USP21-EGFR-Lyn axis drives NSCLC progression and therapeutic potential of USP21 inhibition

Abstract

Non-small cell lung cancer (NSCLC) is a highly aggressive malignancy frequently driven by oncogenic mutations in the epidermal growth factor receptor (EGFR). Although EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have shown clinical efficacy, challenges such as limited response duration and intrinsic mechanisms-such as EGFR amplification-can affect therapeutic outcomes. This study investigates the role of the USP21-EGFR-Lyn axis in NSCLC progression, identifying USP21 as a key regulator of EGFR and Lyn stability. Gene Set Enrichment Analysis (GSEA) of NSCLC patient datasets revealed a strong correlation between USP21 overexpression and poor prognosis. Functional studies using USP21-knockout (USP21-KO) lung cancer cell lines demonstrated reduced proliferation, migration, colony formation, and tumor spheroid growth. Mechanistically, USP21 interacts with EGFR and Lyn, preventing their ubiquitination and degradation, thereby sustaining oncogenic signaling. In vivo, USP21 depletion significantly suppressed tumor growth in xenograft models. Additionally, pharmacological inhibition of USP21 with BAY-805 effectively reduced EGF-induced tumor spheroid formation, highlighting its therapeutic potential. Collectively, these findings position USP21 as a promising target for NSCLC treatment and offer a potential approach to complement existing EGFR-targeted therapies.

Keywords: BAY-805; Epidermal growth factor receptor; Lck/Yes novel tyrosine kinase; Non-small cell lung cancer; Ubiquitin specific peptidase 21.

Fig. 1

The USP21-EGFR-Lyn axis regulates lung cancer progression. A Schematic representation of the experimental workflow used to analyze associations and functional roles of USP21 in non-small cell lung cancer (NSCLC) patients (n = 42). B Differential expression magnitude (△Mag) of USP21 between lung tumor tissues (LTTs, n = 42) and matched lung normal tissues (mLNTs, n = 42). C △Mag of USP21 between USP21^up NSCLCs (n = 32) and USP21^down NSCLCs (n = 10). D CRISPR-Cas9 gene editing strategy for generating USP21-knockout (USP21-KO) lung cancer cells using guide RNAs targeting USP21. E Transwell migration assay was performed on control (Ctrl) H1299 cells and USP21-KO H1299 cells. Results are presented as mean ± SD (n = 7). Statistical significance (Student’s t-test); ***, P < 0.001. F Anchorage-dependent colony formation assay conducted with Ctrl H1299 and USP21-KO H1299 cells. Results are presented as mean ± SD (n = 5). Statistical significance (Student’s t-test); ***, P < 0.001. G 3D tumor spheroid formation assay was performed with Ctrl H1299 and USP21-KO H1299 cells. Results are presented as mean ± SD (n = 3). Statistical significance (Student’s t-test); **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. H and I Xenograft mouse model: NSG mice were subcutaneously injected with either Ctrl H1299 (5 × 10⁶ cells per mouse, n = 5) or USP21-KO H1299 cells (5 × 10⁶ cells per mouse, n = 5) (H). Tumor volume was measured using calipers for up to 58 days post-injection. Tumor volumes (mm³) were calculated as (length × width²) × 0.5. Tumor growth curves are shown as mean ± SEM (I, left). Representative tumor-bearing NSG mice are shown at day 58 post-injection (I, right). Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. J Differential expression magnitude (△Mag) of Lyn, EGFR, and USP21 between LTTs (n = 42) and matched LNTs (n = 42). K-M Co-immunoprecipitation (IP) and immunoblotting (IB) analyses in HEK-293T cells transfected with the indicated constructs: (K) IP using an anti-Myc antibody, followed by IB with anti-Flag or anti-Myc antibodies. (L) IP using an anti-Flag antibody, followed by IB with anti-Flag or anti-Myc antibodies. (M) IP using an anti-Myc antibody, followed by IB with anti-Flag, anti-Myc, or anti-HA antibodies. N and O Western blot analysis of EGFR degradation in Ctrl and USP21-KO lung cancer cells (H1299 in N, H460 in O) treated with cycloheximide at the indicated time points. EGFR degradation was quantified relative to GAPDH using ImageJ. Data are presented as mean ± SD from three independent experiments. Statistical significance (Student’s t-test); *, P < 0.05; **, P < 0.01; ***, P < 0.001. P and Q Co-IP and IB analyses in HEK-293T cells transfected with the indicated constructs. IP was performed using an anti-Myc antibody, followed by IB with anti-Myc or anti-Flag antibodies. R Co-IP and IB assays in HEK-293T cells transfected with the indicated constructs. IP was performed with an anti-Myc antibody, followed by IB with anti-Flag, anti-Myc, or anti-HA antibodies. S and T Western blot analysis of Lyn degradation in Ctrl and USP21-KO lung cancer cells (H1299 in S, H460 in T) treated with cycloheximide at the indicated time points. Lyn degradation was quantified relative to GAPDH using ImageJ. Data are presented as mean ± SD from three independent experiments. Statistical significance (Student’s t-test); *, P < 0.05; **, P < 0.01; ***, P < 0.001. U Schematic model depicting the role of USP21 in stabilizing EGFR and Lyn. Ubiquitinated EGFR and Lyn are typically degraded via the lysosomal pathway. However, USP21 interacts with EGFR and Lyn to mediate their de-ubiquitination, leading to their stabilization. This stabilization enhances EGFR-mediated lung cancer progression

