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ETV4 promotes proliferation and invasion of lung adenocarcinoma by transcriptionally upregulating MSI2
Biochemical and Biophysical Research Communications 516 (2019) 278e284
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ETV4 promotes proliferation and invasion of lung adenocarcinoma by transcriptionally upregulating MSI2 Tingting Cheng a, b, Zijian Zhang b, c, Yuyu Cheng d, Jing Zhang b, c, Jianbing Tang b, c, Zhaohua Tan b, c, Zhan Liang b, c, Taili Chen b, c, Zhiyuan Liu b, c, Jiahui Li b, c, Jin Zhao e, Rongrong Zhou b, c, * a
Department of Preventive Health Care, Xiangya Hospital, Central South University, Changsha, China Department of Oncology, Xiangya Hospital, Central South University, Changsha, China National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China d Chuanshan College, University of South China, Hengyang, China e The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2019 Accepted 20 June 2019 Available online 25 June 2019
The oncogenic roles of ETV4 have been revealed in multiple cancers. However, its expression and functions in lung cancer are rarely explored. Here, we firstly detected the expression of ETV4 in lung adenocarcinoma (LUAD) via online data and local experiment validation. Furthermore, we explored the functions and corresponding mechanisms of ETV4 in LUAD. Upregulation of ETV4 in LUAD is indicated by online data and our results of qPCR, Western blot and immunohistochemistry in collective tissue samples. ETV4 knockdown significantly inhibits proliferation and invasion in LUAD indicated by the outcomes of CCK8, plate clone formation, and Transwell invasion assays. Mechanistically, chromatin immunoprecipitation and luciferase reporter system assays indicated that ETV4 could directly bind at the promoter of MSI2 and promote its transcription. Furthermore, ectopic expression MSI2 can rescue the inhibitory effects caused by ETV4 knockdown in LUAD. Therefore, we proved that upregulation of ETV4 could promote proliferation and invasion of LUAD by transcriptionally upregulating MSI2 offering a potential therapy treatment target of LUAD. © 2019 Elsevier Inc. All rights reserved.
Keywords: Lung adenocarcinoma ETV4 MSI2 Transcription regulation Proliferation and invasion
1. Introduction Lung cancer is the most malignant disease with the highest incidence and mortality worldwide. lung adenocarcinoma (LUAD) is the most common subtype of lung cancer with the comparable incidence in both smoking and non-smoking population. Surgical resection is the ideal treatment for early LUAD with satisfactory prognosis [1e3]. However, consistent with other subtypes of lung cancer, most LUAD patients are diagnosed as the advanced or metastatic stage, and the chance of surgery is lost. Although small molecular drugs targeting EGFR, KRAS, ALK, and BRAF may achieve ideal outcome at the beginning, ultimately, drug resistance and
* Corresponding author. Department of Oncology, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 410078, Changsha, Hunan, China. E-mail address: [email protected] (R. Zhou). https://doi.org/10.1016/j.bbrc.2019.06.115 0006-291X/© 2019 Elsevier Inc. All rights reserved.
tumor re-progression are inevitable [4]. Therefore, it is of great importance to explore new tumor regulators that may provide new markers for early diagnosis and new targets for treatment in LUAD. ETV4 (ETS Variant 4), a transcription factor belonging to EST family, is exclusively involved in the carcinogenesis of multiple tissues including prostate, pancreas, breast, liver, and intestine, and so forth [5e9]. Generally, ETV4 is upregulated in tumors and acts as an oncogenic factor by promoting proliferation and metastasis via distinct mechanisms. By fusing with other genes, ETV4 can regulate the transcriptional pattern of the genome and modulate the carcinogenesis [10]. For example, TMPRSS2:ETV4 gene fusions may be an early event of prostate cancer and defines a new molecular subtype of prostate cancer [11]. Moreover, ETV4 can directly bind the promoter region of the target genes and regulate their transcriptional activity. Cyclin D1 and MMPs are confirmed targets of ETV4 and serve as effectors of ETV4 in promoting proliferation and metastasis in pancreatic cancer [8] and hepatocellular carcinoma
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[12,13]. However, the expression and function of ETV in lung cancers remains unknown. Musashi-2 (MSI2) is an RNA-binding protein and can regulate the expression of target mRNAs at the translation level. Numerous studies have confirmed that MSI2 is a potent oncogene and plays critical roles in carcinogenesis and malignant progression for both hematological and solid tumors [14]. Mechanistically, MSI2 exerts its pro-tumor effects in different cancers by activating the oncogenic signaling pathways, including TGF-b [15], JAK2/STAT3 [16], Wnt/b-catenin and Hedgehog signaling [17], respectively. Notably, the mechanisms accounting for upregulation of MSI2 have rarely revealed. A recent study indicates that KLF4 is a negative regulator of MSI2 transcription in pancreatic cancer [18]. However, positive transcription factors that promote MSI2 transcription remains unknown. Here, we explored the expression and functions of ETV4 in LUAD and further identified its downstream target. The results indicated that ETV4 is upregulated and can promote proliferation and invasion in LUAD. Furthermore, we revealed that MSI2 is a direct target of ETV4 and can mediate the pro-tumor functions of ETV4 in LUAD.
2. Methods and materials
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2.4. Western blot Western blot was applied as previously described [20]. Briefly, RIPA buffer was used to disrupt grinded tissue, and total proteins were obtained via high-speed centrifugation at 4 C. Then, the proteins were denatured and separated by 12% SDS PAGE (30 mg/ lane). Subsequently, the proteins were transferred onto polyvinylidene difluoride membranes (PVDF), and the membranes were successively subjected to the procedure including blocking, primary antibody incubation, TBST washing, secondary antibody incubation, and TBST washing. At last, the membranes were visualized using chemiluminescent HRP substrate (BI, Jerusalem, Israel) via a FluorChem FC3 system (Proteinsimple, CA, USA). The specific antibodies and the dilution ratio were as follow: Anti-ETV4 rabbit polyclonal antibody (dilution: 1:500, BBI, Shanghai, China), mouse-anti-GAPDH monoclonal (dilution: 1:5000, Abclonal, MA, USA), Anti-MSI2 rabbit polyclonal antibody(dilution: 1:500, BBI, Shanghai, China), HRP-conjugated Mouse anti-rabbit IgG (dilution:1:5000, BBI, Shanghai, China), HRP-conjugated Goat AntiMouse IgG (dilution:1:5000, BBI, Shanghai, China). 2.5. Immunohistochemistry (IHC)
Fresh tissue specimens, including 15 cases of LUAD tissues and 8 cases of normal lung tissues, were collected from surgical patients with LUAD and other non-malignant lung diseases in the Xiangya Hospital of Central South University. The specimens were divided into two parts, one part fixed by formaldehyde for paraffin section production, other one frozen by nitrogen liquid for protein and RNA extraction. Informed consent was acquired from all patients. Moreover, this study was approved and supervised by the Ethical Committee of Xiangya Hospital of Central South University.
