Cellular and Molecular Biology
Open Access

Cell migration; Cell invasion; CDK1; Sox2; Esophageal carcinoma

Introduction

Esophageal carcinoma is a fatal malignancy with poor prognosis and high mortality. Esophageal carcinoma is the sixth most common cause of cancer-related deaths and eighth most common cancer in the world [1]. Squamous cell carcinoma and adenocarcinoma are the two major types of esophageal carcinoma [2]. Surgery combined with chemotherapy and radiotherapy is the standard clinical treatment for esophageal carcinoma [3]. However, due to the high migration and invasiveness capability of esophageal carcinoma cell, patients diagnosed with advanced esophageal cancer are usually accompanied with multiple metastatic, which leads to less than 25% five-year survival rate [4]. Therefore, it is of great importance to further explore the mechanism underlying high migration and invasiveness of esophageal carcinoma cell and pave the foundation for the clinical treatment of esophageal carcinoma.

CDK1 is a kinase that belongs to CDK1 family, which can phosphorylate the serine/threonine residues on substrate proteins. The structure of CDK1 contains a bare protein kinase motif that catalyses the covalent binding of phosphate to the oxygen of the hydroxyl serine/ threonine. In addition to the catalytic domain, CDK1 also contains a T-loop structure that prevents the binding of substrate protein in the absence of interacting cycling [5]. CDK1 was originally identified as a key regulator of cell cycle and promoted replicative DNA synthesis [6]. Recently studies indicate that beyond cell cycle regulation, CDK1 also plays an important role in various biological process including autophagy, apoptosis and cell differentiation [7-9]. Interestingly, abnormal CDK1 expression was reported in a variety of disease, such as neurodegenerative diseases, diabetes and cancer [10-12]. However, the role of CDK1 in esophageal carcinoma remains to be elucidated.

The Sox (SRY homology box) family include approximately 20 proteins that are characterized by a conserved DNA-binding domain called HMG-box [13]. Sox family proteins are involved in biological development process including sex determination and differentiation [14]. Sox2 is the best-known member of Sox family for its ability to drive the dedifferentiation of human or murine terminally differentiated somatic cells to Induced Pluripotent Stem Cells (iPSCs) in cooperation with others co-factors [15]. Sox2 is instrumental in the determining of neuronal or epithelial fate, dental development and skin repair [16-18]. In the past decades, aberrant expression of Sox2 was detected in various types of cancers and was associated with the stemness of tumor cells and its malignant phenotype [19].

In this study, CDK1 directly binds to Sox2 and induces the phosphorylation and nuclear localization of Sox2 in ECA109 cells. CDK1 knockdown inhibited the phosphorylation of Sox2, and resulted in the translocation of Sox2 from nucleus to cytoplasm. As a consequence, knockdown of CDK1 inhibited migration and invasion of esophageal carcinoma cells. In summary, our study reveals mechanism underlying CDK1-mediated Sox2 phosphorylation and nuclear localization in the malignant phenotype of esophageal carcinoma.

Materials and Method

Cell culture

Human esophageal carcinoma cell line ECA109 was cultured in RPMI-1640 medium (#PM150110, Procell Life Science & Technology) supplemented with 10% FBS (#164210, Procell Life Science & Technology) and 1% Penicillin-Streptomycin (#PB180120, Procell Life Science & Technology) and maintained at 37℃ containing 5% CO2.

Gene expression profiling interactive analysis (GEPIA)

GEPIA (http://gepia.cancer-pku.cn/) is an interactive online platform that contains tumor and normal tissue information from GTEx and TCGA projects. The expression of CDK1 and Sox2 in esophageal carcinoma and adjacent normal tissue were analyzed from data mined from GEPIA.

Cell migration assay

ECA109 cells were seeded in a 12-well plate at the density of 5×104 cells per well and transfected with indicated siRNAs. 48 h after transfection, a straight line was drawn across cell layer with a pipet tip and the well was washed with PBS twice. Cells were then cultured in RPMI-1640 medium supplemented with 1% FBS for 24 h and photographed with an inverted microscope (CKX31, Olympus).

