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www.nature.com/cddiscovery ARTICLE OPEN ERCC6L facilitates the progression of laryngeal squamous cell carcinoma by the binding of FOXM1 and KIF4A 1,4 2,4 1 1 1 3 Meng Cui , Yu Chang , Jiheng Wang , Junfu Wu , Gang Li and Jie Tan © The Author(s) 2023 The role of excision repair cross-complementation group 6-like (ERCC6L) has been reported in several cancers, but little is known about its expression and function in laryngeal squamous cell carcinoma (LSCC). In this study, the expression of ERCC6L in LSCC was determined by immunohistochemistry and its correlation with prognostic factors was analyzed. Furthermore, cytological functional validation elucidated the role and underlying mechanisms of ERCC6L dysregulation in LSCC. Our data revealed that ERCC6L expression was elevated in LSCC and it’s correlated with TNM stage. In addition, ERCC6L knockdown LSCC cells showed decreased proliferation and migration, increased apoptosis, and reactive oxygen species (ROS). Mechanically, overexpression of ERCC6L promoted nuclear translocation of FOXM1 to facilitate direct binding to the KIF4A promoter and upregulated KIF4A expression. Furthermore, KIF4A knockdown attenuated the role of ERCC6L overexpression in promoting proliferation, migration, and tumorigenesis of LSCC cells. In summary, ERCC6L promoted the binding of FOXM1 and KIF4A in LSCC cells to drive their progression, which may be a promising target for precision therapy in this disease. Cell Death Discovery (2023) 9:41 ; https://doi.org/10.1038/s41420-023-01314-3 INTRODUCTION hepatocellular carcinoma, and non-small cell lung adenocarci- Laryngeal squamous cell carcinoma (LSCC) is one of the most noma [9–13]. Thus, the above evidence demonstrated that the common subtypes of laryngeal cancer. It is aggressive and has expression of ERCC6L was dysregulated in many cancers, which relatively high morbidity and mortality . In addition, the onset may play a crucial role. However, the expression and role of of LSCC is insidious, with approximately 60% of patients ERCC6L in LSCC remains to be investigated. diagnosed in advanced (III or IV) stage . Despite substantial In this study, the expression of ERCC6L in LSCC was determined improvements in clinical management methods such as radio- by immunohistochemistry, and its correlation with prognostic therapy, chemotherapy, and surgery, the 5-year overall survival factors was analyzed. Furthermore, cytological functional valida- in LSCC has not improved signiﬁcantly over the past 20 years . tion elucidated the role and underlying mechanisms of ERCC6L In recent years, new targeted drugs have the hope of improving dysregulation in LSCC. the prognosis of LSCC patients in the metastatic environment . Therefore, the unraveling of the pathogenesis of LSCC is urgently needed to identify its diagnostic biomarkers and RESULTS effective new therapeutic targets. ERCC6L is abundantly highly expressed in LSCC Excision repair cross-complementation Group 6-Like (ERCC6L) Tissue microarrays composed of LSCC tissue (n = 36) and adjacent belongs to the SNF2 helicase-like ATPase family, also known as normal tissue (n = 33) were used for IHC staining to reveal PICH (Plk1 Interacting Checkpoint Helicase) . Deletion of differences in their ERCC6L expression. The typical IHC images ERCC6L in human or animal cells can lead to marked showed large areas of dark brown in tumor tissue, but little in chromosomal abnormalities, DNA damage, embryonic lethality, normal tissue (Fig. 1A). Quantitative results based on IHC staining apoptosis and TP53 activation . Therefore, ERCC6L is closely indicated that the scores in tumor tissues were signiﬁcantly higher related to cell mitosis and chromatin remodeling. In view of than those in normal tissues (P < 0.001, Fig. 1B). Scores higher than this, the correlation between ERCC6L and tumorigenesis has the median (4.5) were considered to ERCC6L high expression, been concerned and explored. For example, ERCC6L over- otherwise low ERCC6L expression. Statistics