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ZNF224 is a transcriptional repressor of AXL in chronic myeloid leukemia cells
Biochimie 154 (2018) 127e131
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Short communication
ZNF224 is a transcriptional repressor of AXL in chronic myeloid leukemia cells Gaetano Sodaro a, Giancarlo Blasio a, Federica Fiorentino a, Patrick Auberger b, Paola Costanzo a, *, Elena Cesaro a a b
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, 80131, Italy ^te d’Azur, Inserm, Nice, 06204, France Universit e Co
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2018 Accepted 31 August 2018 Available online 31 August 2018
ZNF224 is a KRAB-zinc finger transcription factor that exerts a key tumor suppressive role in chronic myelogenous leukemia. In this study, we identify the receptor tyrosine kinase Axl as a novel target of ZNF224 transcriptional repression activity. Axl overexpression is found in many types of cancer and is frequently associated with drug resistance. Interestingly, we also found that sensitivity to imatinib can be partly restored in imatinib-resistant chronic myelogenous leukemia cells by ZNF224 overexpression and the resulting suppression of Axl expression. These results, in accordance with our previous findings, support the role of ZNF224 in imatinib responsiveness and shed new insights into potential therapeutic use of ZNF224 in imatinib-resistant chronic myelogenous leukemia. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: ZNF224 Transcription Axl Chronic myelogenous leukemia Imatinib-resistance
1. Introduction The KRAB zinc-finger transcription factor ZNF224 plays a proapoptotic and antiproliferative role in chronic myelogenous leukemia (CML), acting as a cofactor of Wilms' tumor 1 (WT1) protein and modulating WT1 dependent transcription of apoptotic genes [1,2]. Interestingly, we also observed that ZNF224 expression is negatively regulated by Bcr-Abl fusion protein in CML cell lines and in CML patients and, consistently, inhibition of Bcr-Abl activity by imatinib and second-generation tyrosine kinase inhibitors (TKIs) triggers upregulation of ZNF224 expression [3]. Moreover, we recently demonstrated that ZNF224 is a novel transcriptional repressor of c-Myc oncogene in CML and mediates the imatinibdependent transcriptional repression on c-Myc. In agreement with the role played by c-myc in imatinib responsiveness [4], we also provided evidence that ZNF224 induction, leading to c-Myc repression could contribute to overcome imatinib resistance [5], that represents a key issue in CML research and challenge in clinical
* Corresponding author. Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via Pansini 5, 80131, Naples, Italy. E-mail address: [email protected] (P. Costanzo).
practice. On the other hand, recent findings reported the overexpression of the receptor tyrosine kinase Axl as a mechanism of resistance to TKIs, independent from Bcr-Abl mutations [6,7]. Axl is a receptor tyrosine kinase of the TAM family and was originally isolated as a transforming gene from patients with chronic myeloid leukemia (CML) [8]. Activation of Axl by growth arrest-specific protein 6 (GAS6) ligand, regulates a number of pro-survival pathways, such as RAS, Pi3K/Akt, and mTOR. Axl is overexpressed in several haematologic and solid malignancies and its overexpression drives a wide range of processes, including epithelial to mesenchymal transition, tumor angiogenesis, resistance to chemotherapeutic and targeted agents [9e13]. However, despite the role of Axl in controlling various malignant properties, the transcription factors involved in the modulation of its expression are still largely unknown. Axl overexpression has been found in Imatinib-resistant CML cell lines and patients [14]. Consistently, it has been demonstrated that Axl knockdown re-sensitized TKI-resistant CML cells to Imatinib, while Axl overexpression confers a refractory response to Imatinib in CML cells [15]. Therefore, understanding of molecular mechanisms involved in Axl overexpression could lead to the development of new
https://doi.org/10.1016/j.biochi.2018.08.011 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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Fig. 1. ZNF224 is a transcriptional repressor of Axl. (A) Western blot analysis of ZNF224 and Axl protein levels in K562 Ima-S and Ima-R, Jurl-MK1 Ima-S and Ima-R. b-actin was used as loading control. (B) Left panel: Western blot analysis of ZNF224 and Axl protein levels in shGFP, shC3 and shE7 cells. GAPDH was used as loading control. Right panel: Axl mRNA levels were measured by RT-qPCR. Error bars represent standard deviations of three independent experiments. (C) Left panel: Western blot analysis of ZNF224-Flag and Axl protein levels in K562 cells transfected with 1,5 mg of ZNF224-Flag expression plasmid or Flag empty vector as control (). b-tubulin was used as loading control. Right panel: Axl mRNA levels were measured by RT-qPCR. Error bars represent standard deviations of three independent experiments. (D) Apoptosis was evaluated after treatment with 1 mM imatinib or vehicle only (DMSO). Error bars represent standard deviations of two independent experiments. (E) X-ChIP assay performed in HEK293 cells with an anti-ZNF224 antibody. The immunoprecipitated DNA was analyzed by qPCR using primers flanking the putative ZNF224 binding region. An unrelated region was used as negative control (UTR). Data shown are the means ± SD of two independent experiments. (F) Upper panel: Schematic representation of Axl promoter region showing the putative ZNF224 binding
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approaches to improve imatinib responsiveness in CML. In the present study, starting from a good anti-correlation between ZNF224 and Axl expression in CML cells resistant to imatinib, we demonstrated that ZNF224 is a transcriptional repressor of Axl gene. Intriguingly, we also showed that ZNF224 induction could contribute to restore imatinib sensitivity of imatinib-resistant CML cells, hampering Axl overexpression. 2. Materials and methods
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2.5. Apoptosis assay Cell death was assayed by annexin V (APC-conjugated) staining as previously described [5]. Apoptosis was measured on FACS flow cytometer (BD Accuri C6 Flow Cytometer, BD Biosciences). For each sample, we acquired a comparable cell number (10,000 cells). 2.6. Statistical analysis Statistical analysis was performed with Student's t-test.
