Cancer Letters 482 (2020) 74–89

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Cancer Letters

journal homepage: www.elsevier.com/locate/canlet

Original Articles ETV4 is a theranostic target in clear cell renal cell carcinoma that promotes metastasis by activating the pro-metastatic FOSL1 in a PI3K-AKT T dependent manner

Liang Xua,b,1, Hao Huc,d,1, Li-Sheng Zhenga, Meng-Yao Wange, Yan Meia, Li-Xia Penga, Yuan-Yuan Qiangf, Chang-Zhi Lia, Dong-Fang Menga, Ming-Dian Wanga, Zhi-Jie Liua, ∗ Xin-Jian Lig, Bi-Jun Huanga, Chao-Nan Qiana,h, a State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, 510060, Guangdong, China b Institute of Gastroenterology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510655, Guangdong, China c Department of Traditional Chinese Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510080, Guangdong, China d Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China e Department of Radiation Oncology, Affiliated Cancer Hospital ＆ Institute of Guangzhou Medical University, Guangzhou, 510095, Guangdong, China f Ningxia Medical University, Ningxia Key Laboratory for Cerebrocranical Disease, Yinchuan, 750001, Ningxia, China g CAS Key Laboratory of Infection and Immunity, CAS Centre for Excellence in Bio-macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China h Department of Nasopharyngeal Carcinoma, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, Guangdong, China

ARTICLE INFO ABSTRACT

Keywords: Distant metastasis is the major cause of short survival in ccRCC patients. However, the development of effective ccRCC therapies for metastatic ccRCC is limited. Herein, we reported that ETV4 was selected from among 150 relevant Metastasis with in vivo evidence of promoting metastasis. In this study, we identified that ETV4 promoted ccRCC cell migration and metastasis in vitro and in vivo, and a positive correlation between ETV4 and FOSL1 expression ETV4 was found in ccRCC tissues and cell lines. Further investigation suggested that ETV4 increase FOSL1 expression FOSL1 through direct binding with the FOSL1 promoter. Furthermore, ETV4/FOSL1 was proved as a novel upstream and downstream causal relationship in ccRCC in an AKT dependent manner. In addition, both ETV4 and FOSL1 serve as an independent, unfavorable ccRCC prognostic indicator, and the accumulation of the ETV4 and FOSL1 in ccRCC patients result in a worse survival outcome in ccRCC patients. Taken together, our results suggest that the ETV4/FOSL1 axis acts as a prognostic biomarker and ETV4 directly up-regulates FOSL1 by binding with its promoter in a PI3K-AKT dependent manner, leading to metastasis and disease progression of ccRCC.

1. Introduction chemotherapy [7,8]. Surgery is the main approach to treat early stage (organ-confined) kidney cancer and is often used in this setting [9–11]. According to the most recent epidemiological statistics, kidney However, like many types of cancers, there is no curative treatment for cancer represents 2.2% of all malignant diseases in adults [1,2]. Com- late stage and metastatic ccRCC [12,13]. pared with other cancers, it remains relatively rare; however, the ETV4 is a member of the E26 transformation-specific (ETS) family of worldwide incidence and mortality rates are reported to be steadily transcription factors. This family has highly similar DNA-binding ETS rising, by approximately 2–3% per decade [3–5]. Clear cell renal cell domains that recognize a purine-rich GGAA core motif within the carcinoma (ccRCC) accounts for 75% of all kidney cancers and is the promoters and enhancers of various target genes [14,15]. The ETS most lethal of all the genitourinary tumors, accounting for most ma- transcription factor family consists of 28 genes in humans [16,17] and lignant kidney tumors [6]. CcRCC is also resistant to radiotherapy and regulates a variety of biological and pathological processes [18,19],

∗ Corresponding author. Department of Experimental Research, State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, 651 Dongfeng East Road, Guangzhou, 510060, Guangdong, China. E-mail address: [email protected] (C.-N. Qian). 1 These authors contributed equally and are co-first authors for this work. https://doi.org/10.1016/j.canlet.2020.04.002 Received 12 January 2020; Received in revised form 22 March 2020; Accepted 2 April 2020 0304-3835/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). L. Xu, et al. Cancer Letters 482 (2020) 74–89 including cellular proliferation, differentiation, lymphocyte develop- of medium were seeded into a 96-well plate (Corning, 3599) and cul- ment and activation, transformation, and apoptosis, by recognizing the tured under normal conditions. At various time points after seeding, the GGAA core motif in the promoter or enhancer of their target genes cells in each well were stained with 20 μl of MTS (Promega, G3580) for [20,21]. Moreover, ETS-domain usually function in colla- 4 h, and the absorbance value 490 (OD490) was determined with a boration with other transcription factors [22,23]. As an ETS-oncogene microplate reader. All experiments were independently repeated at family transcription factor, ETV4 was cloned via its ability to bind the least three times. adenovirus E1A enhancer element [24] and the gene is located on 17q21. More recently, ETV4 was reported to be overexpressed, and its 2.3. RNA extraction and qPCR expression correlates with malignant aggressiveness and poor prog- nosis, in various malignant tumors [23,25]. The function of ETV4 re- The targets of the ETV4 siRNAs were 5′- GCGUUGUCCCUGAGAA mains poorly characterized and its pathological significance needs to be AUUTT -3’ (si#1) and 5′-GCUGGAUGACCCAACAAAUTT -3’ (si#2) determined. from FulenGen Co. Ltd. These procedures were performed as previously Accumulating evidence shows that cancer involves dynamic described [52]. Briefly, total RNA was extracted from cultured cell lines changes in the genome [26,27]. Whereas cancer metastasis is the using the Trizol reagent (Sigma, T9424) according to the manufac- leading cause of death in all types of malignancies tumors [28,29], and turer's instructions and subjected to reverse transcription using a cDNA accounts for the vast majority of cancer-associated deaths, this com- Synthesis Kit (Thermo, K1622), q-PCR was performed using an SYBR plicated process remains the least understood aspect of cancer biology FAST universal qPCR Kit (Vazyme, Q311-02). The relative expression [25,30]. Stated differently, the overwhelming majority of cancer-asso- levels of the target genes were calculated as two power values of ΔCt ciated deaths (about 90%) are caused by metastatic disease rather than (the Ct of β-actin or ETV4 minus the Ct of the target gene). The primers primary tumors [31,32]. It is believed that cancer metastasis is a mul- are listed in supplementary 1. tistep process involving complex genetic alterations that drive the pri- mary tumor to convert into a highly malignant and metastatic tumor 2.4. The transwell assays [33–35]. Many transcription factors play crucial roles in regulating cancer metastasis. For instance, cancer metastasis is inhibited by For the transwell migration assay, 3 × 104 cells in 200 μl of serum- but promoted by ZEB1 [36,37]. Metastatic renal cell carcinoma (mRCC) free DMEM was added to the cell culture inserts with an 8-μm micro- is one of the most treatment-resistant malignancies, whose outcomes porous filter without extracellular matrix coated (Corning. 3428). are inferior and the median survival after diagnosis is less than one year 800 μl DMEM medium containing 10% FBS was added to the bottom [38,39]. About 25–30% of patients with ccRCC have metastatic disease chamber. After 22 h of incubation, the cells in the lower surface of the at diagnosis, and fewer than 5% have solitary metastasis [40,41]. Fre- filter were fixed, stained with 1% crystal violet (TargetMol, T1343L), quent sites of ccRCC metastasis include the lung parenchyma (in and examined. Both experiments were repeated independently three 50–60% of patients with metastases), bone (in 30–40%), liver (in times. 30–40%) and the brain (in 5%) [42,43]. In the present study, we hypothesized that ETV4 might function as 2.5. Colony-formation assays an oncogene and additionally promote metastasis in ccRCC. Therefore, we aimed to explore the molecular mechanisms regulating this phe- For the colony-formation assays, 1000 cells/2 ml were seeded into a nomenon. By performing a series of in vitro and in vivo experiments to 6-well plate (Corning, 3516). The culture medium was subsequently test this hypothesis, we determined the crucial role of ETV4 in ccRCC changed every 3 days. After 2 weeks, the cells were washed with metastasis. Our data demonstrated that FOSL1 is a candidate down- phosphate-buffered saline (PBS), fixed with methanol for 15 min at stream target gene of ETV4 in ccRCC, and many evidence indicates that room temperature, and stained with 1% crystal violet for half an hour. aberrant AP-1 activity is implicated in the pathogenesis of a number of The colonies were counted. All experiments were independently re- diseases, including tumorigenesis [44–46], inflammation [47,48], and peated at least three times. infection [49]. More importantly, elevated ETV4 and FOSL1 expression in primary ccRCC were both independent unfavorable prognostic fac- 2.6. Western blotting tors for overall survival (OS) in patients. In this study, we demonstrate that ETV4 plays a key role in ccRCC metastasis by directly up-regulating Western blotting was performed using the standard protocol. FOSL1, and that this novel axis of ETV4/FOSL1 may be valuable to Briefly, cells were lysed in RIPA buffer (150 mM NaCl, 0.5% EDTA, develop new strategies for treating ccRCC patients with metastasis. 50 mM Tris, 0.5% NP40) containing a protease inhibitor cocktail (Roche, 11697498001) and phosphatase inhibitor mixture (TargetMol, 2. Materials and methods CC0004). concentrations were assessed using the BCA Protein Assay Kit (Absin, 9232). Equal amounts of protein mixed with sample 2.1. Cell lines and cell culture loading buffer (TaKaRa, 9157) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Sigma, The human ccRCC cell lines 1932, A498, Caki-1, Caki-2, ACHN, 3010040001). After blocking with 5% skim milk or BSA in Tris-buffered SN12C and HCC cell lines SK, Huh7, 7721, 7402, 7703, 97L, LM3, 3B, saline-Tween-20 (TBST), the membranes were incubated with the pri- G2, LO2 were routinely cultured in Dulbecco's Modified Eagle Medium mary antibodies overnight at 4 °C and then with the horseradish per- DMEM (Gibco, c11995500bt) supplemented with 10% fetal bovine oxidase-conjugated secondary antibodies at room temperature for 1 h. serum (Invitrogen, 10099141) as well as penicillin (bioss, bs-10687PA- The protein bands were visualized using a chemiluminescence kit 1). All cells were grown at 37 °C in a humidified incubator containing (BioVision, BIV-K4005-500). GAPDH and β-Actin were used as controls 5% CO2. The STR reports of cell line authentication were listed in for equal protein loading. The primary antibodies, including anti-ETV4 supplementary 3–5. (Santa Cruz, sc-113), anti-GAPDH (Proteintech, 60004-1-1g), anti-β- Actin (Proteintech, 60008-1-1g), anti-FOSL1 (Affinity, DF3096), anti-E- 2.2. Cell proliferation assay cadherin (CST, 3195), anti-N-cadherin (CST, 13116), anti-Vimentin (CST, 5741), anti-slug (CST, 9585), anti-Desmoplakin (Proteintech, The colorimetric MTS assays (Cell Titer 96 Aqueous One Solution 25318-1-AP), anti-AKT (CST, 4685), anti-P-AKT-S473 (CST, 4060), Cell Proliferation Assay) were performed to determine cancer cell anti- P-AKT-T308 (CST, 13038) and the anti-mouse and anti-rabbit growth and viability as previously reported [50,51]. 1000 cells/200 μl peroxidase-conjugated secondary antibodies were purchased from Cell