showed that ERCC6L expression is associated with disease progression and poor was highly expressed in 52.8% of tumor tissues, while all normal survival in patients with breast, renal, and hepatocellular tissues were low ERCC6L expression (P < 0.001, Table 1). Consis- carcinoma [7, 8]. In addition, ERCC6L silencing can lead to tently, the results of WB conﬁrmed the high expression of ERCC6L cycle arrest, proliferation inhibition, and reduced invasion of in LSCC (Fig. 1C). Moreover, ERCC6L was abundantly highly tumor cells in renal carcinoma, breast cancer, colorectal cancer, expressed in LSCC cells, such as TU686, TU212, and AMC‐HN‐8 Department of Head and Neck Thyroid, The Afﬁliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, 127 Dongming Road, Zhengzhou 450008, People’s Republic of China. Department of Oncology, The First Afﬁliated Hospital of Zhengzhou University, 1 Jianshe Dong Road, Zhengzhou 450007, People’s Republic of China. Department of Otorhinolaryngology Head and Neck Surgery, Peking University People’s Hospital, Peking University, 133 Fuchengmennei Dajie, Beijing 100044, People’s Republic of China. These authors contributed equally: Meng Cui, Yu Chang. email: email@example.com Received: 28 September 2022 Revised: 4 January 2023 Accepted: 9 January 2023 Ofﬁcial journal of CDDpress 1234567890();,: M. Cui et al. Fig. 1 Expression detection of ERCC6L in LSCC. A Representative IHC staining of ERCC6L expression in LSCC tissues and normal tissues. B Quantitative IHC results of ERCC6L expression in LSCC tissue (n = 36) and adjacent normal tissue (n = 33). C The mRNA expression of ERCC6L in LSCC cells such as TU686, TU212, and AMC‐HN‐8 was detected using qPCR. D The mRNA expression of ERCC6L was detected after shRNA targeting ERCC6L (shERCC6L) was transduced into AMC-HN-8 and TU212 cells. E The protein levels of ERCC6L in shERCC6L-mediated AMC-HN- 8 and TU212 cells was analyzed by western blotting. F The mRNA expression of ERCC6L was detected after shRNA targeting ERCC6L (shERCC6L) was transduced into AMC-HN-8 and TU212 cells. G The protein expression of ERCC6L was detected after shERCC6L was transduced into AMC-HN-8 and TU212 cells. The representative images were selected from at least three independent experiments. Data was shown as mean ± SD. *P < 0.05, ***P < 0.001. (Fig. 1D, E). The above results revealed that ERCC6L was generally of-function assays in vitro. As shown in Fig. 2A, the cell highly expressed in LSCC. Next, the correlation between ERCC6L proliferation ability of AMC-HN-8 and TU212 in the shERCC6 expression and clinicopathological features of LSCC patients was group was attenuated compared to shCtrl (P < 0.01). Not surpris- preliminarily analyzed using Mann-Whitney U. Of note, patholo- ingly, LSCC cells in the shERCC6 group formed fewer and smaller gical staging criteria for LSCC patients were based on the seventh cell clones compared to shCtrl (P < 0.05, Fig. 2B). Furthermore, ﬂow edition of the American Joint Committee on Cancer (AJCC) Cancer cytometry-based data indicated that AMC-HN-8 and TU212 cells Staging Manual. Our data indicated that the expression level of showed a stronger apoptosis rate after ERCC6 was stably knocked ERCC6L was signiﬁcantly positively correlated with T inﬁltrate and down (P < 0.05, Fig. 2C). In addition, the migration ability of LSCC TNM of LSCC (P < 0.001, Table 2). Consistently, the analysis results cells in shCtrl and shERCC6 groups was detected by Transwell and of Spearman correlation coefﬁcient further conﬁrmed the above wound healing assays, respectively. The number of crystal violet- data (P < 0.001, stained cells in the shERCC6 group was signiﬁcantly less than that Table 3). Taken together, the expression of ERCC6L was elevated of shCtrl, suggesting that knockdown of ERCC6 inhibited the in LSCC and correlated with poor prognostic factors, suggesting migration of LSCC cells (P < 0.01, Fig. 2D). Undoubtedly, wound that ERCC6L may be a diagnostic marker for this disease. healing experiments showed the same phenomenon, conﬁrming