TBK1 inhibitor amlexanox exerts anti-cancer effects against endometrial cancer by regulating AKT/NF-κB signaling

Abstract

Endometrial cancer, a common gynecological malignancy, poses significant clinical challenges, particularly in advanced or recurrent cases. TANK-binding kinase 1 (TBK1), a serine/threonine kinase, plays crucial roles in inflammation and immunity by activating nuclear factor (NF)-κB and interferon regulatory factor 3. However, its specific roles in endometrial cancer remain unknown. In this study, we aimed to investigate the anti-cancer effects and underlying mechanisms of amlexanox, a TBK1 inhibitor, against endometrial cancer. The main genetic mutations in TBK1 were found to be mRNA downregulation and missense mutations. Kaplan-Meier plotter analysis revealed that low TBK1 expression was associated with a good prognosis in patients with uterine corpus endometrial carcinoma (UCEC). In vitro experiments demonstrated that TBK1 knockdown or amlexanox significantly inhibited the proliferation, cell cycle progression, and migration of endometrial cancer cells. Furthermore, the inhibitory effects of targeting TBK1 on cancer cell proliferation and migration were mediated by the protein kinase B (AKT)/NF-κB signaling pathway. Xenograft experiments revealed that both amlexanox treatment and TBK1 knockdown effectively suppressed the tumor growth. Overall, this study highlights the potent anti-cancer effects of amlexanox against endometrial cancer by modulating AKT/NF-κB signaling, thus providing a new avenue for the development of novel TBK1-targeting therapeutic strategies for UCEC.

Keywords: NF-κB; TBK1; endometrial cancer; prognosis; shRNA; xenograft.

Figure 1

Genetic mutations in TANK-binding kinase 1 (TBK1) and their association with endometrial cancer prognosis. (A) Oncoprint analysis revealed the proportion and distribution of samples with different types of alterations in TBK1. (B and C) Kaplan-Meier curves of the overall and disease-free survival of patients with endometrial cancer in TBK1-altered and TBK1-unaltered groups. (D) Kaplan-Meier curves of overall survival according to TBK1 expression level in patients with endometrial cancer.

Figure 2

TBK1 knockdown suppresses proliferation and promotes cell cycle arrest in endometrial cancer cells. (A) Immunoblots of p-TBK1 and TBK1 in the cell lysates of three endometrial cancer cell lines. (B) Immunoblots of TBK1 in the cell lysates of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells. Band intensities were quantified and normalized to the control levels. (C) Water-soluble tetrazolium salt (WST)-8 assay of HEC-1A and Ishikawa cells infected with sh-TBK1 or sh-Luc lentiviruses at 24 h, 48 h, and 72 h. (D) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of MKI67 mRNA levels in HEC-1A and Ishikawa cells infected with sh-TBK1 or sh-Luc lentiviruses. (E) Cell cycle analysis of HEC-1A and Ishikawa cells infected with sh-TBK1 or sh-Luc lentiviruses via propidium iodide (PI) staining followed by flow cytometry. (F) Immunoblots of TBK1 in the cell lysates of TBK1 knockdown cells transfected with GFP or TBK1-Flag. (G) WST-8 assay of TBK1 knockdown cells transfected with GFP or TBK1-Flag. Data are represented as the mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

Amlexanox suppresses proliferation and promotes cell cycle arrest in endometrial cancer cells. (A) WST-8 assay of HEC-1A and Ishikawa cells treated with the indicated concentrations of amlexanox (25-400 μM) for 24 h. (B) WST-8 assay of HEC-1A and Ishikawa cells treated with 100 μM amlexanox for 24 h, 48 h, and 72 h. (C) RT-qPCR analysis of MKI67 mRNA levels in HEC-1A and Ishikawa cells treated with 100 μM amlexanox for 24 h. (D) Colony formation assays of HEC-1A and Ishikawa cells treated with the indicated concentrations of amlexanox (50-200 μM). (E) Quantification of the colony formation assay shown in (D). (F) Cell cycle analysis of HEC-1A and Ishikawa cells treated with 100 μM amlexanox for 24 h via PI staining followed by flow cytometry. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