The expression of ETV in lung and LUAD tissues were detected by IHC using UltraSensitive™ streptavidin-peroxidase IHC Kit (MaiXin, Fuzhou, China) as previously described [19]. Briefly, after dewaxing, antigen retrieval and H2O2 treatment, the paraffinembedded tissue slides, with antigen retrieval and endogenous peroxidase activity elimination, were successively incubated with the ETV4 antibody (dilution: 1:100, BBI, Shanghai, China), biocatalytic secondary antibody, and avidin-biotin-peroxidase complex. Finally, sections were treated with 30 , 30 -diaminobenzidine, and countersigned with Harris’ modified hematoxylin. The photos were obtained with a microscopic imaging system (Lecia, Wizla, Germany).
2.2. Cell culture
2.6. siRNAs, plasmids, and transfection
Human LUAD cell lines including A549, H1299, H358, H1650, and H1975, and normal bronchial line NL-20, purchased from American Type Culture Collection, were cultured with DMEM (BI, Jerusalem, Israel) medium supplemented with 10% fetal bovine serum (BI, Jerusalem, Israel) under a humidified cell incubator at 37 C with 5% CO2.
siETV4, specifically targeting ETV4, and siNC, control siRNAs, were purchased from RIBOBIO Inc (Guangzhou, China). The MSI2 expression vector, pENTER-MSI2, and corresponding vector were obtained from Vigene Bioscience Inc (MD, USA). The siRNAs and plasmids were introduced into LUAD cells with Lipofectamine™ 3000 according to the manufacturer's instructions.
2.1. Tissue samples
2.7. CCK-8 2.3. RNA isolation, reverse transcription, and quantitative reverse transcription polymerase chain reaction (qPCR) Total RNA from tissues and cells were extracted by Trizol™ reagent (Thermo, MA, USA) following instruction and previous protocol [19]. Then, the mRNAs were reversely transcripted into cDNA with FastKing gDNA Dispelling RT SuperMix (TIANGEN, Beijing, China) according to attached instruction. Then, the expression of ETV4 in tissues and cells was detected with SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China) via CFX Connect Real-Time System (Bio-Rad, CA, USA). Subsequently, the relative expression of ETV4 and MSI2 was analyzed with CFX Manager 2.0 (Bio-Rad, CA, USA). The specific primer sequences of ETV4 and GAPDH, as a normalized control, are listed as follow: ETV4 sense primer: 50 - CAGTGCCTTTACTCCAGTGCC-30 , ETV4 reverse primer: 50 -CTCAGGAAATTCCGTTGCTCT-30 ; MSI2 sense primer:50 - ATCCCACTACGAAACGCTCC-30 , MSI2 reverse primer:50 GGGGTCAATCGTCTTGGAATC-3’; GAPDH sense primer: 50 - CAGCAAGAGCACAAGAGGAA-30 , GAPDH reverse primer: 50 - ATGGTACATGACAAGGTGCGG-3’.
The cell growth and viability were analyzed by CCK-8 according to the previous description [21]. Briefly, LUAD cells were seeded into 96-well plates (1 103 cells/well, three paralleled wells). For successive 3 days, 10 ml CCK-8 was added, and the absorbance value at 450 nm was detected by using Epoch (Bio-Tek, VT, USA). Eventually, the data were statistically analyzed and demonstrated by the growth curves. 2.8. Plate clone formation assay The cell proliferation were investigated by plate clone formation assay as previously described [22]. Simply, LUAD cells were seeded into 6-well plates (800 cells/well, three paralleled wells) and continuously cultured for 7 days. Then, the cells were fixed by methanol and stained with crystal violet. A clone containing more than 50 cells were named as an effective clone, and the clone number was counted under an inverse microscope (Nikon, Tokyo, Japan).
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2.9. Transwell invasion assay The cell invasion were explored by Transwell invasion assay following the previous description [23]. Simply, 500 ml DMEM medium containing 2.5 104 cells was pipetted into the upper chamber. 750 ml DMEM supplemented 5% FBS was added into the low well. 36 h later, the cells were fixed methanol and stained with crystal violet. Subsequently, the invasive cells were photoed and counted after removing the remained cells in the chamber under an inverse microscope (Nikon, Tokyo, Japan). 2.10. Chromatin immunoprecipitation (ChIP) assay ChIP assay was performed using the Pierce™ Magnetic ChIP Kit (Thermo, MA, USA) following the instruction. Briefly, 4 106 cells were successively subjected to the procedures containing DNAprotein cross-linking, disruption of membrane and cytosol, MNase digestion and sonication, incubation ETV4 antibody, addition protein A/G magnetic beads for binding ETV4 immunoprecipitated complex, wash, elution, crosslink reversal and proteinase K treatment, and DNA clean-up. Subsequently, PCR was performed using specific MSI2 primers: siteA-F 50 -GGCAACATAAAGTTGTGGTTC-30 , siteA-R 50 TGGAAGGCTACAGACATTGTC-3’; siteB-F 50 -TCTCATGTGTTGAACAGCCAG -30 , siteB-R 50 - GTTCACTTCAGCCTCTCACC-3’; siteC-F 50 -
TATAGACCTCCACAAGCTGACAC-30 , siteC-R 50 GGGATTAGATTAGGAAAGGCC-3’. Input DNA (without immunoprecipitation) and normal IgG-precipitated DNA were loaded as positive and negative controls, respectively.
2.11. Dual luciferase reporter assay LUAD cells were seeded into 12-well plates at a density of 2.5 104 cells/well. The cells were transfected with the reporter plasmid containing wild MSI2 promoter and mutant MSI2 promoter at site B(from “ACAGGAAATG” to “ACACCAAATG”), respectively. Renilla luciferase reporter plasmids were also transfected into cells for signal normalization. 24 h later, the luciferase activity was detected using the Dual Luciferase Assay System Kit (Promega, WI, USA).
2.12. Statistical analysis All experiments were independently repeated for 3 times. Statistical analysis and charts were processed with GraphPad Prism version 8. For comparisons between two groups, a Student t-test or chi-square test was used. P < 0.05 were considered to be statistically significant.
Fig. 1. ETV4 is upregulated in LUAD. The level of ETV4 mRNA is upregulated in LUAD tissues indicated by data from TCGA (A), Oncomine (B), and qPCR assay (C). The level of ETV4 protein is upregulated in LUAD tissues demonstrating by west blot (D) and IHC (E). (F) Western blot and qPCR assay indicated that the level of ETV protein and mRNA is upregulated in LUAD cells. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
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3. Results 3.1. ETV4 is upregulated in LUAD. To investigate the expression of ETV4 in LUAD, we initially analyzed the online data from TCGA and GEO by using UALCAN and Oncomine, respectively. ETV4 is significantly upregulated in LUAD supported by the RNA-sequencing data from TCGA (Fig. 1A), which is validated by data from Oncomine (Fig. 1B). Moreover, qPCR (Fig. 1C), Western blot (Fig. 1D) and immunohistochemistry (Fig. 1E) analysis indicated that upregulation of ETV4 is also observed in our LUAD tissues. The expression of ETV4 is higher in LUAD cells than that in normal lung cells manifested by qPCR and Western blot assays (Fig. 1F). Collectively, those results confirmed that ETV4 is upregulated in LUAD, suggesting it may be an oncogenic regulator in lung cancer. 3.2. ETV4 silencing suppresses proliferation and invasion of LUAD cells Next, we explored the function of ETV4 in LUAD. Firstly, the expression of ETV4 was knocked down by transfecting specific siRNA targeting ETV4, indicated by Western blot results (Fig. 2A).
