Asparagine synthetase (ASNS) Drives Tumorigenicity in Small Cell Lung Cancer

Abstract

Objectives: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma characterized by rapid proliferation, early metastasis, and limited therapeutic response. Metabolic reprogramming is increasingly recognized as a key feature of small cell lung cancer progression, yet the contribution of specific metabolic enzymes remains incompletely understood. This study aimed to investigate the role of asparagine synthetase in small cell lung cancer tumorigenicity and disease progression. Methods: Integrative analyses were performed using public transcriptomic datasets, proteomic profiling, and functional assays in vitro and in vivo. Asparagine synthetase expression levels were evaluated in normal lung, non-small cell lung cancer, and small cell lung cancer tissues using public microarray datasets. Loss of function studies were conducted using shRNA mediated knockdown in murine and human small cell lung cancer cell models. Tumor growth and survival were assessed using xenograft mouse models. Results: Asparagine synthetase expression was significantly elevated in small cell lung cancer compared with normal lung and non-small cell lung cancer tissues. Genetic depletion of asparagine synthetase impaired cellular proliferation and colony forming capacity in vitro. In vivo, asparagine synthetase knockdown suppressed tumor growth and was associated with prolonged survival in xenograft mouse models. Conclusions: These findings demonstrate that asparagine synthetase contributes to tumor growth and metabolic adaptability in small cell lung cancer. The results support a functional role for asparagine synthetase in malignant progression and suggest that targeting asparagine metabolism may represent a potential therapeutic approach in aggressive small cell lung cancer.

Keywords: ASNS; SCLC; oncogene; progression; ribosome biogenesis.

Figure 1

Expression of ASNS in normal lung, non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). (A,B) Data were obtained from the NCBI Gene Expression Omnibus (GSE40275). Samples were classified based on the “Source name” annotation: normal lung (n = 43), NSCLC (n = 16), and SCLC (n = 21). The y-axis represents ASNS expression (log2 normalized). Box plots display median values with interquartile ranges, whiskers indicate minimum to maximum values, and dots represent individual samples. Statistical comparisons using unpaired two-tailed Student’s t-tests showed significantly higher ASNS expression in SCLC compared with normal lung (p < 0.0001) and NSCLC (p = 0.0036).

Figure 2

ASNS is required for SCLC development. (A,B) Immunoblots for ASNS in mouse SCLC cells and human SCLC and NSCLC cell lines. β-actin and GAPDH are used as a loading control. (C) Representative H&E-stained sections of tumors (left) derived from Rb/p53/p130 (RPP) mice and immunostaining for ASNS (middle; red) and DAPI (right; blue). (D) Whole-mount X-gal-stained lungs (left) from Rb/p53/p130/Asns^lox/+ (RPP) mice 9 months after Ad-Cre infection. The interior of cut lungs is shown, and arrows and arrowheads indicate tumors and small lesions, respectively. H&E-stained section of tumors (middle) and immunostaining for CGRP marking neuroendocrine cells (right; green). Scale bars, (C) 50 µm; (D) 5 mm (left), 200 µm (middle and right).

Figure 3

ASNS induces tumorigenic progression of SCLC cells. (A–E) Results of MTT assay measuring the viability of cells treated with ASNS knockdown for 8 days. mSCLC-1 and mSCLC-2 are mouse SCLC cells, and H524, H82, and H69 are human SCLC cell lines. MTT assays were repeated with similar results at least once. **, p < 0.001. Statistical tests were performed using an unpaired t test. Error bar, SD.

Figure 4

ASNS is required for the expansion of human SCLC lines and mouse tumor cells. (A–H) Representative images (left) and quantification (right) of soft agar colonies formed by mouse and human SCLC cell lines (n = 3 per cell type). *, p < 0.01; **, p < 0.001. Statistical tests were performed using an unpaired t test. Error bar, SD. Scale bars, (A,C,E,G), 5 mm.

Figure 5

Knockdown of ASNS decreased ribosomal transcription programs. (A,B) Immunoblots for puromycin incorporation in nascent proteins. Two-hundred-thousand cells were treated with 10 µg of puromycin for 10 min. GAPDH is used as a loading control.

Figure 6

Overall survival by ASNS amplification in the TCGA PanCancer Atlas cohort. (A) Immunoblots for ASNS in mouse SCLC cells. GAPDH is used as a loading control. (B,C) Quantification of allograft tumors over time, following injection of mice with control and Asns-knockdown mouse SCLC cells (right) and of subcutaneous tumors >1.5 cm in diameter, where relative tumor growth represents tumor weight (g, grams) divided by latency (days after allograft; left). (D) Overall survival (OS) curves were generated using cBioPortal https://www.cbioportal.org/ (accessed on 15 September 2025). based on the TCGA PanCancer Atlas dataset (32 studies, 10,967 samples). Groups were defined as ASNS Amplified (cBioPortal “amplified”; 7 homozygous deletions removed; n = 161) versus Unaltered (diploid; n = 10,454). KM curves were generated with the cBioPortal Comparison/Survival module using GISTIC CNV data only. OS in months is shown for the two groups (red: Amplified, blue: Unaltered). ASNS amplification was associated with significantly worse overall survival (log-rank p = 0.0123; median OS, 48.69 vs. 80.74 months; HR = 1.44, 95 percent CI 1.08 to 1.92). **, p < 0.001. Statistical tests were performed using an unpaired t test. Error bar, SD.