that ERCC6 may drive the migration of LSCC cells to some extent ERCC6L drives progression of LSCC cells (P < 0.05, Fig. 2E). Interestingly, ROS have been detected in nearly ERCC6L expression was detected after shRNA targeting ERCC6L all cancers, and they contribute to tumor development and (shERCC6L) was transduced into AMC-HN-8 and TU212 cells. The progression . The present study indicated that the ROS mRNA level of ERCC6L in the shERCC6L group was signiﬁcantly content in the shERCC6L group was increased compared to the lower than that in the shCtrl group in LSCC cells (P < 0.001, Fig. 1F). shCtrl group (P < 0.01, Fig. 2F). Moreover, RAD51 and γH2A.X are As expected, protein level of ERCC6L in shERCC6L-mediated AMC- involved in ROS generation and redox stress [15, 16]. Our results HN-8 and TU212 cells was decreased relative to shCtrl (Fig. 1G). indicated that knockdown of ERCC6L downregulated RAD51 and Subsequently, the effects of ERCC6L on LSCC cell proliferation, upregulated γH2A.X (Fig. 2G). Collectively, ERCC6L drove progres- clone formation, apoptosis, and migration were assessed by loss- sion of LSCC cells. Cell Death Discovery (2023) 9:41 M. Cui et al. Table 1. Expression patterns in laryngocarcinoma tissues and normal tissues was revealed by immunohistochemistry analysis. ERCC6L expression Tumor tissue Normal tissue p value Cases Percentage Cases Percentage Low 17 47.2% 33 100.0% <0.001 High 19 52.8% 0 0% with ERCC6 (Fig. 3B). In addition, the expression levels of KIF4A and ERCC6L were signiﬁcantly positively correlated (Fig. 3C). To further Table 2. Relationship between ERCC6L expression and tumor verify the effect of ERCC6L on KIF4A, knock downed ERCC6L in LSCC characteristics in patients with laryngocarcinoma. cells to detect the expression of KIF4A at mRNA and protein levels. Features No. of ERCC6L p value The results indicated that knockdown of ERCC6L downregulated the patients expression expression of KIF4A at mRNA and protein levels (P <0.001, Fig. 3D, E). On the other hand, KIF4A had been demonstrated to be a direct Low High transcriptional target of FOXM1, which binds to the KIF4A promoter All patients 36 17 19 . In view of the above results, we conducted further veriﬁcation. Age (years) 1.000 Our data showed that FOXM1 overexpression upregulated the <62 17 8 9 expression of KIF4A (P <0.001, Fig. 3F, G). Furthermore, the wild-type (WT) and mutant (MUT) KIF4A promoter regions were constructed in ≥62 19 9 10 HEK293T cells according to the predicted binding sites (2Kb Gender 0.778 upstream of the TSS site, chrX: 70288104-70420886) of FOXM1 and Male 35 16 19 KIF4A. The results showed that FOXM1 overexpression enhanced the Female 1 1 0 dual-luciferase activity of WT-KIF4A but not MUT-KIF4A, suggesting that FOXM1 directly associated with the KIF4A promoter (P <0.001, TInﬁltrate 0.001 Fig. 3H). In addition, we performed Ch-IP assay and veriﬁed that T1 8 8 0 ERCC6L overexpression can promote the binding of transcription T2 16 8 8 factor FOXM1 to the promoter region of KIF4A (P <0.01, Fig. 3I). More T3 7 1 6 interestingly, WB analysis showed that ERCC6L overexpression promoted nuclear translocation of FOXM1 (Fig. 3J). Collectively, our T4 5 0 5 ﬁndings supported the view that overexpression of ERCC6L Lymphatic 0.175 promoted nuclear translocation of FOXM1 to facilitate direct binding metastasis (N) to the KIF4A promoter and upregulated KIF4A expression. N0 23 13 10 N1 4 2 2 ERCC6L drives LSCC progression dependent on the presence of KIF4A N2 9 2 7 Given that the molecular mechanisms of ERCC6L and KIF4A had Maximum tumor 0.271 been initially revealed, their coordinated roles in cells required diameter further veriﬁcation. Firstly, the data indicated that KIF4A was ≤2cm 17 10 7 abundantly expressed in LSCC cells (Fig. 4A). Subsequently, >2 cm 19 7 12 ERCC6L overexpression and KIF4A knockdown were separately TNM 0.001 constructed in AMC-HN-8 and TU212 cells for loss/gain function assays. As shown in Fig. 4B, the cell proliferation ability of AMC- I7 7 0 HN-8 and TU212 in the shKIF4A group