75 L. Xu, et al. Cancer Letters 482 (2020) 74–89

Fig. 1. Elevated ETV4 mRNA level correlated with shorter overall survival, relapse-free survival in ccRCC and HCC patients. (A).The 11 overlapped genes are commonly associated with a poor prognosis both in ccRCC and HCC cohorts. (B). Among the 11 overlapped genes, 3 genes are driver genes (TF), 8 genes are passenger genes, respectively. (C) and (D).The overall survival rate and the relapse-free survival rate were both significantly higher in the low ETV4 group in ccRCC and HCC cohorts. (E) and (F). Time-to-death analysis of ETV4 in the ccRCC and HCC TCGA data set.

Signaling Technology (Danvers, MA). 2.8. The chromatin immunoprecipitation (ChIP) assay

All of the procedures were following the Magna ChIP™ A- 2.7. Lentiviral transduction studies Chromatin Immunoprecipitation Kit (Millipore, 17–610). 10 cm plates for each cell line to be tested were seeded with cells that were allowed Cell lines stably expressing ETV4 short hairpin RNA (shRNA) or a to grow to 70–80% confluence (about 3 x 106 per plates). 400 μl of 37% negative control shRNA were purchased from FulenGen Co. Ltd. formaldehyde (Amresco, 0493-1) was added to 20 ml of growth media (Guangzhou, China). Lentivirus-expressing ETV4 shRNAs were pro- for crosslinking, and the dish was gently swirled and incubated at room duced after co-transfection of 293T cells with PLKD-CMV vectors car- temperature for 10 min 2 ml of 10 X Glycine (Millipore, 20–282) was rying the shRNAs. According to the manufacturer instructions. added to each dish to quench the unreacted formaldehyde, and the Infectious lentiviruses were harvested 48 h after transfection and fil- dishes were swirled and incubated at room temperature for 5 min. After tered through the 0.45 μm filter (Bedford, MA). Cells were transduced the formaldehyde fixation and PBS washing, 2 ml of cold PBS con- with lentiviruses ETV4 shRNA or negative control shRNA, then cultured taining 1X Protease Inhibitor Cocktail II was added to each dish to in medium containing 3 mg/ml puromycin (Biofroxx, 1299MG025) for avoid degradation, and cells were scraped from each dish into a conical 4 days to be used for selection and ETV4 knockdown efficiency was tube. The tube was spun at 1000 x g at 4 °C for 5 min to pellet the cells. determined by Western blotting. The full-length cDNAs of human ETV4 After the supernatant was removed, each cell pellet was resuspended were cloned into the pLenti-CMV vector. ETV4 was constructed into the into 1 ml of SDS Lysis Buffer (Millipore, 20–163) containing 1X pLenti-CMV vector by inserting a FLAG tag into the ETV4 N-terminal. Protease Inhibitor Cocktail II (Millipore, 20–283), and the suspension Infectious lentiviruses were harvested 48 h after transfection and fil- was sonicated to shear the DNA. 60 μl of Protein G Agarose (Millipore, tered through a 0.45 μm filter (Bedford, MA). Cells were transduced 16-201D) was added for each IP, and the tubes were incubated for 1 h with lentiviruses containing ETV4 or the vector and cultured in medium at 4 °C with rotation. The ETV4 antibody (Santa cruz, sc-113x) was containing 3 μg/mL puromycin for 4 days. Real-time PCR and western added to the supernatant fraction, and the fraction was incubated blotting were performed to evaluate the efficiency of ETV4 over- overnight at 4 °C with rotation. 60 μl of Protein G Agarose was added to expression. The ORF sequence information for ETV4 (EX-T8074-Lv121) each IP, and the fractions were incubated for 1 h at 4 °C with rotation. is described in supplementary 1. Finally, the immune complexes were extracted (1 X TE containing 20% The targets of ETV4 shRNA-1 and shRNA-2 were 5′- GCGGTAGGC SDS) and processed by reverse crosslinking, proteinase K (Millipore, GTGTACGGT' and 5′- ATCCACGCTGTTTTGACC -3′, respectively. Real- 20–298) digestion, and DNA precipitation. Purified DNA was re- time PCR and immunoblotting were performed to evaluate ETV4 suspended in TE buffer (Millipore, 20–157) for PCR. The primers used knockdown efficiency. for qPCR amplification are described in supplementary 1.

