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CDK16 overexpressed in non-small cell lung cancer and regulates cancer cell growth and apoptosis via a p27-dependent mechanism
Biomedicine & Pharmacotherapy 103 (2018) 399–405
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CDK16 overexpressed in non-small cell lung cancer and regulates cancer cell growth and apoptosis via a p27-dependent mechanism
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Hongtao Wangb, Hongli Liuc, Shengping Mina, Yuanbing Shena, Wei Lia, Yuqing Chena, , ⁎ Xiaojing Wanga, a Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, No. 287 Changhuai Road, Bengbu, 233000, Anhui Province, China b Department of Immunology, Bengbu Medical College, Bengbu, 233000, Anhui Province, China c Department of Gynecological Oncology, First Affiliated Hospital, Bengbu Medical College, Bengbu, 233000, Anhui Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung cancer NSCLC Cyclindependent kinase 16 CDK16 Cyclin-dependent kinase inhibitor 1B
Cyclin-dependent kinase 16 (CDK16, PCTAIRE1) expression is upregulated in a wide variety of human malignancies. However, the function(s) of CDK16 in non-small cell lung cancer (NSCLC) remain unknown. Therefore, here we investigated the role of CDK16 in NSCLC. From 43 NSCLC tumors and matching healthy control lung tissues, immunohistochemistry revealed significantly greater CDK16 and phospho-p27Ser10 staining levels in NSCLC samples relative to healthy controls. The NSCLC cell line EKVX was transfected with a control siRNA, a CDK16-siRNA, or CDK16-siRNA + p27-siRNA. We found significantly decreased proliferation levels and significantly increased apoptosis levels in CDK16-silenced NSCLC cells. However, these effects were abrogated in cells treated with both the CDK16-siRNA and the p27-siRNA. In CDK16-silenced NSCLC cells, we found upregulated p27 and downregulated phospho-p27Ser10 protein expression but downregulated ubiquitinated p27 and ubiquitinated phospho-p27Ser10 protein expression. Cycloheximide-treated CDK16-silenced NSCLC cells displayed a much milder reduction in p27 protein expression over time relative to untreated CDK16-silenced NSCLC cells. In summary, CDK16 is significantly upregulated in human NSCLC tumor tissue and plays an oncogenic role in NSCLC cells via promoting cell proliferation and inhibiting apoptosis in a p27-dependent manner. Moreover, CDK16 negatively regulates expression of the p27 via ubiquination and protein degradation.
1. Introduction Lung cancer is one of the most common type of cancer and the leading cause of death among malignancies worldwide [1–3]. Nonsmall cell lung cancer (NSCLC) is the most common lung malignancy, accounting for over 80% of all lung cancer cases [1]. Unfortunately, the mortality rates associated with NSCLC have remained unchanged over the last 30 years, indicating a pressing need for a better understanding of this devastating condition [1–3]. Cyclin-dependent kinases (CDKs) are key enzymes that regulate cell cycle transitions in eukaryotic cells [4]. Many cancers, including NSCLC, exhibit features of cell cycle dysregulation, and numerous chemotherapeutics targeting CDK activity (e.g., CDK4 and CDK6) in cancer cells have been introduced over the past few decades [4,5]. One lesser-known CDK – cyclin-dependent kinase 16 (CDK16, PCTAIRE1, PCTK1) – has been shown to be critically involved in neuronal vesicular transport and spermatogenesis [6–8]. The central kinase domain of CDK16 is similar to other CDKs; however, the N- and C-terminal regions ⁎
Corresponding authors. E-mail addresses: [email protected] (Y. Chen), [email protected] (X. Wang).
https://doi.org/10.1016/j.biopha.2018.04.080 Received 29 November 2017; Received in revised form 2 April 2018; Accepted 10 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
are unique, and their functions remain unknown. That being said, structural analysis suggests that CDK16 may be involved in the phosphorylation of other kinases [9]. Through yeast two-hybrid screening, researchers have identified that CDK16 interacts directly with cyclindependent kinase inhibitor 1B (CDKN1B, p27, Kip1), a key cell cycle regulator and tumor suppressor [10–12]. Specifically, CDK16 negatively regulates p27 through phosphorylation of the Ser10 residue on p27, an action facilitating p27 degradation [13,14]. These findings suggest that CDK16 may play an important role in cell cycle regulation and oncogenesis. With respect to cancer, CDK16 expression is significantly upregulated in a wide variety of human malignancies, including breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma (HCC), colon cancer, brain cancer, melanoma, and squamous cell carcinoma (SCC) [11,12,15,16]. Furthermore, significant positive correlations between CDK16 expression and tumor invasiveness (or aggressive phenotype) have been observed in ovarian cancer, prostate cancer, and HCC [10,12,16]. Despite this previous evidence linking CDK16 to
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oncogenesis, the function(s) of CDK16 in NSCLC remain unknown. Therefore, here we investigated the role of CDK16 in NSCLC using both primary human NSCLC tumor cells and the NSCLC cell line EKVX. 2. Materials and methods 2.1. Ethics statement The Institutional Review Board (IRB) of the First Affiliated Hospital of Bengbu Medical College (Bengbu, China) approved the protocols of this study. All patients have provided informed consent prior to inclusion in this study. 2.2. Patients and specimens Surgical pneumonectomy or lobectomy were performed at the Department of Surgery at the First Affiliated Hospital of Bengbu Medical College. The paraffin-embedded tissue samples of lung tumor cores and matching healthy control lung tissue were stored at −80 °C immediately after surgical resection. Clinical records (Department of Pathology, the First Affiliated Hospital of Bengbu Medical College) for all lung tumor resection cases performed between January 2013 and December 2015 were screened. After screening, a total of 43 NSCLC cases (mean age: 56 years, range: 45–70 years; sex: 30 male and 13 female) were finally enrolled in this study. 2.3. EKVX cell line and siRNA transfection The NSCLC cell line EKVX was obtained from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). As recommended, EKVX cells were grown in complete growth medium and cultured in a humidified incubator (5% CO2) at 37 °C. Pre-designed small interfering RNAs (siRNAs) against human CDK16 and p27 as well as a non-coding scrambled siRNA were purchased from Life Technologies (Carlsbad, CA, USA). For transfection, EKVX cells were seeded in six-well plates at a density of 5 × 105 cells per well, cultured overnight, and then transfected with the CDK16siRNA alone or a combination of the CDK16-siRNA plus the p27-siRNA. The non-coding scrambled siRNA served as a negative control. According to the manufacturer’s protocol, cells were transfected with 20 nM of each siRNA with the Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Validation of CDK16 silencing and p27 silencing in EKVX cells was validated by immunoblotting (Supplementary Fig. S1, Supplementary Information).
Fig. 1. CDK16 Upregulated in NSCLC Tumor Cells. CDK16 protein expression was assessed by immunohistochemical analysis of tissue samples. (A) Representative images of healthy control lung tissue samples (left panel) and matching NSCLC tumor tissue samples (right panel) are provided. Scale bars, 50 μm. (B) CDK16 immunoreactivity scores were determined through blinded analysis. Box plots display the median (center line) and the interquartile range (top and bottom lines). **p < 0.01 versus control group.
randomly chosen from different areas of each specimen for immunoreactivity scoring, and the average immunoreactivity score derived from the ten visual fields was calculated as the final value for each patient. 2.5. GFP fluorescence proliferation assay Analysis of proliferation was determined using a Thermo Scientific Cellomics ArrayScan HCS reader (Thermo Fisher Scientific) with the Spot detector V3 Cellomics Bioapplication (Cellomics, Pittsburgh, PA, USA). GFP-positive cells were detected using the FITC channel at a 20× magnification, and images were acquired on a high-resolution chargecoupled device (CCD) camera on days one, two, three, four, and five (GRAS-14S5M; Point Grey, Richmond, Canada). Cells were identified based on the presence of nuclei and GFP fluorescence intensity. Cell count values were reported as the fold-change in cell count from day one using ArrayScan software (Thermo Fisher Scientific). A total of 1 × 103 cells were counted per sample, and experiments were repeated three times.