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Subsequently, CCK-8, plate clone formation, and Transwell invasion assays were conducted to analyze the role of ETV4. As Fig. 2B, C, and D showed, the proliferation and invasion ability of LUAD cells were constrained revealed by the lower growth rate, fewer clones, and less invasive cells. Thus, ETV4 depletion can inhibit proliferation and invasion of LUAD. 3.3. MSI2 is a direct transcriptional target of ETV4 in LUAD. Considering ETV4 is a transcript factor, we proposed that ETV4 may exert its functions by regulating the transcription of target genes. Thus, we analyzed the potential targets of ETV4 using the GTRD (Gene Transcription Regulation database). Six candidate genes, with sitecounts being more massive than or equal to 15, were obtained. Then, we analyzed the expression of candidate genes and their promoter methylation level by UALCAN, and the correlation between candidate genes and ETV4 by GEPIA in LUAD. The results indicated that MSI2 is upregulated and positively correlated with ETV4 and the promoter methylation level of MSI2 is comparable between normal and lung cancer tissues (Supplementary Fig. 1) indicating MSI2 may be a target of ETV4 in LUAD. Therefore, we detected the expression of MSI2 in LUAD cells with ETV4 silencing. Indeed, the MSI2 mRNA is significantly
Fig. 2. ETV4 silencing suppresses growth, proliferation, and invasion of LUAD cells. (A) Western blot indicated that ETV4 was significantly inhibited by siETV4 in A549 and H1299 cells. (B) ETV knockdown markedly inhibited the growth of A549 and H1299 cells indicated by CCK8. (C) ETV knockdown markedly inhibited proliferation of A549 and H1299 cells indicated by plate clone formation. (D) ETV knockdown markedly inhibited invasion of A549 and H1299 cells indicated by Transwell invasion assay. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
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downregulated in LUAD cells upon ETV4 depletion (Fig. 3A). Thus, we further predicted the bind sites of ETV4 at the promoter region, approximately 1.5 kb DNA region 5’ upstream of coding DNA sequence, of MSI2 using JASPAR. Three candidate binding sites with the highest score were selected for validation by ChIP assays (Fig. 3B). The gDNAs precipitated by ETV4 antibody were subjected to qPCR amplification using primer sets covering the region of putative binding sites. As Fig. 3C shown, strong binding signals were detected at site B (1123 to 1132), suggesting this site may be the binding site of ETV4 at MSI2 promoter. Whereas, no signal or weak signals were detected at site A (641 to 650) and site C (1461 to 1470), respectively. Subsequently, dual luciferase reporter assay was performed to determine further whether siteB is the functional binding site of ETV4 at the promoter of MSI2. Indeed, the relative luciferase activity of cells transfected with the mutant promoter of MSI2 reporter plasmids was remarkably lower than that in cells transfected with wild one (Fig. 3D). Therefore, the above results manifested that ETV4 can directly regulate the transcription of MSI2 in LUAD. 3.4. ETV4 promotes proliferation and invasion through upregulating MSI2 in LUAD. To reveal the role of MSI2 in ETV4 associated functions, ectopic
expression of MSI2 was applied in LUAD cells with ETV4 knockdown, and the effects on proliferation and invasion were detected. As Fig. 4A indicated, MSI2 was significantly upregulated. Next, the role of MSI2 in ETV4 related functions was analyzed. The data of CCK-8 and plate clone formation assays demonstrated ectopic expression of MSI2 markedly abrogated the inhibitory effects of ETV4-silencing on the proliferation (Fig. 4B and C). Accordingly, ectopic expression of MSI2 rescued the invasive ability of LUAD cells with ETV4 silencing indicated by the results of Transwell invasion assays (Fig. 4D). Altogether, our results indicate that ETV4 can promote proliferation and invasion of LUAD cells by upregulating MSI2. 4. Discussion In summary, we confirmed that ETV4 is upregulated in LUAD and proved that ETV4 overexpression could promote proliferation and invasion of LUAD cells. Mechanistically, we demonstrated that ETV could regulate MSI2 transcription via directly binding at its promoter region, which mediates the oncogenic functions of ETV4 in LUAD. Taken together, we indicated that upregulation of ETV4 could promote proliferation and invasion in an MSI2 dependent manner in LUAD. Aberrant expression of ETV4 and its oncogenic roles have been
Fig. 3. ETV4 can transcriptionally regulate MSI2 by direct binding at the promoter region of MSI2 in LUAD cells. (A) qPCR assay indicated that the level of MSI2 mRNA was significantly inhibited by ETV4 knockdown in A549 and H1299 cells. (B) The schematic diagram for predicted bind sites of ETV4 in the promoter region of MSI2. (C) ChIP assay revealed that strong signaling was detected via primers targeting siteB of MSI2. (D) Mutant at predicted siteB of MSI2 significantly decreased the luciferase activity in A549 and H1299 cells indicated by dual luciferase reporter system assay. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
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Fig. 4. Ectopic expression of MSI2 can rescue the inhibitory effects of ETV4 knockdown in LUAD. (A) Western blot indicated that ETV4 knockdown decreased MSI2 in A549 and H1299 cells, which can be rescued by ectopic expression of MSI2. (B) Ectopic expression of MSI2 released the inhibitory effects of ETV4 knockdown on growth in A549 and H1299 cells. (C) Ectopic expression of MSI2 eliminated the inhibitory effects of ETV4 knockdown on proliferation in A549 and H1299 cells. (D) Ectopic expression of MSI2 eliminated the inhibitory effects of ETV4 knockdown on invasion in A549 and H1299 cells. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
revealed in various cancers. For example, ETV4 attracts many attentions in prostate cancer. Overexpression of ETV4, serving as a negative indicator of prognosis, could promote tumor progression involving proliferation, metastasis, and therapy sensitization [9,24]. Moreover, ETV4 related fusion genes such as TMPRSS2-ETV4, KLK2ETV4, and CANTT-ETV4, define a new molecular subtype of prostate cancer [10,11]. Meanwhile, upregulation of ETV4 and pro-tumor effects have also been revealed in other tumors including breast cancer [7], pancreatic cancer [8], colorectal cancer [6], sarcomas [25], hepatocellular carcinoma [12], as well as esophageal squamous cell carcinoma [26], etc. Upregulation of ETV4 has been implied by online data from TCGA and Oncomine without further verification. Indeed, we confirmed that ETV4 is upregulated in LUAD, demonstrated by qPCR and Western blot analysis in our collected specimens. Moreover, consistent with existing studies, ETV4 knockdown notably inhibited LUAD proliferation and invasion as well, further confirming the universality of tumorigenic effects of ETV4. Regulating the transcription of target genes is the most common mechanism by which transcription factors exert their biological functions. For example, numerous studies have indicated that
