The inhibitory effect and targets of CRM197 on tumor growth in arsenic-transformed cells and lung cancer cells.

Lung squamous cell carcinoma (LSCC) is among the most prevalent and deadly malignancies in arsenic-exposed populations, yet effective targeted therapies remain elusive. Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin that binds heparin-binding EGF‑like growth factor (HB‑EGF), has demonstrated anti‑tumor activity, but its efficacy against arsenic-induced LSCC has not been characterized. In this study, we assessed CRM197's effects on proliferation (CCK‑8 and colony‑formation assays), cell‑cycle progression (flow cytometry), comparing its activity to cetuximab and afatinib in arsenic‑transformed BEAS‑2B cells obtained by 6-month treatment on human bronchial epithelial cells BEAS-2B with low-dose sodium arsenite (As‑T) and lung cancer cells(NCI‑H226 and PC‑9). Next, we evaluated in vivo efficacy in nude mice bearing As‑T and NCI‑H226 xenografts treated daily with CRM197 (5 mg/kg) or afatinib. Finally, tandem‑mass‑tag proteomics and network pharmacology identified 179 upregulated proteins in As‑T cells and 11 overlapping CRM197 targets-including AKR1B1, PTPN1, PPARA, and SERPINE1-which were validated by molecular docking and 100‑ns dynamics simulations. CRM197 markedly inhibited cell proliferation, induced G₀/G₁ arrest, and outperformed cetuximab and matched or exceeded afatinib in vitro; in vivo, CRM197 reduced tumor volume and weight more effectively than afatinib, with extensive necrosis. These data establish CRM197 as a potent, multi‑targeted inhibitor of arsenic‑driven LSCC and highlight novel therapeutic targets for further drug development.