running title: ARF3 Promotes the Proliferation of Breast Cancer Danping Huang1*, Yuanyuan Pei 2*, Changping Dai1, Yun Huang3, Han Chen4, Xuhong Chen4, Xiaolan Zhang4, Chun Lin4, Hongying Wang1, Rui Zhang1, Xinhong Wan2, Lan Wang3# 1
Department of Ultrasonography, Guangzhou Women and Children's Medical Center,
Guangzhou Medical University, Guangzhou 510623, China 2
Shenzhen Long-gang Maternal and Child Health Hospital Centralab, Shenzhen 518172,
China 3
Department of Pathogen Biology and Immunology, School of Basic Courses, Guangdong
Pharmaceutical University, Guangzhou 510006, China 4
Key Laboratory of Protein Modification and Degradation, Department of Pathophysiology,
School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China * These authors contributed equally to this work. #
Corresponding author
Correspondence to: Lan Wang, Department of Pathogen Biology and Immunology, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China. e-mail: [email protected]. Abbreviations: ARF=ADP-ribosylation factor; FOXO1=forkhead box O1; Ras=KRAS proto-oncogene, GTPase; ER=estrogen receptor; PR=progesterone receptor; HER2=human epidermal growth factor type 2 receptor; GSEA=Gene Set Enrichment Analysis; TNBC=triple negative breast cancer; TCGA=The Cancer Genome Atlas; ANT=adjacent normal tissues; RT-qPCR=Real time-quantitative polymerase chain reaction; NBEC=normal breast epithelial cells; AKT=AKT serine/threonine kinase 1; ERK=extracellular signal-regulated kinase.
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Abstract ADP-ribosylation factor 3 (ARF3) is a member of the KRAS proto-oncogene, GTPase(Ras) super-family of guanine nucleotide-binding proteins that mediates Golgi-related mitosis, but its role in malignant cells is unclear. In the present study, we found that mRNA and protein expression of ARF3 is up-regulated in breast cancer cells. Immunohistochemical analysis of 167 paraffin-embedded archived breast cancer tissues showed that ARF3 expression was localized primarily in the cytoplasm and was significantly up-regulated in malignant specimens compared to benign specimens. There were strong associations between ARF3 expression and clinicopathological characteristics in breast cancer. We also found that overexpressing ARF3 promoted, while silencing endogenous ARF3 inhibited, the proliferation of breast cancer cells by regulating cell cycle G1-S transition. Moreover, the pro-proliferative effect of ARF3 on breast cancer cells was associated with inactivation of the forkhead box O1 (FOXO1) transcription factor. ARF3 promotes breast cancer cell proliferation through the participation of FOXO1 and represents as a novel prognostic marker and therapeutic target for breast cancer. Keywords: ARF3; FOXO1; Breast cancer; Proliferation; G1-S transition
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Introduction Breast cancer, the most common tumor in women, causes more than 400,000 deaths every year worldwide [1]. The best treatments are based largely on the growth rate and probability of recurrence in breast cancer, which was always first assessed by imaging [2, 3]. With positive expression of the estrogen receptor (ER) and progesterone receptor (PR), tumor tissue may grow more quickly and can be inhibited from growing by use of estrogen and progesterone blocking agents [4, 5]. Similarly, tumor tissue may grow more quickly and is more likely to spread to other parts of the body with high levels of human epidermal growth factor type 2 receptor (HER2) protein, which is targeted by Trastuzumab and Pertuzumab treatment [6-8]. However, numerous breast cancer patients classified as having triple negative breast cancer (TNBC), which is ER negative, PR negative and HER2 negative, have the highest recurrence rates and mortality rate due to lack of effective therapeutic targets[9]. Therefore, obtaining earlier diagnosis and identifying new prognostic indicators are critical for ameliorating poor survival outcomes in breast cancer. ADP-ribosylation factors (ARFs) include six related gene products, ARF1-ARF6, which are ubiquitously expressed in almost all human tissues, such as the breast, brain, bladder, etc [10-13]. As members of the Ras super-family of 20-kDa guanine nucleotide-binding proteins,