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Fig. 2. ETV4 promotes migration in ccRCC cell lines. (A) and (B). The relative mRNA and protein levels were determined by qPCR and immunoblotting analysis, GAPDH was used as a loading control. Showing that ETV4 is differentially expressed in ccRCC cell lines. (C) and (E). Transient suppression and overexpression of ETV4 in ccRCC cell lines were determined by immunoblotting analysis, GAPDH was used as a loading control. (D) and (F). Cell proliferation were determined by the MTS assay. (G) and (H). Representative micrographs of colony formation ability in overexpression and suppression of ETV4. (I) and (J). Suppression of ETV4 dramatically reduced the migration ability of the cells as determined by a wound-healing assay. Yellow dashed lines denote the margins of the wound. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.9. The luciferase assay relationship between ETV4 and FOSL1 expression and clin- icopathological characteristics. The Spearman correlation test (2-tailed) 293T cells were seeded into 96-well plates at the density of was used to calculate the correlation coefficient (r) and p value between 5000 cells per well, to normalize transfection efficiency, cells were also the ETV4 and FOSL1 expression, or between the ETV4 and insulin like co-transfected with the pRL-CMV Renilla luciferase (ZoomaBio, growth factor binding protein 2 (IGFBP2) expression. The significance ZK489), and co-transfected with Firefly luciferase plasmid FOSL1 pro- of several variables for survival was analyzed by using the Cox re- moter (Transheep, TSB-20171906-1), ETV4 plasmid (OBiO, gression model in a multivariate analysis. The p-value < 0.05 was HYFW20170717001), using the X-tremeGENE™ HP DNA (Roche, considered statistically significant in all cases. The authenticity of this 6366546001), in transfection reagent according to the manufacturer's article has been validated by uploading the key raw data onto the protocols. The ratio of ETV4 plasmid: FOSL1 luciferase: Renilla: trans- Research Data Deposit public platform (www.researchdata.org.cn), fection reagent was 0.2ug: 0.2ug: 0.004ug: 0.25uL (per well), every test with the approval RDD number as RDDB2020000832. with 5 duplicates. Plasmid luciferase activities were normalized against the Renilla luciferase activity of a co-transfected internal control plasmid. Cells were harvested 48 h after transfection and luciferase 3. Results activities measured by using the Dual-Luciferase Reporter Assay System (Promega, E1910). The final data = Firefly luciferase/Renilla luci- 3.1. ETV4 was selected for its significant prognostic role in ccRCC and HCC ferase. Transfection and Luciferase assay was performed in duplicate and repeated independently at least three times. The FOSL1 promoter A pro-metastatic gene signature, containing 150 genes that can sequence is listed in supplementary 2. promote metastasis, was collected by filtering the literature that pre- sented convincingly in vivo evidence [53]. When we re-analyzed these 150 genes, 11 of them overlapped in both ccRCC and HCC tumor types 2.10. Animal experiments (Fig. 1A). These 11 overlapping genes were BIRC5 (Survivin), CXCL1, CXCL8 (IL8), , ETV4, EZH2, MMP1, MMP9, MYB, PTTG1, and All animal experiments were performed in accordance with the YBX1. Among them, three genes transcription factors: E2F1, protocols approved by the Research Animal Resource Center of Sun Yat- ETV4, and MYB (Fig. 1B). In consideration of that transcription factors Sen University (IACUC approval number: L201501077). All animal are potential drivers of cancer metastasis, and there is many literature studies were conducted in accordance with the principles and proce- have reported the function and mechanisms of E2F1 [54,55] and MYB dures outlined in the guidelines of the Institutional Animal Care and [56–58] in many different types of cancer. In our study, we also found Use Committee at Sun Yat-Sen University Cancer Center. Male athymic out E2F1 and MYB was significant in ccRCC and HCC survival outcome mice between 4 and 5 weeks of age were obtained from the Shanghai ( Figs. S1A–B). However, the mechanism of ETV4 remains poorly un- Institutes for Biological Sciences (Shanghai, China) and maintained derstood [59], so we focused our attention on ETV4, one of the ETS under a specific pathogen-free environment. family members. Other ETS family members (FLI1, ERG, ETV1/ER81, For the tumor xenograft experiments, all nude mice were randomly FEV) also play key roles in cancer metastasis [14,60,61]. TCGA data- divided into 3 groups of 12 mice each, respectively are control, KD#1, base analyzed that high ETV4 expression was associated with sig- KD#2 (there was one mouse died during the injection process in control nificantly shorter time to death (Fig. 1E and F), indicating a potential group). Tumor cells (1 x 106 cells) were suspended in 50 μl phosphate- role of ETV4 in ccRCC and HCC cancer progression. The survival curves buffered saline (PBS) and intravenously injected into the tail vein of of OS and RFS in ccRCC or HCC are presented in Fig. 1C and D. In the each mouse. The mice were monitored twice per week. Euthanized 3 Kaplan-Meier analysis, OS and RFS were much longer for the patients months after injection of the cancer cells, and distant metastases in with lower ETV4 expression than in those with higher ETV4 expression. lungs were assessed and counted. Lungs were excised and embedded in This indicated that ETV4 expression significantly correlated with paraffin for further study. shorter OS and RFS in patients with the two types of cancer. Moreover, different pathological stages, neoplasm histological grades, and patho- 2.11. Wound healing assay logical T stages were associated with the differential expression of ETV4 (Figs. S5A, S5C, and S5E). Taken together, these analyses revealed that Cells were seeded into 6-well plate and cultured until 90% con- a high ETV4 level is significantly associated with more aggressive tu- fl uence. After 24 h starvation of the cells in serum-free medium, we mors. Therefore, we hypothesized that ETV4 might play a crucial role in μ fi used a sterile 200 l pipette tip to create arti cial wounds in the cell ccRCC and HCC tumorigenesis and progression. fl monolayer, and the oating cells were removed by washing with PBS. To determine the characteristics of the ETV4 alteration in ccRCC Respective images were captured at 0 h and 24 h by using an inverted and HCC, we used the TCGA database and revealed the differential fi microscope. The width of the scratch was quanti ed in microscopic distribution of ETV4 between ccRCC and HCC (Fig. S1). The alteration images for evaluating the migration ability of the cells. rate of ETV4 in ccRCC and HCC is 2.5% and 1.6%, respectively (Figs. S1E, H, and J). Most alteration of ETV4 was mutated in ccRCC, while in 2.12. Statistical analysis HCC most alteration of ETV4 was amplification. Owing to the limited number of mutation group, ETV4 mutation groups and non-mutation All statistical analyses were performed using the SPSS (version 24) group showed no significant in OS and RFS (Figs. S1F, G, and K), but package. Student's t-test was used to compare two independent groups there was a trend to be significant. As shown in Fig. S1I, there was an of data, data are represented as the mean ± S.D. Optimized cut-off illustration of the distribution of ETV4 different mutated sites in ccRCC. value was used to divide the patients into low and high ETV4 and Above all, it indicated that ETV4 alteration in ccRCC and HCC were FOSL1 expression groups. Chi-squared tests were applied to analyze the quite di fferent, and there is a high possibility for ETV4 mutation to have

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Fig. 3. ETV4 promotes EMT and distant metastasis in vitro and in vivo. (A). Localization of E-cadherin in A498 vector, A498 overexpressed cells by confocal immunofluorescence analysis ( × 400). (B). Localization of E-cadherin in SN12C control, SN12C knockdown si#1 and si#2 cells by confocal immunofluorescence analysis ( × 400). (C) and (D). Transient suppression and overexpression of ETV4 in ccRCC cells and whole-cell lysates were immunoblotted for Slug, N-cadherin, Vimentin, E-cadherin, Desmoplakin at 72 h after transfection. GAPDH was used as a loading control. (E) and (H). Silencing ETV4 could inhibit cell migration in ACHN cells compared with negative control cells. Data represent the mean ± S.D. *p < 0.05. (G). Histological image of lung metastasis in nude mice after tail vein injection of ACHN cells. (F) and (I). Suppression of ETV4 decreased the metastatic rate of ACHN cells in vivo. Data represent the mean ± S.D. *p < 0.05.

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Fig. 4. FOSL1 is a candidate downstream molecule in ETV4 signaling. (A).Overlapped the differentially expressed genes from 3 data sets, respectively are ChIP- seq dataset, pro-metastatic genes dataset, and co-expression genes dataset, FOSL1 and IGFBP2 were identified to be candidate target genes in ccRCC. (B).The correlation between candidate target genes and ETV4 in normal and tumor tissues. (C) and (D). The overall survival and relapse-free survival rate of FOSL1 and IGFBP2 were significantly higher in the low FOSL1 and IGFBP2 groups. (E) and (F). The time to death of low and high expression groups in IGFBP2 and FOSL1 genes. Data represent the mean ± S.D. *p < 0.05, **p < 0.01. (G). The mRNA levels of ETV4 in ccRCC tissues and noncancerous tissues, p values were calculated using the Student t-test. Data represent the mean ± S.D. **p < 0.01, ***p < 0.001. (H). ETV4 expression were positively corrected with FOSL1 and IGFBP2 expression in ccRCC tissues. A significant positive correction between ETV4 expression and FOSL1 expression was found. (I) and (J). Combined Kaplan-Meier analysis indicated up-regulation of ETV4 and FOSL1 were significantly associated with poorer overall survival and relapse-free survival of ccRCC patients.