2.1. Cell culture, transfection and luciferase reporter assays
3. Results and discussion
HEK293, K562 and Ima-R K562 cells were grown and transfected with 3X-Flag ZNF224 expression plasmid or 3X-Flag empty vector as previously described [5]. K562 cells stably knocked-down for ZNF224 were previously described [5]. P6-AXL reporter plasmid containing the Axl promoter region - 614/þ7 was a gift from Heike Allgayer [16]. Luciferase activity was measured using the DualLuciferase Reporter Assay System (Promega Corporation, WI, USA), according to the manufacturer's instructions.
3.1. ZNF224 expression inversely correlates with Axl expression in CML cells
2.2. RNA isolation, reverse transcription, and real-time PCR The procedures for extraction of total RNA, reverse transcription and real-time PCR have been described elsewhere [5,17]. The sequences of the primers were: Axl Fw: 50 -GGTGGCTGTGAAGACGATGA-30 , Axl Rw: 50 -CTCAGATACTCCATGCCACT-3’; Abl Fw: 50 GATGTAGTTGCTTGGGACCCA-30 , Abl Rw: 50 -TGGAGATAACACTCT AAGCATACT-3’; b2 microglobulin Fw: 50 eCCGTGGCCTTAGCTGTG CT-30 , b2 microglobulin Rw: 50 -TCGGATGGATGAAACCCAGA-3’. The relative quantification in gene expression was determined using the DDCT method and Abl or b2 microglobulin as housekeeping genes.
Our recent findings showed that ZNF224 mediates imatinibinduced cell death in CML cells. Overexpression of Axl tyrosine kinase receptor is one of the causes of imatinib resistance in CML cell lines [15]. Interestingly, by western blot analysis we observed that the higher expression of Axl in imatinib-resistant (Ima-R) cell lines compared to their imatinib-sensitive (Ima-S) counterpart was associated with a lower expression of ZNF224 (Fig. 1A). Prompted by this observation, we evaluated the effect of ZNF224 knocking-down or overexpression on Axl expression in K562 cells. As shown in Fig. 1B, Axl protein and mRNA levels were increased in K562 cells stably knocked-down for ZNF224 (shC3 and shE7 cells), while they were sensibly reduced by ZNF224 overexpression (Fig. 1C). Consistently and in agreement with our previous data [5], we observed that ZNF224 overexpression increased imatinib sensitivity of K562 cells (Fig. 1D). These results suggest that ZNF224 could be involved in imatinib responsiveness of CML cells also through the repression of Axl. 3.2. ZNF224 is a transcriptional repressor of Axl gene
2.3. Chromatin immunoprecipitation Chromatin immunoprecipitation assay (X-ChIP) was performed as previously described [5]. Immunoprecipitated DNA and input controls were analyzed by quantitative real-time PCR using a Master Mix SYBRGreen (Bio-Rad) and the following primer pairs for Axl promoter (Fw: 50 -GAGAGGGGAGTGGAGTTCTG-30 , Rev:50 -AGG GAGGGAGCTGAGGAG-30 ) and unrelated region (50 -GAAGCGGAAA TTGCAGTGAG-30 , Rev:50 -AGGGATAGGGTCTTGCTACG-30 ). The percentage of DNA immunoprecipitated with anti-ZNF224 antibody was calculated relative to the ChIP input DNA. 2.4. Cell lysates and western blot assays Total cell lysates were prepared by homogenization in modified RIPA buffer [18]. The membranes were incubated with the following antibodies: anti-ZNF224 (rabbit polyclonal, T3) diluted 1:300 in Super-Block Blocking Buffer (Thermo Scientific, Waltham, MA, USA), anti-AXL, anti-c-myc and anti-PCNA (Santa Cruz Biotechnology, CA, USA) diluted 1:500, anti-GAPDH (Santa Cruz Biotechnology, CA, USA) diluted 1:1000, anti- b-tubulin (Upstate, Lake Placid, NY) diluted 1:1000, anti-Flag and anti-b-actin (Sigma-Aldrich) diluted 1:1000. Signals were detected with ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology).
