Abstract
Purpose
Cervical cancer remains a major cause of cancer-related death in women, especially in developing countries. Previously, we found that the acetylation levels of chloride intracellular channel 1 (CLIC1) at lysine 131 were increased in cervical cancer tissues using a label-free proteomics approach. The aim of this study was to further determine the role of CLIC1 expression and its acetylation in cervical cancer.
Methods
CLIC1 expression and its implications for the prognosis of cervical cancer were analyzed using primary patient samples and cells, and the Gene Expression Profiling Interactive Analysis (GEPIA) database (gepia.cancer-pku.cn). The effect of CLIC1 on cervical cancer cells was evaluated using Cell Counting Kit (CCK)-8, flow cytometry, scratch wound healing, transwell, Western blotting and co-immunoprecipitation (Co-IP) assays. In vivo tumor growth was assessed using mouse xenograft models.
Results
We found that CLIC1 expression was increased in cervical cancer tissues and cells and that patients with a high CLIC1 expression tended to have a shorter overall survival time. Knockdown of CLIC1 significantly reduced in vitro cervical cancer cell proliferation, migration and invasion, and in vivo tumorigenesis. At the molecular level, we found that nuclear factor kappa B (NF-κB) activity was positively regulated by CLIC1. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, attenuated the tumor-promoting effect of CLIC1. Moreover, we found that CLIC1 acetylation at K131 was upregulated in cervical cancer cells, which stabilized CLIC1 by inhibiting its ubiquitynation. Substitution of K131 inhibited CLIC1 ubiquitynation and promoted in vitro cervical cancer cell proliferation, migration and invasion, and in vivo tumor growth. In addition, we found that acetyltransferase HAT1 was responsible for CLIC1 acetylation at K131.
Conclusion
Our data indicate that CLIC1 acts as a tumor promoter in cervical cancer, suggesting a potential treatment strategy for cervical cancer by regulating CLIC1 expression and/or acetylation.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13402-020-00582-w.
Keywords: Cervical cancer, Chloride intracellular channel 1, Nuclear factor kappa B, Acetylation
Introduction
Cervical cancer ranks as the fourth malignancy both in incidence and mortality in females worldwide [1]. The mortality of cervical cancer in low-income and middle-income countries is 18 times higher than that in developed countries [2]. In spite of improvements that have been made in therapeutic strategies in the past few decades, the prognosis of patients with advanced or recurrent metastatic cervical cancer remains poor [3]. Thus, a further understanding of the molecular mechanisms involved in cervical cancer development is urgently needed.
Chloride intracellular channel 1 (CLIC1), a member of the p64 family, is a small protein composed of 241 amino acids, with a molecular weight of 27 kDa [4]. A proteomics database search (https://www.proteomicsdb.org/proteomicsdb/#overview), which contains high-quality shotgun proteomics data of the whole human proteome [5], shows that CLCI1 is ubiquitously expressed in various organs. CLIC1 has been found to exert crucial functions in multiple physiological and pathological processes, such as osteoblastic differentiation [6], neurotoxicity [7] and neurite elongation [8]. Moreover, increasing evidence indicates that CLIC1 is involved in tumor progression. The expression of CLIC1 has been found to be upregulated in a variety of different cancers, including hepatocellular carcinoma [9], gallbladder cancer [10], pancreatic cancer [11] and glioma [12], and increased CLIC1 levels have been associated with a poor prognosis in these patients. CLIC1 expression has also been found to be related to clear cell renal cell carcinoma tumor grade and invasion [13], to serve as a biomarker of epithelial ovarian cancer and to be necessary for tumor cell proliferation and migration [14]. LncRNA UCA1 has been reported to promote tumor cell migration and invasion by regulating the miR-122/CLIC1 signaling axis [15]. Katayama et al. reported that CLIC1 is part of a TGFβ signature that serves as a prognostic biomarker in triple negative breast cancer [16]. CLIC1 knockout has been found to inhibit medulloblastoma growth in xenograft and genetically engineered mouse models [17], whereas CLIC1 overexpression has been found to promote the growth of oral squamous cell carcinoma cells by activating integrin/ERK pathways [18]. CLIC1 silencing has been found to repress the viability and invasive potential of hepatocarcinoma cells by regulating maspin expression [19]. As yet, however, the function and mode of action of CLIC1 in cervical cancer are still unclear.
Lysine acetylation is a common post‐translational modification (PTM) regulating protein stability, subcellular localization and function [20]. Acetylation of histone and nonhistone proteins has been reported to be involved in several diseases, including cardiomyopathy [21], cancer [22–24], colitis [25] and Alzheimer’s disease [26]. Previously, we assessed the lysine acetylproteome in primary cervical cancer tissues and corresponding adjacent normal tissues using a label-free proteomics approach and found that the acetylation levels of CLIC1 at lysine 131 were significantly increased in the cancer tissues [27]. Hence, we hypothesized that CLIC1 acetylation at lysine 131 may play a crucial role in cervical cancer development. In the present study, we show that both the level of CLIC1 and its acetylation at lysine 131 are upregulated in cervical cancer tissues. We also found that CLIC1 can promote in vitro cervical cancer cell proliferation, migration and invasion, and in vivo tumorigenesis by regulating nuclear factor kappa B (NF-κB) activity. In addition, we found that acetylation of CLIC1 at lysine 131 inhibits its ubiquitination and degradation, thus leading to an increase in the CLIC1 protein level. Finally, we found that acetyltransferase 1 (HAT1) can acetylate CLIC1 at lysine 131 and, thereby, increase its stability.