functions of c-Myc and p53, two important transcription factors with antagonistic function, are almost dependent on downstream targets in carcinogenesis and progression [27,28]. Several targets of ETV4 have been identified in multiple cancers. ETV4 could promote proliferation via upregulating cyclin proteins like cyclin D1 [8] and cyclin D3 [29] by directly transcriptional regulation in pancreatic cancer and breast cancers. MMPs like MMP13 and urokinase plasminogen activator (uPA) are also transcriptionally regulated by ETV4 and involved in ETV4-drive metastasis in prostate cancer [24] and breast cancer [7]. Here, our results demonstrated that ETV4 could directly bind at the promoter of MSI2 and subsequently activate MSI2 transcription. Moreover, ectopic MSI2 expression can rescue the inhibitory effects induced by ETV4 silencing, suggesting oncogenic roles of ETV4 are, at least partially, dependent on MSI2 in LUAD. Similarly, the oncogenic roles of MSI2, involving regulations of proliferation, metastasis, treatment sensitivity, and prognosis evaluation, have been indicated by dozens of studies in multiple cancer types [16,17], including lung cancer [15]. MSI2 can promote non-small cell lung cancer metastasis via inhibiting claudins in a TGF-b signaling related manner [15]. Compared with numerous
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studies focusing on the downstream mechanisms of MSI2, there are few studies focusing on the mechanism of abnormal expression of MSI2 itself. The comparable promoter methylation level of MSI2 has been indicated by methylation sequencing data in multiple cancers, suggesting transcription factors may account for the aberrant expression of MSI2 in cancers. Accordingly, a recent study has confirmed that KLF4 can negatively regulate MSI2 transcription by directly binding at its promoter in pancreatic cancer [18]. In this study, maybe for the first time, we further revealed that ETV4 is a decisive transcription factor in the regulation of MSI2 transcription in LUAD. Collectively, our studies revealed that upregulation of ETV4 could promote malignant progression in LUAD via directly regulating MSI2 in transcriptional level. ETV4/MSI2 axis may be a potential diagnosis indicator and therapy target in LUAD. Conflicts of interest All authors declare no conflict of interest for this study. Acknowledgment The present study was supported by the National Natural Science Foundation of China (nos. 81770928). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.115. References [1] M.M. Jacobsen, S.C. Silverstein, M. Quinn, L.B. Waterston, C.A. Thomas, J.C. Benneyan, P.K.J. Han, Timeliness of access to lung cancer diagnosis and treatment: a scoping literature review, Lung Cancer 112 (2017) 156e164. [2] G.A. Rivera, H. Wakelee, Lung cancer in never smokers, Adv. Exp. Med. Biol. 893 (2016) 43e57. [3] A.C. Borczuk, Prognostic considerations of the new world health organization classification of LUAD, Eur. Respir. Rev. 25 (2016) 364e371. [4] K. Inamura, Clinicopathological characteristics and mutations driving development of early LUAD: tumor initiation and progression, Int. J. Mol. Sci. 19 (2018). [5] S. Oh, S. Shin, R. Janknecht, ETV1, 4 and 5: an oncogenic subfamily of ETS transcription factors, Biochim. Biophys. Acta 1826 (2012) 1e12. [6] J. Xiao, S. Yang, P. Shen, Y. Wang, H. Sun, F. Ji, D. Zhou, Phosphorylation of ETV4 at Ser73 by ERK kinase could block ETV4 ubiquitination degradation in colorectal cancer, Biochem. Biophys. Res. Commun. 486 (2017) 1062e1068. [7] M. Dumortier, F. Ladam, I. Damour, S. Vacher, I. Bieche, N. Marchand, Y. de Launoit, D. Tulasne, A. Chotteau-Lelievre, ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis, Breast Cancer Res. 20 (2018) 73. [8] N. Tyagi, S.K. Deshmukh, S.K. Srivastava, S. Azim, A. Ahmad, A. Al-Ghadhban, A.P. Singh, J.E. Carter, B. Wang, S. Singh, ETV4 facilitates cell-cycle progression in pancreatic cells through transcriptional regulation of cyclin D1, Mol. Cancer Res. 16 (2018) 187e196. [9] A. Aytes, A. Mitrofanova, C.W. Kinkade, C. Lefebvre, M. Lei, V. Phelan, H.C. LeKaye, J.A. Koutcher, R.D. Cardiff, A. Califano, M.M. Shen, C. Abate-Shen, ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) E3506eE3515.
[10] J.D. Barros-Silva, P. Paulo, A.C. Bakken, N. Cerveira, M. Lovf, R. Henrique, C. Jeronimo, R.A. Lothe, R.I. Skotheim, M.R. Teixeira, Novel 5' fusion partners of ETV1 and ETV4 in prostate cancer, Neoplasia 15 (2013) 720e726. [11] S.A. Tomlins, R. Mehra, D.R. Rhodes, L.R. Smith, D. Roulston, B.E. Helgeson, X. Cao, J.T. Wei, M.A. Rubin, R.B. Shah, A.M. Chinnaiyan, TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer, Cancer Res. 66 (2006) 3396e3400. [12] Q.X. Yang, S. Zhong, L. He, X.J. Jia, H. Tang, S.T. Cheng, J.H. Ren, H.B. Yu, L. Zhou, H.Z. Zhou, F. Ren, Z.W. Hu, R. Gong, A.L. Huang, J. Chen, PBK overexpression promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR signaling pathway, Cancer Lett. 452 (2019) 90e102. [13] E. Kim, D. Kim, J.S. Lee, J. Yoe, J. Park, C.J. Kim, D. Jeong, S. Kim, Y. Lee, Capicua suppresses hepatocellular carcinoma progression by controlling the ETV4MMP1 axis, Hepatology 67 (2018) 2287e2301. [14] A.E. Kudinov, J. Karanicolas, E.A. Golemis, Y. Boumber, Musashi RNA-binding proteins as cancer drivers and novel therapeutic targets, Clin. Cancer Res. 23 (2017) 2143e2153. [15] A.E. Kudinov, A. Deneka, A.S. Nikonova, T.N. Beck, Y.H. Ahn, X. Liu, C.F. Martinez, F.A. Schultz, S. Reynolds, D.H. Yang, K.Q. Cai, K.M. Yaghmour, K.A. Baker, B.L. Egleston, E. Nicolas, A. Chikwem, G. Andrianov, S. Singh, H. Borghaei, I.G. Serebriiskii, D.L. Gibbons, J.M. Kurie, E.A. Golemis, Y. Boumber, Musashi-2 (MSI2) supports TGF-beta signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 6955e6960. [16] C. Yang, W. Zhang, L. Wang, G. Kazobinka, X. Han, B. Li, T. Hou, Musashi-2 promotes migration and invasion in bladder cancer via activation of the JAK2/ STAT3 pathway, Lab. Investig. 96 (2016) 950e958. [17] Z. Li, H. Jin, G. Mao, L. Wu, Q. Guo, Msi2 plays a carcinogenic role in esophageal squamous cell carcinoma via regulation of the Wnt/beta-catenin and Hedgehog signaling pathways, Exp. Cell Res. 361 (2017) 170e177. [18] K. Guo, J. Cui, M. Quan, D. Xie, Z. Jia, D. Wei, L. Wang, Y. Gao, Q. Ma, K. Xie, The novel KLF4/MSI2 signaling pathway regulates growth and metastasis of pancreatic cancer, Clin. Cancer Res. 23 (2017) 687e696. [19] J.F. Zhu, W. Huang, H.M. Yi, T. Xiao, J.Y. Li, J. Feng, H. Yi, S.S. Lu, X.H. Li, R.H. Lu, Q.Y. He, Z.Q. Xiao, Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation, Cell Death Dis. 9 (2018) 1154. [20] H. Peng, Z. Li, J. Fu, R. Zhou, Growth and differentiation factor 15 regulates PDL1 expression in glioblastoma, Cancer Manag. Res. 11 (2019) 2653e2661. [21] Y. Dai, H. Duan, C. Duan, R. Zhou, Y. He, Q. Tu, L. Shen, Down-regulation of TCF21 by hypermethylation induces cell proliferation, migration and invasion in colorectal cancer, Biochem. Biophys. Res. Commun. 