Subsequently, an in silico analysis revealed one putative binding site for ZNF224 on the promoter of Axl gene at -213/-206 bp upstream of the transcription start site (TSS). We evaluated the ZNF224 binding on this site performing XChIP assays in HEK293 cells. Chromatin was immunoprecipitated with a specific antibody for endogenous ZNF224 protein and then analyzed by RT-qPCR using oligonucleotides flanking the ZNF224 putative binding region. The results, shown in Fig. 1E, confirmed in vivo ZNF224 occupancy on the Axl promoter. To explore whether Axl transcriptional activity was repressed by ZNF224, a luciferase reporter plasmid containing the Axl promoter region -614/þ7 (P6-AXL) (Fig. 1F upper panel) was introduced into HEK293 cells and luciferase activity was measured in the presence of increasing amounts of the ZNF224 expression vector. As shown in Fig. 1F (lower panel), Axl promoter activity was reduced in a dose-dependent manner by ZNF224 overexpression. 3.3. ZNF224 suppresses Axl expression and restores imatinibsensitivity in imatinib-resistant CML cells At first, by X-ChIP assays we demonstrated that ZNF224 is able to bind Axl promoter in both imatinib-sensitive and -resistant K562 cells (Fig. 2A, left panel). We also observed a reduced ZNF224 binding on Axl promoter in K562 Ima-R cells compared to Ima-S
site (underlined nucleotides). Lower panel shows the luciferase reporter assay of Axl promoter P6 activity. 0,2 mg of P6 construct were transfected into HEK293 cells together with increasing amounts of ZNF224-Flag or Flag empty vector as control (). The promoter activity was determined 24 h post-trasfection by normalizing Firefly to Renilla luciferase activity. Error bars represent standard deviations of three independent experiments. Expression of ZNF224-Flag recombinant protein was verified by western blot analysis. GAPDH was used as loading control.
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Fig. 2. ZNF224 represses Axl in CML cells and contributes to override imatinib-resistance. (A) Left panel: X-ChIP assay performed in K562 Ima-S and K562 Ima-R cells with an anti-ZNF224 antibody. qPCR analysis was performed using primers flanking the putative ZNF224 binding region. An unrelated region was used as negative control (UTR). Data shown are the means ± SD of two independent experiments. Right panel: Axl mRNA levels were measured by RT-qPCR. Error bars represent standard deviations of three independent experiments. (B) 0,5 mg of P6 construct were transfected into K562 Ima-S and K562 Ima-R cells together with 1 mg of ZNF224-Flag or Flag empty vector as control (). The promoter activity was determined 24 h post-transfection by normalizing Firefly to Renilla luciferase activity. Error bars represent standard deviations of three independent experiments. Expression of ZNF224-Flag recombinant protein was verified by western blot analysis. GAPDH was used as loading control. (C) Western blot analysis of ZNF224-Flag, cmyc, Axl, PCNA levels in K562 ima-R cells transfected with 1,5 mg of ZNF224-Flag or Flag empty vector (). (D) Apoptosis was evaluated after treatment with imatinib 2 mM or DMSO () for 24 h. Error bars represent standard deviations of two independent experiments.
that is associated with an increased Axl mRNA expression (Fig. 2A, right panel). Then, we confirmed the transcriptional repressive activity of ZNF224 on Axl promoter in both leukemia cell lines by luciferase assays, as shown in Fig. 2B. These data strongly indicate that the down-regulation of ZNF224 in K562 Ima-R cells (see Fig. 1A) is one of the factors involved in Axl overexpression. Our findings contribute to extend the current knowledge on the transcriptional regulation of Axl gene [16,19,20], that is still poorly understood, and shed light on the mechanisms of Axl overexpression in leukemia. Importantly, recent findings indicate that the transcriptional regulation plays a relevant role in Axl expression in imatinib-resistant CML cells, through the AP1 transcription factor [15]. Finally, we found that ZNF224 overexpression in Ima-R K562 cells was accompanied by decreased protein levels of Axl, PCNA and c-myc (Fig. 2C) and increased imatinib sensitivity in these cells (Fig. 2D).