Materials and methods
Clinical samples and animal models
In total 30 paired cervical cancer tissues and corresponding adjacent normal tissues were obtained from Harbin Medical University Cancer Hospital. Each patient signed informed consent prior to participating in the study. All procedures involving human subjects were approved by the Ethics Committee of the Harbin Medical University and were in accordance with the Declaration of Helsinki.
Sixty BALB/c nude mice were purchased from Beijing Huafukang Bioscience Co. Inc. (Beijing, China) and housed in cages with a 12 h light/dark cycle. Food and water were freely available. All procedures involving animal experiments were approved by the Animal Care and Use Committee of Harbin Medical University and were in compliance with the Guide for the Care and Use of Laboratory Animals.
Cells and culture conditions
Human cervical epithelial cells (HCerEpiC) and cervical cancer cells (Hela, Siha, C-33A and CaSki) were purchased from Procell (Wuhan, China). Hcerepic, Siha, Hela and C-33A cells were cultured in minimal essential medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Caski cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) supplemented with 10% FBS (Hyclone). All cells were maintained in an incubator set to 37 °C and 5% CO2. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, was obtained from Aladdin Reagent Company (Shanghai, China). After transfection for 24 h, 200 μmol/L PDTC was added to the cells for 24 h.
Transient transfection and stable cell line generation
Specific small interfering RNAs (siRNAs) directed against CLIC1 and HAT1 were obtained from JTS Scientific (Hubei, Wuhan, China). Recombinant plasmids expressing CLIC1, P300/CBP-associated factor (PCAF), E1A-binding protein p300 (P300), CREB-binding protein (CBP), General Control Of Amino Acid Synthesis Protein 5-Like 2 (GCN5), Tat-interacting protein 60 kDa (Tip60) and HAT1 were purchased from GenScript (Piscataway, NJ, USA). Recombinant plasmids expressing Flag-tagged CLIC1 or Flag-tagged mutant CLIC1 (lysine-to-arginine at position 131, CLIC1 K131R) were obtained from ViGene Biosciences (Shandong, China). The plasmids were transiently transfected into Hela or Siha cells for 48 h (except for cell proliferation assays) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For in vivo experiments, cells with stable overexpression or knockdown of CLIC1 were selected and used.
Protein isolation and Western blot analysis
Total protein was extracted from cells or tumor tissues using RIPA lysis buffer (Solarbio, Beijing, China). Nuclear extracts were obtained from cells using a Nuclear Protein Extraction Kit (Solarbio). Protein concentrations were determined using a BCA Protein Assay kit (Solarbio). The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes and blocked with 5% skimmed milk. Next, the membranes were incubated with primary antibodies directed against CLIC1 (1:1000, Abcam, Cambridge, MA, USA), P-p65 (1:500, Cell Signaling Technology, CST, Danvers, MA, USA), p65 (1:1000, CST), P-IkB (1:500, CST), IkB (1:1000, CST), PCAF (1:3000, CST), P300 (1:3000, CST), CBP (1:3000, CST), GCN5 (1:3000, ABclonal, Boston, MA, USA), Tip60 (1:3000, ABclonal), HAT1 (1:3000, ABclonal), Histone H3 (1:5000, Gene Tex, CA, USA), GAPDH (1:10000, Proteintech, Wuhan, China), Ubiquitin (1:1000, Abcam) and Flag (1:1000, KangWei, Beijing, China) overnight 4 °C. Protein bands were visualized using an ECL detection method (Solarbio) and quantified by densitometry.
Immunohistochemistry
Tissue sections were deparaffinized with xylene, rehydrated in graded alcohol and subjected to antigen retrieval with citric acid buffer. Endogenous peroxidase activity was blocked by 3% H2O2 treatment. Next, the sections were incubated with a primary antibody directed against CLIC1 (1:50, ABclonal) overnight at 4 °C, followed by incubation with a HRP-labeled secondary antibody for 1 h at 37 °C. The resulting tissue sections were examined using a microscope (Olympus, Tokyo, Japan) at 400× magnification after DAB and hematoxylin staining.
Cell proliferation assay
Cell proliferation was evaluated using a Cell Counting Kit (CCK)-8 assay. Briefly, cells were seeded at a density of 4 × 103/well in 96-well plates in quintuplicate. After treatment, the plates were incubated for 0, 24, 48, 72, 96 or 120 h at 37 °C. Next, 10 μl CCK-8 solution was added to each well at the indicated time points for 2 h, after which the absorbance of each well was determined at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
Apoptosis assay
Cell apoptosis was assessed by flow cytometry after annexin V-FITC and propidium iodide (PI) staining (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer’s instructions.
Xenograft assay
BALB/c nude mice were randomly divided into 10 groups (n = 6/group). Both Siha and Hela cells with stable expression of control shRNA, CLIC1 shRNA1, CLIC1 shRNA2, CLIC1 WT and CLIC1 K131R were subcutaneously injected into the mice. Tumor volumes were measured every two days. Nineteen days after inoculation, the mice were sacrificed and the tumor tissues collected for Western blot analysis.