ARFs play important roles in the regulation of normal human cellular physiology [14]. Jackson CL clarified that ARF1 is involved in the progression of cell mitosis [15]. Transfection of cells with epitope-tagged ARFs revealed that ARF2 displays perinuclear Golgi localization [16]. ARF3 mediates cytidine phosphate-guanosine oligodeoxynucleotide-induced responses by regulating Toll-like receptor 9 trafficking [17]. ARF4 is associated with a variety of human pathogens, such as Chlamydia trachomatis and Shigella flexneri, invading human target cells [18]. ARF5 mediates cell adhesion by regulating integrin surface expression [19, 20]. ARF6 is locally activated around the ingressing cleavage furrow and recruited to the Flemming body during late phases of cytokinesis and is involved in faithful completion of cytokinesis. As malignant cells are characterized by enhanced and uncontrolled cell proliferation and migration [21], it follows that ARFs play important roles in the development of cancer, which has already been confirmed by several studies. In prostate cancer, ARF1 promotes cell proliferation by activating mitogen-activated protein kinases signaling pathways [22]. In ovarian cancer,
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overexpression of ARF1 is associated with cell proliferation and migration mediated by the phosphatidylinositol 3-kinase signaling pathway [23]. However, the role of ARF3 in breast cancer is unknown. Herein, we demonstrate that ARF3 mRNA and protein expression is up-regulated in human breast cancer cells and tissues. Expression of ARF3 is positively correlated with the proliferation of breast cancer cells. Moreover, the phosphorylation status, localization and activity of the forkhead box O1 (FOXO1) transcription factor are modified by ARF3 protein. These findings suggest that ARF3 plays an important role in the proliferation of human breast cancer cells and identifies a potentially valuable therapeutic target for breast cancer.
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Material and methods Immunohistochemistry Immunohistochemistry and scoring of ARF3 expression were performed as previously described [24]. Normal breast tissues and 167 paraffin-embedded breast cancer samples were obtained from patients first diagnosis by ultrasonic examination, with final resection of the tumor performed at Guangzhou Women and Children’s Medical Center. Clinicopathological classification and staging were determined according to criteria of the American Joint Committee on Cancer (AJCC). This study was approved by the Ethics Committee of Southern Medical University and meets the standards of the Declaration of Helsinki. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay The 2,000 cells/well was seeded in seven 96-well plates, with each well in triplicate. Every 24 hours, a plate was taken out randomly and every well of this plate was incubated with 100 µl of 0.5 mg/ml sterile MTT (Sigma) for 4 h at 37 ºC. One hundred fifty microliters DMSO (Sigma, St. Louis, MO, USA) was added to dissolve the residue after pipetting of MTT solution gently into every well. Absorbance values were measured by a spectrophotometer according to the instructions provided. Anchorage-independent growth assay Two milliliters serum-free medium with 1% agar (Sigma) was smoothly plated into each well of a six-well plate to form a transparent solidified layer. Five hundred single cells were mixed with 2 ml complete media containing 0.3% agar and were placed on the top of this layer. When magnified 200 times under a microscope, colonies that were larger than 0.1 mm or that were greater than 50 cells were counted[25]. BrdU labeling and immunofluorescence BrdU (Abcam, Cambridge, MA) was diluted to be a final concentration of 10 µM and was incubated with cells grown on coverslips (Fisher, Pittsburgh, PA) for 1 h followed by staining with an anti-BrdU antibody as previously described [24]. Colony formation assay Five hundred single cells were evenly plated into each well of 6-well plates. Seven days later, adherent cells were fixed with 10% formaldehyde for 5 min and stained with 1% crystal violet for 30 s. After natural drying, plates were placed on the scanner to obtain images and
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count the number of clones. Xenograft tumor model NOD/SCID mice (female, 4-5 weeks old, 18-20 g) were purchased from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, Hunan, China). Each mouse was injected in the mammary pads with vector-transfected cells (5,000,000 cells) on the left side with ARF3-RNAi2 cells (5,000,000 cells) on the right side. Every three days, the length (L) and width (W) of tumors were measured using calipers, and volumes were calculated using the equation (L×W2)/2. On day 18, animals were euthanized, and tumors were excised and weighed. All animal experiments should comply with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