2.4. Immunohistochemistry and immunoreactivity scoring Specimens had been fixed in 10% formalin and embedded in paraffin wax immediately after resection. Three-micrometer sections were then cut from the paraffin blocks for immunohistochemical (IHC) analysis. The sections were stained with an anti-human CDK16 antibody (diluted 1:500, Invitrogen, Carlsbad, CA, USA) or an anti-human phospho-p27Ser10 antibody (diluted 1:50, Invitrogen) at 4 °C overnight. Following application of the secondary antibody, the immunoreactions were visualized with the avidin-biotin-peroxidase complex method according to the manufacturer’s recommendations (Vectastain Elite ABC Kit, Vector Labs, Burlingame, CA, USA). Secondary-only negative controls were used to control for non-specific binding. Immunoreactivity scoring in IHC samples was performed as previously described by Fromowitz et al. with minor modifications [17]. Briefly, the formula for the immunoreactivity score was as follows: immunoreactivity score = staining intensity (SI) + percentage of positive cells (PP). SI was tabulated as follows: negative staining = 0; weak staining = 1; moderate staining = 2; and strong staining = 3. PP was tabulated as follows: 0% = 0; 1–20% positive cells = 1; 21–50% positive cells = 2; and 51–100% positive cells = 3. Ten visual fields were
2.6. MTT proliferation assay The capacity for cellular proliferation was measured with a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A total of 3 × 103 cells were seeded in 96-well culture plates for one, two, three, four, and five days. The cells were then incubated with 20 μl of MTT (5 mg/ml) for four hours at 37 °C, and 150 μl of DMSO was added to solubilize the crystals for 20 min at room temperature. The optical density was determined with a spectrophotometer (Shanghai 400
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driver-oncogene, a microarray based lung cancer dataset obtained from the GEO database (GSE31210), which includes 226 lung cancer patients from Japan were downloaded. The correlation in expression between CDK16 and several oncogenes, including EGFR, KRAS, ALK, BRAF, and NF1, was computed by Spearman’s rank correlation test. Using the Japan cohort, the expression pattern of CDK16 between the patients without mutations in several oncogenes, i.e. EGFR, KRAS, and ALK (EGFR/KRAS/ALK mutation-), and the patients with mutations in EGFR (EGFR mutation+), KRAS (KRAS mutation+), or ALK (ALK mutation +) were investigated. 2.10. Statistical analysis The SPSS 17.0 software package (SPSS, Inc., Chicago, IL, USA) was used for all statistical analysis. Data were analyzed with Student ttesting or one-way analysis of variance (ANOVA) followed by Bonferroni testing where appropriate. P < 0.05 was considered to indicate a statistically significant difference. Data are reported as means ± standard deviations (SDs) as absolute values or percentages of controls. 3. Results 3.1. Upregulation of CDK16 and Phospho-p27 in human NSCLC tumors CDK16 expression is significantly upregulated in a wide variety of human malignancies [11,12,15,16]; however, its expression in NSCLC remains unknown. In the GEO lung cancer cohort, we found the expression of CDK16 was significantly and positively correlated with that of EGFR and BRAF (P < 0.05) (Supplementary Fig. S2). On the other hand, we tried to address whether the expression of CDK16 is affected by the somatic mutation status in the other oncogenes mutation. Comparison of CDK16 expression between lung cancer patients with and without oncogene mutations, we didn’t find significant difference in CDK16 among these patient categories (Supplementary Fig. S3). The roles of EGFR and BRAF overexpression in cancer progression have been well established, the role of CDK16 is much less clear. Here, a total of 43 NSCLC tumor samples were comparatively analyzed against matching healthy control lung tissue. IHC analysis revealed significantly greater levels of CDK16 staining in NSCLC samples relative to healthy controls (Fig. 1A). Scoring revealed significantly greater CDK16 immunoreactive scores in NSCLC samples relative to healthy controls (Fig. 1B, **p < 0.01). IHC analysis also revealed significantly greater levels of phospho-p27Ser10 staining in NSCLC samples relative to healthy controls (Fig. 2A). Scoring revealed significantly greater phospho-p27Ser10 immunoreactive scores in NSCLC samples relative to healthy controls (Fig. 2B, **p < 0.01).
Fig. 2. p27 Phosphorylation Upregulated in NSCLC Tumor Cells. Phospho-p27Ser10 protein expression was assessed by immunohistochemical analysis of tissue samples. (A) Representative images of healthy control lung tissue samples (left panel) and matching NSCLC tumor tissue samples (right panel) are provided. Scale bars, 50 μm. (B) Phospho-p27Ser10 immunoreactivity scores were determined through blinded analysis. Box plots display the median (center line) and the interquartile range (top and bottom lines). **p < 0.01 versus control group.
Spectrophotometer Co. Ltd., China) at a wavelength of 490 nm. 2.7. Flow cytometric apoptotic analysis Cells were cultured for 48 h post-transfection. Cells were then harvested, washed with phosphate buffered saline (PBS), and then fixed with 70% ethanol overnight at 4 °C. Cells were stained with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, BioVision, Mountain View, CA, USA) for 5 min before flow cytometric analysis. 2.8. Immunoprecipitation and immunoblotting
3.2. CDK16 silencing reduces NSCLC cell proliferation in a p27-dependent manner
Immunoprecipitation (IP) was performed on 800–1000 μg of cell lysate separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the manufacturer’s instructions (Thermo Fisher Scientific). Immunoblots were performed on cells prepared in lysis buffer (containing 150 mM NaCl, 100 mM Tris, pH 8.0, 1% Tween 20, 50 mm diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethyl-sulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml trypsin inhibitor, and 10 μg/ml leupeptin). Proteins were separated using SDSPAGE under reducing conditions on 10% polyacrylamide gels. Resolved proteins were transferred to nitrocellulose membranes and incubated with the indicated antisera followed by a horseradish peroxidase-conjugated secondary antibody.
As CDK16 has been shown to promote cellular proliferation through a p27-dependent mechanism [15], here we assessed the roles of CDK16 and p27 in NSCLC cell proliferation via gene silencing experimentation. Specifically, NSCLC cells were initially treated with CDK16-siRNA and/ or p27-siRNA and then followed over a period of five days to assess differences in cell proliferation (Fig. 3A). Via fluorescence microscopy, we found proliferation levels were significantly decreased in NSCLC cells treated with the CDK16-siRNA on days three, four, and five (Fig. 3B, **p < 0.01). However, decreased proliferation was not observed in cells treated with both the CDK16-siRNA and the p27-siRNA (Fig. 3B, p > 0.05). To validate these results, MTT assays were also performed. A similar significant decrease in proliferation was observed in NSCLC cells treated with CDK16-siRNA on days four and five (Fig. 3C, *p < 0.05). Moreover, decreased proliferation was not observed in cells treated
2.9. GEO analysis To check the relationship between CDK16 and other single-gene 401
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Fig. 3. Fluorescence Microscopy of NSCLC Cell Proliferation Following CDK16 Silencing and/or p27 Silencing. Following transfection with siRNAs specific to CDK16 and/or p27, EVKX cells were analyzed daily over a period of five days. (A) Representative fluorescent images from the three experimental groups over the five-day period are provided. (B) GFP+ cells were counted using Cellomics ArrayScan technology, and fold-change values were calculated from the GRP+ cell counts. Three independent experiments were performed. Data are presented as the means ± standard deviations of the fold-change in cell count normalized to day one. **p < 0.01 versus control group. (C) Following transfection with siRNAs specific to CDK16 and/or p27, EVKX cells were analyzed daily by MTT assay over a period of five days. Three independent experiments were performed. Data are presented as means ± standard deviations of the fold-change in 490-nm optical density (OD490) values normalized to day one. *p < 0.05 versus control group.