Cell migration assay
Cell migration was determined using a scratch wound healing assay. In short, the cells were grown to confluence after which 1 μg/ml mitomycin C was added to induce growth arrest. Then a 200 μl pipette tip was used to create a scratch (‘wound’) in the monolayer, after which the plate was washed once and replenished with the desired medium. Images were captured using an inverted phase-contrast microscope (Olympus).
Cell invasion assay
Cell invasion was evaluated using a transwell assay. In brief, cells at a density of 1.5 × 104 were seeded into transwell chambers (Corning Inc., Corning, NY, USA) coated with Matrigel. The bottom chamber was filled with 800 μl medium containing 10% FBS. After 48 h, the invaded cells were immobilized with 4% paraformaldehyde, stained with 0.4% crystal violet and counted using a microscope (Olympus).
Co-immunoprecipitation (co-IP) assay
Total protein was isolated and quantified as described above. Equal amounts of cell lysates were subjected to incubation with specific primary antibodies overnight at 4 °C after which 60 μl protein A/G agarose was added for 2 h at 4 °C. The complexes formed were pelleted by centrifuging and analyzed by Western blotting with the indicated antibodies.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 (La Jolla, CA, USA). The data are expressed as mean ± SD of three independent experiments for the in vitro and six animals in each group for the in vivo studies. Student’s t test was used to analyze differences between two groups, and one- or two-way ANOVA to analyze differences between more than two groups. The differences were considered statistically significant at p < 0.05.
Results
CLIC1 expression is frequently upregulated in cervical cancer
The expression levels of CLIC1 in 30 pairs of cervical cancer and matched normal cervix tissue samples were determined by Western blot analysis. We found that CLIC1 expression in the tumor tissues was increased in 21 of the 30 cases (Fig. 1a). In addition, we found that the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/), a web-based tool to deliver fast and customizable functionalities based on TCGA and GTEx data, indicated that CLIC1 mRNA levels were significantly up-regulated in 306 cervical cancer tissues (297 HPV-positive and 9 HPV-negative cases) compared to 13 normal tissues (Fig. 1b), and that cervical cancer patients with a high CLIC1 expression tended to have shorter overall survival times (Supplementary file 1A). Using Western blot analyses, we additionally found that the expression of CLIC1 was increased in human cervical carcinoma cell lines (Hela, Siha, C-33A and CaSki) compared to normal human cervical epithelial cells (Hcerepic). Immunohistochemistry analysis of the cervical cancer tissues revealed that CLIC1 was mainly located in the nucleus (Supplementary file 1B).
Fig. 1.
CLIC1 expression is increased in cervical cancer tissues and cells. (a) Western blot analysis of CLIC1 expression in 30 pairs of cervical tumor tissues (T) and corresponding normal tissues (N; six examples are shown). (b) CLIC1 mRNA expression in cervical cancer tissues (left) and normal tissues (right) from the TCGA and GTEx databases assessed by the GEPIA web tool. (c) Western blot analysis of CLIC1 expression in human cervical epithelial (Hcerepic) cells and human cervical carcinoma cell lines (Hela, Siha, C-33A, and CaSki). Error bars represent SDs of three independently prepared samples. *P < 0.05
CLIC1 knockdown inhibits cervical cancer cell malignant phenotypes
Subsequently, the function of CLIC1 in cervical cancer cells was investigated. Two siRNAs targeting CLIC1 were used to silence its expression in Siha and Hela cells. The knockdown efficiencies were validated by real-time PCR (Fig. 2a). Using a CCK-8 assay we found that the proliferative ability was markedly inhibited in the cervical cancer cells transfected with CLIC1 siRNAs (Fig. 2b). Next, the occurrence of apoptosis was determined using flow cytometry after annexin V-FITC/PI staining. We found that the numbers of apoptotic cells were significantly increased in the CLIC1-silenced Siha and Hela cells (Fig. 2c). A subsequent scratch wound healing assay revealed that the migratory activity of the CLIC1-silenced cervical cancer cells was markedly attenuated (Fig. 2d). A transwell assay additionally indicated that CLIC1 silencing inhibited cervical cancer cell invasiveness (Fig. 2e). Finally, we found that stable CLIC1 knockdown suppressed the in vivo tumorigenesis of cervical cancer cells in mouse xenograft models (Fig. 2f and g).
Fig. 2.
CLIC1 knockdown inhibits cervical cancer cell malignant phenotypes. Siha and Hela cells were transfected with two siRNAs targeting different regions of the CLIC1 mRNA. (a) Knockdown efficiencies of the siRNAs determined by Western blot analysis. (b) Proliferation abilities of the respective cells evaluated by CCK-8 assay. (c) Apoptotic rates of the respective cells determined by flow cytometry after Annexin V-FITC/PI staining. (d) Scratch wound healing assays showing the migratory capacities of Siha and Hela cells transfected with CLIC1 siRNAs. (e) Transwell assays showing the invasive abilities of Siha and Hela cells with or without CLIC1 silencing. Stably CLIC1-silenced Siha and Hela cells were constructed and inoculated subcutaneously in nude mice. (f) Photographs of Siha and Hela xenograft tumors excised from the nude mice. (g) Xenograft Siha and Hela tumor growth curves. Error bars represent the SD of three independently prepared samples. N = 6 per group for animal studies. *P < 0.05
CLIC1 knockdown inactivates the NF-κB pathway
Next, the effect of CLIC1 knockdown on NF-κB, a critical regulator of oncogenic processes, was investigated. We found that the expression levels of P-p65, P-IΚBα and nuclear p65 were reduced, while that of IΚBα was increased in Siha and Hela cells transfected with CLIC1 siRNAs (Fig. 3a and b). Similar results for P-p65, P-IΚBα and IΚBα were obtained in xenograft tumor tissues derived from Siha or Hela cells with stable knockdown of CLIC1 (Fig. 3c).