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Results ARF3 is up-regulated in breast cancer cells and tissues Analysis of mRNA expression of various genes from TCGA indicates that ARF3 exhibits expression differences between normal and malignant breast tissues. After grouping and comparison, ARF3 was up-regulated in 1,092 malignant breast tissues (Tumor) compared to 111 paracancerous normal breast tissue samples (Normal, P < 0.001, Fig. 1A). To eliminate the impact of individual differences, we further compared expression of ARF3 in the 111 paracancerous tissues and their corresponding cancerous tissues. As shown in Fig. 1B, ARF3 was significantly up-regulated in malignant tissues compared to their corresponding paracancerous tissues (P < 0.001). To verify the above expression differences, normal breast epithelial cells (NBEC1 and NBEC2) and six breast cancer cell lines were experimentally examined. Real time-quantitative polymerase chain reaction (RT-qPCR) analysis and western blot demonstrated that mRNA and the protein levels of ARF3 were indeed up-regulated in cultured breast cancer cell lines compared to NBEC1 (Fig. 1C and D). Primary human tissues removed directly from patients reflect the clinical trend of ARF3 expression more accurately. Therefore, we assessed expression levels of ARF3 in five paired malignant tissues (T) and matched adjacent normal tissues (ANT) by RT-qPCR and Western blot. As shown in Fig. 1E and F, ARF3 was markedly overexpressed in malignant tissues compared to adjacent normal tissues. ARF3 is related to clinicopathological characteristics in breast cancer patients To investigate the frequency of ARF3 up-regulation in breast cancer, we examined ARF3 expression by immunohistochemistry staining of 167 paraffin-embedded, archived human breast cancer tissues. Tissue sections were stained using an ARF3 antibody, followed by scoring and grouping. As shown in Fig. 2A, ARF3 protein was localized primarily in the cytoplasm and up-regulated in 92.8% (155 cases) of malignant specimens compared to benign specimens. Dividing specimens into high-group and low-group ARF3 expression revealed that all 167 malignant specimens were positively associated with clinical stage (P < 0.001, Fig. 2B), representing ARF3’s potential for use in clinical diagnosis. Summarized analyses between ARF3 expression and other clinical characteristics are listed in Supplemental Table 1. Briefly, there were strong associations between ARF3 expression and T classification (P < 0.001), N classification (P < 0.001), M classification (P = 0.013) and
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histologic grade (P < 0.001). In contrast, expression of ARF3 did not correlate with ER (P = 0.55), PR (P = 0.178) or HER2 (P = 0.398) status. Also shown in Fig. 2C, Kaplan-Meier survival curves demonstrate that the overall survival of patients with high expression of ARF3 was significantly shorter than those with low ARF3 expression (P < 0.001). In vitro assays reveal the role of ARF3 in promoting proliferation To investigate the biological role of ARF3 in the malignant progression of breast cancer, MCF-7 and SKBR-3 cell lines were transduced to stably overexpress exogenous ARF3 or silence endogenous ARF3 (Fig. 3A and B). MTT assays revealed that growth levels in ARF3-transfected cells were significantly increased, while ARF3-silenced cells (Ri1 and Ri2) exhibited significantly decreased levels of growth compared to negative-control-transduced cells (Vector or Ri-vector, Fig. 3C). Overexpression of ARF3 also increased mean colony number, while silencing of ARF3 decreased mean colony number in colony formation assay (Fig. 3D and E) compared to negative-control-transfected cells. Moreover, overexpressing ARF3 increased anchorage-independent growth ability, while silencing endogenous ARF3 reduce this ability (Fig. 3F and G). In vivo assay reveals ARF3 promotes proliferation To validate this pro-proliferative effect of ARF3 in vivo, we created a xenograft model in NOD/SCID nude mice. As shown in Fig. 4A, ARF3-tranfected cells showed a pro-proliferative tendency in nude mice (M1, mouse 1; M2, mouse 