downregulated phospho-p27Ser10 protein expression in CDK16-silenced NSCLC cells (Fig. 5A). Notably, via IP, we also observed decreased levels of ubiquitinated p27 protein expression and ubiquitinated phospho-p27Ser10 in CDK16-silenced NSCLC cells (Fig. 5A). We next analyzed the role of protein degradation in p27 downregulation by CDK16 using the protein synthesis inhibitor cycloheximide (CHX), a compound which potently suppresses protein degradation [18]. Control and CDK16-silenced NSCLC cells treated with CHX were followed for a period of 12 h to assess changes in p27 protein expression (Fig. 5B). In control cells, p27 protein levels were dramatically reduced following 12 h of CHX treatment (Fig. 5B). However, a much milder reduction in p27 expression was observed in CDK16-silenced NSCLC cells (Fig. 5B). The associated densitometric analysis revealed significantly higher levels of p27 protein expression in CDK16silenced cells relative to controls (Fig. 5C, ***p < 0.001). Taken together, these results indicate that CDK16 ubiquinates the tumor suppressor p27 and reduces p27 expression via protein degradation (Supplementary Fig. S4).
with both the CDK16-siRNA and the p27-siRNA (Fig. 3C, p > 0.05). Taken together, these results indicate that CDK16 promotes NSCLC cell proliferation in a p27-dependent manner. 3.3. CDK16 silencing increases NSCLC cell apoptosis in a p27-dependent manner As CDK16 has been shown to regulate cancer cell apoptosis through a p27-dependent mechanism [15], here we assessed the roles of CDK16 and p27 in NSCLC cell apoptosis via gene silencing experimentation. Specifically, NSCLC cells were initially treated with CDK16-siRNA and/ or p27-siRNA and then followed over a period of 72 h to assess differences in apoptosis levels. Via Annexin V/PI flow cytometric analysis (Fig. 3A), we found significantly higher apoptosis levels at 48 and 72 h in NSCLC cells treated with the CDK16-siRNA relative to controls (Fig. 4B, *p < 0.05). However, the pro-apoptotic effect of CDK16 silencing was abrogated by p27 silencing (Fig. 4B, p > 0.05). Taken together, these results indicate that CDK16 inhibits NSCLC cell apoptosis in a p27-dependent manner.
4. Discussion 3.4. Regulation of p27 by CDK16 NSCLC remains one of the most deadly forms of cancer globally. Therefore, new targeted molecular therapeutic strategies for NSCLC are still needed to aid patients with this disease. To this end, here we demonstrate a novel oncogenic role for the cyclin-dependent kinase CDK16 in NSCLC. Namely, we first showed that CDK16 expression is significantly upregulated in human NSCLC tumor tissue. We also demonstrated that CDK16 promotes NSCLC cell proliferation and inhibits NSCLC cell apoptosis in a p27-dependent manner. We finally revealed that CDK16 ubiquinates the tumor suppressor p27 and reduces p27
CDK16 has been shown to negatively regulate the tumor suppressor p27 through phosphorylation of the Ser10 residue on p27, an action facilitating p27 degradation [13,14]. To further investigate the negative regulation of p27 by CDK16, NSCLC cell lysates were subjected to simple immunoblotting for p27 and phospho-p27Ser10 as well as IP with agarose-conjugated anti-ubiquitin antibody followed by immunoblotting for p27 and phospho-p27Ser10 (Fig. 5A). As expected, simple immunoblotting revealed upregulated p27 protein expression and 402
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Fig. 4. Flow Cytometric Analysis of NSCLC Cell Apoptosis Following CDK16 Silencing and/or p27 Silencing. (A) Apoptosis levels were assessed by flow cytometric analysis of annexin V and propidium iodide (PI)-stained EVKX cells. Red points in the upper-right quadrant (annexin V+/PI+) represent late apoptotic cells, while those in the lower-right quadrant (annexin V+/PI−) represent early apoptotic cells. (B) Apoptotic cell percentages were derived from doublepositive cells (annexin V+/PI+) plus singlepositive cells (annexin V+/PI−). Three independent experiments were performed. Data are expressed as means ± standard deviations. *p < 0.05 versus control group.
Fig. 5. Analysis of p27 Ubiquitination and Degradation in NSCLC Cells Following CDK16 Silencing. (A) Control and CDK16-siRNA EVKX cell lysates were subjected to simple immunoblotting for p27 and phospho-p27Ser10 expression (Inputs). These cell lysates were then immunoprecipitated with an agarose-conjugated anti-ubiquitin antibody, and Western blots for p27 and phospho-p27Ser10 were performed on the ubiquitin-immunoprecipitated lysates (IP: Ub). GAPDH was used as a loading control. (B) Control or CDK16-siRNA EVKX cells were exposed to the protein synthesis inhibitor CHX for 0, 4, 8, and 12 h. Then, p27 expression was analyzed by immunoblotting with GAPDH as a loading control. (C) Relative p27 protein expression (normalized to GAPDH) was measured by densitometry. Three independent experiments were performed. Results are expressed as means ± standard deviations. ***p < 0.001 versus control group.
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Research of Higher Education of Anhui Province (grant nos. KJ2017A241 and KJ2016A472), the Key Program for Excellent Young Talent in College & University of Anhui Province (grant no. gxyqZD2016168), the National Natural Science Foundation of China (grant no. 81772493), and the Science and Technology Program of Anhui Province (Key Laboratories grant nos. 1606c08225, 2016080503B035, and 2017070503B037).