Fig. 3.
CLIC1 knockdown suppresses NF-κB activation in cervical cancer cells. (a) Expression of CLIC1, P-p65, p65, P-IΚBα and IΚBα in Siha and Hela cells with stably silenced expression of CLIC1 evaluated by Western blotting. (b) Expression of p65 in cytoplasmic and nuclear fractions of CLIC1-silenced Siha and Hela cells evaluated by Western blotting. (c) Expression of CLIC1, P-p65, p65, P-IΚBα and IΚBα in xenograft tumor tissues originating from Siha and Hela cells with stable CLIC1 silencing evaluated by Western blotting. Error bars represent the SD of three independently prepared samples. *P < 0.05
CLIC1 overexpression promotes malignant phenotypes of cervical cancer cells by activating the NF-κB pathway
Next, we explored the effect of CLIC1 upregulation on cervical cancer cells and the putative role of NF-κB in CLIC1-mediated actions. In contrast to the effects caused by CLIC1 downregulation, we found that CLIC1 overexpression induced NF-κB activity (Fig. 4a and b) and promoted cervical cancer cell proliferation (Fig. 4c), migration (Fig. 4d) and invasion (Fig. 4e). The tumor-promoting effects driven by exogenous CLIC1 overexpression could be attenuated by treatment with PDTC, a NF-κB inhibitor (Fig. 4c-e).
Fig. 4.
CLIC1 overexpression promotes the growth of cervical cancer cells by activating NF-κB. Siha and Hela cells were transfected with a CLIC1-overexpression plasmid for 24 h, followed by treatment with PDTC. (a) Expression of CLIC1, P-p65, p65, P-IΚBα and IΚBα evaluated by Western blotting. (b) Expression of p65 in cytoplasmic and nuclear fractions evalueted by Western blotting. (c) Proliferative abilities evaluated by CCK-8 assay. (d) Migratory abilities evaluated by scratch wound healing assay. (e) Invasive capabilities evaluated by transwell assay. Error bars represent the SD of three independently prepared samples. *P < 0.05
CLIC1 acetylation at K131 stabilizes the protein by blocking its ubiquitination
Previously, we found that the acetylation levels of CLIC1 at lysine 131 were dramatically augmented in primary cervical cancer tissues compared to corresponding adjacent normal tissues using a label-free proteomics approach. In order to further validate acetylation of this site, we generated an antibody specific to acetylated K131 by immunization of rabbits with a synthetic peptide. The characteristics of the antibody obtained were as follows: (1) the antibody preferentially detected the acetylated peptide, but not the unmodified peptide, (2) the antibody detected a strong signal of exogenously expressed wild-type CLIC1, but not K131R mutant CLIC1 and (3) recognition of CLIC1 by the antibody was competed by an acetylation-modified peptide, but not by the unmodified peptide.
Using this antibody, we found that the acetylation levels of CLIC1 at K131 were increased in cervical cancer tissues and cells (Fig. 5a and b). Moreover, we observed a significant decrease in the CLIC1 acetylation level when K131 was mutated to arginine (CLIC1 K131R) in Hela cells (Fig. 6a). We also found that the stability of CLIC1 K131R was increased compared to wild type CLIC1 (CLIC1 WT) (Fig. 6a) and that the CLIC1 ubiquitynation levels were reduced in Hela cells expressing CLIC1 K131R (Fig. 6b). Subsequently, the effect of the CLIC1 K131R mutant on cell function in vitro and in vivo was studied. We found that exogenous CLIC1 K131R overexpression markedly promoted in vitro cervical cancer cell proliferation, migration and invasion, and in vivo tumorigenesis (Fig. 6c-g).
Fig. 5.
Acetylation of CLIC1 at K131 is increased in cervical cancer. (a) Acetylation levels of CLIC1 at K131 in 30 pairs of cervical tumor tissues and corresponding normal tissues evaluated by Western blotting. (b) Acetylation levels of CLIC1 at K131 in human cervical epithelial (Hcerepic) cells and human cervical carcinoma (Hela, Siha, C-33A and CaSki) cells determined by Western blotting. Error bars represent the SD of three independently prepared samples. *P < 0.05
Fig. 6.
