p53 overexpression counteracts the pro-survival effect of Bcl-2 by restoring MAMs function in cutaneous squamous cell...

Cutaneous squamous cell carcinoma (cSCC) is a prevalent type of skin cancer, and its development is strongly associated with impaired regulation of apoptosis. The anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and the tumor suppressor p53 play pivotal roles in controlling apoptotic signaling. Although Bcl-2 expression in cSCC has been reported to be heterogeneous, its role in mitochondria-associated membranes (MAMs)-mediated apoptosis remains unclear. This study aims to clarify how Bcl-2 and p53 regulate cSCC cell apoptosis by modulating the structure and function of MAMs.

We constructed stable Bcl-2 overexpression (oe-Bcl-2) and Bcl-2/p53 co-overexpression (oe-Bcl-2 + oe-p53) A431 cell lines using lentiviral transduction. Transcriptome sequencing (RNA-seq) was performed, followed by pathway enrichment and protein-protein interaction (PPI) network analyses to screen for critical signaling routes. Cell apoptosis, growth, migratory capacity, and invasive potential were evaluated using flow cytometry, CCK-8, EdU incorporation, colony formation, and Transwell-based assays. Key MAMs functions, including Ca²⁺ flux, mitochondrial membrane potential (MMP), ROS, and ATP levels, were measured. Subcutaneous tumor models in nude mice were used for in vivo validation.

RNA-seq analysis revealed that Bcl-2 regulates multiple apoptosis- and calcium signaling-related pathways, significantly affecting key MAM-associated proteins, including VDAC2, IP3R, SERCA2, as well as the p53 signaling pathway. Bcl-2 overexpression markedly enhanced proliferation and migration of A431 cells while reducing apoptosis. It also modulated mitochondrial membrane potential, ATP production, and ROS generation, suggesting that Bcl-2 exerts anti-apoptotic effects through MAMs. In contrast, Bcl-2 knockdown significantly suppressed proliferation and invasion while inducing apoptosis in A431 cells, further supporting its oncogenic role. Notably, p53 overexpression reversed these effects and significantly inhibited tumor growth in vivo.

Our findings demonstrate that Bcl-2 promotes cSCC progression by modulating MAMs structure and function to inhibit p53-mediated mitochondrial apoptosis. The study identifies the novel Bcl-2-MAMs-p53 signaling axis that plays a pivotal role in determining cSCC cell fate, highlighting a promising target for therapeutic intervention.