CASP2 Knockout Cell Lines

Investigate the role of caspase-2 (CASP2) in regulating ferroptosis, a non-apoptotic cell death pathway, and its potential as a therapeutic target in mutant-p53 (mut-p53) cancer cells using CRISPR/Cas9-generated CASP2 knockout (KO) cell lines.

1. Caspase-2 and Cellular Stress Responses

Caspase-2 is a conserved caspase family member with established roles in apoptosis and tumor suppression, but its function in non-apoptotic cell death pathways remains unclear. Mutant-p53 cancer cells, which account for over 50% of human cancers, exhibit deregulated oxidative stress responses and heightened sensitivity to ferroptosis .

2. Ferroptosis and Cancer Therapy

Ferroptosis is an iron-dependent cell death driven by lipid peroxidation, suppressed by antioxidant pathways like glutathione peroxidase 4 (GPX4). Targeting ferroptosis holds promise for treating cancers resistant to conventional apoptosis-inducing therapies, but the role of caspase-2 in this process is unexplored .

3. Gaps in Knowledge

Does caspase-2 influence ferroptosis in mut-p53 cancer cells?

Can CASP2 KO cell lines serve as a model to identify novel therapeutic strategies for mut-p53 cancers?

1. Generation of CASP2 KO Cell Lines

Method: CRISPR/Cas9 genome editing was used to disrupt CASP2 in human lung cancer (H1299p53R273H) and esophageal cancer (Flo-1) cell lines with mut-p53, as well as wild-type p53 mouse embryonic fibroblasts (MEFs). KO was confirmed by immunoblotting and DNA sequencing, showing complete loss of caspase-2 protein .

Validation: CASP2 KO cells maintained normal viability and morphology but exhibited enhanced sensitivity to ferroptosis inducers (erastin, RSL3) compared to wild-type cells .

2. Functional Assays in CASP2 KO Cells

Ferroptosis Sensitization: CASP2 KO mut-p53 cells showed a >2-fold decrease in IC50 for erastin and RSL3, with enhanced lipid peroxidation (C11-BODIPY staining) and reduced glutathione levels, characteristic of ferroptosis .

Mut-p53 Dependence: Sensitivity to ferroptosis was specific to mut-p53 cells, as CASP2 KO in wild-type p53 MEFs did not enhance ferroptosis .

Therapeutic Rescue: Ferroptosis inhibitors (ferrostatin-1, deferoxamine) rescued cell death in CASP2 KO cells, confirming ferroptosis as the mechanism of cell death .

3. Mechanistic Insights

Transcriptomic Changes: RNA sequencing revealed downregulation of ferroptosis regulators (SLC7A11, SESN2, HMOX1) in CASP2 KO cells, linking caspase-2 to oxidative stress response pathways .

Non-Proteolytic Function: Catalytically inactive caspase-2 mutants (C320G, D135A, D330A) rescued ferroptosis sensitivity in CASP2 KO cells, indicating caspase-2 acts via protein-protein interactions, not proteolytic activity .

Chaperone-Mediated Autophagy (CMA): CASP2 KO promoted GPX4 degradation via CMA, as shown by reduced GPX4 levels and rescue of cell death by the HSP90 inhibitor 17AAG .

1. Mechanistic Insights

Caspase-2 suppresses ferroptosis in mut-p53 cancer cells through two key mechanisms:

Regulation of Oxidative Stress Genes: Caspase-2 maintains expression of antioxidant genes (e.g., SLC7A11) to limit lipid peroxidation .

Inhibition of GPX4 Degradation: Caspase-2 interacts with chaperone machinery (HSPA8, DNAJB1) to prevent CMA-mediated degradation of GPX4, preserving antioxidant capacity .

2. Translational Significance

Therapeutic Target: CASP2 KO sensitizes mut-p53 cancer cells to ferroptosis, suggesting caspase-2 inhibitors could enhance ferroptosis-based therapies