469 (2016) 430e436. [22] J. Liu, W. Huang, C. Ren, Q. Wen, W. Liu, X. Yang, L. Wang, B. Zhu, L. Zeng, X. Feng, C. Zhang, H. Chen, W. Jia, L. Zhang, X. Xia, Y. Chen, Flotillin-2 promotes metastasis of nasopharyngeal carcinoma by activating NF-kappaB and PI3K/ Akt3 signaling pathways, Sci. Rep. 5 (2015) 11614. [23] J.Y. Li, T. Xiao, H.M. Yi, H. Yi, J. Feng, J.F. Zhu, W. Huang, S.S. Lu, Y.H. Zhou, X.H. Li, Z.Q. Xiao, S897 phosphorylation of EphA2 is indispensable for EphA2dependent nasopharyngeal carcinoma cell invasion, metastasis and stem properties, Cancer Lett. 444 (2019) 162e174. [24] M. Qi, Z. Liu, C. Shen, L. Wang, J. Zeng, C. Wang, C. Li, W. Fu, Y. Sun, B. Han, Overexpression of ETV4 is associated with poor prognosis in prostate cancer: involvement of uPA/uPAR and MMPs, Tumour Biol 36 (2015) 3565e3572. [25] Y.P. Hung, C.D. Fletcher, J.L. Hornick, Evaluation of ETV4 and WT1 expression in CIC-rearranged sarcomas and histologic mimics, Mod. Pathol. 29 (2016) 1324e1334. [26] T.M. Fung, K.Y. Ng, M. Tong, J.N. Chen, S. Chai, K.T. Chan, S. Law, N.P. Lee, M.Y. Choi, B. Li, A.L. Cheung, S.W. Tsao, Y.R. Qin, X.Y. Guan, K.W. Chan, S. Ma, Neuropilin-2 promotes tumourigenicity and metastasis in oesophageal squamous cell carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation, J. Pathol. 239 (2016) 309e319. [27] Z.E. Stine, Z.E. Walton, B.J. Altman, A.L. Hsieh, C.V. Dang, MYC, metabolism, and cancer, Cancer Discov. 5 (2015) 1024e1039. [28] C.M. Roake, S.E. Artandi, Control of cellular aging, tissue function, and cancer by p53 downstream of telomeres, Cold Spring Harb Perspect Med 7 (2017). [29] J. Jiang, Y. Wei, D. Liu, J. Zhou, J. Shen, X. Chen, S. Zhang, X. Kong, J. Gu, E1AF promotes breast cancer cell cycle progression via upregulation of Cyclin D3 transcription, Biochem. Biophys. Res. Commun. 358 (2007) 53e58.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ETV4 promotes proliferation and invasion of lung adenocarcinoma by transcriptionally upregulating MSI2 Tingting Cheng a, b, Zijian Zhang b, c, Yuyu Cheng d, Jing Zhang b, c, Jianbing Tang b, c, Zhaohua Tan b, c, Zhan Liang b, c, Taili Chen b, c, Zhiyuan Liu b, c, Jiahui Li b, c, Jin Zhao e, Rongrong Zhou b, c, * a
Department of Preventive Health Care, Xiangya Hospital, Central South University, Changsha, China Department of Oncology, Xiangya Hospital, Central South University, Changsha, China National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China d Chuanshan College, University of South China, Hengyang, China e The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2019 Accepted 20 June 2019 Available online 25 June 2019
The oncogenic roles of ETV4 have been revealed in multiple cancers. However, its expression and functions in lung cancer are rarely explored. Here, we firstly detected the expression of ETV4 in lung adenocarcinoma (LUAD) via online data and local experiment validation. Furthermore, we explored the functions and corresponding mechanisms of ETV4 in LUAD. Upregulation of ETV4 in LUAD is indicated by online data and our results of qPCR, Western blot and immunohistochemistry in collective tissue samples. ETV4 knockdown significantly inhibits proliferation and invasion in LUAD indicated by the outcomes of CCK8, plate clone formation, and Transwell invasion assays. Mechanistically, chromatin immunoprecipitation and luciferase reporter system assays indicated that ETV4 could directly bind at the promoter of MSI2 and promote its transcription. Furthermore, ectopic expression MSI2 can rescue the inhibitory effects caused by ETV4 knockdown in LUAD. Therefore, we proved that upregulation of ETV4 could promote proliferation and invasion of LUAD by transcriptionally upregulating MSI2 offering a potential therapy treatment target of LUAD. © 2019 Elsevier Inc. All rights reserved.
Keywords: Lung adenocarcinoma ETV4 MSI2 Transcription regulation Proliferation and invasion
1. Introduction Lung cancer is the most malignant disease with the highest incidence and mortality worldwide. lung adenocarcinoma (LUAD) is the most common subtype of lung cancer with the comparable incidence in both smoking and non-smoking population. Surgical resection is the ideal treatment for early LUAD with satisfactory prognosis [1e3]. However, consistent with other subtypes of lung cancer, most LUAD patients are diagnosed as the advanced or metastatic stage, and the chance of surgery is lost. Although small molecular drugs targeting EGFR, KRAS, ALK, and BRAF may achieve ideal outcome at the beginning, ultimately, drug resistance and
* Corresponding author. Department of Oncology, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 410078, Changsha, Hunan, China. E-mail address: [email protected] (R. Zhou). https://doi.org/10.1016/j.bbrc.2019.06.115 0006-291X/© 2019 Elsevier Inc. All rights reserved.
tumor re-progression are inevitable [4]. Therefore, it is of great importance to explore new tumor regulators that may provide new markers for early diagnosis and new targets for treatment in LUAD. ETV4 (ETS Variant 4), a transcription factor belonging to EST family, is exclusively involved in the carcinogenesis of multiple tissues including prostate, pancreas, breast, liver, and intestine, and so forth [5e9]. Generally, ETV4 is upregulated in tumors and acts as an oncogenic factor by promoting proliferation and metastasis via distinct mechanisms. By fusing with other genes, ETV4 can regulate the transcriptional pattern of the genome and modulate the carcinogenesis [10]. For example, TMPRSS2:ETV4 gene fusions may be an early event of prostate cancer and defines a new molecular subtype of prostate cancer [11]. Moreover, ETV4 can directly bind the promoter region of the target genes and regulate their transcriptional activity. Cyclin D1 and MMPs are confirmed targets of ETV4 and serve as effectors of ETV4 in promoting proliferation and metastasis in pancreatic cancer [8] and hepatocellular carcinoma
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[12,13]. However, the expression and function of ETV in lung cancers remains unknown. Musashi-2 (MSI2) is an RNA-binding protein and can regulate the expression of target mRNAs at the translation level. Numerous studies have confirmed that MSI2 is a potent oncogene and plays critical roles in carcinogenesis and malignant progression for both hematological and solid tumors [14]. Mechanistically, MSI2 exerts its pro-tumor effects in different cancers by activating the oncogenic signaling pathways, including TGF-b [15], JAK2/STAT3 [16], Wnt/b-catenin and Hedgehog signaling [17], respectively. Notably, the mechanisms accounting for upregulation of MSI2 have rarely revealed. A recent study indicates that KLF4 is a negative regulator of MSI2 transcription in pancreatic cancer [18]. However, positive transcription factors that promote MSI2 transcription remains unknown. Here, we explored the expression and functions of ETV4 in LUAD and further identified its downstream target. The results indicated that ETV4 is upregulated and can promote proliferation and invasion in LUAD. Furthermore, we revealed that MSI2 is a direct target of ETV4 and can mediate the pro-tumor functions of ETV4 in LUAD.