was attenuated (P < 0.01), II 7 5 2 but enhanced in the ERCC6L overexpression group compared to III 9 3 6 NC (P < 0.01). Moreover, KIF4A knockdown can alleviate the IV 13 2 11 promoting effect of ERCC6L overexpression on LSCC cell proliferation (P < 0.01, Fig. 4B). Not surprisingly, ERCC6L over- expression enhanced the migration of LSCC cells, and this effect was partially reversed by KIF4A knockdown (P < 0.05, Fig. 4C). Table 3. Relationship between ERCC6L expression and tumor Taken together, ERCC6L promoted LSCC progression may characteristics in patients with laryngocarcinoma. dependent on the presence of KIF4A. ERCC6L TInﬁltrate Spearman correlation coefﬁcient 0.672 KIF4A knockdown attenuates the role of ERCC6L overexpression in promoting tumorigenesis of LSCC cells Signiﬁcance (two tails) 0.000 In vivo mice xenografts were established to further validate the N 36 role of ERCC6L and KIF4A in regulating LSCC. The mice were TNM Spearman correlation coefﬁcient 0.638 subcutaneously inoculated with AMC-HN-8 cells and divided into Signiﬁcance (two tails) 0.000 the following four groups: NC, ERCC6L, shKIF4A and ERCC6L + shKIF4A. Mice were monitored for 28 days and data were N 36 collected on the intensity of ﬂuorescence expression of the xenografts. Figure 5A showed the ﬂuorescent pictures of the ERCC6L promotes the binding of FOXM1 and KIF4A in LSCC tumors of mice in each group on day 28. The ﬂuorescence cells intensity of tumor was ERCC6L group, ERCC6L + shKIF4A group Bioinformatics analysis revealed that KIF4A and ERCC6L were co- and shKIF4A group in order from high to bottom. Consistently, the expressed genes (Fig. 3A). Moreover, we used the string online same was true for the size of tumors taken from mice (Fig. 5B). On database (https://cn.string-db.org/)toanalyze andpredict the day 28, the mean tumor volume was 1125.08 mm in the ERCC6L proteins interacting with ERCC6L, and found that KIF4A interacted Cell Death Discovery (2023) 9:41 M. Cui et al. Fig. 2 ERCC6L knockdown inhibits LSCC progression in vitro. A Celigo cell counting assay was employed to show the effects of ERCC6L on cell proliferation of AMC-HN-8 and TU212 cells. B Colony forming ability of the shERCC6L-mediated AMC-HN-8 and TU212 cells was detected. C Flow cytometry was performed to detect cell apoptosis of AMC-HN-8 and TU212 cells with or without ERCC6L knockdown. The shERCC6L- mediated LSCC cell migration ability was accessed by (D) Transwell assay and (E) wound-healing assay. F The ROS content in the shCtrl group and shERCC6L group was analyzed by Reactive Oxygen Species (ROS) assay. G The protein levels of RAD51 and γH2A.X in AMC-HN-8 and TU212 cells was analyzed by western blotting. The representative images were selected from at least 3 independent experiments. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. 3 3 group, only 305.02 mm in the shKIF4A group, and 589.77 mm in support for clinical treatment. On the other hand, the role of the ERCC6L + shKIF4A group (Fig. 5C). As expected, tumor weight ERCC6L has been reported in several cancers, but little is known showed the same trend (Fig. 5D). In addition, IHC staining was about its expression and function in LSCC. In this study, we found performed to determine the expression of ERCC6L, KIF4A, Ki67, that ERCC6L was signiﬁcantly upregulated in LSCC and was RAD51 and γH2A.X (Fig. 5E). These results are consistent with positively associated with clinicopathological features, such as T in vitro data, suggesting that KIF4A knockdown attenuated the inﬁltrate and TNM. Taken together, our ﬁndings suggested that role of ERCC6L overexpression in promoting tumorigenesis of ERCC6L may be a diagnostic marker for this disease. LSCC cells. However, the exact mechanism by which ERCC6L regulated tumor cell development and progression in LSCC remained unclear. The present study demonstrated that ERCC6L knockdown DISCUSSION LSCC cells showed decreased proliferation and migration, and LSCC has relatively high morbidity and mortality, and current increased apoptosis. Interestingly, ROS