Transwell invasion assay

After indicated treatment, ECA109 cells were suspended in culture medium and mixed with Matrigel matrix (#356234, BD Biocoat). Matrigel-coated cells were transferred to the invasion chamber, which was then inserted into a 24-well plate containing culture medium supplemented with 10% FBS. 48 h after incubation, the invasion chamber was fixed in 4% paraformaldehyde and then stained with crystal-violet solution. Photos were acquired with an inverted microscope (CKX31, Olympus).

Cell apoptosis assay

Cell apoptosis assay was carried out with Annexin V-FITC/PI apoptosis kit (#40302ES20, Yeasen Biotechnology). Briefly, ECA109 cells were washed with PBS and then re-suspended in binding buffer. Then, cells were incubated with Annexin V-FITC and PI and processed with a flow cytometry.

RNA extraction and RT-qPCR analysis

Total RNA in ECA109 cells was extracted with Trizol reagent (#9108, Takara) and then reverse transcribed with PrimeScript™ RT reagent Kit (#RR047A, Takara). RT-qPCR was performed using SYBR® Premix Ex Taq (#RR820A, Takara). The primers used were as followings. CDK1-F: GGATGTGCTTATGCAGGATTCC. CDK1-R: CATGTACTGACCAGGAGGGATAG. Sox2-F: TGGACAGTTACGCGCACAT. Sox2-R: CGAGTAGGACATGCTGTAGGT. GAPDH-F: CCATCAATGACCCCTTCATTGACC. GAPDH-R: CCATCAATGACCCCTTCATTGACC.

Western blot

Total proteins were extracted and separated on 10% SDS-PAGE gel, which were then transferred to PVDF membranes. The PDVF membranes were blocked with 5% BSA and incubated with following antibodies at 4°C overnight: Sox2 antibody (#ab92494, Abcam), p-Sox2 antibody (#PA5-105708, Invitrogen), Lamin B (#ab16048, Abcam), β-actin antibody (#ab8227, Abcam). The membranes were then washed with TBST buffer three times and incubated with HRPconjugated secondary antibody (#ab6721, Abcam) for an hour at room temperature. Protein bands were obtained with chemiluminescence detection kit (#180-5001, Tanon) and digital gel image analysis system (#5200, Tanon).

Statistical analysis

The results were processed with GraphPad software and analyzed by Student’s t-test or one-way ANOVA. All data were shown as Mean ± SEM. p<0.05 was considered as statistically significant.

Results

CDK1 promoted the migration and invasion of ECA109 cells.

GEPIA database was used to analyze RNA expression in TCGA. We analyzed the expression of CDK1 in esophageal carcinoma (n=182) and normal tissues (n=286). Generally, it was found that the expression of CDK1 was higher in esophageal carcinoma compared with those in normal tissue, suggesting that high CDK1 expression is correlated with esophageal carcinoma (Figure S1A).

To explore the biological function of CDK1 in the malignant phenotype of esophageal carcinoma, CDK1 siRNA was used to knockdown the expression of CDK1 in ECA109 cells. Then, RT-qPCR was performed to determine the knockdown efficiency of CDK1 siRNA. The results showed that compared with control group, transfection of scramble siRNA had no effect on the expression of CDK1, while the transfection of CDK1 siRNA significantly down-regulated the level of CDK1 mRNA, indicating CDK1 was successfully knocked down (Figure S2A). We then performed wound healing assay to evaluate the effect of CDK1 knockdown on the migration of ECA109 cells. The results showed that following CDK1 knockdown, cell migration ability was remarkably inhibited (Figure 1A). Statistical analysis showed that migration rate of cell in CDK1 knockdown group decreased approximately 60% (Figure 1B). Subsequently, transwell invasion analysis was performed to determine the ability of cell invasion following CDK1 knockdown. It was found that CDK1 knockdown inhibited cell invasion, and the invaded cell number decreased about 72% (Figure 2A, B). These data suggest that CDK1 plays an important role in the migration and invasion of esophageal carcinoma cells.

CDK1 knockdown induced apoptosis in ECA109 cells

Apoptosis is a programmed process of cell death that happens in normal physiological conditions as well as in the pathogenesis of diseases [20]. Apoptosis is a popular target of drugs and treatment strategies and is proved to be a practical measure clinically [21]. Here, apoptotic cells were detected by Annexin V-FITC/PI staining and cytometry flow. As shown in Figure 3A, B, transfection of scramble siRNA had no effect on cell apoptosis. However, the ratio of apoptotic cells increased significantly following CDK1 knockdown. These results indicate that down-regulation of CDK1 induces apoptosis in esophageal carcinoma cells.