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Table 1 in the ccRCC cell lines (Fig. 2A and B). To explore the role of ETV4 in Association between expression of FOSL1 and clinical characteristics in ccRCC cell growth, we transfected ACHN and SN12C cell lines with siRNAs patients. (ETV4 si#1 and si#2) or a negative control siRNA. The siRNA sup- ffi fi Characteristics No. FOSL1 expression P value pression e ciency of ETV4 protein levels was con rmed by using im- munoblotting (Fig. 2C). We observed that ETV4 suppression did not Low High inhibit ccRCC and HCC cell proliferation (Fig. 2D and Fig. S2C). Meanwhile, we transfected cell lines (A498 and 1932, with a low basal Gender Female 181 112 69 0.200 expression of ETV4) and overexpressed ETV4. The ETV4 protein level Male 340 196 144 was confirmed by using immunoblotting (Fig. 2E). Overexpression of Age ETV4 in A498 and 1932 cells did not affect ccRCC cell proliferation as ≤ 55 178 103 75 0.104 well (Fig. 2F). Moreover, suppression and overexpression of ETV4 also > 55 343 205 138 ff – T stagea have no e ect on colony formation ability (Fig. 2E H). We performed

T1-2 332 207 125 0.026 wound-healing assays, showed that ETV4 suppression dramatically re- T3-4 187 101 86 duced the migratory ability of the cells (Fig. 2I and J). It indicated that N stage ETV4 specifically work on promoting migration in ccRCC cell lines. N0 237 153 84 0.127

N1-X 284 155 127 M stage

M0 420 250 170 0.039 3.3. ETV4 promotes EMT and distant metastasis in vitro and in vivo

M1 101 58 43 Clinical stage To examine the causal role of ETV4 in ccRCC metastasis, we per- I-II 315 197 118 0.059 fl III-IV 206 111 95 formed confocal immuno uorescence assay, the analysis of ETV4 Gradea knockdown and overexpression cells revealed that ETV4 have an effect I-II 237 146 91 0.439 on E-cadherin expression significantly. We found that suppression of III-IV 276 155 121 ETV4 expression resulted in the downregulation of the levels of EMT- a Recurrence promoting factors, such as Slug, N-cadherin, and Vimentin, and upre- No 315 303 112 0.001 Yes 205 104 101 gulation of epithelial markers, such as E-cadherin and Desmoplakin Deatha (Fig. 3C). Conversely, ETV4 overexpression resulted in the upregulation No 356 233 123 < 0.0001 of N-cadherin and Vimentin, and downregulation of E-cadherin and Yes 164 74 90 Desmoplakin in A498 and 1932 cell lines (Fig. 3D). Similar results were obtained in the HCC cell line SK (Fig. S2E). Taken together, these data a Means some data are not available. suggested that ETV4 could induce EMT in ccRCC and HCC cell lines. To confirm this hypothesis, we performed transwell assay, which significant in OS and RFS outcome only if patients’ numbers double or showed that ETV4 suppression dramatically reduced the migratory triple. ability of the cells (Fig. 3E–H). To validate the metastatic function of To investigate the role of ETV4 in ccRCC progression, we further ETV4 in ccRCC cell lines in vivo, ETV4 KD #1, ETV4 KD #2, and control evaluated the clinical implications of ETV4 expression by using the cells were injected into the tail veins of nude mice. After 9 weeks, the TCGA database for 520 ccRCC samples. The correlations between ETV4 mice were euthanized and their lungs were excised and examined. The expression and clinicopathological characteristics are presented in number of metastatic lung nodules was significantly lower in the KD #1 Table S1. A high level of ETV4 in primary tumors was significantly and KD #2 groups compared with that in the control group (Fig. 3F–I). correlated with the advanced T stage, M stage, grade, recurrence, and Using hematoxylin and eosin (H&E) staining, we found that the meta- high risk of death. Importantly, Multivariate analyses of different static nodules in the lung tissue of ETV4 KD#1 and ETV4 KD#2 mice prognostic parameters revealed that high ETV4 expression was an in- were fewer and smaller compared with those in the control mice dependent, unfavorable prognostic indicator for OS (Table S2). Taken (Fig. 3G). In clinical data, we found that the ETV4 mRNA was upre- together, these analyses revealed that high ETV4 expression levels in gulated in ccRCC primary tissues with lymph node metastases, while ccRCC significantly correlated with adverse patient outcomes. downregulated in ccRCC primary tissues without lymph node metas- tases (Fig. S3C). Kaplan-Meier analysis indicated that high ETV4 ex- 3.2. ETV4 promotes migration in ccRCC cell lines pression with metastasis group results in significantly poorer overall survival outcomes than low ETV4 expression without metastasis group To explore the roles of the transcriptional factors ETV4 in ccRCC (Fig. S3A). Taken together, these results demonstrated the importance progression. Firstly, we tested ETV4 expression in ccRCC cell lines. The of ETV4 in the promotion of ccRCC and HCC cell migration in vitro and mRNA and protein expression levels were differentially expressed metastasis in vivo. among different ccRCC and HCC cell lines (Figs. S2A and B), especially

Table 2 Univariate and multivariate analysis of different prognostic parameters in ccRCC patients.

Variables Univariate analysis Multivariate analysis

HR CI p value HR CI p value

Age 1.793 1.255-2.561 0.001 1.506 1.043-2.175 0.029 T stage 3.457 2.523-4.735 < 0.0001 –– – M stage 4.067 2.956-5.598 < 0.0001 –– – Clinical-stage 4.311 3.098-6.000 < 0.0001 2.051 1.406-2.991 < 0.0001 Grade 2.712 1.903-3.864 < 0.0001 1.534 1.050-2.241 0.027 Tumora 5.309 3.809-7.399 < 0.0001 3.457 2.396-4.989 < 0.0001 FOSL1 1.864 1.370-2.535 0.0001 1.912 1.390-2.630 0.001

a Tumor: person neoplasm cancer status. No: tumor free, Yes: with tumor.