Identification of the oncogenic receptor tyrosine kinase Axl as a novel target of ZNF224 transcriptional repression strengthens our belief that strategies based on ZNF224 induction could represent a new and promising therapeutic approach in CML. Indeed, ZNF224 expression leads to the simultaneous repression of c-Myc and Axl oncogenes, which are involved in different mechanisms of resistance to imatinib. 4. Conclusions In this study, we demonstrate that ZNF224 is a transcriptional repressor of Axl and suggest that it may represent a new therapeutic target in CML. Indeed, inhibition of Axl expression could contribute to bypass resistance to conventional therapies, leading to the inactivation of Axl mediated downstream pathways such as PI3K, MAPK, and PKC involved in increased proliferation and survival of CML cells [11]. Since Axl overexpression is linked to imatinib resistance in other
G. Sodaro et al. / Biochimie 154 (2018) 127e131
malignancies, such as gastrointestinal stromal tumors [6] and nonsmall cell lung carcinoma [21], dissecting the ZNF224/Axl transcriptional regulatory pathway in various cancer types could lead to a better definition of ZNF224 role in cancer resistance and progression. Conflicts of interest The authors declare no conflict of interest. Author contributions G.S. designed and performed experiments, analyzed data and wrote the first draft of the manuscript; G.B., F.F., performed experiments; P.A. contributed to the experimental design and provided scientific advice. P.C. designed experiments and wrote the manuscript. E.C developed the concept, designed and analyzed experiments. Acknowledgement We are grateful to Prof. Heike Allgayer for providing the P6-AXL reporter plasmid. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] F. Florio, E. Cesaro, G. Montano, P. Izzo, C. Miles, P. Costanzo, Biochemical and functional interaction between ZNF224 and ZNF255, two members of the Kruppel-like zinc-finger protein family and WT1 protein isoforms, Hum. Mol. Genet. 19 (2010) 3544e3556. [2] G. Montano, E. Cesaro, L. Fattore, K. Vidovic, C. Palladino, R. Crescitelli, et al., Role of WT1-ZNF224 interaction in the expression of apoptosis-regulating genes, Hum. Mol. Genet. 22 (2013) 1771e1782. [3] G. Montano, K. Vidovic, C. Palladino, E. Cesaro, G. Sodaro, C. Quintarelli, et al., WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene, Oncotarget 6 (29) (2015) 28223e28237. [4] M. Albajr, M.T. Gomer-Cesares, J. Lorca, I. Mauleon, J.P. Vaque, Myc in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib, Mol. Canc. Res. 9 (2011) 564e576. [5] G. Sodaro, E. Cesaro, G. Montano, G. Blasio, F. Fiorentino, S. Romano, et al., Role of ZNF224 in c-Myc repression and imatinib responsiveness in chronic
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myeloid leukemia, Oncotarget 9 (2018) 3417e3431. [6] D. Mahadevan, L. Cooke, C. Riley, R. Swart, B. Simons, K. Della Croce, et al., A novel tyrosine kinase switch is a mechanism of imatinib resistance in gastrointestinal stromal tumors, Oncogene 26 (2007) 3909e3919. [7] Z. Zhang, J.C. Lee, L. Lin, V. Olivas, V. Au, T. LaFramboise, et al., Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer, Nat. Genet. 44 (8) (2012) 852e860. [8] J.P. O'Bryan, R.A. Frye, P.C. Cogswell, A. Neubauer, B. Kitch, C. Prokop, et al., Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase, Mol. Cell Biol. 11 (1991) 5016e5031. [9] S. Hafizi, B. Dahlback, Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases, Cytokine Growth Factor Rev. 17 (4) (2006) 295e304. [10] D.K. Graham, D. DeRyckere, K.D. Davies, H.S. Earp, The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer, Nat.Rev. Cancer. 14 (2014) 769e785. [11] M. Scaltriti, M. Elkabets, J. Baselga, Molecular pathways: AXL, a membrane receptor mediator of resistance to therapy, Clin. Canc. Res. 22 (6) (2016) 1313e1317. [12] M.K. Asiedu, F.D. Beauchamp-Perez, J.N. Ingle, M.D. Behrens, D.C. Radisky, K.L. Knutson, AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells, Oncogene 33 (2014) 1316e1324. [13] E.B. Rankin, K.C. Fuh, T.E. Taylor, A.J. Krieg, M. Musser, J. Yuan, et al., AXL is an essential factor and therapeutic target for metastatic ovarian cancer, Canc. Res. 70 (19) (2010) 7570e7579. [14] S. Grosso, A. Puissant, M. Dufies, P. Colosetti, A. Jacquel, k. Lebrigand, et al., Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors, Mol. Canc. Therapeut. 8 (7) (2009) 1924e1933. [15] M. Dufies, A. Jacquel, N. Belhacene, G. Robert, T. Cluzeau, F. Luciano, J. P, et al., Mechanisms of AXL overexpression and function in Imatinib-resistant chronic myeloid leukemia cells, Oncotarget 2 (2011) 874e885. [16] G. Mudduluru, H. Allgayer, The human receptor tyrosine kinase Axl gene promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation, Biosci. Rep. 28 (2008) 161e176. [17] T. Busiello, M. Ciano, S. Romano, G. Sodaro, O. Garofalo, D. Bruzzese, et al., Role of ZNF224 in cell growth and chemoresistance of chronic lymphocitic leukemia, Hum. Mol. Genet. 26 (2) (2017) 344e353. [18] S. Romano, Y. Xiao, M. Nakaya, A. D'Angelillo, M. Chang, J. Jin, FKBP51 employs both scaffold and isomerase functions to promote NF-jB activation in melanoma, Nucleic Acids Res. 43 (2015) 6983e6993. [19] G. Mudduluru, J.H. Leupold, P. Stroebel, H. Allgayer, PMA up-regulates the transcription of Axl by AP-1 transcription factor binding to TRE sequences via the MAPK cascade in leukaemia cells, Biol. Cell. 103 (1) (2010) 21e33. [20] G. Mudduluru, P. Vajkoczy, H. Allgayer, Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer, Mol. Canc. Res. 8 (2) (2010) 159e169. [21] R.M.A. Linger, R.A. Cohen, C.T. Cummings, S. Sather, J. Migdall-Wilson, D.H.G. Middleton, et al., Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer, Oncogene 32 (2013) 3420e3431.
