Background
Laryngeal carcinoma is the most common cancer in the larynx and the second most common malignant tumor of the respiratory system. The incidence of laryngeal cancer is generally low, accounting for 1–5% of all cancer sites, and in the United States, there were 13,430 new cases of laryngeal carcinoma and 3620 cancer deaths in 2016 [
1]. Laryngeal squamous cell carcinoma (LSCC) is the major type for laryngeal carcinoma (about 95%) [
2]. Epidemiological investigations show that smoking is considered as the primary risk factor for laryngeal cancer and alcohol drinking is also an important pathogenic factor [
3]. The mechanism of smoking may be due to a large amount of carcinogen in tobacco smoke, trigging cellular DNA damage and eventually leading to carcinogenesis [
4]. Genetic studies found that oncogene c-Myc is related to the event of laryngeal cancer [
5]. c-Myc is a transcription factor containing a leukine zipper and regulates a large number of target genes [
6]. Overexpression of c-Myc promotes cell growth, proliferation, apoptosis, transformation, pre-messenger-RNA splicing and genomic instability [
7]. It has been reported that c-Myc is highly amplified in LSCC tissues, and the positive rate of c-Myc amplification and copy-number change increased with the increasing severity of laryngeal lesions [
8]. MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene and a direct downstream gene of c-Myc in LSCC. DNA methylation of MYCT1 altered the promoter activity by interfering with its binding to c-Myc in LSCC [
9]. Overexpression of MYCT1 could inhibit cell proliferation and invasion and promote apoptosis in LSCC cells [
10]. In spite of therapeutic advances, the 5-year survival rate for LSCC patients remains relatively low [
1]. Thus, it is warranted to better understand the molecular mechanisms underlying LSCC tumorigenesis and to identify the novel therapeutic targets for LSCC treatment.
The human Evi5 gene was originally isolated in a patient with stage 4S neuroblastoma with a constitutional chromosome translocation [
11]. Evi5 belongs to a small subfamily of the Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, functions as a GTPase-activating protein for the Rab11 GTPase [
12‐
14]. Evi5 is also required to allow the accumulation of the anaphase promoting complex, cyclosome (APC/C) inhibitor Emi1 in late G1. In the absence of Evi5, Emi1 is rapidly destructed in a Polo-like Kinase 1 (PLK1)- and β-transducin repeat-containing protein (β-TrCP)-dependent manner, resulting in a failure to inactivate the APC/C, which in turn leads to a failure in S phase entry. Silencing of Evi5 caused increased centrosome number and a multinucleation phenotype, reflecting a failed cytokinesis event [
15,
16]. The deregulated expression of Evi5 has also been observed in several kinds of cancers, indicating a tumor promoting function of Evi5 [
17,
18]. However, whether Evi5 plays a role in LSCC is completely unknown.
In this study, we found that the Evi5 oncogene is an upstream regulator of c-Myc oncogene in LSCC. Evi5 antagonized FBXW7-mediated c-Myc destruction, leading to c-Myc accumulation and activation in LSCC.
Materials and methods
Cell cultures and transfection
Laryngeal cancer cell line TU212 cell line and HEK293T cells were purchased from the Shanghai Cell Collection (Shanghai, China). Cells were maintained in a humidified incubator at 37 °C with 5% CO2, grown in RPMI 1640 or DMEM supplemented with 10% FBS (Hyclone, UT, USA) and 100 IU/ml penicillin/streptomycin. All cell lines used were negative for mycoplasma. All plasmids were purchased from Shanghai Genechem Co., LTD. Shanghai, China. For transfection, TU212 cells were cultured as described to 70–80% confluence in a 6-well plate. 1 µg Flag-Evi5 and/or 1 µg HA-FBXW7 plasmids were co-transfected using Lipofectamine 2000 (Thermo Fisher Scientific, USA.) following the manufacturer’s instructions.
CRISPR/Cas9 knock out (KO) cell lines
TU212 cells were transfected with Evi5 KO plasmids (sc-407739, Santa Cruz Biotechnology, USA) using Lipofectamine 2000 (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Cells were selected with 1 µg/ml puromycin over 1 week. Single clones were then selected and the knockout efficiency was verified by western blot assay.
