Silencing myeloid cell leukemia-1 improves apoptotic and ferroptotic responses in melanoma

Melanoma is a highly malignant skin cancer characterized by strong metastatic and invasive capabilities. Integrative transcriptomic profiling offers a systematic route to uncover tumor-specific regulatory hubs that could expose new therapeutic vulnerabilities. By combining multi-cohort bioinformatics screening with mechanistic cell-based assays, we sought to delineate critical drivers of disease progression in melanoma.

An integrative analysis of three GEO microarray cohorts (GSE3189, GSE46517, GSE114445) identified hub genes through differential-expression filtering, protein-protein interaction mapping, and CytoHubba ranking. MCL1 was prioritized as the top network hub, prompting its selection for functional validation. B16F1 murine melanoma cells were transfected with MCL1-specific siRNA or a non-targeting control. Cell viability (CCK-8), scratch-wound migration, adhesion, Annexin V/PI apoptosis, cell-cycle distribution, and reactive-oxygen species (ROS) were quantified. Western blots probed apoptosis, cell cycle, and ferroptosis-related proteins; glutathione (GSH) and malondialdehyde (MDA) were measured by ELISA; mitochondrial ultrastructure was examined by transmission electron microscopy. Ferrostatin-1 (Fer-1) was used to assess the functional relevance of ferroptosis.

In silico analysis prioritized MCL1 as the top hub within a 136-gene melanoma consensus signature. siRNA achieved > 80% knock-down of Mcl-1 mRNA and protein in B16F1 cells, resulting in: (i) significant reductions in cell viability, migration, and adhesion; (ii) substantial elevation of apoptosis with upregulating Bax, downregulating Bcl-2 and caspase-9/3 activation; (iii) G1-phase arrest accompanied by p53/p21 upregulation; and (iv) a ferroptotic phenotype typified by GSH depletion, MDA/ROS accumulation, mitochondrial swelling, reduced GPX4/SLC7A11 ratio, and higher ACSL4/NCOA4 ratio. Fer-1 partially rescued these molecular alterations yet did not fully restore growth, indicating that Mcl-1 coordinates both caspase-dependent apoptosis and lipid peroxidation-driven ferroptosis in melanoma.

Overall, our findings pinpoint that Mcl-1 operates as a multifaceted survival node in melanoma, concurrently sustaining proliferation, migration, and redox homeostasis while suppressing apoptotic and ferroptotic death programs. Dual targeting of Mcl-1 and the ferroptosis pathway may offer a robust therapeutic strategy against melanoma.