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Short communication
ZNF224 is a transcriptional repressor of AXL in chronic myeloid leukemia cells Gaetano Sodaro a, Giancarlo Blasio a, Federica Fiorentino a, Patrick Auberger b, Paola Costanzo a, *, Elena Cesaro a a b
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, 80131, Italy ^te d’Azur, Inserm, Nice, 06204, France Universit e Co
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2018 Accepted 31 August 2018 Available online 31 August 2018
ZNF224 is a KRAB-zinc finger transcription factor that exerts a key tumor suppressive role in chronic myelogenous leukemia. In this study, we identify the receptor tyrosine kinase Axl as a novel target of ZNF224 transcriptional repression activity. Axl overexpression is found in many types of cancer and is frequently associated with drug resistance. Interestingly, we also found that sensitivity to imatinib can be partly restored in imatinib-resistant chronic myelogenous leukemia cells by ZNF224 overexpression and the resulting suppression of Axl expression. These results, in accordance with our previous findings, support the role of ZNF224 in imatinib responsiveness and shed new insights into potential therapeutic use of ZNF224 in imatinib-resistant chronic myelogenous leukemia. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: ZNF224 Transcription Axl Chronic myelogenous leukemia Imatinib-resistance
1. Introduction The KRAB zinc-finger transcription factor ZNF224 plays a proapoptotic and antiproliferative role in chronic myelogenous leukemia (CML), acting as a cofactor of Wilms' tumor 1 (WT1) protein and modulating WT1 dependent transcription of apoptotic genes [1,2]. Interestingly, we also observed that ZNF224 expression is negatively regulated by Bcr-Abl fusion protein in CML cell lines and in CML patients and, consistently, inhibition of Bcr-Abl activity by imatinib and second-generation tyrosine kinase inhibitors (TKIs) triggers upregulation of ZNF224 expression [3]. Moreover, we recently demonstrated that ZNF224 is a novel transcriptional repressor of c-Myc oncogene in CML and mediates the imatinibdependent transcriptional repression on c-Myc. In agreement with the role played by c-myc in imatinib responsiveness [4], we also provided evidence that ZNF224 induction, leading to c-Myc repression could contribute to overcome imatinib resistance [5], that represents a key issue in CML research and challenge in clinical
* Corresponding author. Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via Pansini 5, 80131, Naples, Italy. E-mail address: [email protected] (P. Costanzo).
practice. On the other hand, recent findings reported the overexpression of the receptor tyrosine kinase Axl as a mechanism of resistance to TKIs, independent from Bcr-Abl mutations [6,7]. Axl is a receptor tyrosine kinase of the TAM family and was originally isolated as a transforming gene from patients with chronic myeloid leukemia (CML) [8]. Activation of Axl by growth arrest-specific protein 6 (GAS6) ligand, regulates a number of pro-survival pathways, such as RAS, Pi3K/Akt, and mTOR. Axl is overexpressed in several haematologic and solid malignancies and its overexpression drives a wide range of processes, including epithelial to mesenchymal transition, tumor angiogenesis, resistance to chemotherapeutic and targeted agents [9e13]. However, despite the role of Axl in controlling various malignant properties, the transcription factors involved in the modulation of its expression are still largely unknown. Axl overexpression has been found in Imatinib-resistant CML cell lines and patients [14]. Consistently, it has been demonstrated that Axl knockdown re-sensitized TKI-resistant CML cells to Imatinib, while Axl overexpression confers a refractory response to Imatinib in CML cells [15]. Therefore, understanding of molecular mechanisms involved in Axl overexpression could lead to the development of new
https://doi.org/10.1016/j.biochi.2018.08.011 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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Fig. 1. ZNF224 is a transcriptional repressor of Axl. (A) Western blot analysis of ZNF224 and Axl protein levels in K562 Ima-S and Ima-R, Jurl-MK1 Ima-S and Ima-R. b-actin was used as loading control. (B) Left panel: Western blot analysis of ZNF224 and Axl protein levels in shGFP, shC3 and shE7 cells. GAPDH was used as loading control. Right panel: Axl mRNA levels were measured by RT-qPCR. Error bars represent standard deviations of three independent experiments. (C) Left panel: Western blot analysis of ZNF224-Flag and Axl protein levels in K562 cells transfected with 1,5 mg of ZNF224-Flag expression plasmid or Flag empty vector as control (). b-tubulin was used as loading control. Right panel: Axl mRNA levels were measured by RT-qPCR. Error bars represent standard deviations of three independent experiments. (D) Apoptosis was evaluated after treatment with 1 mM imatinib or vehicle only (DMSO). Error bars represent standard deviations of two independent experiments. (E) X-ChIP assay performed in HEK293 cells with an anti-ZNF224 antibody. The immunoprecipitated DNA was analyzed by qPCR using primers flanking the putative ZNF224 binding region. An unrelated region was used as negative control (UTR). Data shown are the means ± SD of two independent experiments. (F) Upper panel: Schematic representation of Axl promoter region showing the putative ZNF224 binding
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approaches to improve imatinib responsiveness in CML. In the present study, starting from a good anti-correlation between ZNF224 and Axl expression in CML cells resistant to imatinib, we demonstrated that ZNF224 is a transcriptional repressor of Axl gene. Intriguingly, we also showed that ZNF224 induction could contribute to restore imatinib sensitivity of imatinib-resistant CML cells, hampering Axl overexpression. 2. Materials and methods
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2.5. Apoptosis assay Cell death was assayed by annexin V (APC-conjugated) staining as previously described [5]. Apoptosis was measured on FACS flow cytometer (BD Accuri C6 Flow Cytometer, BD Biosciences). For each sample, we acquired a comparable cell number (10,000 cells). 2.6. Statistical analysis Statistical analysis was performed with Student's t-test.