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Fig. 5. The correlation of ETV4 and FOSL1 in cell lines and other tumor type tissues. (A). The clinical distribution of ETV4 and FOSL1 were analyzed by using the TCGA ccRCC data set. (B). The protein level were determined by western blotting showing that ETV4 and FOSL1 were correlatively expressed in ccRCC cell lines. (C), (E) and (G). Transient suppression of ETV4 in ACHN, ETV4 and FOSL1 mRNA level was detected by qPCR. Data represent the mean ± S.D. ***p < 0.0001. ETV4 and FOSL1 expression were measured by immunoblotting. (D), (F) and (H). Transient suppression of ETV4 in SN12C, ETV4 and FOSL1 mRNA level was detected by qPCR. Data represent the mean ± S.D. ***p < 0.0001. ETV4 and FOSL1 expression were measured by immunoblotting. (I) Localization of Vimentin in ACHN NC, ACHN si# 1 cells by confocal immunofluorescence analysis. (J). Suppression of FOSL1 dramatically reduced the migration ability of the cells as de- termined by a wound-healing assay. Yellow dashed lines denote the margins of the wound. (K). The quantified data of wound width of transwell. Columns, average of three independent experiments; bars, SD. *p < 0.05, Student t-test. (L) and (M). The migratory ability of overexpressed ETV4 and suppressed FOSL1 in ACHN cells. Data represent the mean ± S.D. *p < 0.05. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. FOSL1 is a candidate downstream molecule in ETV4 signaling FOSL1 were significantly associated with poorer OS and RFS of patients with ccRCC (Fig. 4I and J). Consistently, there was a positive correla- As mentioned before, our laboratory identified about 150 pro-me- tion between ETV4 and FOSL1 expression distribution in clin- tastatic genes from the literature that prominently serve as important icopathological characteristics (Fig. 5A). Based on ETV4 and FOSL1 regulators in other cancer types in vivo. Based on previous ChIP-se- expression level, we divide the patients into 3 groups, respectively were quence in literature [62] and microarray analyses by other researchers, low ETV4 and low FOSL1 group, high ETV4 and high FOSL1 group, low which identified more than 3000 binding sequences, after bioinfor- ETV4 and high FOSL1 or high ETV4 and low FOSL1 group. We found out matics analysis, we discovered 1350 genes, which included 21 tran- grade I and grade II most happened in low ETV4 and low FOSL1 group; scription factors (Supplementary 1) among the 3000 binding sequences. On the contrary, grade III and grade IV most happened in high ETV4 The application of a variety of high-throughput technologies has ac- and high FOSL1 group (Fig. S3D). It indicated that both high expres- cumulated big data, which enables researchers to perform further re- sions of ETV4 and FOSL1 could result in poor survival outcome in levant analyses. Using C-bioportal (https://www.cbioportal.org/), it is ccRCC. Taken together, the results suggested that the ETV4/FOSL1 axis a widely used database, we identified 250 genes that are significantly is clinically relevant in patients with ccRCC. co-expressed with ETV4 in ccRCC. Thus, we overlapped the differen- tially expressed genes from three different groups comprising pro-me- tastatic genes, ChIP-sequence data, and the co-expressed genes 3.5. The effects of down-regulating ETV4 on ccRCC metastasis depend on (Fig. 4B), which identified two differentially expressed genes: FOSL1 the down-regulation of FOSL1 and IGFBP2 (Fig. 4A). We were trying to verify whether these two candidate genes were Interestingly, the immunoblotting results showed there is also a the direct target for ETV4 in ccRCC. By reading literature, we found out positive correlation between ETV4 and FOSL1 in ccRCC and HCC cell insulin like growth factor binding protein 2 (IGFBP2) can drive the EMT lines (Fig. 5B and S2B). FOSL1 expression was decreased in both ACHN and is associated with the invasive character of pancreatic ductal ade- and SN12C cells after ETV4 suppression at both the mRNA and protein nocarcinoma [62–64]. Previous studies also showed that AP-1 tran- levels (Fig. 5C and D and 5G-H). The decrease in FOSL1 expression at scription factor subunit (FOSL1) promotes metastasis in breast cancer the mRNA and protein levels after the suppression of ETV4 was vali- [65,66]. Subsequently, we carried out FOSL1 and IGFBP2 bioinfor- dated by using qPCR and western blotting, respectively (Fig. 5E and F). matics analysis from the TCGA database, which showed that the OS and The function of FOSL1 in ccRCC has not been reported, to find out RFS rate was significantly higher in the low IGFBP2 group compared to more about downstream target gene of ETV4, we performed wound high IGFBP2 group (Fig. 4C). The relative expression level of IGFBP2 healing and immunofluorescent staining assays in ACHN cells. The re- was lower expression in ccRCC tissues compared with the noncancerous sults showed suppressing FOSL1 could decrease the expression of vi- tissues (Fig. 4G), there was a positive correlation between ETV4 and mentin (Fig. 5I), which is a mesenchymal marker, as well as reduce the IGFBP2 expression distribution in clinicopathological characteristics migratory ability of the cells (Fig. 5J). To dig out whether FOSL1 is the (Fig. S3E), a weak positive correlation between ETV4 and IGFBP2 also necessary target gene for ETV4 to promote metastasis, we over- found in the TCGA dataset (Fig. 4H). Meanwhile, univariate and mul- expressed ETV4 and then suppressed FOSL1 simultaneously. The tivariate analyses revealed that IGFBP2 acts as an independent and transwell assay showed that ETV4 overexpression could largely in- unfavorable prognostic indicator for overall survival in ccRCC (Table crease the number of migrated cells (Fig. 5L), which was consistent S3). with the above-mentioned data, while suppressing FOSL1 after over- By contrast, we further evaluated the clinical implications of FOSL1 expressing ETV4 could suppress the migratory ability of the cells expression using the TCGA dataset. The correlations between FOSL1 compared to the overexpressing ETV4 cells. It revealed that FOSL1 expression and clinicopathological characteristics are presented in knocking-down could reverse the function of ETV4 overexpression Table 1. A high level of FOSL1 in primary tumors was significantly upon promoting cellular motility. correlated with advanced T stage, M stage, recurrence, and high risk of To determine whether the ETV4/FOSL1 axis is important in other death. Multivariate analyses of different prognostic parameters re- cancers, we tested the ETV4 and FOSL1 mRNAs level. To determine vealed that high FOSL1 expression was an independent, unfavorable whether the ETV4/FOSL1 axis is important in our collected tissues, we prognostic indicator for overall survival (Table 2). Taken together, tested the ETV4 and FOSL1 mRNAs level in 34 ccRCC tissues, 36 Colon these analyses revealed that a high FOSL1 level in ccRCC correlated Cancer tissues, and 31 Nasopharyngeal Carcinoma (NPC) tissues. The significantly with poor patient outcomes. FOSL1 was associated with results showed a positive correlation between ETV4 and FOSL1 ex- poor survival (OS and RFS) (Fig. 4D). Whether in the metastatic group pression in ccRCC, COAD, and NPC tumor tissues (Fig. S5G). Moreover, or non-metastatic group, the high FOSL1 level can result in poor sur- there was a positive correlation between ETV4 and FOSL1 expression vival outcomes (Fig. S3B). The data showed a higher FOSL1 expression distribution in ACC, LGG, SARC cancers (Figs. S5H and I) and HNSC, in ccRCC tissues compared with the noncancerous tissues (Fig. 4G). LGG cancers (Fig. S5K). Taken together, the results showed that the Meanwhile, different pathological stages, neoplasm histological grades, ETV4/FOSL1 axis is important not just in ccRCC, other types of cancer and pathological T stages were associated with the differential ex- as well. The combined Kaplan-Meier analysis of ETV4 and FOSL1 pression of ETV4 (Figs. S5B, S5D, and S5F). And there was a strong showed similar results in ccRCC (Fig. 5J and Fig. S5L). Taken together, positive correlation between ETV4 and FOSL1 in ccRCC (Fig. 4H). In the these results showed there is a positive correlation between ETV4 and combined Kaplan-Meier analysis, both upregulations of ETV4 and FOSL1 in ccRCC cell lines and tissues.

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Fig. 6. ETV4 directly binds to and activates the FOSL1 promoter activity. (A).The indicated cells were analyzed by ChIP using an anti-ETV4 antibody as described in the Methods section. (B).Schematic illustration for FOSL1 promoter primers location. (C). The indicated cells that were stably transfected with vector or ETV4 as indicated were analyzed by ChIP. FOSL1 DNA was quantified by using DNA agarose gel electrophoresis and qPCR. (D).The FOSL1 DNA was quantified by using DNA agarose gel electrophoresis and PCR with different primers. (E). Schematic illustration of the luciferase activity for upstream ETV4 and downstream FOSL1. (F). The schematic illustration of the region described within wild-type FOSL1 promoter and its mutants at motif 1, 2, 3, 4, 5. (G) and (H). Overexpressed ETV4 and analyzed FOSL1-luciferase activity in 293T cells. GLuc activities in buffers with a stabilizer, Data represent the mean ± S.D.; *p < 0.05, **p < 0.01, ***p < 0.01.