expression via protein degradation. There have been several recent studies investigating the role of CDK16 in oncogenesis. Mirroring our findings in NSCLC cells, CDK16 silencing via siRNA has been demonstrated to inhibit the cellular proliferation of breast, cervical, prostate, melanoma, and medulloblastoma cancer cells but does not affect cellular proliferation in non-malignant cells [10,19,20]. In addition, Yanagi et al.’s study examined the oncogenic role of CDK16 in colorectal cell tumor xenografts using CDK16siRNA-lipid nanoparticles [11]. Consistent with our findings, they found that CDK16 silencing was able to reduce subcutaneous xenograft tumor size accompanied by heightened apoptosis of tumor cells [11]. Moreover, Yanagi et al.’s follow-on 2017 work in SCC cells revealed that CDK16 silencing reduces cell proliferation, induces G2/M arrest followed by apoptosis, and promotes accumulation of the tumor suppressor p27 [15]. In their associated tumor xenograft experiments, the conditional knockdown of CDK16 restored p27 expression while suppressing tumor growth [15]. Wang et al.’s recent work in HCC cells also demonstrated that CDK16 silencing suppresses cellular proliferation, the epithelial-mesenchymal transition (EMT), and invasiveness while inducing cell cycle arrest and promoting apoptosis [16]. Taken together, these previous findings all suggest that CDK16 plays an oncogenic role via promoting cell proliferation, inhibiting apoptosis, and reducing expression of the tumor suppressor p27. Our study is the first to demonstrate that CDK16 plays an oncogenic role in NSCLC and its actions in promoting NSCLC cell proliferation and inhibiting NSCLC cell apoptosis are dependent upon p27. Based on our findings and those from previous studies, CDK16 suppresses expression of the tumor suppressor p27 via ubiquination and protein degradation and this p27suppressive action likely promotes NSCLC cell proliferation and inhibits NSCLC cell apoptosis via cell cycle checkpoint dysregulation. That being said, further mechanistic studies are still needed to better elucidate the role of the CDK16-p27 axis in NSCLC tumorigenesis. Recent work has revealed that the intracellular regulator 14-3-3 protein (complexed with cyclin Y) binds to and activates CDK16 [21]. Human lung cancer tissue displays a 7.2× upregulation in total 14-3-3protein content relative to normal lung tissue [22]. More specifically, lung cancer cell lines display significant overexpression of four 14-3-3 isoforms (β, ε, θ and ζ) [23], two of which (β and ζ) have been shown to produce oncogenic effects [22,24]. Notably, 14-3-3ζ directly activates oncogenic RAF in human prostate, breast, and fibroblastic cell lines [25]. On this basis, 14-3-3ζ upregulation may be an underlying driver to both CDK16 activation and RAF activation in lung cancer cells. Further research on the putative relationships between 14-3-3ζ, CDK16, and RAF in NSCLC is still needed to elucidate this issue. In conclusion, CDK16 is significantly upregulated in human NSCLC tumor tissue and plays an oncogenic role in NSCLC cells via promoting cell proliferation and inhibiting apoptosis in a p27-dependent manner. Moreover, CDK16 negatively regulates expression of the tumor suppressor p27 via ubiquination and protein degradation. Our results suggest that targeting the CDK16-p27 axis may provide an effective therapeutic strategy for NSCLC.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.04.080. References [1] L.A. Torre, R.L. Siegel, A. Jemal, Lung cancer statistics, Adv. Exp. Med. Biol. 893 (2016) 1–19. [2] P.G. Thill, P. Goswami, G. Berchem, B. Domon, Lung cancer statistics in Luxembourg from 1981 to 2008, Bull. Soc. Sci. Med. Grand Duche Luxemb. (2) (2011) 43–55. [3] M. Zwitter, Dutch statistics on lung cancer: sobering experience for a new approach, J. Thorac. Oncol. 7 (2) (2012) 269–271. [4] U. Asghar, A.K. Witkiewicz, N.C. Turner, E.S. Knudsen, The history and future of targeting cyclin-dependent kinases in cancer therapy, Nat. Rev. Drug Discov. 14 (2) (2015) 130. [5] S. Singhal, A. Vachani, D. Antin-Ozerkis, L.R. Kaiser, S.M. Albelda, Prognostic implications of cell cycle, apoptosis, and angiogenesis biomarkers in non–small cell lung cancer: a review, Clin. Cancer Res. 11 (11) (2005) 3974–3986. [6] P. Cwiek, Z. Leni, F. Salm, V. Dimitrova, B. Styp-Rekowska, G. Chiriano, M. Carroll, K. Holand, V. Djonov, L. Scapozza, P. Guiry, A. Arcaro, RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification, Oncotarget 6 (1) (2015) 116–129. [7] K. Shimizu, A. Uematsu, Y. Imai, T. Sawasaki, Pctaire1/Cdk16 promotes skeletal myogenesis by inducing myoblast migration and fusion, FEBS Lett. 588 (17) (2014) 3030–3037. [8] Z. Zi, Z. Zhang, Q. Li, W. An, L. Zeng, D. Gao, Y. Yang, X. Zhu, R. Zeng, W.W. Shum, J. Wu, CCNYL1, but not CCNY, cooperates with CDK16 to regulate spermatogenesis in mouse, PLoS Genet. 11 (8) (2015) e1005485. [9] S.N. Shehata, M. Deak, N.A. Morrice, E. Ohta, R.W. Hunter, V.M. Kalscheuer, K. Sakamoto, Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16, Biochem. J. 469 (3) (2015) 409–420. [10] T. Yanagi, S. Matsuzawa, PCTAIRE1/PCTK1/CDK16: a new oncotarget? Cell Cycle 14 (4) (2015) 463–464. [11] T. Yanagi, K. Tachikawa, R. Wilkie-Grantham, A. Hishiki, K. Nagai, E. Toyonaga, P. Chivukula, S. Matsuzawa, Lipid nanoparticle-mediated siRNA transfer against PCTAIRE1/PCTK1/Cdk16 inhibits in vivo cancer growth, molecular therapy, Nucleic Acids 5 (6) (2016) e327. [12] Q. Zhou, Y. Yu, Upregulated CDK16 expression in serous epithelial ovarian cancer cells, Med. Sci. Monit. 21 (2015) 3409–3414. [13] S. Matsuda, K. Kominato, S. Koide-Yoshida, K. Miyamoto, K. Isshiki, A. Tsuji, K. Yuasa, PCTAIRE kinase 3/cyclin-dependent kinase 18 is activated through association with cyclin A and/or phosphorylation by protein kinase A, J. Biol. Chem. 289 (26) (2014) 18387–18400. [14] P. Mikolcevic, R. Sigl, V. Rauch, M.W. Hess, K. Pfaller, M. Barisic, L.J. Pelliniemi, M. Boesl, S. Geley, Cyclin-dependent kinase 16/PCTAIRE kinase 1 is activated by cyclin Y and is essential for spermatogenesis, Mol. Cell. Biol. 32 (4) (2012) 868–879. [15] T. Yanagi, H. Hata, E. Mizuno, S. Kitamura, K. Imafuku, S. Nakazato, L. Wang, H. Nishihara, S. Tanaka, H. Shimizu, PCTAIRE1/CDK16/PCTK1 is overexpressed in cutaneous squamous cell carcinoma and regulates p27 stability and cell cycle, J. Dermatol. Sci. 86 (2) (2017) 149–157. [16] Y. Wang, X. Qin, T. Guo, P. Liu, P. Wu, Z. Liu, Up-regulation of CDK16 by multiple mechanisms in hepatocellular carcinoma promotes tumor progression, J. Exp. Clin. Cancer Res. 36 (1) (2017) 97. [17] F.B. Fromowitz, M.V. VIOlA, S. Chao, S. Oravez, Y. Mishriki, G. Finkel, R. Grimson, J. Lundy, Ras p21 expression in the progression of breast cancer, Hum. Pathol. 18 (12) (1987) 1268–1275. [18] T. Watanabe-Asano, A. Kuma, N. Mizushima, Cycloheximide inhibits starvationinduced autophagy through mTORC1 activation, Biochem. Biophys. Res. Commun. 445 (2) (2014) 334–339. [19] T. Yanagi, J.C. Reed, S.-i. Matsuzawa, PCTAIRE1 regulates p27 stability, apoptosis and tumor growth in malignant melanoma, Oncoscience 1 (10) (2014) 624. [20] P. Ćwiek, Z. Leni, F. Salm, V. Dimitrova, B. Styp-Rekowska, G. Chiriano, M. Carroll, K. Höland, V. Djonov, L. Scapozza, RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification, Oncotarget 6 (1) (2015) 116. [21] S.E. Dixon-Clarke, S.N. Shehata, T. Krojer, T.D. Sharpe, F. von Delft, K. Sakamoto, A.N. Bullock, Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16, Biochem. J. 474 (5) (2017) 699–713. [22] K. Nakanishi, S. Hashizume, M. Kato, T. Honjoh, Y. Setoguchi, K. Yasumoto, Elevated expression levels of the 14-3-3 family of proteins in lung cancer tissues,
Author contributions Conceived and designed the study: YQC and XJW. Performed the experimental procedures: HTW, HLL, SPM, and YBS. Analyzed the data: WL. Drafted the manuscript: HTW. Conflicts of interest None. Acknowledgments This work was supported by the Key Program of Natural Science 404
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proliferation and oncogenic transformation, Carcinogenesis 21 (11) (2000) 2073–2077. [25] Y. Aghazadeh, V. Papadopoulos, The role of the 14-3-3 protein family in health, disease, and drug development, Drug Discov. Today 21 (2) (2016) 278–287.