2.1. Cell culture, transfection and luciferase reporter assays
3. Results and discussion
HEK293, K562 and Ima-R K562 cells were grown and transfected with 3X-Flag ZNF224 expression plasmid or 3X-Flag empty vector as previously described [5]. K562 cells stably knocked-down for ZNF224 were previously described [5]. P6-AXL reporter plasmid containing the Axl promoter region - 614/þ7 was a gift from Heike Allgayer [16]. Luciferase activity was measured using the DualLuciferase Reporter Assay System (Promega Corporation, WI, USA), according to the manufacturer's instructions.
3.1. ZNF224 expression inversely correlates with Axl expression in CML cells
2.2. RNA isolation, reverse transcription, and real-time PCR The procedures for extraction of total RNA, reverse transcription and real-time PCR have been described elsewhere [5,17]. The sequences of the primers were: Axl Fw: 50 -GGTGGCTGTGAAGACGATGA-30 , Axl Rw: 50 -CTCAGATACTCCATGCCACT-3’; Abl Fw: 50 GATGTAGTTGCTTGGGACCCA-30 , Abl Rw: 50 -TGGAGATAACACTCT AAGCATACT-3’; b2 microglobulin Fw: 50 eCCGTGGCCTTAGCTGTG CT-30 , b2 microglobulin Rw: 50 -TCGGATGGATGAAACCCAGA-3’. The relative quantification in gene expression was determined using the DDCT method and Abl or b2 microglobulin as housekeeping genes.
Our recent findings showed that ZNF224 mediates imatinibinduced cell death in CML cells. Overexpression of Axl tyrosine kinase receptor is one of the causes of imatinib resistance in CML cell lines [15]. Interestingly, by western blot analysis we observed that the higher expression of Axl in imatinib-resistant (Ima-R) cell lines compared to their imatinib-sensitive (Ima-S) counterpart was associated with a lower expression of ZNF224 (Fig. 1A). Prompted by this observation, we evaluated the effect of ZNF224 knocking-down or overexpression on Axl expression in K562 cells. As shown in Fig. 1B, Axl protein and mRNA levels were increased in K562 cells stably knocked-down for ZNF224 (shC3 and shE7 cells), while they were sensibly reduced by ZNF224 overexpression (Fig. 1C). Consistently and in agreement with our previous data [5], we observed that ZNF224 overexpression increased imatinib sensitivity of K562 cells (Fig. 1D). These results suggest that ZNF224 could be involved in imatinib responsiveness of CML cells also through the repression of Axl. 3.2. ZNF224 is a transcriptional repressor of Axl gene
2.3. Chromatin immunoprecipitation Chromatin immunoprecipitation assay (X-ChIP) was performed as previously described [5]. Immunoprecipitated DNA and input controls were analyzed by quantitative real-time PCR using a Master Mix SYBRGreen (Bio-Rad) and the following primer pairs for Axl promoter (Fw: 50 -GAGAGGGGAGTGGAGTTCTG-30 , Rev:50 -AGG GAGGGAGCTGAGGAG-30 ) and unrelated region (50 -GAAGCGGAAA TTGCAGTGAG-30 , Rev:50 -AGGGATAGGGTCTTGCTACG-30 ). The percentage of DNA immunoprecipitated with anti-ZNF224 antibody was calculated relative to the ChIP input DNA. 2.4. Cell lysates and western blot assays Total cell lysates were prepared by homogenization in modified RIPA buffer [18]. The membranes were incubated with the following antibodies: anti-ZNF224 (rabbit polyclonal, T3) diluted 1:300 in Super-Block Blocking Buffer (Thermo Scientific, Waltham, MA, USA), anti-AXL, anti-c-myc and anti-PCNA (Santa Cruz Biotechnology, CA, USA) diluted 1:500, anti-GAPDH (Santa Cruz Biotechnology, CA, USA) diluted 1:1000, anti- b-tubulin (Upstate, Lake Placid, NY) diluted 1:1000, anti-Flag and anti-b-actin (Sigma-Aldrich) diluted 1:1000. Signals were detected with ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology).