RNA isolation and real-time PCR
Total RNA of tissues or cell lysates were extracted by using TRIzol reagent (Invitrogen, Shanghai). The cDNA was synthesized by using PrimeScript RT reagent Kit (TaKaRa, Dalian, China), quantitative real-time PCR was performed by using a SYBR Green Premix Ex Taq (TaKaRa) on Light Cycler480 (Roche, Switzerland). GAPDH was used for normalization of mRNAs expression. The PCR condition is first an initial holding period at 95 °C for 5 min, followed by a two-step PCR program consisting of 95 °C for 5 s and 60 °C for 45 cycles. Relative quantitation analysis of gene expression results was performed according to the 2
−∆∆Ct method [
19]. The primers used were as follows: Cyclin D1, forward, 5′-GCTGCGAAGTGGAAACCATC-3′; reverse, 5′-CCTCCTTCTGCACACATTTGAA-3′. P21, forward, 5′-TGTCCGTCAGAACCCATGC-3′; reverse, 5′-AAGTCGAAGTTCCATCGCTC-3′. Evi5, forward, 5′-AGAAACCCTAGTGGGAAACAGG-3′; reverse, 5′-TGACTGTATGCGATACTGTGTTC-3′. C-Myc, forward, 5′-GGCTCCTGGCAAAAGGTCA-3′; reverse, 5′-CTGCGTAGTTGTGCTGATGT-3′.
GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse 5′-TGGTGAAGACGCCAGTGGA-3′.
Immunoprecipitation and TUBEs pulldown
Cells were lysed in RIPA buffer to extract total cellular protein. Lysates were cleared by centrifugation (12,000g for 30 min at 4 °C) and then filtered through 0.22 μM spin filters to further remove cell debris. The resulting lysates were clarified by centrifugation at 15,000g for 20 min at 4 °C before immunoprecipitation with antibodies and resin. Resin-containing immune complexes were washed 3 times with RIPA buffer washes and eluted with SDS loading buffer by boiling at 100 °C for 5 min. The immune complexes were then subjected to western blot assay. Ubiquitin immunoprecipitation was performed under denaturing conditions. Lysates were harvested in RIPA buffer, followed by sonication Ubiquitinated substrates were precipitated from lysates using agarose-bound Tandem Ubiquitin Binding Entities (TUBEs, Life Sensors, UM401) following the manufacturer’s protocol.
Western blot analysis
Cells were lysed in RIPA buffer to extract total cellular protein. Protein concentration was determined according to the BCA quantitative method, and 30 µg of each protein sample was resolved by SDS-PAGE and the protein bands were transferred to a nitrocellulose membrane. Following protein transfer, the membrane was blocked for 1 h in the presence of 5% skimmed milk proteins, following by incubation at 4 °C overnight with the primary antibodies targeted against Evi5, EMI1, c-Myc, cyclin D1 and p21 (Abcam, Cambridge, USA), Flag, HA and GAPDH (Sigma-Aldrich, St. Louis, MO, USA). On the following day, the blots were incubated with a secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h, and specific protein bands were visualized by an enhanced chemiluminescence (ECL) assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA).
Cycloheximide inhibition test
5 × 105 cells were cultured to 70–80% confluence in a 6-well plate and treated with 20 µg/ml cycloheximide (CHX; Sigma-Aldrich; Merck KGaA) for 0, 2, 4 or 8 h and. c-Myc protein expression was measured by western blot, using GAPDH as loading control.
FACS assay
Cells were harvested and fixed by 70% ice-cold ethanol for 1 h and then incubated with propidium iodide (PI) (Beyotime, Shanghai, China) in the presence of 0.2 mg/ml RNase A (Beyotime, Shanghai, China) for 15 min, at 37 °C. DNA content was measured on flow cytometry (Beckman, CA, USA).
Cells were seeded into 6-well plates (5 × 103 cells per well). Cells were then cultured in the in complete media for 1–2 weeks. Cells were fixed with methanol (1%) and formaldehyde (1%), stained with 0.5% crystal violet. All experiments were performed at least three times. Representative experiments are shown.
Luciferase reporter assays
To monitor the transfection activity of c-Myc, a c-Myc-dependent luciferase reporter plasmid (p4 ×E-SVP-Luc) was used. The p4 × E-SVP-Luc and the pRL-TK plasmid encoding Renilla luciferase were co-transfected with other plasmids into 293T cells for 48 h. Luciferase activity was measured using the Dual Luciferase Reporter Assay System. Results are expressed relative to the activity in vector control.
Xenograft assays
Animal study was approved by Animal Care and Use Committee of Jing Zhou Central Hospital, the Second Clinical Medical College, Yangtze University, Jing Zhou, Hubei, China. Eight-week-old male nude mice were kept in a specific pathogen-free facility. Cells at a density of 1 × 107 were suspended in 50 µl of DMEM medium, mixed with Matrigel (Corning; 1:1) and injected into the flanks of male nude mice. Tumor sizes were measured by a caliper and volumes were calculated using the formula length × width 2 × 1/2. Tumor weights were measured after mice were sacrificed.
