Elevated TAB182 enhances the radioresistance of esophageal squamous cell carcinoma through G2-M checkpoint modulation

Abstract

Background: Radiotherapy is one of the main strategies for the treatment of esophageal squamous cell carcinoma (ESCC). However, treatment failure often occurs due to the emergence of radioresistance. In this study, we report a key regulator of radiation sensitivity, termed TAB182 that may become an ideal biomarker and therapeutic target to overcome radioresistance.

Materials and methods: By applying qRT-PCR and immunohistochemical staining, the expression of TAB182 was detected in patient tissues. We next assessed the influence of TAB182 downregulation to radiosensitivity using clonogenic survival assay and γ-H2A.X foci analysis in TE-1, TE-10, and radioresistant TE-1R cell lines after ionizing radiation. To unveil the mechanism underlying, TAB182 interacting proteins were identified by mass spectrometry following co-immunoprecipitation. Furthermore, flow cytometry and western blot assay were applied to validate the identified proteins.

Results: Our results demonstrated that the expression of TAB182 is higher in cancer tissues than normal tissues and elevated expression of TAB182 correlates with poor outcomes of postoperative radiotherapy. Downregulation of TAB182 sensitized cancer cells to ionizing radiation, particularly in radioresistant TE-1R cells that spontaneously overexpress TAB182. Mechanically, TAB182 interacts with FHL2 to induce G2-M arrest through wiring the CHK2/CDC25C/CDC2 signaling pathway. Finally, overexpression of shRNA-resistant TAB182 restored the checkpoint and radioresistance.

Conclusion: TAB182 potentiates the radioresistance of ESCC cells by modulating the G2-M checkpoint through its interaction with FHL2. Thus, TAB182 may become an ideal biomarker and therapeutic target of ESCC radiotherapy.

Keywords: FHL2; G2-M cell cycle checkpoint; TAB182; esophageal squamous cell carcinoma; radioresistance.

FIGURE 1

Expression of TAB182 in clinical samples. (A) The mRNA level of TAB182 in patient samples from the TCGA database and (B) from The Affiliated Suzhou Hospital of Nanjing Medical University. *p < 0.05, **p < 0.01. (C) Immunohistochemical staining of TAB182 in paraffin‐embedded ESCC tissues. (D) Kaplan–Meier survival analysis based on different IHC scores of TAB182

FIGURE 2

Radiosensitivity of ESCC cells after TAB182 downregulation. Western blot confirmed TAB182 downregulation in TE‐1 (A) and TE‐10 (D) cells. Clonogenic survival assay of TE‐1 (B) and TE‐10 cells (E) after ionizing radiation (n = 3). (C) and (F) Multitarget click model analysis after IR. Surviving fraction (SF) =1 − (1 − e^−DK) ^N. The quasi‐threshold dose (D _q) and the mean lethal dose (D ₀) were calculated: D _q = LN(N) *D ₀; D ₀ = 1/K. (G) Immunofluorescent staining of γ‐H2A.X foci in control group and TAB182 downregulated group in TE‐1 and TE‐10 cells at different time points after 4 Gy IR. *p < 0.05, ***p < 0.001

FIGURE 3

TAB182 affects radioresistance in radioresistant TE‐1R cells. (A) Scheme of radioresistant TE‐1R cells generation. (B) Western blot confirmed TAB182 upregulation in TE‐1R cells compared with parent TE‐1 cells and TAB182 expression was successfully downregulated by shRNA. (C) Clonogenic survival assay of TE‐1, TE‐1R, and TAB182 knockdown TE‐1R cells after ionizing radiation (n = 3). (D) Parameters of multitarget click model analysis after IR. (E) Immunofluorescent staining of γ‐H2A.X foci in the control group and TAB182 knockdown group at different time points after 4 Gy IR. *p < 0.05, ***p < 0.001 versus parent TE‐1 cells. ^# p < 0.05 versus control TE‐1R cells

FIGURE 4

TAB182 regulates the G2‐M checkpoint through interaction with FHL2. (A) Co‐IP confirmed the interaction between TAB182 and FHL2 in both TE‐1 and TE‐10 cells. Mouse or rabbit IgG was used as a negative control. (B) The proportion of mitotic cells after 8 h of 4 Gy IR were analyzed by flow cytometry using the mitotic marker phospho‐histone H3 (Ser 10) and PI double staining. The mitotic cells were marked in the box. Western blot was employed to study the activation of the G2‐M checkpoint in TE‐1 (C), TE‐10 (D), and TE‐1R (E) cells. Cells were irradiated by 4 Gy IR and were harvested at different time points. The expression of β‐actin was determined as a loading control. *p < 0.05, **p < 0.01

FIGURE 5

Overexpression of shRNA‐resistant TAB182 (synonymous mutation, sm) restores the G2‐M checkpoint in TAB182 downregulated TE‐1 cells. (A) Clonogenic survival assay after ionizing radiation (n = 3). (B) Parameters of multitarget click model analysis after IR. (C) The proportion of mitotic cells after 8 h of 4 Gy IR. (D) Western blot confirmed the restored G2‐M checkpoint after TAB182 overexpression. *p < 0.05