K131R mutant increases the stability of CLIC1. (a) Flag-tagged wild-type (WT) and K131R mutant CLIC1 were co-transfected into Hela cells, after which CLIC1 acetylation was assessed by co-immunoprecipitation using a Pan-Ac antibody. (b) Flag-tagged wild-type and K131R mutant CLIC1, and HA-tagged ubiquitin were co-transfected into Hela cells, after which ubiquitynation of CLIC1 was assessed using co-immunoprecipitation and Western blot assays. (c) Assessment of Siha and HeLa CLIC1 WT and CLIC1 K121R mutant cell proliferation using CCK-8 assays. (d) Assessment of Siha and HeLa CLIC1 WT and CLIC1 K121R mutant cell invasion using transwell assays. (e) Assessment of Siha and HeLa CLIC1 WT and CLIC1 K121R mutant cell migration using scratch wound healing assays. Siha and HeLa cells stably expressing wild type or K131R mutant CLIC1 were established and subcutaneously inoculated in nude mice. (f) Photographs of Siha and HeLa xenograft tumors excised from the nude mice. (g) Siha and HeLa xenograft tumor growth curves. Error bars represent the SD of three independently prepared samples. N = 6 per group for the animal studies. *P < 0.05
HAT1 acetylates CLIC1
To identify the acetyltransferase responsible for CLIC1 K131 acetylation, Hela cells were transfected with six acetyltransferases, PCAF, P300, CBP, GCN5, Tip60 or HAT1. Subsequent Western blot analysis showed that HAT1 overexpression upregulated K131 acetylation of CLIC1, whereas the others did not (Fig. 7a). Additional co-IP and Western blotting assays confirmed the binding of HAT1 to CLIC1 (Fig. 7b). Concordantly, we found that HAT1 silencing by two siRNAs led to decreased CLIC1 and CLIC1 K131 acetylation levels (Fig. 7c).
Fig. 7.
HAT-1 can acetylate CLIC1. (a) Expression levels of CLIC1 acetylation at K131 in Hela cells transfected with PCAF, P300, CBP, GCN5, Tip60 or HAT1 overexpression plasmids evaluated by Western blotting. (b) In vivo interaction of HAT-1 with CLIC1 in Hela cells evaluated by co-IP and Western blotting. (c) Expression levels of CLIC1 and CLIC1 acetylation at K131 in HAT1 silenced Hela cells evaluated by Western blotting. Error bars represent the SD of three independently prepared samples. *P < 0.05
Discussion
A tumor-promoting role of CLIC1 has been reported for a variety of tumors [11, 17, 18]. It has also been reported that, indirectly, miR-124 can inhibit the proliferation, migration and invasion of liver cancer cells by targeting CLIC1 [28] and that PA28β can enhance the invasion and metastasis of gastric cancer cells via upregulating CLIC1 [29]. Whether CLIC1 may also play a role in cervical cancer has not been resolved yet. Here, we found that increased expression of CLIC1 frequently occurs in cervical cancer tissues and cells. Analysis of loss- and gain-of-function phenotypes showed that CLIC1 positively regulates in vitro cervical cancer cell proliferation, migration and invasion, and in vivo tumorigenesis, indicating that CLIC1 acts as a tumor promoter in cervical cancer. A high nuclear CLIC1 expression has been reported to serve as an independent factor for a poor prognosis in pancreatic cancer [11], which is concordant with our current results. We also found that CLIC1 was predominantly located in the nucleus in cervical cancer tissues, suggesting that its nuclear expression may play a role in cervical cancer development.
The transcription factor NF-κB was first identified in B cells as a nuclear factor that binds to the enhancer element of the immunoglobulin kappa light-chain [30]. The NF-κB family comprises five members, including p50, p52, p65 (RelA), c-Rel and RelB, all of which contain an N-terminal Rel homology domain (RHD) that mediates DNA contacts as well as homo- and heterodimerization [31]. Under physiological conditions, NF-κB homo- and heterodimers are kept inactive in the cytoplasm by interactions with IκB inhibitors (existing in three isoforms IκBα, IκBβ and IκBε) [32]. Cell stimulation can lead to activation of the IκB kinase (IKK) complex, which is responsible for IκB phosphorylation, marking it for degradation via the β-transducin repeat-containing protein (β-TrCP)-dependent E3 ubiquitin ligase-mediated proteasomal degradation machinery. This in turn may lead to NF-kB nuclear translocation and subsequent activation of target genes associated with various biological processes, including cancer development [33]. Here, we found that CLIC1 inhibited IκBα expression and promoted phosphorylation and translocation of p65 and p-IκBα expression. The pro-tumor effects mediated by CLIC1 could be suppressed by an NF-κB inhibitor (PDTC), suggesting a key role of the CLIC1/NF-κB axis in tumor progression. Although the mechanism underlying NF-κB activation by CLIC1 needs to be further clarified, it has been reported that CLIC1 can induce transcription of IL-1β, which is capable of activating NF-κB [34, 35], suggesting a possible mechanism for CLIC1-mediated NF-κB activation.
Post-transcriptional modification (PTM) of proteins is emerging as a regulatory mechanism in diverse cellular processes, including metabolism, protein degradation, protein trafficking, signal transduction and DNA damage responses. Lysine acetylation is one of the most versatile PTMs and has been shown to play a critical role in several diseases, including cancer [36]. In addition, it has been reported that acetylation may prevent ubiquitination of the same lysine residues [37]. Protein PTMs can be detected by various techniques, including mass spectrometry (MS), liquid chromatography, radioactive chemical and chromatin immune precipitation (ChIP) methods [38]. Previously, we found that lysine acetylation of CLIC1 at K131 was significantly upregulated in cervical cancer tissues using a label-free proteomics approach. Here, the ubiquitination levels of K131R mutant CLIC1 were found to be decreased, leading to increased CLIC1 protein stability and, thereby, more aggressive cervical cancer cell phenotypes.