3.6. ETV4 directly binds to and activates FOSL1 promoter 4. Discussion

Since ETV4 is a transcription factor, we surmised that the FOSL1 Metastatic RCC is one of the most treatment-resistant malignancies, promoter would be regulated by ETV4. To perform mechanistic re- reducing the life quality of patients and representing an important search, we used the chromatin immunoprecipitation (ChIP) assay. ETV4 socio-economic problem [67]. However, our knowledge concerning the was detected in cell lines stably overexpressing ETV4 via im- mechanism of mRCC development and metastasis are not complete. The munoblotting by using a specific anti-ETV4 antibody. Using the ChIP present recommendations are nephrectomy might be justified in pa- assay, we successfully pulled down ETV4 protein (Fig. 6A). Based on tients with metastatic disease when the intention is to improve their the FOSL1 promoter region and previous ChIP assay results, we de- quality of life by alleviating local symptoms. However, patients who signed 5 pair primers for PCR assay (Fig. 6B). The results showed that have solitary metastases at the time of their initial diagnosis generally the FOSL1 DNA motif could be analyzed by ChIP assays by using an have a poor prognosis, even when the primary and metastatic sites are anti-ETV4 antibody as described in the Methods section (Fig. 6C). both resected aggressively [68]. Nowadays, the main obstacle to the FOSL1 was detected by qPCR and normalized to the input level clinical management of ccRCC is metastasis. Thus, preventing, pre- (Fig. 6D). These observations indicated that ETV4 could bind to the dicting and inhibiting metastasis is crucial to improving treatment FOSL1 gene. In order to find out the specific binding sites, we used the outcomes. Jasper database (http://jaspar.binf.ku.dk/) to predict 5 possible In the present study, we confirmed that ETV4 is an oncogene and a binding sites (Figs. S4A and B). To verify the binding sites, we designed promising theranostic target for ccRCC. Moreover, elevated ETV4 ex- 5 binding sites that were mutated and then cloned into luciferase pro- pression in primary ccRCC was found to be an independent unfavorable moter plasmid. As shown in Fig. 6G, using a luciferase assay, we de- prognostic factor for patients (Tables S1–2). ETV4 is associated with termined the region of the FOSL1 promoter that was transcriptionally poor survival in patients with ccRCC and those with HCC. Therefore, we activated by ETV4. Furthermore, among the constructed mutant FOSL1 used two different cancer cell lines to verify ETV4's function in pro- promoters, we found that mutant 2 and mutant 3 were not the direct moting metastasis. Functional studies using both cancer cell lines binding sites, mutant 1 and mutant 4 were the effective mutants, and showed similar results (Fig. 2I–J and Figs. S2C–D), demonstrating that mutant 1 seems to be more important than mutant 4(Fig. 6H and I). ETV4 is not only associated with poor survival in patients with ccRCC Fig. 6E showed a schematic illustration of the layout of ETV4 target and HCC but also promoted metastasis in both cell lines. sites in the FOSL1 promoter. Fig. 6F showed a schematic diagram of the Transcription factors play vital roles in regulating cancer metastasis, binding region within the predicted binding site and its five mutants. In and individual transcription factors could have different or even op- summary. The data indicated that ETV4 directly binds to the FOSL1 posite functions among different cancer types [69,70]. Thus, it was promoter and activates the transcription of FOSL1, and there were two necessary to possess a suitable tool to easily elucidate the role of any binding sites in FOSL1 promoter when regulated by ETV4. transcription factor in metastasis for a certain cancer type. Thus, we used a combination of ChIP, TCGA database, and pro-metastatic genes filters. Based on previous ChIP-sequence analyses [62], 1350 genes 3.7. PI3K/AKT signaling triggers ETV4 promotes ccRCC cellular migration were identified as having a binding site with ETV4, 250 genes were and target downstream FOSL1 in positive feedback significantly co-expressed with ETV4, and 150 pro-metastatic genes were identified in our laboratory [53]. Among them, FOSL1 and IGFBP2 To further explore the possible underlying mechanism about ETV4 were the most di fferential target genes of ETV4 from the three over- and FOSL1, we treated with LY294002, it is a PI3K inhibitor, showed lapping groups. Based on clinical correlation analysis (Fig. 4H), we fi- LY294002 treatment decreased the phosphorylation of AKT. nally focused on FOSL1 for further analyses. Surprisingly, LY294002 treatment also can reduce the expression of The transcription factor FOSL1 controls both early and later events ETV4 and FOSL1 (Fig. 7A), Therefore, we speculated that ETV4 and in the establishment of distant metastases in breast cancer [66]. In the FOSL1 were downstream of PI3K/AKT pathway. To confirm this hy- present study, we showed that higher expression of FOSL1 is an in- pothesis, we examined the expression of the EMT marker after treated dicator of poor overall survival and relapse-free survival for patients with LY294002, it showed LY294002 treatment restored the expression with ccRCC (Table 1). Furthermore, elevated FOSL1 expression in pri- of epithelial markers and decreased the expression of the mesenchymal mary ccRCC was found to be an independent, unfavorable prognostic marker and EMT-promoting transcription factor in ETV4-over- factor for patients (Table 2), indicating that the FOSL1 level might be a expressing A498 and 1932 cells (Fig. 7B). The transwell assay showed good marker for patients with ccRCC. The analysis showed ETV4 was the LY294002 treatment restored the migratory ability in ETV4-over- positively correlated with FOSL1 in the clinical data (Figs. 4H and 5A). expressing A498 and 1932 cells (Fig. 7C and D, 7E and F). Our novel Moreover, the downregulation of ETV4 also decreased FOSL1 expres- findings are summarized in Fig. 8. These significant findings revealed sion at both the mRNA and protein levels (Fig. 5C-H). Interestingly, we that ETV4 and FOSL1 might be downstream of PI3K/AKT pathway and also found a correlation between ETV4 and FOSL1 in ccRCC cell lines at through this pathway, ETV4 and FOSL1 can promote metastasis in the protein level. In addition, the ChIP assay showed that FOSL1 is a ccRCC. direct downstream target gene of ETV4 because ETV4 could activate In summary, our study is the first to elucidate the crucial role of FOSL1 by directly binding to its promoter in ccRCC cells (Fig. 6). These ETV4 in promoting cancer metastasis and predicting unfavorable pa- data showed that ETV4 and FOSL1 are novel oncogenes and promising tient prognosis. Moreover, ETV4 severs as a transcription factor, it can theranostic targets for ccRCC. Moreover, the clinical data showed that binds to downstream target gene FOSL1 through PI3K/AKT pathway both of them could promote metastasis (Table 1 and Table S1). Previous signaling. We believe that ETV4 is a potential molecular target for studies [66,71] showed that both of ETV4 and FOSL1 could promote predicting, preventing and treating ccRCC metastasis.

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Fig. 7. ETV4 promotes ccRCC cellular migration and target downstream FOSL1 through PI3K/AKT pathway. (A). Western blotting analysis of p-AKT(S473), p- AKT(T308), t-AKT, ETV4 and FOSL1 expression levels after treated with LY294002. (B). Western blotting analysis of p-AKT(S473), p-AKT(T308), t-AKT, Desmoplakin, E-cadherin, N-cadherin and Vimentin expression levels after overexpressed ETV4 and then treated with LY294002. (C) and (D). Cells were treated with LY294002 or DMSO for 48h, and subjected to migration, representative images and quantification of LY294002 attenuate the effects of ETV4 overexpressing on the migratory abilities of A498 cells as determined by migration assays. Columns, average of three independent experiments; bars, SD. *p < 0.05, **p < 0.01, Student t-test. (E) and (F). Cells were treated with LY294002 or DMSO for 48h, and subjected to migration, representative images and quantification of LY294002 attenuate the effects of ETV4 overexpressing on the migratory abilities of 1932 cells as determined by migration assays. Columns, average of three independent experiments; bars, SD. *p < 0.05, Student t-test.

Fig. 8. Schematic illustration of the ETV4 directly binding to FOSL1 in PI3K-AKT dependent manner in ccRCC. metastasis in vivo and are probably associated with phosphatidylino- Consent for publication sitol-4,5-bisphosphate 3-Kinase (PI3)-kinase and Ras signaling. How- ever, although we showed that FOSL1 is a target gene of ETV4, that Not applicable. ETV4 is positively correlated with FOSL1 in the in vivo and clinical data, and that the combination ETV4 and FOSL1 can result in a worse Availability of data and materials survival outcome via promoting EMT, there is insufficient evidence to show that ETV4 directly binds to FOSL1 to cooperatively promote All data generated or analyzed during this study are included in this metastasis in ccRCC. published article and its supplementary information files. FOSL1 also known as Fra-1, it can enhance growth and metastasis of human prostate cancer cells through epithelial mesenchymal transition Funding pathway [72]. In epithelioid adenocarcinoma cells, FOSL1 has a proven function of regulating the expression of a subset of genes including This work was supported by grants from the National Natural mts1, HMGI (Y), MMP-3, MMP-9, uPA, uPAR, PAI-1, tPA, and TIMP [73]. Science Foundation of China (No. 81472386, No. 81672872, and No. The most pronounced effect has been found in HMGI(Y) expression. 81872384 to C.Q., No. 81972785, No. 81773162and No. 81572901to Ectopic FOSL1 expression can also induce multiple changes in the B.H.), the Science and Technology Planning Project of Guangdong genotype of malignant glioma cells including IGFBP6, JunB, IGFBP3, Province, China (No. 2014B020212017, No. 2014B050504004 and No. RBBP4, EGFR3, PLK, and Furin et al. [74]. About 21 relevant genes were 2015B050501005 to C.Q., and No. 2014A020209024 to B.H.), and the up-regulated, one of the most intriguing is the fact that junB is being Provincial Natural Science Foundation of Guangdong, China (No. highly up-regulated, and lack of junB has been documented to be the 2016A030311011 to C.Q.), and a research program from Sun Yat-sen only one to produce identical abnormality to FOSL1 knockout in mice. University (No. 84000-18843409 to C.Q.). While, in our study we find out the FOSL1 was a target gene of the ETV4, and both of them were the downstream genes of PI3K/AKT Authors’ contributions pathway. In this report, we have demonstrated for the first time that ETV4 is a Conceptualization: CN-Q, BJ-H, LS-Z, XJ-L. key player in ccRCC metastasis. Moreover, by directly upregulating pro- Methodology: LX, HH, LS-Z. metastatic gene FOSL1, the combination of high ETV4 expression and Software: LS-Z, HH, CZ-L. high FOSL1 expression correlated more significantly with overall sur- Validation: LX, HH. vival and relapse-free survival. This novel ETV4/FOSL1 axis may be Formal analysis: LX, MY-W. useful to develop new strategies to treat patients with ccRCC metastasis. Investigation: HH, DF-M. Resources: LX, YM, MY-W. Data curation: LX, LX-P, YY-Q. Declarations Writing (original draft preparation): LX, HH, LS-Z. Writing (review and editing): CN-Q, BJ-H, LS-Z, XJ-L. The study was approved by the Ethics Committee of Sun Yat-sen Visualization: LX, MD-W, ZJ-L. University Cancer Center. Supervision: CN-Q, BJ-H, LS-Z.