Hum. Antibodies 8 (4) (1997) 189–194. [23] W. Qi, J.D. Martinez, Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation, Radiat. Res. 160 (2) (2003) 217–223. [24] Y. Takihara, Y. Matsuda, J. Hara, Role of the β isoform of 14-3-3 proteins in cellular
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CDK16 overexpressed in non-small cell lung cancer and regulates cancer cell growth and apoptosis via a p27-dependent mechanism
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Hongtao Wangb, Hongli Liuc, Shengping Mina, Yuanbing Shena, Wei Lia, Yuqing Chena, , ⁎ Xiaojing Wanga, a Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, No. 287 Changhuai Road, Bengbu, 233000, Anhui Province, China b Department of Immunology, Bengbu Medical College, Bengbu, 233000, Anhui Province, China c Department of Gynecological Oncology, First Affiliated Hospital, Bengbu Medical College, Bengbu, 233000, Anhui Province, China
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A B S T R A C T
Keywords: Lung cancer NSCLC Cyclindependent kinase 16 CDK16 Cyclin-dependent kinase inhibitor 1B
Cyclin-dependent kinase 16 (CDK16, PCTAIRE1) expression is upregulated in a wide variety of human malignancies. However, the function(s) of CDK16 in non-small cell lung cancer (NSCLC) remain unknown. Therefore, here we investigated the role of CDK16 in NSCLC. From 43 NSCLC tumors and matching healthy control lung tissues, immunohistochemistry revealed significantly greater CDK16 and phospho-p27Ser10 staining levels in NSCLC samples relative to healthy controls. The NSCLC cell line EKVX was transfected with a control siRNA, a CDK16-siRNA, or CDK16-siRNA + p27-siRNA. We found significantly decreased proliferation levels and significantly increased apoptosis levels in CDK16-silenced NSCLC cells. However, these effects were abrogated in cells treated with both the CDK16-siRNA and the p27-siRNA. In CDK16-silenced NSCLC cells, we found upregulated p27 and downregulated phospho-p27Ser10 protein expression but downregulated ubiquitinated p27 and ubiquitinated phospho-p27Ser10 protein expression. Cycloheximide-treated CDK16-silenced NSCLC cells displayed a much milder reduction in p27 protein expression over time relative to untreated CDK16-silenced NSCLC cells. In summary, CDK16 is significantly upregulated in human NSCLC tumor tissue and plays an oncogenic role in NSCLC cells via promoting cell proliferation and inhibiting apoptosis in a p27-dependent manner. Moreover, CDK16 negatively regulates expression of the p27 via ubiquination and protein degradation.
1. Introduction Lung cancer is one of the most common type of cancer and the leading cause of death among malignancies worldwide [1–3]. Nonsmall cell lung cancer (NSCLC) is the most common lung malignancy, accounting for over 80% of all lung cancer cases [1]. Unfortunately, the mortality rates associated with NSCLC have remained unchanged over the last 30 years, indicating a pressing need for a better understanding of this devastating condition [1–3]. Cyclin-dependent kinases (CDKs) are key enzymes that regulate cell cycle transitions in eukaryotic cells [4]. Many cancers, including NSCLC, exhibit features of cell cycle dysregulation, and numerous chemotherapeutics targeting CDK activity (e.g., CDK4 and CDK6) in cancer cells have been introduced over the past few decades [4,5]. One lesser-known CDK – cyclin-dependent kinase 16 (CDK16, PCTAIRE1, PCTK1) – has been shown to be critically involved in neuronal vesicular transport and spermatogenesis [6–8]. The central kinase domain of CDK16 is similar to other CDKs; however, the N- and C-terminal regions ⁎
Corresponding authors. E-mail addresses: [email protected] (Y. Chen), [email protected] (X. Wang).
https://doi.org/10.1016/j.biopha.2018.04.080 Received 29 November 2017; Received in revised form 2 April 2018; Accepted 10 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
are unique, and their functions remain unknown. That being said, structural analysis suggests that CDK16 may be involved in the phosphorylation of other kinases [9]. Through yeast two-hybrid screening, researchers have identified that CDK16 interacts directly with cyclindependent kinase inhibitor 1B (CDKN1B, p27, Kip1), a key cell cycle regulator and tumor suppressor [10–12]. Specifically, CDK16 negatively regulates p27 through phosphorylation of the Ser10 residue on p27, an action facilitating p27 degradation [13,14]. These findings suggest that CDK16 may play an important role in cell cycle regulation and oncogenesis. With respect to cancer, CDK16 expression is significantly upregulated in a wide variety of human malignancies, including breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma (HCC), colon cancer, brain cancer, melanoma, and squamous cell carcinoma (SCC) [11,12,15,16]. Furthermore, significant positive correlations between CDK16 expression and tumor invasiveness (or aggressive phenotype) have been observed in ovarian cancer, prostate cancer, and HCC [10,12,16]. Despite this previous evidence linking CDK16 to
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oncogenesis, the function(s) of CDK16 in NSCLC remain unknown. Therefore, here we investigated the role of CDK16 in NSCLC using both primary human NSCLC tumor cells and the NSCLC cell line EKVX. 2. Materials and methods 2.1. Ethics statement The Institutional Review Board (IRB) of the First Affiliated Hospital of Bengbu Medical College (Bengbu, China) approved the protocols of this study. All patients have provided informed consent prior to inclusion in this study. 2.2. Patients and specimens Surgical pneumonectomy or lobectomy were performed at the Department of Surgery at the First Affiliated Hospital of Bengbu Medical College. The paraffin-embedded tissue samples of lung tumor cores and matching healthy control lung tissue were stored at −80 °C immediately after surgical resection. Clinical records (Department of Pathology, the First Affiliated Hospital of Bengbu Medical College) for all lung tumor resection cases performed between January 2013 and December 2015 were screened. After screening, a total of 43 NSCLC cases (mean age: 56 years, range: 45–70 years; sex: 30 male and 13 female) were finally enrolled in this study. 2.3. EKVX cell line and siRNA transfection The NSCLC cell line EKVX was obtained from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). As recommended, EKVX cells were grown in complete growth medium and cultured in a humidified incubator (5% CO2) at 37 °C. Pre-designed small interfering RNAs (siRNAs) against human CDK16 and p27 as well as a non-coding scrambled siRNA were purchased from Life Technologies (Carlsbad, CA, USA). For transfection, EKVX cells were seeded in six-well plates at a density of 5 × 105 cells per well, cultured overnight, and then transfected with the CDK16siRNA alone or a combination of the CDK16-siRNA plus the p27-siRNA. The non-coding scrambled siRNA served as a negative control. According to the manufacturer’s protocol, cells were transfected with 20 nM of each siRNA with the Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Validation of CDK16 silencing and p27 silencing in EKVX cells was validated by immunoblotting (Supplementary Fig. S1, Supplementary Information).
Fig. 1. CDK16 Upregulated in NSCLC Tumor Cells. CDK16 protein expression was assessed by immunohistochemical analysis of tissue samples. (A) Representative images of healthy control lung tissue samples (left panel) and matching NSCLC tumor tissue samples (right panel) are provided. Scale bars, 50 μm. (B) CDK16 immunoreactivity scores were determined through blinded analysis. Box plots display the median (center line) and the interquartile range (top and bottom lines). **p < 0.01 versus control group.
randomly chosen from different areas of each specimen for immunoreactivity scoring, and the average immunoreactivity score derived from the ten visual fields was calculated as the final value for each patient. 2.5. GFP fluorescence proliferation assay Analysis of proliferation was determined using a Thermo Scientific Cellomics ArrayScan HCS reader (Thermo Fisher Scientific) with the Spot detector V3 Cellomics Bioapplication (Cellomics, Pittsburgh, PA, USA). GFP-positive cells were detected using the FITC channel at a 20× magnification, and images were acquired on a high-resolution chargecoupled device (CCD) camera on days one, two, three, four, and five (GRAS-14S5M; Point Grey, Richmond, Canada). Cells were identified based on the presence of nuclei and GFP fluorescence intensity. Cell count values were reported as the fold-change in cell count from day one using ArrayScan software (Thermo Fisher Scientific). A total of 1 × 103 cells were counted per sample, and experiments were repeated three times.