2, etc.). ARF3-silenced SKBR-3 cells showed an anti-proliferative tendency in nude mice. As tumor volume measurements revealed, the growth rate of ARF3-tranfected cells was faster than that of vector-transfected cells, while the growth rate of ARF3-silenced cells was slower than that of vector-transfected cells (Fig. 4B). Final xenograft tumor weight also reflected a positive correlation between the growth rate of transplanted tumor and expression of ARF3 (Fig. 4C). ARF3 promotes cell-cycle G1-S transition The percentage of cells being in a certain portion of the cell cycle generally reflects the proliferation rate of a cell population. In terms of biological function, overexpression of ARF3 significantly reduced the percentage of cells in G0/G1 phase, i.e. MCF-7 cells decreased from 63.07% to 43.60%, and SKBR-3 cells decreased from 58.37% to 47.12%. At the same time, the proportion of cells in S phase increased significantly when ARF3 overexpressed, i.e. MCF-7 cells increased from 23.15% to 42.43%; SKBR-3 cells increased
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from 25.47% to 41.67%. Conversely, silencing ARF3 significantly increased the percentage of cells in the G0/G1 phase, but the cells in the S phase decreased dramatically (Fig. 5A). BrdU can be integrated into the DNA in the process of replication, so the amount of BrdU incorporation can be detected using an anti-BrdU antibody to detect the proliferative activity of the cells. As shown in Fig. 5B and 5C, the percentage of cells in or over the S phase in cells overexpressing exogenous ARF3 increased by at least 15%, but decreased by approximately 65% in endogenous ARF3 silenced cells. Compared with vector-transfected cells, protein and mRNA expression of p21Cip1 and p27Kip1, 2 G1-S transition inhibitor, was decreased in ARF3-transfected cells but increased in ARF3-silenced cells (Fig. 5D and E). FOXO1 activity is modified by ARF3 FOXO1 is the most well known cell cycle inhibitor associated with p21Cip1 and p27Kip1. As shown in Fig. 6A, luciferase reporter assay demonstrated that transcriptional activity of FOXO1 was decreased in ARF3-transfected cells but increased in ARF3-silenced cells, suggesting that FOXO1 may be involved in the regulation of ARF3. Transcriptional activity of FOXO1 is dependent on its phosphorylation status and subsequent protein localization. Phosphorylated FOXO1 is transported from the nucleus to the cytoplasm, and its tumor suppressor activity is also downregulated. As shown in Fig. 6B, phosphorylation of FOXO1 was increased in ARF3-transfected cells but decreased in ARF3-silenced cells (Fig. 6B). Cell immunofluorescence assay showed that FOXO1 protein was very rarely found in the nucleus of ARF3-transfected cells. Conversely, FOXO1 protein was almost entirely in the nucleus of ARF3-silenced cells (Fig. 6C). To confirm that ARF3 functions through FOXO1, overexpression of FOXO1 and an empty control plasmid (mock) were used to treat ARF3 overexpressing cells. Both FOXO1 protein levels and phosphorylation were up-regulated in FOXO1-treated cells (Fig. 6D). MTT analysis showed that overexpression of FOXO1 significantly inhibited ARF3-induced cell growth (Fig. 6E). At the same time, in the BrdU assay, overexpression of FOXO1 abolished the effect of ARF3 on the proportion of S phase cells (Fig. 6F). These results suggest that ARF3 may phosphorylate FOXO1 and promote translocation of FOXO1 protein from the nucleus to the cytoplasm.
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Discussion Each intracellular compartment involved in the biosynthetic/secretory pathway of eukaryotic cells bears at least one small GTP-binding protein [26, 27]. As important parts of small GTP-binding proteins, ARFs (ARF1-ARF6) play multiple roles in the regulation of cellular functions and are regarded as providing overlapping functions [11]. Studies have found that ARF1 binds to the microtubule minus-end motor protein dynein and control Golgi reorganization [28]. Activation of ARF1 triggers plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling [29]. ARF6 expression is enriched in human podocytes, and it modulates podocyte cytoskeletal dynamics [30]. As to the malignant process, more and more studies have shown the important role of