have been detected in clinical treatment options are limited . Therefore, the elucida- nearly all cancers, and they contribute to tumor development and tion of the molecular mechanism of LSCC progression and the progression . Moreover, RAD51 and γH2A.X are involved in identiﬁcation of potential therapeutic targets provide theoretical ROS generation and redox stress [15, 16]. Our results indicated Cell Death Discovery (2023) 9:41 M. Cui et al. Fig. 3 ERCC6L promotes the binding of FOXM1 and KIF4A in LSCC cells. A Bioinformatics analysis revealed that KIF4A and ERCC6L were co- expressed genes. B We used the string online database (https://cn.string-db.org/) to analyze and predict the proteins interacting with ERCC6L, and found that KIF4A interacted with ERCC. C The expression levels of KIF4A and ERCC6L were signiﬁcantly positively correlated. D, E The expression of ERCC6L and KIF4A at mRNA and protein levels in shERCC6L-mediated AMC-HN-8 and TU212 cells was analyzed. F, G The expression of KIF4A at mRNA and protein levels in FOXM1-mediated AMC-HN-8 cells was analyzed. H The wild-type (WT) and mutant (MUT) KIF4A promoter regions were constructed in HEK293T cells and the dual-luciferase activity of KIF4A was analyzed. I We performed Ch-IP assay and veriﬁed that ERCC6L overexpression can promote the binding of transcription factor FOXM1 to the promoter region of KIF4A. J The protein expression of FOXM1 was detected in the cytoplasm and nucleus of ERCC6L-overexpressing AMC-HN-8 cells. WB analysis showed that ERCC6L overexpression promoted nuclear translocation of FOXM1. The representative images were selected from at least three independent experiments. Data was shown as mean ± SD. **P < 0.01, ***P < 0.001. that knockdown of ERCC6L increased ROS content, upregulated we performed dual-luciferase assay and Ch-IP assay, verifying that γH2A.X and downregulated RAD51 in LSCC cells. These results ERCC6L overexpression can promote the binding of transcription suggested that ERCC6L was involved in ROS generation in LSCC factor FOXM1 to the promoter region of KIF4A. More interestingly, cells and contributed to the malignant progression of this it has been reported that FOXM1d is located in the cytoplasm and tumor cells. does not directly control transcription . In this study, our data Bioinformatics analysis revealed that KIF4A and ERCC6L were showed that ERCC6L overexpression promoted nuclear transloca- co-expressed genes. In addition, the expression levels of KIF4A tion of FOXM1. Collectively, our ﬁndings supported the view that and ERCC6L were signiﬁcantly positively correlated. KIF4A had overexpression of ERCC6L promoted nuclear translocation of been demonstrated to be a direct transcriptional target of FOXM1 to facilitate direct binding to the KIF4A promoter and Forkhead Box M1 (FOXM1), which binds to the KIF4A promoter upregulated KIF4A expression. . FOXM1 is an important regulator of many biological KIF4A is overexpressed in most tumors, but also low in a processes, and dysregulation of FOXM1 leads to carcinogenesis minority . Yang et al., found that KIF4A is abnormally highly and tumor progression . In view of the above results, we expressed in human clear cell renal cell carcinoma tissues and conducted further veriﬁcation. Our data showed that FOXM1 can act as a tumor-inducing gene . Zhu et al., proposed that overexpression upregulated the expression of KIF4A. In addition, enhanced KIF4A expression in osteosarcoma predicts poor Cell Death Discovery (2023) 9:41 M. Cui et al. Fig. 4 ERCC6L drives LSCC progression dependent on the presence of KIF4A. A The mRNA expression of KIF4A in LSCC cells such as TU686, TU212, and AMC‐HN‐8 was detected using qPCR. B, C ERCC6L overexpression and KIF4A knockdown were separately constructed in AMC-HN- 8 and TU212 cells for loss/gain function assays. The cells models were subjected to the (B) MTT assay and (C) wound-healing assay. The representative images were selected from at least three independent experiments. