Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway

Abstract

Background: Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC.

Methods: We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings.

Results: Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway.

Conclusions: Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

Keywords: N‐acetyltransferase 10; Remodelin; epithelial‐to‐mesenchymal transition; invasion and metastasis; lung cancer; mouse xenograft; patients‐derived organoids; proliferation; tumorigenicity.

FIGURE 1

High NAT10 Expression in NSCLC. (A) Representative immunohistochemical images depicting NAT10 expression in NSCLC tissues (10 cases) and normal lung tissues (10 cases) from the Tissue Microarray (TMA). (B) Correlation between high NAT10 expression and Overall Survival (OS) in NSCLC patients. (C) Quantitative analysis of NAT10 expression levels in human bronchial epithelial cells (BECs) and NSCLC cancer cell lines (A549 and NCI‐H1975) conducted via qRT‐PCR. β‐Actin was used as the reference gene for normalization. (D) Western blot analysis illustrating NAT10 expression levels in BECs and NSCLC cell lines. GAPDH was utilized as a loading control in the western blotting assays. Values are depicted as the mean ± SD of three independent experiments.

FIGURE 2

Modulation of NAT10 Expression in Lung Cancer Cell Lines. (A) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis illustrating NAT10 expression levels in NSCLC cell lines following transfection with NAT10siRNA *p < 0.05 versus Control. (B) Western blotting showing NAT10 expression levels in NSCLC cell lines post‐transfection with NAT10siRNA. *p < 0.05 versus Control. (C) qRT‐PCR analysis demonstrating NAT10 expression levels in NSCLC cell lines transfected with OE‐NAT10. *p < 0.05 versus Vector. (D) Western blotting depicting NAT10 expression levels in NSCLC cell lines after transfection with OE‐NAT10. *p < 0.05 versus Vector. β‐actin was utilized as the reference gene for qRT‐PCR, while GAPDH served as the loading control in Western blotting assays. The depicted values represent the mean ± standard deviation (SD) obtained from three independent experiments.

FIGURE 3

NAT10 Promotes Proliferation, Tumorigenicity, Invasion, and Migration in NSCLC Cell Lines. (A and B) Quantification of Cell Counting Kit‐8 (CCK8) assays depicting the viability of NSCLC cell lines post‐transfection with NCsiRNA, NAT10siRNA, Vector, and OE‐NAT10. *p < 0.05. (C and D) Images and quantification of clone formation assays representing the clonogenic potential of NSCLC cell lines following transfection with NCsiRNA, NAT10siRNA, Vector, and OE‐NAT10. *p < 0.05. (E and F) Images and quantification of transwell assays demonstrating the invasive capacity of NSCLC cell lines subsequent to transfection with NCsiRNA, NAT10siRNA, Vector, and OE‐NAT10. *p < 0.05. (G–J) Images and quantification of wound healing assays illustrating the migratory ability of NSCLC cell lines post‐transfection with NCsiRNA, NAT10siRNA, Vector, and OE‐NAT10. *p < 0.05. GAPDH served as a loading control. The depicted values denote the mean ± standard deviation (SD) obtained from three independent experiments.

FIGURE 4

NAT10's Association with the EMT Phenotype in Human NSCLC Cell Lines. (A, C) Quantification of E‐ cadherin, N‐cadherin, and Vimentin expression in NSCLC cell lines assessed via qRT‐PCR analysis. *p < 0.05, indicating statistically significant differences. (B, D). Western blotting results displaying the expression levels of E‐cadherin, N‐cadherin, and Vimentin following NAT10 knockdown with NAT10‐siRNA and overexpression of NAT10 using OE‐NAT10. β‐Actin served as a reference gene in the qRT‐PCR analysis, while GAPDH was used as a loading control in the Western blotting. Values depicted are the mean ± SD obtained from three independent experiments.

FIGURE 5

Remodelin's Impact on NSCLC Progression and EMT Phenotype (A) Quantification of CCK8 assays showing the effect of Remodelin treatment on NSCLC cell lines' proliferation. Statistical significance denoted by *p <0.05. (B) Images and quantification from clone formation assays depicting the influence of Remodelin treatment on NSCLC cell lines' clone formation ability. Statistical significance denoted by *p < 0.05. (C) Images and quantification demonstrating the impact of Remodelin treatment on transwell assays in NSCLC cell lines. Statistical significance denoted by *p < 0.05. (D and E) Images and quantification displaying the results of wound healing assays indicating the effect of Remodelin treatment on the migration capability of NSCLC cell lines. Statistical significance denoted by *p < 0.05. (F and G) Evaluation of E‐cadherin, N‐cadherin, and vimentin expression levels in NSCLC cell lines through qRT‐PCR and western blotting. β‐Actin served as the reference gene for qRT‐PCR, while GAPDH served as the loading control for western blotting. Values represented as mean ± SD from three independent experiments. Statistical analysis conducted using the Student's t‐test.

FIGURE 6

Remodelin's Effect on NSCLC Patient‐Derived Organoids. (A) Hematoxylin and eosin (HE) stained images depicting representative sections from two NSCLC patients. (B) Successful cultivation and growth of organoids derived from two NSCLC patients, establishing a platform for further experimentation. (C) Evaluation of proliferation and tumorigenesis inhibition efficiency of Remodelin in two distinct NSCLC patient‐derived organoids treated with varying concentrations. (D) Representative images displaying the impact of Remodelin treatment on the growth of two NSCLC patient‐derived organoids after a 96‐h period, comparing treated versus untreated conditions.

FIGURE 7

Remodelin's Effect on NSCLC Inhibition via NAT10 in Vivo. (A) Images displaying subcutaneous xenograft tumors (n = 6 for each group) derived from A549 cells under various treatment conditions. (B) Images of excised tumors from six BALB/c nude mice at 29 days after various treatment conditions. (C) Hematoxylin and eosin (HE) staining alongside Immunohistochemistry (IHC) of xenograft tumors, depicting the expression levels of Ki67 and Vimentin. (D, E) Evaluation of tumor volumes measured every 4 days and analysis of tumor weights in different experimental groups. (F, G) Examination of NAT10, E‐cadherin, N‐cadherin, and vimentin expression in xenograft tumors using both qRT‐PCR and western blotting techniques. β‐Actin served as the reference gene for qRT‐PCR, while GAPDH acted as the loading control in western blotting. Values represented as mean ± SD from three independent experiments. Statistical analysis conducted using Student's t‐test.