ARFs in the malignant progression of tumor cells. ARF1 promotes prostate tumorigenesis via targeting oncogenic MAPK signaling[31]. ARF1 also regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion [32]. Knockdown of ARF6 increases drug sensitivity in gastric cancer cells[33]. Expression of ARF6 is enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues [34]. It is well known that the growth rate of malignant cells is extremely critical for the prognosis of tumor patients [35]. In ovarian cancer, immunoreactivity of ARF1 is positively correlated with Ki-67
expression [36]. Knockdown of ARF1 expression notably inhibited cell proliferation of ovarian cancer cells [36], and knockdown of ARF6 inhibits proliferation in gastric cancer cells[33]. In the present study, we identified that ARF3 is up-regulated in breast cancer cells. Statistical analysis of 167 clinical cases showed that there were positive associations between ARF3 expression and tumor growth in breast cancer. Cell function experiments also verified the proto oncogenic properties of ARF3 in promoting tumor growth. As a central link of many signal transduction pathways, GTP-binding proteins, including ARFs, have become target sites for many drugs and toxin attacks [37]. Investigation of ARF3 protein will aid in the effort to discover new tumor therapeutic targets. FOXO1 changes in response to cellular stimulation and maintains tissue homeostasis during cell proliferation, apoptosis, stem cell division, oxidative stress and metabolic dysregulation in many diseases [38]. It primarily remains in the inactive phosphorylated state in the cytosol, thus maintaining normal cellular proliferation and survival [39]. Translocation of non-phosphorylated FOXO1 into the nucleus promotes inhibition of the transcription of genes such as p27Kip1, thus inhibiting cell growth [40]. Phosphorylated FOXO1 translocates 10
into the cytoplasm and is inactivated. Low expression of FOXO1 is crucial for the malignant process of many tumors, such as tumorsphere formation capacity of gastric cancer, cell invasion of prostate cancer and the proliferation of hepatocellular carcinoma [41-43]. In the present study, we found that the pro-proliferative effect of ARF3 on breast cancer cells was associated with inactivation of FOXO1. Through construction of reporter plasmids and quantitative detection, we found that transcriptional activity of FOXO1 was enhanced in ARF3-silenced breast cancer cells, suggesting a relationship between ARF3 and FOXO1. Subsequently, we demonstrated by ELISA assay that phosphorylation levels of FOXO1 were up-regulated in response to silencing of ARF3. More accurately, cell in situ staining by immunofluorescent assay showed the distribution and localization of FOXO1 protein. However, the mechanism by which ARF3 regulates FOXO1 transcriptional activity is unclear. A large number of studies have confirmed that AKT1 is a direct upstream regulator of FOXO1 activity[44]. Our current research indicates a correlation between ARF3 and AKT. Jing-Yiing Wu et al found that ARF3 did not affect the expression of AKT protein, but positively correlated with the phosphorylation of AKT1 in mouse macrophages and human embryonic kidney cells[45]. However, it is unclear how non-enzymatic ARF3 acts on AKT1 remains unclear. The potential similarity between ARF3 and ARF1 functions, based on similar sequences and structures, suggests a meaningful research direction. Co-immunoprecipitation indicated that ARF1 interacts with PI3K in ovarian cancer cells. Furthermore, the loss of ARF1 significantly inhibited the activation of the PI3K pathway by AKT1 phosphorylation[46]. We speculate and will test in future studies that ARF3 may alter the phosphorylation of AKT1 by binding to PI3K protein, ultimately regulating FOXO1 activity. In conclusion, ARF3 status upon diagnosis and treatment is even more important, as it serves as an upstream control gene of the potential therapeutic target gene FOXO1. Conclusion we demonstrate that the frequently up-regulated ARF3 promotes the proliferation of breast cancer cells by inhibiting the activity of FOXO1.
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Funding: This work was supported by Natural science Foundation of Guangdong Province, China (grant numbers 2017A030313549, 2017) , Guangzhou Institute of Pediatrics/ Guangzhou Women and Children’s Medical Center (grant numbers YIP-2019-049); Shenzhen Science and Technology Project (grant numbers JCYJ20140414124506130, 2016; grant numbers, JCYJ20170307144612471, 2017). Disclosure of Potential Conflicts of Interest Conflicts of interest: none.