HAT1 is the first lysine acetyltransferase identified in yeast [39]. Subsequently, it was found that HAT1 is cytoplasmic and involved in histone deposition [40]. In addition to acetylation of histones, HAT1 has been found to acetylate nonhistone proteins, such as promyelocytic leukemia zinc finger protein (PLZF) [41] and p53 [42]. Here, we show that HAT1 can acetylate CLIC1 at K131 and, hence, increase its stability by inhibiting its ubiquitination.
Taken together, our results indicate that the expression of CLIC1 is frequently increased in cervical cancer and that it acts as a tumor promoter. Acetylation of CLIC1 at K131 by HAT1 stabilizes the protein and protects it from ubiquitination-mediated degradation. Our results suggest a potential strategy for the treatment of cervical cancer by regulating the (anomalous) expression and acetylation of CLIC1.
Supplementary Information
(PNG 777 kb)
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China (No. 81772274), the Natural Science Foundation of Heilongjiang Province (No. H2017045) and the Key Project of the Petrel Foundation of Harbin Medical University Cancer Hospital (No. JJZD2020-07). In addition, the study was supported by the Beijing Medical Award Foundation (No.YXJL-2020-0417-0043 and No.YXJL-2020-0510-0044) and the Heilongjiang University student innovation and Entrepreneurship training project (No.202011230002).
Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author upon reasonable request.
Authors’ contributions
WYX and CXW conceived and designed the study. WWY, LX and XY conducted the experiments. WWY, GWK, YH and ZL analyzed the data. WWY and LX wrote the manuscript. WYX and CXW modified the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Harbin Medical University and written informed consent was obtained from all patients. All animal experiments were performed in line with the Guide for the Care and Use of Laboratory Animal by the National Institutes of Health.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
This article title is present on Research Square as pre-print and can be accessed on https://www.researchsquare.com/article/rs-26424/v1.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Wanyue Wang, Xin Li and Ye Xu contributed equally to this work.
Contributor Information
Yaoxian Wang, Email: wyxxs012@126.com.
Xiuwei Chen, Email: chenxiuwei1023@163.com.
References
- 1.F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68, 394–424 (2018) [DOI] [PubMed] [Google Scholar]
- 2.P.A. Cohen, A. Jhingran, A. Oaknin, L. Denny, Cervical cancer. Lancet 393, 169–182 (2019) [DOI] [PubMed] [Google Scholar]
- 3.R. Wei, G. Jiang, M. Lv, S. Tan, X. Wang, Y. Zhou, T. Cheng, X. Gao, X. Chen, W. Wang, C. Zou, F. Li, X. Ma, J. Hu, D. Ma, D. Luo, L. Xi, TMTP1-modified indocyanine green-loaded polymeric micelles for targeted imaging of cervical cancer and metastasis sentinel lymph node in vivo. Theranostics 9, 7325–7344 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.S. Averaimo, R.H. Milton, M.R. Duchen, M. Mazzanti, Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress. FEBS Lett 584, 2076–2084 (2010) [DOI] [PubMed] [Google Scholar]
- 5.P. Samaras, T. Schmidt, M. Frejno, S. Gessulat, M. Reinecke, A. Jarzab, J. Zecha, J. Mergner, P. Giansanti, H.C. Ehrlich, S. Aiche, J. Rank, H. Kienegger, H. Krcmar, B. Kuster, M. Wilhelm, ProteomicsDB: A multi-omics and multi-organism resource for life science research. Nucleic Acids Res 48, D1153–D1163 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.J.Y. Yang, J.Y. Jung, S.W. Cho, H.J. Choi, S.W. Kim, S.Y. Kim, H.J. Kim, C.H. Jang, M.G. Lee, J. Han, C.S. Shin, Chloride intracellular channel 1 regulates osteoblast differentiation. Bone 45, 1175–1185 (2009) [DOI] [PubMed] [Google Scholar]
- 7.G. Novarino, C. Fabrizi, R. Tonini, M.A. Denti, F. Malchiodi-Albedi, G.M. Lauro, B. Sacchetti, S. Paradisi, A. Ferroni, P.M. Curmi, S.N. Breit, M. Mazzanti, Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity. J Neurosci 24, 5322–5330 (2004) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.S. Averaimo, M. Gritti, E. Barini, L. Gasparini, M. Mazzanti, CLIC1 functional expression is required for cAMP-induced neurite elongation in post-natal mouse retinal ganglion cells. J Neurochem 131, 444–456 (2014) [DOI] [PubMed] [Google Scholar]
- 9.X. Jiang, Y. Liu, G. Wang, Y. Yao, C. Mei, X. Wu, W. Ma, Y. Yuan, Up-regulation of CLIC1 activates MYC signaling and forms a positive feedback regulatory loop with MYC in hepatocellular carcinoma. Am J Cancer Res 10, 2355–2370 (2020) [PMC free article] [PubMed] [Google Scholar]