2. Methods and materials
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2.4. Western blot Western blot was applied as previously described [20]. Briefly, RIPA buffer was used to disrupt grinded tissue, and total proteins were obtained via high-speed centrifugation at 4 C. Then, the proteins were denatured and separated by 12% SDS PAGE (30 mg/ lane). Subsequently, the proteins were transferred onto polyvinylidene difluoride membranes (PVDF), and the membranes were successively subjected to the procedure including blocking, primary antibody incubation, TBST washing, secondary antibody incubation, and TBST washing. At last, the membranes were visualized using chemiluminescent HRP substrate (BI, Jerusalem, Israel) via a FluorChem FC3 system (Proteinsimple, CA, USA). The specific antibodies and the dilution ratio were as follow: Anti-ETV4 rabbit polyclonal antibody (dilution: 1:500, BBI, Shanghai, China), mouse-anti-GAPDH monoclonal (dilution: 1:5000, Abclonal, MA, USA), Anti-MSI2 rabbit polyclonal antibody(dilution: 1:500, BBI, Shanghai, China), HRP-conjugated Mouse anti-rabbit IgG (dilution:1:5000, BBI, Shanghai, China), HRP-conjugated Goat AntiMouse IgG (dilution:1:5000, BBI, Shanghai, China). 2.5. Immunohistochemistry (IHC)
Fresh tissue specimens, including 15 cases of LUAD tissues and 8 cases of normal lung tissues, were collected from surgical patients with LUAD and other non-malignant lung diseases in the Xiangya Hospital of Central South University. The specimens were divided into two parts, one part fixed by formaldehyde for paraffin section production, other one frozen by nitrogen liquid for protein and RNA extraction. Informed consent was acquired from all patients. Moreover, this study was approved and supervised by the Ethical Committee of Xiangya Hospital of Central South University.
The expression of ETV in lung and LUAD tissues were detected by IHC using UltraSensitive™ streptavidin-peroxidase IHC Kit (MaiXin, Fuzhou, China) as previously described [19]. Briefly, after dewaxing, antigen retrieval and H2O2 treatment, the paraffinembedded tissue slides, with antigen retrieval and endogenous peroxidase activity elimination, were successively incubated with the ETV4 antibody (dilution: 1:100, BBI, Shanghai, China), biocatalytic secondary antibody, and avidin-biotin-peroxidase complex. Finally, sections were treated with 30 , 30 -diaminobenzidine, and countersigned with Harris’ modified hematoxylin. The photos were obtained with a microscopic imaging system (Lecia, Wizla, Germany).
2.2. Cell culture
2.6. siRNAs, plasmids, and transfection
Human LUAD cell lines including A549, H1299, H358, H1650, and H1975, and normal bronchial line NL-20, purchased from American Type Culture Collection, were cultured with DMEM (BI, Jerusalem, Israel) medium supplemented with 10% fetal bovine serum (BI, Jerusalem, Israel) under a humidified cell incubator at 37 C with 5% CO2.
siETV4, specifically targeting ETV4, and siNC, control siRNAs, were purchased from RIBOBIO Inc (Guangzhou, China). The MSI2 expression vector, pENTER-MSI2, and corresponding vector were obtained from Vigene Bioscience Inc (MD, USA). The siRNAs and plasmids were introduced into LUAD cells with Lipofectamine™ 3000 according to the manufacturer's instructions.
2.1. Tissue samples
2.7. CCK-8 2.3. RNA isolation, reverse transcription, and quantitative reverse transcription polymerase chain reaction (qPCR) Total RNA from tissues and cells were extracted by Trizol™ reagent (Thermo, MA, USA) following instruction and previous protocol [19]. Then, the mRNAs were reversely transcripted into cDNA with FastKing gDNA Dispelling RT SuperMix (TIANGEN, Beijing, China) according to attached instruction. Then, the expression of ETV4 in tissues and cells was detected with SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China) via CFX Connect Real-Time System (Bio-Rad, CA, USA). Subsequently, the relative expression of ETV4 and MSI2 was analyzed with CFX Manager 2.0 (Bio-Rad, CA, USA). The specific primer sequences of ETV4 and GAPDH, as a normalized control, are listed as follow: ETV4 sense primer: 50 - CAGTGCCTTTACTCCAGTGCC-30 , ETV4 reverse primer: 50 -CTCAGGAAATTCCGTTGCTCT-30 ; MSI2 sense primer:50 - ATCCCACTACGAAACGCTCC-30 , MSI2 reverse primer:50 GGGGTCAATCGTCTTGGAATC-3’; GAPDH sense primer: 50 - CAGCAAGAGCACAAGAGGAA-30 , GAPDH reverse primer: 50 - ATGGTACATGACAAGGTGCGG-3’.
The cell growth and viability were analyzed by CCK-8 according to the previous description [21]. Briefly, LUAD cells were seeded into 96-well plates (1 103 cells/well, three paralleled wells). For successive 3 days, 10 ml CCK-8 was added, and the absorbance value at 450 nm was detected by using Epoch (Bio-Tek, VT, USA). Eventually, the data were statistically analyzed and demonstrated by the growth curves. 2.8. Plate clone formation assay The cell proliferation were investigated by plate clone formation assay as previously described [22]. Simply, LUAD cells were seeded into 6-well plates (800 cells/well, three paralleled wells) and continuously cultured for 7 days. Then, the cells were fixed by methanol and stained with crystal violet. A clone containing more than 50 cells were named as an effective clone, and the clone number was counted under an inverse microscope (Nikon, Tokyo, Japan).