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Project administration: CN-Q, BJ-H, LX-P. factor, Canc. Lett. 216 (2004) 1–8. Funding acquisition: CN-Q, BJ-H. [25] C.C. Benz, R.C. O'Hagan, B. Richter, G.K. Scott, C.H. Chang, X. Xiong, et al., HER2/ Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer, Oncogene 15 (1997) 1513–1525. Declaration of competing interest [26] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [27] J. Yokota, Tumor progression and metastasis, Carcinogenesis 21 (2000) 497–503. [28] P.S. Steeg, Targeting metastasis, Nat. Rev. Canc. 16 (2016) 201–218. The authors have declared no competing interest. [29] J.S. Vermaat, I.J. Nijman, M.J. Koudijs, F.L. Gerritse, S.J. Scherer, M. Mokry, et al., Primary colorectal cancers and their subsequent hepatic metastases are genetically ff Acknowledgments di erent: implications for selection of patients for targeted treatment, Clin. Canc. Res. 18 (2012) 688–699. [30] C.N. Qian, Y. Mei, J. Zhang, Cancer metastasis: issues and challenges, Chin. J. Canc. Not applicable. 36 (2017) 38. [31] A.W. Lambert, D.R. Pattabiraman, R.A. Weinberg, Emerging biological principles of metastasis, Cell 168 (2017) 670–691. Appendix A. Supplementary data [32] J.S. Vermaat, I.J. Nijman, M.J. Koudijs, F.L. Gerritse, S.J. Scherer, M. Mokry, et al., Primary colorectal cancers and their subsequent hepatic metastases are genetically ff Supplementary data to this article can be found online at https:// di erent: implications for selection of patients for targeted treatment, Clin. Canc. Res. 18 (2012) 688–699. doi.org/10.1016/j.canlet.2020.04.002. [33] L. Gomez-Cuadrado, N. Tracey, R. Ma, B. Qian, V.G. Brunton, Mouse models of metastasis: progress and prospects, Dis Model Mech 10 (2017) 1061–1074. References [34] D. Wirtz, K. Konstantopoulos, P.C. Searson, The physics of cancer: the role of physical interactions and mechanical forces in metastasis, Nat. Rev. Canc. 11 (2011) 512–522. [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer [35] C.L. Chaffer, R.A. Weinberg, A perspective on cancer cell metastasis, Science 331 statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 (2011) 1559–1564. cancers in 185 countries, Ca - Cancer J. Clin. 68 (2018) 394–424. [36] G. Moreno-Bueno, E. Cubillo, D. Sarrio, H. Peinado, S.M. Rodriguez-Pinilla, S. Villa, [2] J. Ferlay, M. Colombet, I. Soerjomataram, C. Mathers, D.M. Parkin, M. Pineros, et al., Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a et al., Estimating the global cancer incidence and mortality in 2018: GLOBOCAN distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition, sources and methods, Int. J. Canc. 144 (2019) 1941–1953. Canc. Res. 66 (2006) 9543–9556. [3] W.H. Chow, S.S. Devesa, J.L. Warren, J.J. Fraumeni, Rising incidence of renal cell [37] W.L. Tam, R.A. Weinberg, The epigenetics of epithelial-mesenchymal plasticity in cancer in the United States, J. Am. Med. Assoc. 281 (1999) 1628–1631. cancer, Nat. Med. 19 (2013) 1438–1449. [4] J.M. Hollingsworth, D.C. Miller, S. Daignault, B.K. Hollenbeck, Five-year survival [38] S.K. Pal, S. Williams, D.Y. Josephson, C. Carmichael, N.J. Vogelzang, D.I. Quinn, after surgical treatment for kidney cancer, Cancer-Am Cancer Soc 109 (2007) Novel therapies for metastatic renal cell carcinoma: efforts to expand beyond the 1763–1768. VEGF/mTOR signaling paradigm, Mol. Canc. Therapeut. 11 (2012) 526–537. [5] M. Sorbellini, M.W. Kattan, M.E. Snyder, V. Reuter, R. Motzer, M. Goetzl, et al., A [39] G. Fyfe, R.I. Fisher, S.A. Rosenberg, M. Sznol, D.R. Parkinson, A.C. Louie, Results of postoperative prognostic nomogram predicting recurrence for patients with con- treatment of 255 patients with metastatic renal cell carcinoma who received high- ventional clear cell renal cell carcinoma, J. Urol. 173 (2005) 48–51. dose recombinant interleukin-2 therapy, J. Clin. Oncol. 13 (1995) 688–696. [6] M. Rydzanicz, T. Wrzesiński, H.A.R. Bluyssen, J. Wesoły, Genomics and epige- [40] R.J. Motzer, J. Bacik, B.A. Murphy, P. Russo, M. Mazumdar, Interferon-alfa as a nomics of clear cell renal cell carcinoma: recent developments and potential ap- comparative treatment for clinical trials of new therapies against advanced renal plications, Canc. Lett. 341 (2013) 111–126. cell carcinoma, J. Clin. Oncol. 20 (2002) 289–296. [7] W. Yang, K. Yoshigoe, X. Qin, J.S. Liu, J.Y. Yang, A. Niemierko, et al., Identification [41] N.J. Vogelzang, W.M. Stadler, Kidney cancer, Lancet 352 (1998) 1691–1696. of genes and pathways involved in kidney renal clear cell carcinoma, BMC Bioinf. [42] K. Gupta, J.D. Miller, J.Z. Li, M.W. Russell, C. Charbonneau, Epidemiologic and 15 (Suppl 17) (2014) S2. socioeconomic burden of metastatic renal cell carcinoma (mRCC): a literature re- [8] U.N. Vaishampayan, H. Do, M. Hussain, K. Schwartz, Racial disparity in incidence view, Canc. Treat Rev. 34 (2008) 193–205. patterns and outcome of kidney cancer, Urology 62 (2003) 1012–1017. [43] J.S. Lam, J.T. Leppert, A.S. Belldegrun, R.A. Figlin, Novel approaches in the therapy [9] M.W. Kattan, V. Reuter, R.J. Motzer, J. Katz, P. Russo, A postoperative prognostic of metastatic renal cell carcinoma, World J. Urol. 23 (2005) 202–212. nomogram for renal cell carcinoma, J. Urol. 166 (2001) 63–67. [44] C.P. Matthews, N.H. Colburn, M.R. Young, AP-1 a target for cancer prevention, [10] M. Sorbellini, M.W. Kattan, M.E. Snyder, V. Reuter, R. Motzer, M. Goetzl, et al., A Curr. Cancer Drug Targets 7 (2007) 317–324. postoperative prognostic nomogram predicting recurrence for patients with con- [45] C. Zhao, Y. Qiao, P. Jonsson, J. Wang, L. Xu, P. Rouhi, et al., Genome-wide profiling ventional clear cell renal cell carcinoma, J. Urol. 173 (2005) 48–51. of AP-1-regulated transcription provides insights into the invasiveness of triple- [11] G.H. Mickisch, R.H. Mattes, Combination of surgery and immunotherapy in meta- negative breast cancer, Canc. Res. 74 (2014) 3983–3994. static renal cell carcinoma, World J. Urol. 23 (2005) 191–195. [46] D. Kamide, T. Yamashita, K. Araki, M. Tomifuji, Y. Tanaka, S. Tanaka, et al., [12] W.J. Kaelin, Kidney cancer: now available in a new flavor, Canc. Cell 14 (2008) Selective activator protein-1 inhibitor T-5224 prevents lymph node metastasis in an 423–424. oral cancer model, Canc. Sci. 107 (2016) 666–673. [13] L.M. Hock, J. Lynch, K.C. Balaji, Increasing incidence of all stages of kidney cancer [47] H.B. Schonthaler, J. Guinea-Viniegra, E.F. Wagner, Targeting inflammation by in the last 2 decades in the United States: an analysis of surveillance, epidemiology modulating the Jun/AP-1 pathway, Ann. Rheum. Dis. 70 (2011) i109–i112. and end results program data, J. Urol. 167 (2002) 57–60. [48] R. Zenz, R. Eferl, L. Kenner, L. Florin, L. Hummerich, D. Mehic, et al., Psoriasis-like [14] A. Kar, A. Gutierrez-Hartmann, Molecular mechanisms of ETS transcription factor- skin disease and arthritis caused by inducible epidermal deletion of Jun proteins, mediated tumorigenesis, Crit. Rev. Biochem. Mol. Biol. 48 (2013) 522–543. Nature 437 (2005) 369–375. [15] I. Bieche, S. Tozlu, I. Girault, P. Onody, K. Driouch, M. Vidaud, et al., Expression of [49] A. Duverger, F. Wolschendorf, M. Zhang, F. Wagner, B. Hatcher, J. Jones, et al., An PEA3/E1AF/ETV4, an Ets-related transcription factor, in breast tumors: positive AP-1 binding site in the enhancer/core element of the HIV-1 promoter controls the links to MMP2, NRG1 and CGB expression, Carcinogenesis 25 (2004) 405–411. ability of HIV-1 to establish latent infection, J. Virol. 87 (2013) 2264–2277. [16] A.D. Sharrocks, The ETS-domain transcription factor family, Nat. Rev. Mol. Cell [50] L. Xu, T.J. Huang, H. Hu, M.Y. Wang, S.M. Shi, Q. Yang, et al., The developmental Biol. 2 (2001) 827–837. transcription factor IRF6 attenuates ABCG2 and distinctively re- [17] R. Cangemi, A. Mensah, V. Albertini, A. Jain, M. Mello-Grand, G. Chiorino, et al., verses stemness phenotype in nasopharyngeal carcinoma, Canc. Lett. 431 (2018) Reduced expression and tumor suppressor function of the ETS transcription factor 230–243. ESE-3 in prostate cancer, Oncogene 27 (2008) 2877–2885. [51] C. Yang, L. Peng, T. Huang, G. Yang, Q. Chu, Y. Liang, et al., Cancer stem-like cell [18] B. Wasylyk, S.L. Hahn, A. Giovane, The Ets family of transcription factors, Eur. J. characteristics induced by EB virus-encoded LMP1 contribute to radioresistance in Biochem. 211 (1993) 7–18. nasopharyngeal carcinoma by suppressing the p53-mediated apoptosis pathway, [19] G. Alipov, T. Nakayama, M. Ito, K. Kawai, S. Naito, M. Nakashima, et al., Canc. Lett. 344 (2014) 260–271. Overexpression of Ets-1 proto-oncogene in latent and clinical prostatic carcinomas, [52] S. Xu, Z. Feng, M. Zhang, Y. Wu, Y. Sang, H. Xu, et al., hSSB1 binds and protects p21 Histopathology 46 (2005) 202–208. from ubiquitin-mediated degradation and positively correlates with p21 in human [20] R. Li, H. Pei, D.K. Watson, Regulation of Ets function by protein - protein interac- hepatocellular carcinomas, Oncogene 30 (2011) 2219–2229. tions, Oncogene 19 (2000) 6514–6523. [53] Y. Mei, J.P. Yang, C.N. Qian, For robust big data analyses: a collection of 150 im- [21] H.C. Crawford, B. Fingleton, M.D. Gustavson, N. Kurpios, R.A. Wagenaar, portant pro-metastatic genes, Chin. J. Canc. 36 (2017) 16. J.A. Hassell, et al., The PEA3 subfamily of Ets transcription factors synergizes with [54] L. Clijsters, C. Hoencamp, J. Calis, A. Marzio, S.M. Handgraaf, M.C. Cuitino, et al., beta-catenin-LEF-1 to activate matrilysin transcription in intestinal tumors, Mol. Cyclin F controls cell-cycle transcriptional outputs by directing the degradation of Cell Biol. 21 (2001) 1370–1383. the three activator E2Fs, Mol. Cell. 74 (2019) 1264–1277 e7. [22] B. Wasylyk, J. Hagman, A. Gutierrez-Hartmann, Ets transcription factors: nuclear [55] T. Palm, K. Hemmer, J. Winter, I.B. Fricke, K. Tarbashevich, S.F. Sadeghi, et al., A effectors of the Ras-MAP-kinase signaling pathway, Trends Biochem. Sci. 23 (1998) systemic transcriptome analysis reveals the regulation of neural stem cell main- 213–216. tenance by an E2F1-miRNA feedback loop, Nucleic Acids Res. 41 (2013) [23] K. Hida, M. Shindoh, M. Yasuda, M. Hanzawa, K. Funaoka, T. Kohgo, et al., 3699–3712. Antisense E1AF transfection restrains oral cancer invasion by reducing matrix [56] S.R. Lee, Y.G. Roh, S.K. Kim, J.S. Lee, S.Y. Seol, H.H. Lee, et al., Activation of EZH2 metalloproteinase activities, Am. J. Pathol. 150 (1997) 2125–2132. and SUZ12 regulated by E2F1 predicts the disease progression and aggressive [24] M. Shindoh, F. Higashino, T. Kohgo, E1AF, an ets-oncogene family transcription characteristics of bladder cancer, Clin. Canc. Res. 21 (2015) 5391–5403.