- 10.Q. Ding, M. Li, X. Wu, L. Zhang, W. Wu, Q. Ding, H. Weng, X. Wang, Y. Liu, CLIC1 overexpression is associated with poor prognosis in gallbladder cancer. Tumour Biol 36, 193–198 (2015) [DOI] [PubMed] [Google Scholar]
- 11.J. Lu, Q. Dong, B. Zhang, X. Wang, B. Ye, F. Zhang, X. Song, G. Gao, J. Mu, Z. Wang, F. Ma, J. Gu, Chloride intracellular channel 1 (CLIC1) is activated and functions as an oncogene in pancreatic cancer. Med Oncol 32, 616 (2015) [DOI] [PubMed] [Google Scholar]
- 12.M. Setti, N. Savalli, D. Osti, C. Richichi, M. Angelini, P. Brescia, L. Fornasari, M.S. Carro, M. Mazzanti, G. Pelicci, Functional role of CLIC1 ion channel in glioblastoma-derived stem/progenitor cells. J Natl Cancer Inst 105, 1644–1655 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.A. Nesiu, A.M. Cimpean, R.A. Ceausu, A. Adile, I. Ioiart, C. Porta, M. Mazzanti, T.C. Camerota, M. Raica, Intracellular chloride ion channel protein-1 expression in clear cell renal cell carcinoma. Cancer Genomics Proteomics 16, 299–307 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.B. Singha, S.L. Harper, A.R. Goldman, B.G. Bitler, K.M. Aird, M.E. Borowsky, M.G. Cadungog, Q. Liu, R. Zhang, S. Jean, R. Drapkin, D.W. Speicher, CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer. Sci Rep 8, 14725 (2018) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.L. Kong, Q. Wu, L. Zhao, J. Ye, N. Li, H. Yang, Upregulated lncRNA-UCA1 contributes to metastasis of bile duct carcinoma through regulation of miR-122/CLIC1 and activation of the ERK/MAPK signaling pathway. Cell Cycle 18, 1212–1228 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.H. Katayama, P. Tsou, M. Kobayashi, M. Capello, H. Wang, F. Esteva, M.L. Disis, S. Hanash, A plasma protein derived TGFbeta signature is a prognostic indicator in triple negative breast cancer. NPJ Precis Oncol 3, 10 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.M.A. Francisco, S. Wanggou, J.J. Fan, W. Dong, X. Chen, A. Momin, N. Abeysundara, H.K. Min, J. Chan, R. McAdam, M. Sia, R.J. Pusong, S. Liu, N. Patel, V. Ramaswamy, N. Kijima, L.Y. Wang, Y. Song, R. Kafri, M.D. Taylor, X. Li, X. Huang, Chloride intracellular channel 1 cooperates with potassium channel EAG2 to promote medulloblastoma growth. J Exp Med 217, e20190971 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.J. Feng, J. Xu, Y. Xu, J. Xiong, T. Xiao, C. Jiang, X. Li, Q. Wang, J. Li, Y. Li, CLIC1 promotes the progression of oral squamous cell carcinoma via integrins/ERK pathways. Am J Transl Res 11, 557–571 (2019) [PMC free article] [PubMed] [Google Scholar]
- 19.X. Wei, J. Li, H. Xie, H. Wang, J. Wang, X. Zhang, R. Zhuang, D. Lu, Q. Ling, L. Zhou, X. Xu, S. Zheng, Chloride intracellular channel 1 participates in migration and invasion of hepatocellular carcinoma by targeting maspin. J Gastroenterol Hepatol 30, 208–216 (2015) [DOI] [PubMed] [Google Scholar]
- 20.E. Verdin, M. Ott, 50 years of protein acetylation: From gene regulation to epigenetics, metabolism and beyond. Nat Rev Mol Cell Biol 16, 258–264 (2015) [DOI] [PubMed] [Google Scholar]
- 21.S.S. Vadvalkar, C.N. Baily, S. Matsuzaki, M. West, Y.A. Tesiram, K.M. Humphries, Metabolic inflexibility and protein lysine acetylation in heart mitochondria of a chronic model of type 1 diabetes. Biochem J 449, 253–261 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.H. Kozuka-Hata, A. Kitamura, T. Hiroki, A. Aizawa, K. Tsumoto, J.I. Inoue, M. Oyama, System-wide analysis of protein acetylation and ubiquitination reveals a diversified regulation in human cancer cells. Biomolecules 10, 411 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.S. Nagarajan, S.V. Rao, J. Sutton, D. Cheeseman, S. Dunn, E.K. Papachristou, J.G. Prada, D.L. Couturier, S. Kumar, K. Kishore, C.S.R. Chilamakuri, S.E. Glont, E. Archer Goode, C. Brodie, N. Guppy, R. Natrajan, A. Bruna, C. Caldas, A. Russell, R. Siersbaek, K. Yusa, I. Chernukhin, J.S. Carroll, ARID1A influences HDAC1/BRD4 activity, intrinsic proliferative capacity and breast cancer treatment response. Nat Genet 52, 187–197 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.E.M. Da Costa, G. Armaos, G. McInnes, A. Beaudry, G. Moquin-Beaudry, V. Bertrand-Lehouillier, M. Caron, C. Richer, P. St-Onge, J.R. Johnson, N. Krogan, Y. Sai, M. Downey, M. Rafei, M. Boileau, K. Eppert, E. Flores-Diaz, A. Haman, T. Hoang, D. Sinnett, C. Beausejour, S. McGraw, N.J. Raynal, Heart failure drug proscillaridin a targets MYC overexpressing leukemia through global loss of lysine acetylation. J Exp Clin Cancer Res 38, 251 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.C. Li, Y. Chen, H. Zhu, X. Zhang, L. Han, Z. Zhao, J. Wang, L. Ning, W. Zhou, C. Lu, L. Xu, J. Sang, Z. Feng, Y. Zhang, X. Lou, X. Bo, B. Zhu, C. Yu, M. Zheng, Y. Li, J. Sun, Z. Shen, Inhibition of histone deacetylation by MS-275 alleviates colitis by activating the vitamin D receptor. J Crohns Colitis 14, 1103–1118 (2020) [DOI] [PubMed] [Google Scholar]