TBK1 expression is correlated with the epithelial-mesenchymal transition (EMT)-related marker expression and cell migration in endometrial cancer. (A) Spearman correlation analysis between TBK1 and EMT-related gene expression in The Cancer Genomic Atlas (TCGA) endometrial cancer database. (B) Immunoblots of N-cadherin, vimentin, snail, and TBK1 in the cell lysates of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells treated with 10 ng/mL transforming growth factor (TGF)-β1 or vehicle. Band intensities were quantified and normalized to the control levels. (C) Wound-healing assay of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or vehicle. Images were taken 24 h after the scratch wound. (D) Quantification of cell migration expressed as a percentage of control values. (E) Transwell migration assay of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or vehicle. Images were taken after 24 h under a light microscope. Scale bars, 200 μm. (F) Quantification of cell migration expressed as a percentage of control values. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Amlexanox inhibits the migration of endometrial cancer cells. (A) Immunoblots of N-cadherin, vimentin, and snail in the cell lysates of HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or 100 μM amlexanox for 36 h. Band intensities were quantified and normalized to the control levels. (B) Wound-healing assay of HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or 100 μM amlexanox. Images were taken 24 h after the scratch wound. (C) Quantification of cell migration expressed as a percentage of control values. (D) Transwell migration assay of HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or 100 μM amlexanox. Images were taken after 24 h under a light microscope. Scale bars, 200 μm. (E) Quantification of cell migration expressed as a percentage of control values. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6

Targeting TBK1 attenuates nuclear factor (NF)-κB activation by inhibiting the protein kinase B (AKT) pathway in endometrial cancer cells. (A, B) Spearman correlation analysis between TBK1 and AKT- or NF-κB-related gene expression in TCGA endometrial cancer database. (C) Immunoblots of p-AKT, AKT, p-NF-κB, and NF-κB in the cell lysates of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells. (D) Immunoblots of p-AKT, AKT, p-NF-κB, and NF-κB in the cell lysates of HEC-1A and Ishikawa cells treated with amlexanox (50-200 μM) for 18 h. (E) Immunofluorescence staining for NF-κB in lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells. (F) Immunofluorescence staining for NF-κB in HEC-1A and Ishikawa cells treated with 100 μM amlexanox for 24 h. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 μm. (G) Immunoblots of p-NF-κB and NF-κB in the cell lysates of HEC-1A and Ishikawa cells treated with MK-2206 (25-100 μM) for 4 h. Band intensities were quantified and normalized to the control levels. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7

Targeting TBK1 inhibits the proliferation and migration of endometrial cancer cells via the AKT/NF-κB pathway. (A) WST-8 assay of HEC-1A and Ishikawa cells treated with 100 μM MK-2206 or 100 μM amlexanox for 24 h. (B) Immunoblots of N-cadherin and snail in the cell lysates of HEC-1A and Ishikawa cells pretreated with 100 μM MK-2206 or 100 μM amlexanox for 36 h in the presence of 10 ng/mL TGF-β1. Band intensities were quantified and normalized to the control levels. (C) Wound-healing assay of HEC-1A and Ishikawa cells pretreated with 100 μM MK-2206 or 100 μM amlexanox in the presence of 10 ng/mL TGF-β1. Images were taken 24 and 48 h after the scratch wound. (D) Quantification of cell migration expressed as a percentage of control values. (E) Transwell migration assay of HEC-1A and Ishikawa cells pretreated with 100 μM MK-2206 or 100 μM amlexanox in the presence of 10 ng/mL TGF-β1. Images were taken after 24 h under a light microscope. Scale bars, 200 μm. (F) Quantification of cell migration expressed as a percentage of control values. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 8

Targeting TBK1 inhibits tumor growth in vivo. (A) Representative images of subcutaneous xenograft tumors in nude mice treated with 5 mg/kg amlexanox or vehicle. Red arrows indicate the xenograft tumor. (B) Tumor growth curve in each group. (C) Tumor weights in each group. (D, E) RT-qPCR analysis of MKI67, CDKN1A, and CDKN1B mRNA levels in the xenograft tissues of each group. (F, G) Immunoblots of N-cadherin, vimentin, snail, p-AKT, AKT, p-NF-κB, and NF-κB in the xenograft tumor lysates of nude mice of each group. Band intensities were quantified and normalized to the control levels. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. (H) Schematic diagram showing the anti-cancer mechanism of TBK1 inhibition, leading to decreased tumor growth, migration, and EMT in endometrial cancer cells.