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Figure legends Fig. 1. ARF3 is up-regulated in breast cancer. (A) ARF3 analyses in 1,092 breast cancer tissues (Tumor) compared with 111 normal breast tissue samples (Normal) in TCGA profile. ** P < 0.01 versus Normal. (B) ARF3 mRNA analyses in 111 paired breast tumor tissues (Tumor) and their adjacent normal tissues (Normal) in TCGA profile. ** P < 0.01 versus Normal. (C) RT-qPCR analysis of ARF3 expression in normal breast epithelial cells (NBEC1, NBEC2) and breast cancer cell lines (T47D, MCF-7, SKBR-3, ZR-75-30, MDA-MB-231, BT549). GAPDH gene was used as a normalized gene. Values are expressed as the means ± SD; n= 3. * p < 0.05 versus NBEC 1. (D) Western blot of ARF3 expression in normal breast epithelial cells (NBEC1, NBEC2) and breast cancer cell lines. β-actin was used as a loading control. (E) RT-qPCR analysis of ARF3 in five paired breast cancer tissues (T) and their adjacent normal tissues (ANT). P1 stand for Patient 1, and so on. GAPDH gene was used as a normalized gene. Values are expressed as the means±SD; * P < 0.05 versus ANT, respectively. (F) Western blot of ARF3 in five paired breast cancer tissues (T) and their adjacent normal tissues (ANT). β-actin was used as a loading control. Fig. 2. High ARF3 expression correlates with clinical characteristics and poor patient survival in breast cancer patients. (A) Representative images of ARF3 expression in normal breast tissues and breast cancer at different clinical stages. Magnification of the original images is 200×. (B) Quantification of the average mean optical density (MOD) for ARF3 in tissues at different stages. Values are expressed as the means±SD; * P < 0.05 versus Normal. (C) Kaplan–Meier overall survival curves for patients with breast cancer stratified by ARF3-high-expressing (93) and ARF3-low-expressing (74) cases. ** P < 0.01 versus ARF3-low. Fig. 3. ARF3 regulates the proliferation of breast cancer cells in vitro. (A) RT-qPCR analysis of ARF3 mRNA in indicated stable cell lines. Values are expressed as the means ± SD; n= 3. * P < 0.05 versus Vector & Ri-vector. (B) Western blot of ARF3 protein in indicated stable cell lines. β-actin was used as a loading control. (C) Cell growth curve by MTT assays. (D) Representative image of colony formation assay. (E) Mean count of colony numbers in colony formation assay. *P < 0.05. (F) Representative micrographs in anchorage-independent growth assay. (G) Average colony (>0.1 mm/colony) numbers of three independent experiments in the anchorage-independent growth assay. Each bar represents the mean of 16
three independent experiments. Values are expressed as the means±SD; * P < 0.05 versus Vector & Ri-vector, respetively. Fig.4. ARF3 promotes cell growth rate in vivo. (A) Images of excised tumors from five NOD/SCID mice 18 days after injection with indicated cells. M1 stand for mouse 1, and so on. (B) Measured tumor volumes at corresponding time points. (C) Average weight of excised tumors. *P < 0.05. Each bar represents the mean of three independent experiments. Values are expressed as the means±SD; * P < 0.05 versus Vector & Ri-vector, respetively. Fig. 5. ARF3 promotes cell-cycle G1-S transition. (A) Flow cytometric analysis of indicated cells, Ri represents RNAi, 2N represents amphiploid, 4N represents fortetraploid. Values are expressed as the means±SD; n= 3. * P < 0.05 versus Vector & Ri-vector, respetively. (B) Representative micrographs of BrdU incorporation in indicated cells. (C) Average quantification of BrdU incorporation in indicated cells from three independent experiments. Each bar represents the mean of three independent experiments. Values are expressed as the means±SD; * P < 0.05 versus Vector & Ri-vector. (D) Western blot analysis G1-S-transition-associated genes in indicated cells. β-actin was used as a loading control. (E) RT-qPCR analysis of key negative regulator p21Cip1and p27Kip1 in indicated cells. Each bar represents the mean of three independent experiments. Values are expressed as the means±SD; n= 3. * P < 0.05 versus Vector & Ri-vector. Fig. 6. FOXO1 activity is modified by ARF3. (A) Relative FOXO1 reporter activity in the indicated cells. Values are expressed as the means±SD; n= 3. * P < 0.05 versus Vector & Ri-vector. (B) Western blot analysis of phospho-FOXO1 and total-FOXO1. β-actin was used as a loading control. (C) Represented images of immunofluorescence staining. (D) Western blot analysis of phospho-FOXO1 and total-FOXO1. (E) Cell growth curve by MTT assays. (F) Average quantification of BrdU incorporation in indicated cells from three independent experiments. Each bar represents the mean of three independent experiments. * P < 0.05.
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Declaration of interests √ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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