- 26.S.J. Marzi, S.K. Leung, T. Ribarska, E. Hannon, A.R. Smith, E. Pishva, J. Poschmann, K. Moore, C. Troakes, S. Al-Sarraj, S. Beck, S. Newman, K. Lunnon, L.C. Schalkwyk, J. Mill, A histone acetylome-wide association study of Alzheimer's disease identifies disease-associated H3K27ac differences in the entorhinal cortex. Nat Neurosci 21, 1618–1627 (2018) [DOI] [PubMed] [Google Scholar]
- 27.L. Zhang, W. Wang, S. Zhang, Y. Wang, W. Guo, Y. Liu, Y. Wang, Y. Zhang, Identification of lysine acetylome in cervical cancer by label-free quantitative proteomics. Cancer Cell Int 20, 182 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.X. Yue, Y. Cui, Q. You, Y. Lu, J. Zhang, MicroRNA124 negatively regulates chloride intracellular channel 1 to suppress the migration and invasion of liver cancer cells. Oncol Rep 42, 1380–1390 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.D.L. Zheng, Q.L. Huang, F. Zhou, Q.J. Huang, J.Y. Lin, X. Lin, PA28beta regulates cell invasion of gastric cancer via modulating the expression of chloride intracellular channel 1. J Cell Biochem 113, 1537–1546 (2012) [DOI] [PubMed] [Google Scholar]
- 30.R. Sen, D. Baltimore, Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705–716 (1986) [PubMed] [Google Scholar]
- 31.S. Tilborghs, J. Corthouts, Y. Verhoeven, D. Arias, C. Rolfo, X.B. Trinh, P.A. van Dam, The role of nuclear factor-kappa B signaling in human cervical cancer. Crit Rev Oncol Hematol 120, 141–150 (2017) [DOI] [PubMed] [Google Scholar]
- 32.J.D. Kearns, S. Basak, S.L. Werner, C.S. Huang, A. Hoffmann, IkappaBepsilon provides negative feedback to control NF-kappaB oscillations, signaling dynamics, and inflammatory gene expression. J Cell Biol 173, 659–664 (2006) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Y. Xia, S. Shen, I.M. Verma, NF-kappaB, an active player in human cancers. Cancer Immunol Res 2, 823–830 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.R. Domingo-Fernandez, R.C. Coll, J. Kearney, S. Breit, L.A.J. O'Neill, The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1beta transcription and activate the NLRP3 inflammasome. J Biol Chem 292, 12077–12087 (2017) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.C. Rebe, F. Ghiringhelli, Interleukin-1beta and cancer. Cancers (Basel) 12, 1791 (2020) [DOI] [PMC free article] [PubMed]
- 36.S. Kaypee, D. Sudarshan, M.K. Shanmugam, D. Mukherjee, G. Sethi, T.K. Kundu, Aberrant lysine acetylation in tumorigenesis: Implications in the development of therapeutics. Pharmacol Ther 162, 98–119 (2016) [DOI] [PubMed] [Google Scholar]
- 37.R. Lin, R. Tao, X. Gao, T. Li, X. Zhou, K.L. Guan, Y. Xiong, Q.Y. Lei, Acetylation stabilizes ATP-citrate lyase to promote lipid biosynthesis and tumor growth. Mol Cell 51, 506–518 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.W. He, L. Wei, Q. Zou, Research progress in protein posttranslational modification site prediction. Brief Funct Genomics 18, 220–229 (2018) [DOI] [PubMed] [Google Scholar]
- 39.S. Kleff, E.D. Andrulis, C.W. Anderson, R. Sternglanz, Identification of a gene encoding a yeast histone H4 acetyltransferase. J Biol Chem 270, 24674–24677 (1995) [DOI] [PubMed] [Google Scholar]
- 40.M.R. Parthun, J. Widom, D.E. Gottschling, The major cytoplasmic histone acetyltransferase in yeast: Links to chromatin replication and histone metabolism. Cell 87, 85–94 (1996) [DOI] [PubMed] [Google Scholar]
- 41.A.J. Sadler, B.A. Suliman, L. Yu, X. Yuan, D. Wang, A.T. Irving, S.T. Sarvestani, A. Banerjee, A.S. Mansell, J.P. Liu, S. Gerondakis, B.R. Williams, D. Xu, The acetyltransferase HAT1 moderates the NF-kappaB response by regulating the transcription factor PLZF. Nat Commun 6, 6795 (2015) [DOI] [PubMed] [Google Scholar]
- 42.P.A. Agudelo Garcia, P. Nagarajan, M.R. Parthun, Hat1-dependent lysine acetylation targets diverse cellular functions. J Proteome Res 19, 1663–1673 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
(PNG 777 kb)
Data Availability Statement
The analyzed data sets generated during the study are available from the corresponding author upon reasonable request.