CDK1 directly interacted with Sox2

Transcriptional factor Sox2 was reported to regulate tumor aggression and metastasis, and the association of Sox2 and CDK1 was implicated [22, 23]. To confirm this, Co-Immunoprecipitation (Co-IP) assay was performed to detect the interaction between CDK1 and Sox2. As shown in Figure 4, CDK1 was pulled down by CDK1 antibody, but not by pre-immune normal IgG, indicating that immunoprecipitation was successfully performed. Notably, Sox2 was also pulled down by CDK1 antibody. CDK1 knockdown by siRNA decreased the amount of Sox2 immunoprecipitated by CDK1 antibody. All these data indicate that CDK1 directly binds to Sox2.

CDK1 knockdown inhibited phosphorylation and nuclear location of Sox2

We explored the effect of CDK1 knockdown on the expression and function of Sox2, and RT-qPCR and western blot were performed. As shown in Figure 5A, B and Figure S2B, CDK1 knockdown had no effect on the mRNA and protein level of Sox2, indicating that CDK1 didn’t regulate the expression of Sox2. Intriguingly, the phosphorylation of Sox2 decreased significantly following CDK1 knockdown. As a transcriptional factor, it was reported that phosphorylation resulted in nuclear localization and activation of Sox2. Therefore, we further investigated the effect of CDK1 knockdown on subcellular location of Sox2. Surprisingly, the level of Sox2 in cytoplasm increased whiles the level of Sox2 in nuclear decreased significantly following CDK1 knockdown (Figure 6A-D). These data indicate that CDK1 knockdown inhibited the phosphorylation and nuclear localization and Sox2.

Discussion

In recent years, aside from traditional surgery, radiotherapy and chemotherapy, advanced approaches including immunotherapy and gene targeted therapy have been applied in the clinical treatment of esophageal carcinoma [24]. However, esophageal carcinoma remains to be one of the most aggressive cancers worldwide with low 5-year survival rate and early metastasis [25]. Tumor cell migration and invasion are the two crucial biological process involved in the metastasis of esophageal carcinoma. However, molecular mechanism regarding metastasis of esophageal carcinoma remains largely unknown. CDK1 is the only cyclin-dependent kinase that drives the G2-M transition in cell cycle [26]. Recently, it was reported that CDK1 acted not only as a modulator in cell cycle, but also as a key regulator in the malignancy phenotype of cancer cells. CDK1 promoted the tumorigenesis of colorectal cancer by regulating cell apoptosis and proliferation [27]. Inactivation of CDK1 ameliorated immune resistance in the clinical treatment of pancreatic cancer [28]. CDK1 also facilitated Sirt3 activation and promoted tumor radio resistance [29]. In consistency with previous findings that CDK1 acted as an oncogene in multiple tumors, we found that the high expression of CDK1 was correlated with esophageal carcinoma through GEPIA analysis. CDK1 knockdown inhibited the migration and invasion capability of ECA109 cells, indicating that CDK1 is a critical factor in the malignancy of esophageal carcinoma. Therefore, targeting CDK1 could be a promising approach in the clinic.

Several upstream factors were reported to contribute to cell migration and invasion by targeting CDK1, including maternal embryonic leucine zipper kinase (MELK), cyclin-dependent kinase substrate 1 (NUCKS1) and miR-378a-5p [30-32]. Molecular mechanism involved in CDK1-mediated cell migration and invasion is still poorly understood. As a transcriptional factor, Sox2 was upregulated and contributed to the stemness of cancer cells. Intriguingly, association of Sox2 and CDK1 was implicated in the stemness of lung cancer [23]. Therefore, we explored whether similar mechanism participated in the malignancy of esophageal carcinoma. Indeed, we found that Sox2 expression was also up-regulated in esophageal carcinoma compared with normal tissue. Co-immunoprecipitation and western blot analysis showed that CDK1 directly bond to Sox2. Though CDK1 knockdown had no effect on the mRNA and protein level of Sox2, p-Sox2 level decreased significantly following CDK1 knockdown. Previous studies showed that the phosphorylation of Sox2 facilitated its nuclear localization and transcriptional activity [33]. Not surprisingly, CDK1 knockdown resulted in the translocation of Sox2 from nucleus to cytoplasm.