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2.9. Transwell invasion assay The cell invasion were explored by Transwell invasion assay following the previous description [23]. Simply, 500 ml DMEM medium containing 2.5 104 cells was pipetted into the upper chamber. 750 ml DMEM supplemented 5% FBS was added into the low well. 36 h later, the cells were fixed methanol and stained with crystal violet. Subsequently, the invasive cells were photoed and counted after removing the remained cells in the chamber under an inverse microscope (Nikon, Tokyo, Japan). 2.10. Chromatin immunoprecipitation (ChIP) assay ChIP assay was performed using the Pierce™ Magnetic ChIP Kit (Thermo, MA, USA) following the instruction. Briefly, 4 106 cells were successively subjected to the procedures containing DNAprotein cross-linking, disruption of membrane and cytosol, MNase digestion and sonication, incubation ETV4 antibody, addition protein A/G magnetic beads for binding ETV4 immunoprecipitated complex, wash, elution, crosslink reversal and proteinase K treatment, and DNA clean-up. Subsequently, PCR was performed using specific MSI2 primers: siteA-F 50 -GGCAACATAAAGTTGTGGTTC-30 , siteA-R 50 TGGAAGGCTACAGACATTGTC-3’; siteB-F 50 -TCTCATGTGTTGAACAGCCAG -30 , siteB-R 50 - GTTCACTTCAGCCTCTCACC-3’; siteC-F 50 -
TATAGACCTCCACAAGCTGACAC-30 , siteC-R 50 GGGATTAGATTAGGAAAGGCC-3’. Input DNA (without immunoprecipitation) and normal IgG-precipitated DNA were loaded as positive and negative controls, respectively.
2.11. Dual luciferase reporter assay LUAD cells were seeded into 12-well plates at a density of 2.5 104 cells/well. The cells were transfected with the reporter plasmid containing wild MSI2 promoter and mutant MSI2 promoter at site B(from “ACAGGAAATG” to “ACACCAAATG”), respectively. Renilla luciferase reporter plasmids were also transfected into cells for signal normalization. 24 h later, the luciferase activity was detected using the Dual Luciferase Assay System Kit (Promega, WI, USA).
2.12. Statistical analysis All experiments were independently repeated for 3 times. Statistical analysis and charts were processed with GraphPad Prism version 8. For comparisons between two groups, a Student t-test or chi-square test was used. P < 0.05 were considered to be statistically significant.
Fig. 1. ETV4 is upregulated in LUAD. The level of ETV4 mRNA is upregulated in LUAD tissues indicated by data from TCGA (A), Oncomine (B), and qPCR assay (C). The level of ETV4 protein is upregulated in LUAD tissues demonstrating by west blot (D) and IHC (E). (F) Western blot and qPCR assay indicated that the level of ETV protein and mRNA is upregulated in LUAD cells. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
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3. Results 3.1. ETV4 is upregulated in LUAD. To investigate the expression of ETV4 in LUAD, we initially analyzed the online data from TCGA and GEO by using UALCAN and Oncomine, respectively. ETV4 is significantly upregulated in LUAD supported by the RNA-sequencing data from TCGA (Fig. 1A), which is validated by data from Oncomine (Fig. 1B). Moreover, qPCR (Fig. 1C), Western blot (Fig. 1D) and immunohistochemistry (Fig. 1E) analysis indicated that upregulation of ETV4 is also observed in our LUAD tissues. The expression of ETV4 is higher in LUAD cells than that in normal lung cells manifested by qPCR and Western blot assays (Fig. 1F). Collectively, those results confirmed that ETV4 is upregulated in LUAD, suggesting it may be an oncogenic regulator in lung cancer. 3.2. ETV4 silencing suppresses proliferation and invasion of LUAD cells Next, we explored the function of ETV4 in LUAD. Firstly, the expression of ETV4 was knocked down by transfecting specific siRNA targeting ETV4, indicated by Western blot results (Fig. 2A).
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Subsequently, CCK-8, plate clone formation, and Transwell invasion assays were conducted to analyze the role of ETV4. As Fig. 2B, C, and D showed, the proliferation and invasion ability of LUAD cells were constrained revealed by the lower growth rate, fewer clones, and less invasive cells. Thus, ETV4 depletion can inhibit proliferation and invasion of LUAD. 3.3. MSI2 is a direct transcriptional target of ETV4 in LUAD. Considering ETV4 is a transcript factor, we proposed that ETV4 may exert its functions by regulating the transcription of target genes. Thus, we analyzed the potential targets of ETV4 using the GTRD (Gene Transcription Regulation database). Six candidate genes, with sitecounts being more massive than or equal to 15, were obtained. Then, we analyzed the expression of candidate genes and their promoter methylation level by UALCAN, and the correlation between candidate genes and ETV4 by GEPIA in LUAD. The results indicated that MSI2 is upregulated and positively correlated with ETV4 and the promoter methylation level of MSI2 is comparable between normal and lung cancer tissues (Supplementary Fig. 1) indicating MSI2 may be a target of ETV4 in LUAD. Therefore, we detected the expression of MSI2 in LUAD cells with ETV4 silencing. Indeed, the MSI2 mRNA is significantly
Fig. 2. ETV4 silencing suppresses growth, proliferation, and invasion of LUAD cells. (A) Western blot indicated that ETV4 was significantly inhibited by siETV4 in A549 and H1299 cells. (B) ETV knockdown markedly inhibited the growth of A549 and H1299 cells indicated by CCK8. (C) ETV knockdown markedly inhibited proliferation of A549 and H1299 cells indicated by plate clone formation. (D) ETV knockdown markedly inhibited invasion of A549 and H1299 cells indicated by Transwell invasion assay. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
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downregulated in LUAD cells upon ETV4 depletion (Fig. 3A). Thus, we further predicted the bind sites of ETV4 at the promoter region, approximately 1.5 kb DNA region 5’ upstream of coding DNA sequence, of MSI2 using JASPAR. Three candidate binding sites with the highest score were selected for validation by ChIP assays (Fig. 3B). The gDNAs precipitated by ETV4 antibody were subjected to qPCR amplification using primer sets covering the region of putative binding sites. As Fig. 3C shown, strong binding signals were detected at site B (1123 to 1132), suggesting this site may be the binding site of ETV4 at MSI2 promoter. Whereas, no signal or weak signals were detected at site A (641 to 650) and site C (1461 to 1470), respectively. Subsequently, dual luciferase reporter assay was performed to determine further whether siteB is the functional binding site of ETV4 at the promoter of MSI2. Indeed, the relative luciferase activity of cells transfected with the mutant promoter of MSI2 reporter plasmids was remarkably lower than that in cells transfected with wild one (Fig. 3D). Therefore, the above results manifested that ETV4 can directly regulate the transcription of MSI2 in LUAD. 3.4. ETV4 promotes proliferation and invasion through upregulating MSI2 in LUAD. To reveal the role of MSI2 in ETV4 associated functions, ectopic
expression of MSI2 was applied in LUAD cells with ETV4 knockdown, and the effects on proliferation and invasion were detected. As Fig. 4A indicated, MSI2 was significantly upregulated. Next, the role of MSI2 in ETV4 related functions was analyzed. The data of CCK-8 and plate clone formation assays demonstrated ectopic expression of MSI2 markedly abrogated the inhibitory effects of ETV4-silencing on the proliferation (Fig. 4B and C). Accordingly, ectopic expression of MSI2 rescued the invasive ability of LUAD cells with ETV4 silencing indicated by the results of Transwell invasion assays (Fig. 4D). Altogether, our results indicate that ETV4 can promote proliferation and invasion of LUAD cells by upregulating MSI2. 4. Discussion In summary, we confirmed that ETV4 is upregulated in LUAD and proved that ETV4 overexpression could promote proliferation and invasion of LUAD cells. Mechanistically, we demonstrated that ETV could regulate MSI2 transcription via directly binding at its promoter region, which mediates the oncogenic functions of ETV4 in LUAD. Taken together, we indicated that upregulation of ETV4 could promote proliferation and invasion in an MSI2 dependent manner in LUAD. Aberrant expression of ETV4 and its oncogenic roles have been
Fig. 3. ETV4 can transcriptionally regulate MSI2 by direct binding at the promoter region of MSI2 in LUAD cells. (A) qPCR assay indicated that the level of MSI2 mRNA was significantly inhibited by ETV4 knockdown in A549 and H1299 cells. (B) The schematic diagram for predicted bind sites of ETV4 in the promoter region of MSI2. (C) ChIP assay revealed that strong signaling was detected via primers targeting siteB of MSI2. (D) Mutant at predicted siteB of MSI2 significantly decreased the luciferase activity in A549 and H1299 cells indicated by dual luciferase reporter system assay. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
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Fig. 4. Ectopic expression of MSI2 can rescue the inhibitory effects of ETV4 knockdown in LUAD. (A) Western blot indicated that ETV4 knockdown decreased MSI2 in A549 and H1299 cells, which can be rescued by ectopic expression of MSI2. (B) Ectopic expression of MSI2 released the inhibitory effects of ETV4 knockdown on growth in A549 and H1299 cells. (C) Ectopic expression of MSI2 eliminated the inhibitory effects of ETV4 knockdown on proliferation in A549 and H1299 cells. (D) Ectopic expression of MSI2 eliminated the inhibitory effects of ETV4 knockdown on invasion in A549 and H1299 cells. **P < 0.01; ***P < 0.001. LUAD: lung adenocarcinoma.