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. prognosis and promotes tumor growth by activating the MAPK (50–74%), 4 (75–100%)) and staining intensity scores (scored as 0: signal less color, 1: brown, 2: light yellow, 3: dark brown). pathway . Cao et al., demonstrated that KIF4A plays an important role in the progression of CRPC and is a key determinant of CRPC resistance to endocrine therapy . In Cell culture this study, the results indicated that KIF4A knockdown Human laryngeal cancer cells (TU686, TU212 and AMC‐HN‐8) were attenuated the role of ERCC6L overexpression in promoting obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were kept in a humidiﬁed incubator at 37 °C under an proliferation, migration, and tumorigenesis of LSCC cells. atmosphere with 5% CO in air. The cells were cultured in Dulbecco’s Therefore, our data together with previous studies further modiﬁed Eagle’s medium (DMEM, HyClone) containing 10% fetal bovine conﬁrmed the critical role of FOXM1c in cancer cell progression. serum (FBS, Invitrogen). Of note, all cells were validated by STR. In summary, ERCC6L promoted the binding of FOXM1 and KIF4A in LSCC cells to drive their progression, which may be a Lentiviral transduction promising target for precision therapy in this disease. The small hairpin RNA sequences targeting ERCC6L (shERCC6: 5′-CTGCCCAAA- GAGGGTGAGAAA-3′,5’-TAAAGAAGACGTACAGAAGAA-3′,5′-CAACTAAAGGAT- GATGAGATT-3′), KIF4A (shKIF4A 5′-TATACTGCAGAGCAAGAGAAT-3′,5′-ATTG MATERIALS AND METHODS ATACTGCGGTGGAGCAA-3′,5′-CTTACTGAAGTGCGTGGTCAA-3′)and Scramble Tissue microarray and immunohistochemistry (IHC) staining sequence (negative control, shCtrl: 5′-TTCTCCGAACGTGTCACGT-3′)were This study was approved by the Research Ethics Committee of Afﬁliated ligated into BR-V-108 lentiviral vector (Shanghai Yiberui Biomedical Technol- Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, and ogy), respectively. The packaged lentivirus was transduced into AMC-HN-8 written informed consent was obtained from each participant. LSCC and TU212 cells using Lipofectamine 2000 (Invitrogen) in accordance with the tissue (n = 36) and adjacent normal tissue (n = 33) constitute a tissue manufacturer’s protocol. After cultured for 72 h at 37 °C, green ﬂuorescent microarray for IHC staining. In detail, the tissue microarray was soaked protein (GFP) expression was detected under a microscope and stably in xylene and alcohol in turn for dewaxing and rehydration. After that, transduced cell lines were selected with puromycin . the tissue microarray was boiled in sodium citrate buffer (pH = 6.0) for antigen repair. At room temperature, they were incubated with 5% animal serum in PBST for 30 min, then incubated with the primary Quantitative real‐time PCR (qRT‐PCR) antibody (anti-ERCC6L, 1:200, Abcam, USA) for another 2 h. Tissue The cells total RNA was extracted using Trizol according to the operating microarray and secondary antibody (goat anti-rabbit IgG, 1:400, instructions (Sigma, Cat. No. T9424-100m). Hiscript QRT supermix (Vazyme, Beyotime, USA) were incubated at 4 °C for 2 h, washed with PBST, and Cat. No. R123-01) was used for reverse transcription of RNA to obtain cDNA. then stained with DAB and hematoxylin, respectively. Finally, the tissues SYBR Green mastermixs (Vazyme, Cat. No. Q111-02), target primers, and cDNA were observed with microscopic and IHC scores were determined by were used to conﬁgure the reaction system of qPCR. The relative mRNA staining percentage scores (classiﬁed as:1(1–24%), 2 (25–49%), 3 expression level of the target in each sample relative to the control group was Cell Death Discovery (2023) 9:41 M. Cui et al. Fig. 5 KIF4A knockdown attenuates the role of ERCC6L overexpression in promoting tumorigenesis of LSCC cells. A In vivo imaging was performed to evaluate the tumor burden in mice of NC, ERCC6L, shKIF4A, and ERCC6L + shKIF4A groups post tumor-inoculation. B–D Mice were monitored for 28 days and data were collected on the volume and weight of the xenografts. E IHC staining was performed to determine the expression of ERCC6L, KIF4A, Ki67, RAD51, and