In summary, this study reveals the mechanism underlying CDK1- mediated phosphorylation and nuclear localization of Sox2 in the migration and invasion of esophageal carcinoma cells. Our results shed new light on CDK1-targeted therapy in the clinical treatment of esophageal carcinoma.

Acknowledgement

None applicable

Availability of Data and Materials

The data that support the findings of this study are available upon reasonable request from the corresponding author.

Fund

None applicable

Competing Interest

The authors declare that there are no competing interests.

Author’s Contribution

JS conceived the research and supervised the project. JS, TYL and FW conducted experimental work. FW performed data analysis. JS and TYL wrote the manuscript.

1. Watanabe M, Otake R, Kozuki R, Toihata T, Takahashi K, et al. (2020) Recent progress in multidisciplinary treatment for patients with esophageal cancer. Surg Today 50: 12-20.

Google Scholar, Crossref, Indexed at

2. Napier KJ, Scheerer M, Misra S (2014) Esophageal cancer:  A Review of epidemiology, pathogenesis, staging workup and treatment modalities. World J Gastrointest Oncol 6: 112-120.

Google Scholar, Crossref, Indexed at

3. Kato H, Nakajima M (2013) Treatments for esophageal cancer:  a review. Gen Thorac Cardiovasc Surg 61: 330-335.

Google Scholar, Crossref, Indexed at

4. Then EO, Lopez M, Saleem S, Gayam V, Sunkara T, et al. (2020) Esophageal Cancer:  An Updated Surveillance Epidemiology and End Results Database Analysis. World J Oncol 11: 55-64.

Google Scholar, Crossref, Indexed at

5. Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, et al. (1995) Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature 376: 313-320.

Google Scholar, Crossref, Indexed at

6. Pagano M (2004) Control of DNA synthesis and mitosis by the Skp2-p27-Cdk1/2 axis. Mol Cell 14: 414-416.

Google Scholar, Crossref, Indexed at

7. Odle RI, Walker SA, Oxley D, Kidger AM, Balmanno K, et al. (2020) An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis. Mol Cell 77: 228-240 e227.

Google Scholar, Crossref, Indexed at

8. Tong Y, Huang Y, Zhang Y, Zeng X, Yan M, et al. (2021) DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation, cell apoptosis, and cell migration. Cell Death Dis 12: 529.

Google Scholar, Crossref, Indexed at

9. Li L, Wang J, Hou J, Wu Z, Zhuang Y, et al. (2012) Cdk1 interplays with Oct4 to repress differentiation of embryonic stem cells into trophectoderm. FEBS Lett 586: 4100-4107.

Google Scholar, Crossref, Indexed at

10. Marlier Q, Jibassia F, Verteneuil S, Linden J, Kaldis P, et al. (2018) Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death. Cell Death Discov 4: 43.

Google Scholar, Crossref, Indexed at

11. Gregg T, Sdao SM, Dhillon RS, Rensvold JW, Lewandowski SL, et al. (2019) Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic beta-cells. J Biol Chem 294: 4656-4666.

Google Scholar, Crossref, Indexed at

12. Smith HL, Southgate H, Tweddle DA, Curtin NJ (2020) DNA damage checkpoint kinases in cancer. Expert Rev Mol Med 22: e2.

Google Scholar, Crossref, Indexed at

13. Bowles J, Schepers G, Koopman P (2000) Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev Biol 227:  239-255.

Google Scholar, Crossref, Indexed at

14. She ZY, Yang WX (2015) SOX family transcription factors involved in diverse cellular events during development. Eur J Cell Biol 94: 547-563.

Google Scholar, Crossref, Indexed at

15. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676.

Google Scholar, Crossref, Indexed at

16. Feng R, Wen J (2015) Overview of the roles of Sox2 in stem cell and development. Biol Chem 396: 883-891.

Google Scholar, Crossref, Indexed at

17. Juuri E, Jussila M, Seidel K, Holmes S, Wu P, et al. (2013) Sox2 marks epithelial competence to generate teeth in mammals and reptiles. Development 140: 1424-1432.