revealed in various cancers. For example, ETV4 attracts many attentions in prostate cancer. Overexpression of ETV4, serving as a negative indicator of prognosis, could promote tumor progression involving proliferation, metastasis, and therapy sensitization [9,24]. Moreover, ETV4 related fusion genes such as TMPRSS2-ETV4, KLK2ETV4, and CANTT-ETV4, define a new molecular subtype of prostate cancer [10,11]. Meanwhile, upregulation of ETV4 and pro-tumor effects have also been revealed in other tumors including breast cancer [7], pancreatic cancer [8], colorectal cancer [6], sarcomas [25], hepatocellular carcinoma [12], as well as esophageal squamous cell carcinoma [26], etc. Upregulation of ETV4 has been implied by online data from TCGA and Oncomine without further verification. Indeed, we confirmed that ETV4 is upregulated in LUAD, demonstrated by qPCR and Western blot analysis in our collected specimens. Moreover, consistent with existing studies, ETV4 knockdown notably inhibited LUAD proliferation and invasion as well, further confirming the universality of tumorigenic effects of ETV4. Regulating the transcription of target genes is the most common mechanism by which transcription factors exert their biological functions. For example, numerous studies have indicated that
functions of c-Myc and p53, two important transcription factors with antagonistic function, are almost dependent on downstream targets in carcinogenesis and progression [27,28]. Several targets of ETV4 have been identified in multiple cancers. ETV4 could promote proliferation via upregulating cyclin proteins like cyclin D1 [8] and cyclin D3 [29] by directly transcriptional regulation in pancreatic cancer and breast cancers. MMPs like MMP13 and urokinase plasminogen activator (uPA) are also transcriptionally regulated by ETV4 and involved in ETV4-drive metastasis in prostate cancer [24] and breast cancer [7]. Here, our results demonstrated that ETV4 could directly bind at the promoter of MSI2 and subsequently activate MSI2 transcription. Moreover, ectopic MSI2 expression can rescue the inhibitory effects induced by ETV4 silencing, suggesting oncogenic roles of ETV4 are, at least partially, dependent on MSI2 in LUAD. Similarly, the oncogenic roles of MSI2, involving regulations of proliferation, metastasis, treatment sensitivity, and prognosis evaluation, have been indicated by dozens of studies in multiple cancer types [16,17], including lung cancer [15]. MSI2 can promote non-small cell lung cancer metastasis via inhibiting claudins in a TGF-b signaling related manner [15]. Compared with numerous
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studies focusing on the downstream mechanisms of MSI2, there are few studies focusing on the mechanism of abnormal expression of MSI2 itself. The comparable promoter methylation level of MSI2 has been indicated by methylation sequencing data in multiple cancers, suggesting transcription factors may account for the aberrant expression of MSI2 in cancers. Accordingly, a recent study has confirmed that KLF4 can negatively regulate MSI2 transcription by directly binding at its promoter in pancreatic cancer [18]. In this study, maybe for the first time, we further revealed that ETV4 is a decisive transcription factor in the regulation of MSI2 transcription in LUAD. Collectively, our studies revealed that upregulation of ETV4 could promote malignant progression in LUAD via directly regulating MSI2 in transcriptional level. ETV4/MSI2 axis may be a potential diagnosis indicator and therapy target in LUAD. Conflicts of interest All authors declare no conflict of interest for this study. Acknowledgment The present study was supported by the National Natural Science Foundation of China (nos. 81770928). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.115. References [1] M.M. Jacobsen, S.C. Silverstein, M. Quinn, L.B. Waterston, C.A. Thomas, J.C. Benneyan, P.K.J. Han, Timeliness of access to lung cancer diagnosis and treatment: a scoping literature review, Lung Cancer 112 (2017) 156e164. [2] G.A. Rivera, H. Wakelee, Lung cancer in never smokers, Adv. Exp. Med. Biol. 893 (2016) 43e57. [3] A.C. Borczuk, Prognostic considerations of the new world health organization classification of LUAD, Eur. Respir. Rev. 25 (2016) 364e371. [4] K. Inamura, Clinicopathological characteristics and mutations driving development of early LUAD: tumor initiation and progression, Int. J. Mol. Sci. 19 (2018). [5] S. Oh, S. Shin, R. Janknecht, ETV1, 4 and 5: an oncogenic subfamily of ETS transcription factors, Biochim. Biophys. Acta 1826 (2012) 1e12. [6] J. Xiao, S. Yang, P. Shen, Y. Wang, H. Sun, F. Ji, D. Zhou, Phosphorylation of ETV4 at Ser73 by ERK kinase could block ETV4 ubiquitination degradation in colorectal cancer, Biochem. Biophys. Res. Commun. 486 (2017) 1062e1068. [7] M. Dumortier, F. Ladam, I. Damour, S. Vacher, I. Bieche, N. Marchand, Y. de Launoit, D. Tulasne, A. Chotteau-Lelievre, ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis, Breast Cancer Res. 20 (2018) 73. [8] N. Tyagi, S.K. Deshmukh, S.K. Srivastava, S. Azim, A. Ahmad, A. Al-Ghadhban, A.P. Singh, J.E. Carter, B. Wang, S. Singh, ETV4 facilitates cell-cycle progression in pancreatic cells through transcriptional regulation of cyclin D1, Mol. Cancer Res. 16 (2018) 187e196. [9] A. Aytes, A. Mitrofanova, C.W. Kinkade, C. Lefebvre, M. Lei, V. Phelan, H.C. LeKaye, J.A. Koutcher, R.D. Cardiff, A. Califano, M.M. Shen, C. Abate-Shen, ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) E3506eE3515.
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