Subsequently, an in silico analysis revealed one putative binding site for ZNF224 on the promoter of Axl gene at -213/-206 bp upstream of the transcription start site (TSS). We evaluated the ZNF224 binding on this site performing XChIP assays in HEK293 cells. Chromatin was immunoprecipitated with a specific antibody for endogenous ZNF224 protein and then analyzed by RT-qPCR using oligonucleotides flanking the ZNF224 putative binding region. The results, shown in Fig. 1E, confirmed in vivo ZNF224 occupancy on the Axl promoter. To explore whether Axl transcriptional activity was repressed by ZNF224, a luciferase reporter plasmid containing the Axl promoter region -614/þ7 (P6-AXL) (Fig. 1F upper panel) was introduced into HEK293 cells and luciferase activity was measured in the presence of increasing amounts of the ZNF224 expression vector. As shown in Fig. 1F (lower panel), Axl promoter activity was reduced in a dose-dependent manner by ZNF224 overexpression. 3.3. ZNF224 suppresses Axl expression and restores imatinibsensitivity in imatinib-resistant CML cells At first, by X-ChIP assays we demonstrated that ZNF224 is able to bind Axl promoter in both imatinib-sensitive and -resistant K562 cells (Fig. 2A, left panel). We also observed a reduced ZNF224 binding on Axl promoter in K562 Ima-R cells compared to Ima-S
site (underlined nucleotides). Lower panel shows the luciferase reporter assay of Axl promoter P6 activity. 0,2 mg of P6 construct were transfected into HEK293 cells together with increasing amounts of ZNF224-Flag or Flag empty vector as control (). The promoter activity was determined 24 h post-trasfection by normalizing Firefly to Renilla luciferase activity. Error bars represent standard deviations of three independent experiments. Expression of ZNF224-Flag recombinant protein was verified by western blot analysis. GAPDH was used as loading control.
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Fig. 2. ZNF224 represses Axl in CML cells and contributes to override imatinib-resistance. (A) Left panel: X-ChIP assay performed in K562 Ima-S and K562 Ima-R cells with an anti-ZNF224 antibody. qPCR analysis was performed using primers flanking the putative ZNF224 binding region. An unrelated region was used as negative control (UTR). Data shown are the means ± SD of two independent experiments. Right panel: Axl mRNA levels were measured by RT-qPCR. Error bars represent standard deviations of three independent experiments. (B) 0,5 mg of P6 construct were transfected into K562 Ima-S and K562 Ima-R cells together with 1 mg of ZNF224-Flag or Flag empty vector as control (). The promoter activity was determined 24 h post-transfection by normalizing Firefly to Renilla luciferase activity. Error bars represent standard deviations of three independent experiments. Expression of ZNF224-Flag recombinant protein was verified by western blot analysis. GAPDH was used as loading control. (C) Western blot analysis of ZNF224-Flag, cmyc, Axl, PCNA levels in K562 ima-R cells transfected with 1,5 mg of ZNF224-Flag or Flag empty vector (). (D) Apoptosis was evaluated after treatment with imatinib 2 mM or DMSO () for 24 h. Error bars represent standard deviations of two independent experiments.
that is associated with an increased Axl mRNA expression (Fig. 2A, right panel). Then, we confirmed the transcriptional repressive activity of ZNF224 on Axl promoter in both leukemia cell lines by luciferase assays, as shown in Fig. 2B. These data strongly indicate that the down-regulation of ZNF224 in K562 Ima-R cells (see Fig. 1A) is one of the factors involved in Axl overexpression. Our findings contribute to extend the current knowledge on the transcriptional regulation of Axl gene [16,19,20], that is still poorly understood, and shed light on the mechanisms of Axl overexpression in leukemia. Importantly, recent findings indicate that the transcriptional regulation plays a relevant role in Axl expression in imatinib-resistant CML cells, through the AP1 transcription factor [15]. Finally, we found that ZNF224 overexpression in Ima-R K562 cells was accompanied by decreased protein levels of Axl, PCNA and c-myc (Fig. 2C) and increased imatinib sensitivity in these cells (Fig. 2D).