Google Scholar, Crossref, Indexed at

18. Johnston AP, Naska S, Jones K, Jinno H, Kaplan DR, et al. (2013) Sox2-mediated regulation of adult neural crest precursors and skin repair. Stem Cell Reports 1: 38-45.

Google Scholar, Crossref, Indexed at

19. Novak D, Huser L, Elton JJ, Umansky V, Altevogt P, et al. (2020) SOX2 in development and cancer biology. Semin Cancer Biol 67(Pt 1): 74-82.

Google Scholar, Crossref, Indexed at

20. Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis:  a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257.

Google Scholar, Crossref, Indexed at

21. Kim C, Kim B (2018) Anti-Cancer Natural Products and Their Bioactive Compounds Inducing ER Stress-Mediated Apoptosis:  A Review. Nutrients 10:  1021.

Google Scholar, Crossref, Indexed at

22. Chaudhary S, Islam Z, Mishra V, Rawat S, Ashraf GM, et al. 2019) Sox2:  A Regulatory Factor in Tumorigenesis and Metastasis. Curr Protein Pept Sci 20: 495-504.

Google Scholar, Crossref, Indexed at

23. Huang Z, Shen G, Gao J (2021) CDK1 promotes the stemness of lung cancer cells through interacting with Sox2. Clin Transl Oncol 23: 1743-1751.

Google Scholar, Crossref, Indexed at

24. Yang J, Liu X, Cao S, Dong X, Rao S, et al. (2020) Understanding Esophageal Cancer:  The Challenges and Opportunities for the Next Decade. Front Oncol 10: 1727.

Google Scholar, Crossref, Indexed at

25. Sun X, Niwa T, Ozawa S, Endo J, Hashimoto J (2022) Detecting lymph node metastasis of esophageal cancer on dual-energy computed tomography. Acta Radiol 63: 3-10.

Google Scholar, Crossref, Indexed at

26. Santamaria D, Barriere C, Cerqueira A, Hunt S, Tardy C, et al. (2007) Cdk1 is sufficient to drive the mammalian cell cycle. Nature 448: 811-815.

Google Scholar, Crossref, Indexed at

27. Zhang P, Kawakami H, Liu W, Zeng X, Strebhardt K, et al. (2018) Targeting CDK1 and MEK/ERK Overcomes Apoptotic Resistance in BRAF-Mutant Human Colorectal Cancer. Mol Cancer Res 16: 378-389.

Google Scholar, Crossref, Indexed at

28. Huang J, Chen P, Liu K, Liu J, Zhou B, et al. (2021) CDK1/2/5 inhibition overcomes IFNG-mediated adaptive immune resistance in pancreatic cancer. Gut 70: 890-899.

Google Scholar, Crossref, Indexed at

29. Liu R, Fan M, Candas D, Qin L, Zhang X, et al. (2015) CDK1-Mediated SIRT3 Activation Enhances Mitochondrial Function and Tumor Radioresistance. Mol Cancer Ther 14: 2090-2102.

Google Scholar, Crossref, Indexed at

30. Tang Q, Li W, Zheng X, Ren L, Liu J, et al. (2020) MELK is an oncogenic kinase essential for metastasis, mitotic progression, and programmed death in lung carcinoma. Signal Transduct Target Ther 5: 279.

Google Scholar, Crossref, Indexed at

31. Zhao S, Wang B, Ma Y, Kuang J, Liang J, et al. (2020) NUCKS1 Promotes Proliferation, Invasion and Migration of Non-Small Cell Lung Cancer by Upregulating CDK1 Expression. Cancer Manag Res 12: 13311-13323.

Google Scholar, Crossref, Indexed at

32. Liu S, Yang Y, Jiang S, Xu H, Tang N, et al. (2019) MiR-378a-5p Regulates Proliferation and Migration in Vascular Smooth Muscle Cell by Targeting CDK1. Front Genet 10: 22.

Google Scholar, Crossref, Indexed at

33. Jeong CH, Cho YY, Kim MO, Kim SH, Cho EJ, et al. (2010) Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cells. Stem Cells 28: 2141-2150.

Google Scholar, Crossref, Indexed at