Statistical analysis
All statistical analyses were assessed by the SPSS statistical software package, version 16.0 for Microsoft Windows (SPSS Inc., Chicago, IL, USA). Results are expressed as mean ± standard deviation (SD). The unpaired two-tailed T test was used for the comparison of parameters between two groups. Statistical significance is displayed as *p < 0.05, **p < 0.01 or ***p < 0.001.
Discussion
The ubiquitin–proteasome pathway plays important roles in all cellular functions by regulating the abundance of regulatory proteins. The selectivity of ubiquitin-mediated degradation of substrate proteins is determined by the E3 ligases. Among the E3 ligase family, the Cullin–RING Ligases (CRLs) constitute the largest class of E3 ligases with more than 200 members [
24]. Each of CRLs contains a different cullin subunit, which can be modified by the ubiquitin-like protein NEDD8, to activate CRLs for substrate ubiquitination [
25]. This process can be inhibited by MLN4924, a small molecule inhibitor of NEDD8-activating enzymes that enhance CRL activity [
23]. The CRL1 ligases, or SCF complexes, are the best characterized among all CRLs. SCF complex consists of the scaffolding protein Cullin 1, RBX1, SKP1 and one of ~ 70 various F-box proteins [
26]. Given the relative large number of F-box proteins, only several members have been extensively investigated, including the oncogenic proteins β-TrCP, SKP2 and the tumor suppressor protein FBXW7 [
27,
28]. c-Myc, a critical transcription factor and an well-known oncogene, was previously shown to be ubiquitinated and degraded by FBXW7 [
21]. Given that the protein stability of c-Myc affected its transcriptional activity, characterization of the upstream regulators or mechanisms of c-Myc stability is important for understanding of c-Myc-dependent oncogenic process.
Evi5 belongs to a small subfamily of the Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins with diverse functions [
14]. Evi5 undergoes several post-translational modifications including phosphorylation and ubiquitination, and has a dynamic association with mitotic structures during the M phase [
15]. Evi5 has been identified as a regulator of cyclin accumulation via its effects on EMI1. Overexpression Emi1 is sufficient to drive S phase by stabilizing cyclin A protein [
29], suggesting a promoting role of Evi5 in the regulation of cell proliferation. However, although Evi5 has been found to be overexpressed in certain cancer types, direct evidence for its role as an oncogene is still missing [
12].
In the present study, we uncovered a novel oncogenic role of Evi5 in LSCC. By using shRNA-mediated gene knockdown or CRISPR-mediated gene knockout assay, we found inhibition of Evi5 in LSCC cells caused growth retard phenotype and delayed S phase entry which are coincidence with the decline of c-Myc protein. However, the mRNA level of c-Myc is unaltered, suggesting Evi5 might regulate c-Myc at posttranscriptional level. We further demonstrated that Evi5 positively regulated the stability of c-Myc protein by reducing its ubiquitination form, but Evi5 itself is not a deubiquitinase, suggesting it might not be a direct effect. Evi5 has been reported to be able to antagonize the binding between beta-Trcp and EMI1, and leading to the stabilizing of EMI1 [
15]. We then asked whether this mechanism is also applicable in the regulation of c-Myc. We found that Evi5 could also antagonize the interaction between c-Myc and FBXW7. Thus, we data clearly showed that Evi5 stabilized c-Myc by preventing the binding of FBXW7 to c-Myc. Considering the critical role of c-Myc in LSCC, Evi5 could also be an important player in the development of LSCC by modulating the stability of c-Myc. However, the exact roles of Evi5 in the development of LSCC should be further explored by using large clinic samples and in Evi5 KO mice.
In summary, our data demonstrate that the Evi5-FBXW7-c-Myc regulatory axis serves a role in the regulation of LSCC cell proliferation in vitro and tumorigenesis in vivo. These findings suggest that Evi5 is a potential therapeutic target in LSCC, and inhibition of Evi5 is the prospective strategy for LSCC therapy.
Conclusion
Our study is the first to show that Evi5 is required for LSCC cells proliferation and tumorigenesis both in vitro and in vivo. Our finding further provides a novel molecular mechanism for the positive regulation of c-Myc by Evi5 in LSCC cells. Interference with this regulatory effect of Evi5 significantly inhibited LSCC cells proliferation. Thus, given the critical role of c-Myc in tumorigenesis, our data suggest that Evi5 is a potential therapeutic target in LSCC, and inhibition of Evi5 should be a prospective strategy for LSCC therapy.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.