γH2A.X. Data was shown as mean ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. −△Ct reﬂected by 2 . The upstream and downstream primer sequences for 5′-GTAAAGGCACAAGTCGTATCCA-3′; The upstream and downstream primer amplifying ERCC6L are shown below: 5′-AAAAGTCAAGCAACCCAGAGG-3′ and sequences for amplifying KIF4A are shown below: 5′- CTGCCAACAAGCGTCT Cell Death Discovery (2023) 9:41 M. Cui et al. CAAGG-3′ and 5′-CCTTCCATTCCACGGCTCTGA-3′; The upstream and down- Reactive oxygen species (ROS) assay stream primer sequences of the internal reference GAPDH are shown below: ROS was detected using the ﬂuorescent probe DCFH-DA following the 5’-TGACTTCAACAGCGACACCCA-3′ and 5′-CACCCTGTTGCTGTAGCCAAA-3′. steps provided in the kit (Beyotime Biotechnology). AMC-HN-8 and TU212 cells were suspended in DCFH-DA diluted with serum-free medium (1:1000) and incubated in a cell incubator at 37 °C for 20 min. Probes and Western blotting (WB) analysis cells were mixed by shaking them every 3–5 min. Subsequently, cells were AMC-HN-8 and TU212 cells protein was extracted with 1× cell lysis washed three times with serum-free cell culture medium to sufﬁciently buffer (Promega, USA) and protein concentrations were quantiﬁed by remove excess DCFH-DA and detected by ﬂuorescence microplate reader BCA Protein Assay Kit (Pierce, USA). The 20 µg proteins in each group at OD488. were separated by 10% SDS-PAGE (Invitrogen) and then transferred onto PVDF membranes. After the PVDF membrane was sealed by the blocking solution (TBST solution containing 5% skim milk) at room Dual-luciferase assay temperature for 1 h, incubated with the primary antibody (anti-ERCC6L, Promoter deletion was analyzed using a dual-luciferase reporting system 1:1000, Abcam, USA; anti-KIF4A, 1:1000, Abcam, USA; anti-RAD51, as previously described . The KIF14A promoter region fragment (2Kb 1:1000, Proteintech, China; anti-γH2A.X, 1:1000, Abcam, USA; GAPDH, upstream of the TSS site, chrX: 70288104-70420886) was ampliﬁed and 1:30000, Proteintech, China) overnight at 4 °C. After the membrane was cloned into the luciferase reporter vector GL002 (Promega Madison, USA), washed by TBST, the secondary antibody (goat anti-rabbit IgG, 1:3000, designated as GL002-KIF14A. Mutant construct KIF14A-MUT was generated Beyotime, USA) was added and incubated at room temperature for 2 h. by site-directed mutagenesis and KIF14A-WT as negative control. Finally, the blots were visualized using enhanced chemiluminescence According to the instructions of Promega dual-luciferase system (Cat. No. (ECL) (Amersham). E2940, Madison, USA), Fireﬂy luciferase value and Renilla luciferase signals were determined. Celigo cell counting assay AMC-HN-8 and TU212 cells were laid with 96-well plates at a density of Chromatin immunoprecipitation (ChIP)-qPCR assay 2000 cell/well. From the second day after laying, the Celigo reading board The ChIP-qPCR assay was performed as described previously . AMC-HN- was tested once a day for 5 days continuously. The data were statistically 8 cells with ERCC6L overexpression were cross-linked with formaldehyde, plotted, and the cell proliferation curve for 5 days was drawn. lysed in the SDS buffer and sheared mechanically by sonication to fragment the DNA. Protein–DNA complexes were precipitated with control goat anti-rabbit IgG (Sigma, USA), Histone H3 (D2B12) XP Rabbit mAb MTT assay (CST), and anti-FOXM1 (1:100, Proteintech), respectively. After separating AMC-HN-8 and TU212 cells were laid with 96-well plates at a density of the complex from the antibody, using the primers speciﬁc for KIF14A 2000 cell/well. From the second day after laying, 20 μL 5 mg/mL MTT promoter and SYBR premix (Vazyme) to detect the eluted DNA fragment. (Genview, China, Cat. No. JT343) was added into the well 4 h before the The primer sequence for KIF14A as follows: 5′- CTGCCAACAAGCGTCT- end of culture. After 4 h, the culture medium was completely absorbed, CAAGG-3′ and 5′-CCTTCCATTCCACGGCTCTGA-3′. and 100 μL DMSO solution was added. The OD value of 450/570 nm was detected by