88 L. Xu, et al. Cancer Letters 482 (2020) 74–89

[57] K.H. Yoo, S. Oh, K. Kang, T. Hensel, G.W. Robinson, L. Hennighausen, Loss of EZH2 [65] Y.Z. Luo, P. He, M.X. Qiu, FOSL1 enhances growth and metastasis of human pros- results in precocious mammary gland development and activation of STAT5-de- tate cancer cells through epithelial mesenchymal transition pathway, Eur. Rev. pendent genes, Nucleic Acids Res. 43 (2015) 8774–8789. Med. Pharmacol. Sci. 22 (2018) 8609–8615. [58] Y. Li, K. Jin, G.W. van Pelt, H. van Dam, X. Yu, W.E. Mesker, et al., c-Myb enhances [66] C.J. Desmet, T. Gallenne, A. Prieur, F. Reyal, N.L. Visser, B.S. Wittner, et al., breast cancer invasion and metastasis through the wnt/beta-catenin/axin2 Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting pathway, Canc. Res. 76 (2016) 3364–3375. breast cancer metastasis, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 5139–5144. [59] Q.X. Yang, S. Zhong, L. He, X.J. Jia, H. Tang, S.T. Cheng, et al., PBK overexpression [67] B.I. Rini, S.C. Campbell, B. Escudier, Renal cell carcinoma, Lancet 373 (2009) promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR sig- 1119–1132. naling pathway, Canc. Lett. 452 (2019) 90–102. [68] D.R. Couillard, W.R. DeVere, Surgery of renal cell carcinoma, Urol Clin North Am [60] R. Chakrabarti, J. Hwang, B.M. Andres, Y. Wei, M. Lukacisin, R.A. Romano, et al., 20 (1993) 263–275. Elf5 inhibits the epithelial-mesenchymal transition in mammary gland development [69] L. Yang, Y. Han, S.F. Suarez, M.D. Minden, A tumor suppressor and oncogene: the and breast cancer metastasis by transcriptionally repressing Snail2, Nat. Cell Biol. WT1 story, Leukemia 21 (2007) 868–876. 14 (2012) 1212–1222. [70] A. Bhoumik, Z. Ronai, ATF2: a transcription factor that elicits oncogenic or tumor [61] A. Mukhopadhyay, T. Khoury, L. Stein, P. Shrikant, A.K. Sood, Prostate derived Ets suppressor activities, Cell Cycle 7 (2008) 2341–2345. transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 [71] A. Aytes, A. Mitrofanova, C.W. Kinkade, C. Lefebvre, M. Lei, V. Phelan, et al., ETV4 constitute a highly active oncogenic axis in breast cancer, Oncotarget 4 (2013) promotes metastasis in response to activation of PI3-kinase and Ras signaling in a 610–621. mouse model of advanced prostate cancer, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) [62] P.C. Hollenhorst, M.W. Ferris, M.A. Hull, H. Chae, S. Kim, B.J. Graves, Oncogenic E3506–E3515. ETS proteins mimic activated RAS/MAPK signaling in prostate cells, Genes Dev. 25 [72] Y.Z. Luo, P. He, M.X. Qiu, FOSL1 enhances growth and metastasis of human pros- (2011) 2147–2157. tate cancer cells through epithelial mesenchymal transition pathway, Eur. Rev. [63] S.K. Das, A.K. Pradhan, P. Bhoopathi, S. Talukdar, X.N. Shen, D. Sarkar, et al., The Med. Pharmacol. Sci. 22 (2018) 8609–8615. MDA-9/syntenin/IGF1R/STAT3 Axis directs prostate cancer invasion, Canc. Res. 78 [73] O. Kustikova, D. Kramerov, M. Grigorian, V. Berezin, E. Bock, E. Lukanidin, et al., (2018) 2852–2863. Fra-1 induces morphological transformation and increases in vitro invasiveness and [64] S. Gao, Y. Sun, X. Zhang, L. Hu, Y. Liu, C.Y. Chua, et al., IGFBP2 activates the NF- motility of epithelioid adenocarcinoma cells, Mol. Cell Biol. 18 (1998) 7095–7105. kappaB pathway to drive epithelial-mesenchymal transition and invasive character [74] W. Debinski, D.M. Gibo, Fos-related antigen 1 modulates malignant features of in pancreatic ductal adenocarcinoma, Canc. Res. 76 (2016) 6543–6554. glioma cells, Mol. Canc. Res. 3 (2005) 237–249.

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