Investigating PRMT5 as a therapeutic target in EGFR TKI resistant NSCLC and assessing the role of mucins in NSCLC as blood biomarkers

Lung cancer is on track to cause over 124,000 U.S. deaths in 2025, and about 10-15% of all lung cancers in the United States involve EGFR-resistant NSCLC. Patients with EGFR-mutant NSCLC are initially responsive to TKIs but frequently relapse within 19 months. PRMT5, a type II arginine methyltransferase, drives oncogenic and inflammatory signaling primarily through P13K-Akt signaling, which may be increased by cigarette smoke. We hypothesize that inhibiting PRMT5 by using siRNA will modulate these pathways and restore EGFR-TKI sensitivity. PRMT5 expression was analyzed in EGFR-mutant NSCLC parental cell lines (H3255P, H1975P, PC9) and resistant cell lines (H3255OR, H1975OR, PC9OR) using qPCR and Western blotting following cigarette smoke extract (CSE) exposure and siRNA knockdown using DharmaFECT. Cytokine expression was profiled using qPCR to assess downstream inflammatory effects. Immunohistochemistry (IHC) was performed on normal and tumor tissues from smokers and non-smokers to evaluate PRMT5 levels. The PRMT5 levels were analyzed using a Keyence microscopic software (BZX-800 Analyzer) and statistical analysis was done using Fisher's Exact test. CSE increased PRMT5 mRNA across EGFR-mutant lines by 1.2-11.5 fold at 48 h (p<0.01-0.001). Immunoblotting confirmed PRMT5 upregulation at 24-48 h in resistant cells by 1.3-2.5 fold (p<0.01-0.001). In H3255-OR and PC9-OR cells, siPRMT5 reduced the 48-h CSE-induced cytokine surge (170-760%) by 10-20%. Compared to Mock plus CSE, siPRMT5 plus CSE reduced IL-8 (60-75%), TNF-alpha (30-50%), and IL-1beta (35-55%). Downregulation of these cytokines was also seen after combinatory treatment with osimertinib and siPRMT5. In PC9-P/OR cells, siPRMT5 lowered PRMT5 transcripts by 35-50% compared to Mock and siPRMT5 plus CSE reduced by 15-45% relative to Mock plus CSE. IHC on 21 smoker and 20 non-smoker lung tissues from lung cancer patients showed PRMT5 expression was higher in smokers versus non-smokers (p<0.05). Studies were also conducted on normal lung tissue from 25 smokers and 19 non-smokers and PRMT5 expression was found to be higher in smokers compared to non-smokers using Fisher's exact test (p<0.001). In conclusion, PRMT5 is upregulated by CSE in osimertinib-resistant EGFR-mutant cells and is upregulated in smokers. PRMT5 knockdown suppresses inflammatory cytokines and supports the rationale that inhibiting PRMT5 could help restore EGFR-TKI sensitivity.