Identification of the oncogenic receptor tyrosine kinase Axl as a novel target of ZNF224 transcriptional repression strengthens our belief that strategies based on ZNF224 induction could represent a new and promising therapeutic approach in CML. Indeed, ZNF224 expression leads to the simultaneous repression of c-Myc and Axl oncogenes, which are involved in different mechanisms of resistance to imatinib. 4. Conclusions In this study, we demonstrate that ZNF224 is a transcriptional repressor of Axl and suggest that it may represent a new therapeutic target in CML. Indeed, inhibition of Axl expression could contribute to bypass resistance to conventional therapies, leading to the inactivation of Axl mediated downstream pathways such as PI3K, MAPK, and PKC involved in increased proliferation and survival of CML cells [11]. Since Axl overexpression is linked to imatinib resistance in other
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malignancies, such as gastrointestinal stromal tumors [6] and nonsmall cell lung carcinoma [21], dissecting the ZNF224/Axl transcriptional regulatory pathway in various cancer types could lead to a better definition of ZNF224 role in cancer resistance and progression. Conflicts of interest The authors declare no conflict of interest. Author contributions G.S. designed and performed experiments, analyzed data and wrote the first draft of the manuscript; G.B., F.F., performed experiments; P.A. contributed to the experimental design and provided scientific advice. P.C. designed experiments and wrote the manuscript. E.C developed the concept, designed and analyzed experiments. Acknowledgement We are grateful to Prof. Heike Allgayer for providing the P6-AXL reporter plasmid. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] F. Florio, E. Cesaro, G. Montano, P. Izzo, C. Miles, P. Costanzo, Biochemical and functional interaction between ZNF224 and ZNF255, two members of the Kruppel-like zinc-finger protein family and WT1 protein isoforms, Hum. Mol. Genet. 19 (2010) 3544e3556. [2] G. Montano, E. Cesaro, L. Fattore, K. Vidovic, C. Palladino, R. Crescitelli, et al., Role of WT1-ZNF224 interaction in the expression of apoptosis-regulating genes, Hum. Mol. Genet. 22 (2013) 1771e1782. [3] G. Montano, K. Vidovic, C. Palladino, E. Cesaro, G. Sodaro, C. Quintarelli, et al., WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene, Oncotarget 6 (29) (2015) 28223e28237. [4] M. Albajr, M.T. Gomer-Cesares, J. Lorca, I. Mauleon, J.P. Vaque, Myc in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib, Mol. Canc. Res. 9 (2011) 564e576. [5] G. Sodaro, E. Cesaro, G. Montano, G. Blasio, F. Fiorentino, S. Romano, et al., Role of ZNF224 in c-Myc repression and imatinib responsiveness in chronic
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myeloid leukemia, Oncotarget 9 (2018) 3417e3431. [6] D. Mahadevan, L. Cooke, C. Riley, R. Swart, B. Simons, K. Della Croce, et al., A novel tyrosine kinase switch is a mechanism of imatinib resistance in gastrointestinal stromal tumors, Oncogene 26 (2007) 3909e3919. [7] Z. Zhang, J.C. Lee, L. Lin, V. Olivas, V. Au, T. LaFramboise, et al., Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer, Nat. Genet. 44 (8) (2012) 852e860. [8] J.P. O'Bryan, R.A. Frye, P.C. Cogswell, A. Neubauer, B. Kitch, C. Prokop, et al., Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase, Mol. Cell Biol. 11 (1991) 5016e5031. [9] S. Hafizi, B. Dahlback, Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases, Cytokine Growth Factor Rev. 17 (4) (2006) 295e304. [10] D.K. Graham, D. DeRyckere, K.D. Davies, H.S. Earp, The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer, Nat.Rev. Cancer. 14 (2014) 769e785. [11] M. Scaltriti, M. Elkabets, J. Baselga, Molecular pathways: AXL, a membrane receptor mediator of resistance to therapy, Clin. Canc. Res. 22 (6) (2016) 1313e1317. [12] M.K. Asiedu, F.D. Beauchamp-Perez, J.N. Ingle, M.D. Behrens, D.C. Radisky, K.L. Knutson, AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells, Oncogene 33 (2014) 1316e1324. [13] E.B. Rankin, K.C. Fuh, T.E. Taylor, A.J. Krieg, M. Musser, J. Yuan, et al., AXL is an essential factor and therapeutic target for metastatic ovarian cancer, Canc. Res. 70 (19) (2010) 7570e7579. [14] S. Grosso, A. Puissant, M. Dufies, P. Colosetti, A. Jacquel, k. Lebrigand, et al., Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors, Mol. Canc. Therapeut. 8 (7) (2009) 1924e1933. [15] M. Dufies, A. Jacquel, N. Belhacene, G. Robert, T. Cluzeau, F. Luciano, J. P, et al., Mechanisms of AXL overexpression and function in Imatinib-resistant chronic myeloid leukemia cells, Oncotarget 2 (2011) 874e885. [16] G. Mudduluru, H. Allgayer, The human receptor tyrosine kinase Axl gene promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation, Biosci. Rep. 28 (2008) 161e176. [17] T. Busiello, M. Ciano, S. Romano, G. Sodaro, O. Garofalo, D. Bruzzese, et al., Role of ZNF224 in cell growth and chemoresistance of chronic lymphocitic leukemia, Hum. Mol. Genet. 26 (2) (2017) 344e353. [18] S. Romano, Y. Xiao, M. Nakaya, A. D'Angelillo, M. Chang, J. Jin, FKBP51 employs both scaffold and isomerase functions to promote NF-jB activation in melanoma, Nucleic Acids Res. 43 (2015) 6983e6993. [19] G. Mudduluru, J.H. Leupold, P. Stroebel, H. Allgayer, PMA up-regulates the transcription of Axl by AP-1 transcription factor binding to TRE sequences via the MAPK cascade in leukaemia cells, Biol. Cell. 103 (1) (2010) 21e33. [20] G. Mudduluru, P. Vajkoczy, H. Allgayer, Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer, Mol. Canc. Res. 8 (2) (2010) 159e169. [21] R.M.A. Linger, R.A. Cohen, C.T. Cummings, S. Sather, J. Migdall-Wilson, D.H.G. Middleton, et al., Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer, Oncogene 32 (2013) 3420e3431.