2.4. Immunohistochemistry and immunoreactivity scoring Specimens had been fixed in 10% formalin and embedded in paraffin wax immediately after resection. Three-micrometer sections were then cut from the paraffin blocks for immunohistochemical (IHC) analysis. The sections were stained with an anti-human CDK16 antibody (diluted 1:500, Invitrogen, Carlsbad, CA, USA) or an anti-human phospho-p27Ser10 antibody (diluted 1:50, Invitrogen) at 4 °C overnight. Following application of the secondary antibody, the immunoreactions were visualized with the avidin-biotin-peroxidase complex method according to the manufacturer’s recommendations (Vectastain Elite ABC Kit, Vector Labs, Burlingame, CA, USA). Secondary-only negative controls were used to control for non-specific binding. Immunoreactivity scoring in IHC samples was performed as previously described by Fromowitz et al. with minor modifications [17]. Briefly, the formula for the immunoreactivity score was as follows: immunoreactivity score = staining intensity (SI) + percentage of positive cells (PP). SI was tabulated as follows: negative staining = 0; weak staining = 1; moderate staining = 2; and strong staining = 3. PP was tabulated as follows: 0% = 0; 1–20% positive cells = 1; 21–50% positive cells = 2; and 51–100% positive cells = 3. Ten visual fields were
2.6. MTT proliferation assay The capacity for cellular proliferation was measured with a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A total of 3 × 103 cells were seeded in 96-well culture plates for one, two, three, four, and five days. The cells were then incubated with 20 μl of MTT (5 mg/ml) for four hours at 37 °C, and 150 μl of DMSO was added to solubilize the crystals for 20 min at room temperature. The optical density was determined with a spectrophotometer (Shanghai 400
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driver-oncogene, a microarray based lung cancer dataset obtained from the GEO database (GSE31210), which includes 226 lung cancer patients from Japan were downloaded. The correlation in expression between CDK16 and several oncogenes, including EGFR, KRAS, ALK, BRAF, and NF1, was computed by Spearman’s rank correlation test. Using the Japan cohort, the expression pattern of CDK16 between the patients without mutations in several oncogenes, i.e. EGFR, KRAS, and ALK (EGFR/KRAS/ALK mutation-), and the patients with mutations in EGFR (EGFR mutation+), KRAS (KRAS mutation+), or ALK (ALK mutation +) were investigated. 2.10. Statistical analysis The SPSS 17.0 software package (SPSS, Inc., Chicago, IL, USA) was used for all statistical analysis. Data were analyzed with Student ttesting or one-way analysis of variance (ANOVA) followed by Bonferroni testing where appropriate. P < 0.05 was considered to indicate a statistically significant difference. Data are reported as means ± standard deviations (SDs) as absolute values or percentages of controls. 3. Results 3.1. Upregulation of CDK16 and Phospho-p27 in human NSCLC tumors CDK16 expression is significantly upregulated in a wide variety of human malignancies [11,12,15,16]; however, its expression in NSCLC remains unknown. In the GEO lung cancer cohort, we found the expression of CDK16 was significantly and positively correlated with that of EGFR and BRAF (P < 0.05) (Supplementary Fig. S2). On the other hand, we tried to address whether the expression of CDK16 is affected by the somatic mutation status in the other oncogenes mutation. Comparison of CDK16 expression between lung cancer patients with and without oncogene mutations, we didn’t find significant difference in CDK16 among these patient categories (Supplementary Fig. S3). The roles of EGFR and BRAF overexpression in cancer progression have been well established, the role of CDK16 is much less clear. Here, a total of 43 NSCLC tumor samples were comparatively analyzed against matching healthy control lung tissue. IHC analysis revealed significantly greater levels of CDK16 staining in NSCLC samples relative to healthy controls (Fig. 1A). Scoring revealed significantly greater CDK16 immunoreactive scores in NSCLC samples relative to healthy controls (Fig. 1B, **p < 0.01). IHC analysis also revealed significantly greater levels of phospho-p27Ser10 staining in NSCLC samples relative to healthy controls (Fig. 2A). Scoring revealed significantly greater phospho-p27Ser10 immunoreactive scores in NSCLC samples relative to healthy controls (Fig. 2B, **p < 0.01).
Fig. 2. p27 Phosphorylation Upregulated in NSCLC Tumor Cells. Phospho-p27Ser10 protein expression was assessed by immunohistochemical analysis of tissue samples. (A) Representative images of healthy control lung tissue samples (left panel) and matching NSCLC tumor tissue samples (right panel) are provided. Scale bars, 50 μm. (B) Phospho-p27Ser10 immunoreactivity scores were determined through blinded analysis. Box plots display the median (center line) and the interquartile range (top and bottom lines). **p < 0.01 versus control group.
Spectrophotometer Co. Ltd., China) at a wavelength of 490 nm. 2.7. Flow cytometric apoptotic analysis Cells were cultured for 48 h post-transfection. Cells were then harvested, washed with phosphate buffered saline (PBS), and then fixed with 70% ethanol overnight at 4 °C. Cells were stained with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, BioVision, Mountain View, CA, USA) for 5 min before flow cytometric analysis. 2.8. Immunoprecipitation and immunoblotting
3.2. CDK16 silencing reduces NSCLC cell proliferation in a p27-dependent manner
Immunoprecipitation (IP) was performed on 800–1000 μg of cell lysate separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the manufacturer’s instructions (Thermo Fisher Scientific). Immunoblots were performed on cells prepared in lysis buffer (containing 150 mM NaCl, 100 mM Tris, pH 8.0, 1% Tween 20, 50 mm diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethyl-sulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml trypsin inhibitor, and 10 μg/ml leupeptin). Proteins were separated using SDSPAGE under reducing conditions on 10% polyacrylamide gels. Resolved proteins were transferred to nitrocellulose membranes and incubated with the indicated antisera followed by a horseradish peroxidase-conjugated secondary antibody.
As CDK16 has been shown to promote cellular proliferation through a p27-dependent mechanism [15], here we assessed the roles of CDK16 and p27 in NSCLC cell proliferation via gene silencing experimentation. Specifically, NSCLC cells were initially treated with CDK16-siRNA and/ or p27-siRNA and then followed over a period of five days to assess differences in cell proliferation (Fig. 3A). Via fluorescence microscopy, we found proliferation levels were significantly decreased in NSCLC cells treated with the CDK16-siRNA on days three, four, and five (Fig. 3B, **p < 0.01). However, decreased proliferation was not observed in cells treated with both the CDK16-siRNA and the p27-siRNA (Fig. 3B, p > 0.05). To validate these results, MTT assays were also performed. A similar significant decrease in proliferation was observed in NSCLC cells treated with CDK16-siRNA on days four and five (Fig. 3C, *p < 0.05). Moreover, decreased proliferation was not observed in cells treated
2.9. GEO analysis To check the relationship between CDK16 and other single-gene 401
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Fig. 3. Fluorescence Microscopy of NSCLC Cell Proliferation Following CDK16 Silencing and/or p27 Silencing. Following transfection with siRNAs specific to CDK16 and/or p27, EVKX cells were analyzed daily over a period of five days. (A) Representative fluorescent images from the three experimental groups over the five-day period are provided. (B) GFP+ cells were counted using Cellomics ArrayScan technology, and fold-change values were calculated from the GRP+ cell counts. Three independent experiments were performed. Data are presented as the means ± standard deviations of the fold-change in cell count normalized to day one. **p < 0.01 versus control group. (C) Following transfection with siRNAs specific to CDK16 and/or p27, EVKX cells were analyzed daily by MTT assay over a period of five days. Three independent experiments were performed. Data are presented as means ± standard deviations of the fold-change in 490-nm optical density (OD490) values normalized to day one. *p < 0.05 versus control group.