Fig. 2

USP21 as a potential therapeutic target in EGF-induced lung cancer progression. A-C MTT assays were performed on control (Ctrl) and USP21-knockout (USP21-KO) lung cancer cells, including A549 (A), H460 (B), and H1299 (C) cells, treated with either vehicle or EGF. Results are presented as mean ± SD (n = 5). Statistical significance (Student’s t-test): *, P < 0.05; **, P < 0.01. D-F Transwell migration assays were conducted on Ctrl and USP21-KO cells, including A549 (D), H460 (E), and H1299 (F) cells, following vehicle or EGF treatment. Results are presented as mean ± SD (n = 7). Statistical significance (Student’s t-test): **, P < 0.01; ***, P < 0.001. ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001—comparisons between USP21-KO cells and their respective Ctrl cells. G-I 3D spheroid formation assays were performed using Ctrl and USP21-KO cells, including A549 (G), H460 (H), and H1299 (I), which were seeded in 96-well plates and incubated at 37 °C for 48 h to allow spheroid formation. Spheroids were then treated with either vehicle or EGF and cultured further as indicated. Spheroid size was measured using ImageJ software, and images were captured using phase-contrast microscopy (scale bar = 100 μm). Data are presented as mean ± SD (G and H, n = 7; I, n = 3). Statistical significance (Student’s t-test): *, P < 0.05; **, P < 0.01; ***, P < 0.001. ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001; ^####, P < 0.0001—comparisons between USP21-KO cells and their respective Ctrl cells. J Schematic representation of the experimental protocol used to evaluate the inhibitory effects of BAY-805 on EGF-induced 3D tumor spheroid formation in lung cancer cells. Stabilized spheroids were incubated at 37 °C for 48 h, pre-treated with either vehicle or BAY-805 for 24 h, and then treated with either vehicle or EGF, followed by further culturing for different time points. K-M 3D tumor spheroid formation assays were conducted with A549 (K), H460 (L), and H1299 (M) cells. Spheroids were pre-treated with either vehicle or BAY-805 at specific concentrations (34.6 µM for A549 in K, 12.07 µM for H460 in L, and 16.9 µM for H1299 in M) for 24 h, followed by treatment with either vehicle or EGF. Spheroid size was measured using ImageJ software, and images were captured via phase-contrast microscopy (scale bar = 100 μm). Data are presented as mean ± SD (K, n = 5; L, n = 7; M, n = 5). Statistical significance (Student’s t-test): *, P < 0.05; **, P < 0.01; ***, P < 0.001. ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001—comparisons between cells treated with and without BAY-805. N Schematic model illustrating the role of USP21 in lung cancer progression through the USP21-EGFR-Lyn axis and its potential as a therapeutic target. Normally, ubiquitinated EGFR and Lyn undergo degradation via the lysosomal pathway. However, USP21 interacts with EGFR and Lyn, mediating their de-ubiquitination, leading to their stabilization and subsequent enhancement of EGFR-driven lung cancer progression. Inhibiting USP21 with compounds such as BAY-805 may provide a promising therapeutic strategy to counteract EGF-induced lung cancer progression