Microplate Reader (Tecan inﬁnite). Animal xenograft model The animal experiment was approved and performed according to the Colony formation assay guidelines of Afﬁliated Cancer Hospital of Zhengzhou University & Henan AMC-HN-8 and TU212 cells were cultured in 6-well plates at a density of Cancer Hospital. Male BALB/c-nu mice (4-weeks old, n = 16) were 800 cell/well for 14 days. Notably, a single cell lasts for more than 6 purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, generations in vitro, and the cell population composed of its progeny is China). After 1 week of adaptive feeding, mice were subcutaneously called a clone. At this point each clone contained more than 50 cells, 7 inoculated with 1 × 10 AMC-HN-8 cells to establish a xenograft model and ranging in size from 0.3 to 1.0 mm. Subsequently, 4% paraformaldehyde of divided into the following four groups: NC (n = 4), ERCC6L (n = 4), shKIF4A 1 mL was added to ﬁx cells for 30 min. Later, 500 μL GIEMSA staining (n = 4) and ERCC6L + shKIF4A (n = 4). After a week, data on mouse body solution was used to dye the cells for 20 min and photographed the cell weight and tumor size (tumor volume: π/6 × length × width× width) were clones. collected every 5 days. After continuous feeding for 28 days, the mice were anesthetized by intraperitoneal injection of 0.7% pentobarbital sodium (10 μL/g) and under the IVIS Spectrum (Perkin Elmer) for ﬂuorescence Cell apoptosis analysis by ﬂow cytometry imaging observation. Subsequently, the mice were sacriﬁced by cervical AMC-HN-8 and TU212 cells were inoculated in 6-well plate (2 ml/well) and vertebrae and the tumors were removed for tissue sections. Tissue sections cultured continuously for 5 days. The cells were then centrifuged for 5 min, were stained by IHC to reveal protein expression as previously described. cell precipitates were washed by D-Hanks (pH = 7.2–7.4) precooled at 4 °C. Antibodies were used as follows: primary antibody (anti-ERCC6L, 1:200, After the cell precipitation was resuscitated by 200 μL 1 × binding buffer, Abcam, USA; anti-KIF4A, 1:200, Abcam, USA; anti-KI67, 1:200, Abcam, USA; 10 μL Annexin V-APC was added for cell staining. Cell apoptosis rate was anti-RAD51, 1:200, Proteintech Group, USA; anti-YH2AX, 1:200, Abcam, calculated by ﬂow cytometry in 3 randomly selected visual ﬁelds. USA) and secondary antibody IgG (1:400, Abcam, USA). Transwell assay Statistical analysis AMC-HN-8 and TU212 cells were inoculated on well-hydrated chamber The data came from three separate experiments, expressed as mean ± SD. (3422 corning) at a density of 50,000 cell/well. The inner chamber contains The signiﬁcance differences between groups were determined using the 100 μL serum-free medium and the outer chamber contains 600 μL two-tailed Student’s t test or One-way ANOVA analysis. Statistical analyses containing 30% FBS. 100 μL of cell suspension was diluted in serum-free and graphs were performed by GraphPad Software 8.0 and P value < 0.05 medium and then added to each compartment for 24 h. The migrated cells as statistically signiﬁcant. were ﬁxed by 4% formaldehyde and photographed after Giemsa staining to analyze the cell migration ability. DATA AVAILABILITY Wound-healing assay Data will be made available on request. AMC-HN-8 and TU212 cells were inoculated on 96-well plate (100 μL/well) at a density of 50,000 cell/well. The next day, the low concentration serum medium was replaced, and a scratch meter was used to aim at the center REFERENCES of the lower end of the 96-well plate and nudge upward to form scratches. Cellomics (Thermo) was used to scan the plate and analyze the migration 1. Xia C, Dong X, Li H, Cao M, Sun D, He S, et al. Cancer statistics in China and United area when they were continuously cultured for 0, 24, and 48 h, States, 2022: proﬁles, trends, and determinants. Chin Med J. 2022;135:584–90. respectively. The migration rate was calculated based on the cell migration 2. Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. 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