downregulated phospho-p27Ser10 protein expression in CDK16-silenced NSCLC cells (Fig. 5A). Notably, via IP, we also observed decreased levels of ubiquitinated p27 protein expression and ubiquitinated phospho-p27Ser10 in CDK16-silenced NSCLC cells (Fig. 5A). We next analyzed the role of protein degradation in p27 downregulation by CDK16 using the protein synthesis inhibitor cycloheximide (CHX), a compound which potently suppresses protein degradation [18]. Control and CDK16-silenced NSCLC cells treated with CHX were followed for a period of 12 h to assess changes in p27 protein expression (Fig. 5B). In control cells, p27 protein levels were dramatically reduced following 12 h of CHX treatment (Fig. 5B). However, a much milder reduction in p27 expression was observed in CDK16-silenced NSCLC cells (Fig. 5B). The associated densitometric analysis revealed significantly higher levels of p27 protein expression in CDK16silenced cells relative to controls (Fig. 5C, ***p < 0.001). Taken together, these results indicate that CDK16 ubiquinates the tumor suppressor p27 and reduces p27 expression via protein degradation (Supplementary Fig. S4).
with both the CDK16-siRNA and the p27-siRNA (Fig. 3C, p > 0.05). Taken together, these results indicate that CDK16 promotes NSCLC cell proliferation in a p27-dependent manner. 3.3. CDK16 silencing increases NSCLC cell apoptosis in a p27-dependent manner As CDK16 has been shown to regulate cancer cell apoptosis through a p27-dependent mechanism [15], here we assessed the roles of CDK16 and p27 in NSCLC cell apoptosis via gene silencing experimentation. Specifically, NSCLC cells were initially treated with CDK16-siRNA and/ or p27-siRNA and then followed over a period of 72 h to assess differences in apoptosis levels. Via Annexin V/PI flow cytometric analysis (Fig. 3A), we found significantly higher apoptosis levels at 48 and 72 h in NSCLC cells treated with the CDK16-siRNA relative to controls (Fig. 4B, *p < 0.05). However, the pro-apoptotic effect of CDK16 silencing was abrogated by p27 silencing (Fig. 4B, p > 0.05). Taken together, these results indicate that CDK16 inhibits NSCLC cell apoptosis in a p27-dependent manner.
4. Discussion 3.4. Regulation of p27 by CDK16 NSCLC remains one of the most deadly forms of cancer globally. Therefore, new targeted molecular therapeutic strategies for NSCLC are still needed to aid patients with this disease. To this end, here we demonstrate a novel oncogenic role for the cyclin-dependent kinase CDK16 in NSCLC. Namely, we first showed that CDK16 expression is significantly upregulated in human NSCLC tumor tissue. We also demonstrated that CDK16 promotes NSCLC cell proliferation and inhibits NSCLC cell apoptosis in a p27-dependent manner. We finally revealed that CDK16 ubiquinates the tumor suppressor p27 and reduces p27
CDK16 has been shown to negatively regulate the tumor suppressor p27 through phosphorylation of the Ser10 residue on p27, an action facilitating p27 degradation [13,14]. To further investigate the negative regulation of p27 by CDK16, NSCLC cell lysates were subjected to simple immunoblotting for p27 and phospho-p27Ser10 as well as IP with agarose-conjugated anti-ubiquitin antibody followed by immunoblotting for p27 and phospho-p27Ser10 (Fig. 5A). As expected, simple immunoblotting revealed upregulated p27 protein expression and 402
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Fig. 4. Flow Cytometric Analysis of NSCLC Cell Apoptosis Following CDK16 Silencing and/or p27 Silencing. (A) Apoptosis levels were assessed by flow cytometric analysis of annexin V and propidium iodide (PI)-stained EVKX cells. Red points in the upper-right quadrant (annexin V+/PI+) represent late apoptotic cells, while those in the lower-right quadrant (annexin V+/PI−) represent early apoptotic cells. (B) Apoptotic cell percentages were derived from doublepositive cells (annexin V+/PI+) plus singlepositive cells (annexin V+/PI−). Three independent experiments were performed. Data are expressed as means ± standard deviations. *p < 0.05 versus control group.
Fig. 5. Analysis of p27 Ubiquitination and Degradation in NSCLC Cells Following CDK16 Silencing. (A) Control and CDK16-siRNA EVKX cell lysates were subjected to simple immunoblotting for p27 and phospho-p27Ser10 expression (Inputs). These cell lysates were then immunoprecipitated with an agarose-conjugated anti-ubiquitin antibody, and Western blots for p27 and phospho-p27Ser10 were performed on the ubiquitin-immunoprecipitated lysates (IP: Ub). GAPDH was used as a loading control. (B) Control or CDK16-siRNA EVKX cells were exposed to the protein synthesis inhibitor CHX for 0, 4, 8, and 12 h. Then, p27 expression was analyzed by immunoblotting with GAPDH as a loading control. (C) Relative p27 protein expression (normalized to GAPDH) was measured by densitometry. Three independent experiments were performed. Results are expressed as means ± standard deviations. ***p < 0.001 versus control group.
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Research of Higher Education of Anhui Province (grant nos. KJ2017A241 and KJ2016A472), the Key Program for Excellent Young Talent in College & University of Anhui Province (grant no. gxyqZD2016168), the National Natural Science Foundation of China (grant no. 81772493), and the Science and Technology Program of Anhui Province (Key Laboratories grant nos. 1606c08225, 2016080503B035, and 2017070503B037).
