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. 2020 Jul 6:14:2613-2622.
doi: 10.2147/DDDT.S245267. eCollection 2020.

The Expression and Therapeutic Potential of Checkpoint Kinase 2 in Laryngeal Squamous Cell Carcinoma

Ying Tian  1 , Yan Wang  1 , Shan Xu  1 , Chao Guan  1 , Qingfu Zhang  2 , Wei Li  1
Affiliations

The Expression and Therapeutic Potential of Checkpoint Kinase 2 in Laryngeal Squamous Cell Carcinoma

Ying Tian et al. Drug Des Devel Ther. .

Abstract

Introduction: Laryngeal squamous cell carcinoma (LSCC) is the most common histological subtype of laryngeal cancer. The involved molecular mechanisms and suitable therapeutic targets for LSCC still need to be further investigated. Checkpoint kinase 2 (CHK2) participates in several cellular physiology pathways and plays a role in tumor progression. However, the roles of CHK2 in LSCC remain unclear.

Methods: mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) database, and bioinformatic analysis was performed. Western blot and immunohistochemical analyses were conducted to detect protein expression. MTS assays were performed to examine cell growth of LSCC-derived cell lines.

Results: In the present study, we found that both active form of CHK2 and total CHK2 protein expressions were up-regulated in LSCC tissues. Positive expression of CHK2 was closely associated with advanced clinical features and poor prognosis. Moreover, potential CHK2-involving bioprocesses and signaling pathways were analyzed. In addition, repressed proliferation of LSCC cells was induced by CHK2 inhibitor.

Discussion: Taken together, our findings elucidated that CHK2 may act as an oncogenic factor in LSCC, suggesting a potential target for clinical treatment.

Keywords: BML-277; checkpoint kinase 2; laryngeal squamous cell carcinoma; tumor growth.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
CHK2 is overexpressed in LSCC. (A) The mRNA expression of CHEK2 in HNSC tissues and control normal tissues in TCGA database. *P<0.05. (B) Western blot analysis for CHK2 expression in 19 paired LSCC tissues and matched adjacent nontumor tissues. N, adjacent nontumor tissues; Ca, LSCC tissues. GAPDH was used as the loading control. (C) Data from (B) were quantified by densitometry, with GAPDH as the reference. Student’s t-test was used for statistical significance. Gray lines indicate the samples with higher expression of p-CHK2 or CHK2 in cancer tissues, and black lines indicate the samples with lower expression of CHK2 in cancer tissues. *P<0.05.
Figure 2
Figure 2
CHK2 expression is associated with poor prognosis in LSCC patients. (A) Representative images of CHK2 expression in LSCC tissues and adjacent noncancerous tissues. NE, normal epithelium; BC, basal cells; LSCC, laryngeal squamous cell carcinoma. Scale bar, 200 µm. (B) Kaplan–Meier curve analysis of differential expression of CHK2 mRNA for the overall survival in HNSC patients. FPKM cutoff of 2.11 was chosen to define high expression or low expression of CHK2. (C) Kaplan–Meier curve analysis of differential expression of CHK2 for the overall survival in LSCC patients.
Figure 3
Figure 3
Functional analysis of genes correlated with CHK2 in squamous cell carcinoma. (A) Significant bioprocesses and pathways represented the functions of genes positively correlated with CHK2. The horizontal axis and the intensity of color represent corrected P-value. The number of enriched genes is shown behind the columns. (B) Significant bioprocesses and pathways represented the functions of genes negatively correlated with CHK2. The horizontal axis and the intensity of color represent corrected P-value. The number of enriched genes is shown behind the columns.
Figure 4
Figure 4
CHK2 inhibition represses LSCC cell proliferation. (A) Tu212 cells were separately transfected with siCHK2 or control siRNA (siCtrl). After 48 hours, cells were harvested, and Western blot analysis was performed with the indicated antibodies. GAPDH was used as internal control. (B) Tu212 cells were separately transfected with siCHK2 or siCtrl. Relative cell numbers were determined using MTS assay by absorbance at 490 nm at indicated times. P-value refers to two-sided Student’s t-test. *P<0.05. (C) Tu212 cells were treated with indicated doses of BML-277 or vehicle. After 24 hours, cells were harvested, and Western blot analysis was performed with the indicated antibodies. GAPDH was used as internal control. (D and F) Tu212 cells or HEP-2 cells were treated with indicated doses of BML-277 or DMSO. After 24 hours, relative cell numbers were determined using MTS assay by absorbance at 490 nm. Data are represented as mean±SD from three independent experiments. P-value refers to two-sided Student’s t-test. *P<0.05. (E and G) Tu212 cells or HEP-2 cells were treated with BML-277 (5 μM) or DMSO. Relative cell numbers were determined at various times using MTS assay by absorbance at 490 nm. Data are represented as mean±SD from three independent experiments. P-value refers to two-sided Student’s t-test. *P<0.05.
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Grants and funding

This work was supported by educational commission of Liaoning Province in China (Numbers: QN2019009).