expression via protein degradation. There have been several recent studies investigating the role of CDK16 in oncogenesis. Mirroring our findings in NSCLC cells, CDK16 silencing via siRNA has been demonstrated to inhibit the cellular proliferation of breast, cervical, prostate, melanoma, and medulloblastoma cancer cells but does not affect cellular proliferation in non-malignant cells [10,19,20]. In addition, Yanagi et al.’s study examined the oncogenic role of CDK16 in colorectal cell tumor xenografts using CDK16siRNA-lipid nanoparticles [11]. Consistent with our findings, they found that CDK16 silencing was able to reduce subcutaneous xenograft tumor size accompanied by heightened apoptosis of tumor cells [11]. Moreover, Yanagi et al.’s follow-on 2017 work in SCC cells revealed that CDK16 silencing reduces cell proliferation, induces G2/M arrest followed by apoptosis, and promotes accumulation of the tumor suppressor p27 [15]. In their associated tumor xenograft experiments, the conditional knockdown of CDK16 restored p27 expression while suppressing tumor growth [15]. Wang et al.’s recent work in HCC cells also demonstrated that CDK16 silencing suppresses cellular proliferation, the epithelial-mesenchymal transition (EMT), and invasiveness while inducing cell cycle arrest and promoting apoptosis [16]. Taken together, these previous findings all suggest that CDK16 plays an oncogenic role via promoting cell proliferation, inhibiting apoptosis, and reducing expression of the tumor suppressor p27. Our study is the first to demonstrate that CDK16 plays an oncogenic role in NSCLC and its actions in promoting NSCLC cell proliferation and inhibiting NSCLC cell apoptosis are dependent upon p27. Based on our findings and those from previous studies, CDK16 suppresses expression of the tumor suppressor p27 via ubiquination and protein degradation and this p27suppressive action likely promotes NSCLC cell proliferation and inhibits NSCLC cell apoptosis via cell cycle checkpoint dysregulation. That being said, further mechanistic studies are still needed to better elucidate the role of the CDK16-p27 axis in NSCLC tumorigenesis. Recent work has revealed that the intracellular regulator 14-3-3 protein (complexed with cyclin Y) binds to and activates CDK16 [21]. Human lung cancer tissue displays a 7.2× upregulation in total 14-3-3protein content relative to normal lung tissue [22]. More specifically, lung cancer cell lines display significant overexpression of four 14-3-3 isoforms (β, ε, θ and ζ) [23], two of which (β and ζ) have been shown to produce oncogenic effects [22,24]. Notably, 14-3-3ζ directly activates oncogenic RAF in human prostate, breast, and fibroblastic cell lines [25]. On this basis, 14-3-3ζ upregulation may be an underlying driver to both CDK16 activation and RAF activation in lung cancer cells. Further research on the putative relationships between 14-3-3ζ, CDK16, and RAF in NSCLC is still needed to elucidate this issue. In conclusion, CDK16 is significantly upregulated in human NSCLC tumor tissue and plays an oncogenic role in NSCLC cells via promoting cell proliferation and inhibiting apoptosis in a p27-dependent manner. Moreover, CDK16 negatively regulates expression of the tumor suppressor p27 via ubiquination and protein degradation. Our results suggest that targeting the CDK16-p27 axis may provide an effective therapeutic strategy for NSCLC.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.04.080. References [1] L.A. Torre, R.L. Siegel, A. Jemal, Lung cancer statistics, Adv. Exp. Med. Biol. 893 (2016) 1–19. [2] P.G. Thill, P. Goswami, G. Berchem, B. Domon, Lung cancer statistics in Luxembourg from 1981 to 2008, Bull. Soc. Sci. Med. Grand Duche Luxemb. (2) (2011) 43–55. [3] M. Zwitter, Dutch statistics on lung cancer: sobering experience for a new approach, J. Thorac. Oncol. 7 (2) (2012) 269–271. [4] U. Asghar, A.K. Witkiewicz, N.C. Turner, E.S. Knudsen, The history and future of targeting cyclin-dependent kinases in cancer therapy, Nat. Rev. Drug Discov. 14 (2) (2015) 130. [5] S. Singhal, A. Vachani, D. Antin-Ozerkis, L.R. Kaiser, S.M. Albelda, Prognostic implications of cell cycle, apoptosis, and angiogenesis biomarkers in non–small cell lung cancer: a review, Clin. Cancer Res. 11 (11) (2005) 3974–3986. [6] P. Cwiek, Z. Leni, F. Salm, V. Dimitrova, B. Styp-Rekowska, G. Chiriano, M. Carroll, K. Holand, V. Djonov, L. Scapozza, P. Guiry, A. Arcaro, RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification, Oncotarget 6 (1) (2015) 116–129. [7] K. Shimizu, A. Uematsu, Y. Imai, T. Sawasaki, Pctaire1/Cdk16 promotes skeletal myogenesis by inducing myoblast migration and fusion, FEBS Lett. 588 (17) (2014) 3030–3037. [8] Z. Zi, Z. Zhang, Q. Li, W. An, L. Zeng, D. Gao, Y. Yang, X. Zhu, R. Zeng, W.W. Shum, J. Wu, CCNYL1, but not CCNY, cooperates with CDK16 to regulate spermatogenesis in mouse, PLoS Genet. 11 (8) (2015) e1005485. [9] S.N. Shehata, M. Deak, N.A. Morrice, E. Ohta, R.W. Hunter, V.M. Kalscheuer, K. Sakamoto, Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16, Biochem. J. 469 (3) (2015) 409–420. [10] T. Yanagi, S. Matsuzawa, PCTAIRE1/PCTK1/CDK16: a new oncotarget? Cell Cycle 14 (4) (2015) 463–464. [11] T. Yanagi, K. Tachikawa, R. Wilkie-Grantham, A. Hishiki, K. Nagai, E. Toyonaga, P. Chivukula, S. Matsuzawa, Lipid nanoparticle-mediated siRNA transfer against PCTAIRE1/PCTK1/Cdk16 inhibits in vivo cancer growth, molecular therapy, Nucleic Acids 5 (6) (2016) e327. [12] Q. Zhou, Y. Yu, Upregulated CDK16 expression in serous epithelial ovarian cancer cells, Med. Sci. Monit. 21 (2015) 3409–3414. [13] S. Matsuda, K. Kominato, S. Koide-Yoshida, K. Miyamoto, K. Isshiki, A. Tsuji, K. Yuasa, PCTAIRE kinase 3/cyclin-dependent kinase 18 is activated through association with cyclin A and/or phosphorylation by protein kinase A, J. Biol. Chem. 289 (26) (2014) 18387–18400. [14] P. Mikolcevic, R. Sigl, V. Rauch, M.W. Hess, K. Pfaller, M. Barisic, L.J. Pelliniemi, M. Boesl, S. Geley, Cyclin-dependent kinase 16/PCTAIRE kinase 1 is activated by cyclin Y and is essential for spermatogenesis, Mol. Cell. Biol. 32 (4) (2012) 868–879. [15] T. Yanagi, H. Hata, E. Mizuno, S. Kitamura, K. Imafuku, S. Nakazato, L. Wang, H. Nishihara, S. Tanaka, H. Shimizu, PCTAIRE1/CDK16/PCTK1 is overexpressed in cutaneous squamous cell carcinoma and regulates p27 stability and cell cycle, J. Dermatol. Sci. 86 (2) (2017) 149–157. [16] Y. Wang, X. Qin, T. Guo, P. Liu, P. Wu, Z. Liu, Up-regulation of CDK16 by multiple mechanisms in hepatocellular carcinoma promotes tumor progression, J. Exp. Clin. Cancer Res. 36 (1) (2017) 97. [17] F.B. Fromowitz, M.V. VIOlA, S. Chao, S. Oravez, Y. Mishriki, G. Finkel, R. Grimson, J. Lundy, Ras p21 expression in the progression of breast cancer, Hum. Pathol. 18 (12) (1987) 1268–1275. [18] T. Watanabe-Asano, A. Kuma, N. Mizushima, Cycloheximide inhibits starvationinduced autophagy through mTORC1 activation, Biochem. Biophys. Res. Commun. 445 (2) (2014) 334–339. [19] T. Yanagi, J.C. Reed, S.-i. Matsuzawa, PCTAIRE1 regulates p27 stability, apoptosis and tumor growth in malignant melanoma, Oncoscience 1 (10) (2014) 624. [20] P. Ćwiek, Z. Leni, F. Salm, V. Dimitrova, B. Styp-Rekowska, G. Chiriano, M. Carroll, K. Höland, V. Djonov, L. Scapozza, RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification, Oncotarget 6 (1) (2015) 116. [21] S.E. Dixon-Clarke, S.N. Shehata, T. Krojer, T.D. Sharpe, F. von Delft, K. Sakamoto, A.N. Bullock, Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16, Biochem. J. 474 (5) (2017) 699–713. [22] K. Nakanishi, S. Hashizume, M. Kato, T. Honjoh, Y. Setoguchi, K. Yasumoto, Elevated expression levels of the 14-3-3 family of proteins in lung cancer tissues,
Author contributions Conceived and designed the study: YQC and XJW. Performed the experimental procedures: HTW, HLL, SPM, and YBS. Analyzed the data: WL. Drafted the manuscript: HTW. Conflicts of interest None. Acknowledgments This work was supported by the Key Program of Natural Science 404
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proliferation and oncogenic transformation, Carcinogenesis 21 (11) (2000) 2073–2077. [25] Y. Aghazadeh, V. Papadopoulos, The role of the 14-3-3 protein family in health, disease, and drug development, Drug Discov. Today 21 (2) (2016) 278–287.
Hum. Antibodies 8 (4) (1997) 189–194. [23] W. Qi, J.D. Martinez, Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation, Radiat. Res. 160 (2) (2003) 217–223. [24] Y. Takihara, Y. Matsuda, J. Hara, Role of the β isoform of 14-3-3 proteins in cellular
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