RETREG1 Knockout Cell Lines

Investigate the function of RETREG1 (Reticulophagy Regulator 1) in endoplasmic reticulum (ER) stress-induced reticulophagy and autophagic cell death (ACD) using CRISPR/Cas9-generated RETREG1 knockout (KO) cell lines in glioblastoma models.

1. Reticulophagy and Glioblastoma

Reticulophagy, the selective degradation of ER fragments via autophagy, is critical for maintaining ER homeostasis. Glioblastoma (GBM) cells often exhibit heightened ER stress due to metabolic dysregulation, making reticulophagy a potential therapeutic target. However, the specific role of RETREG1, a key reticulophagy receptor, in GBM remains unclear .

2. RETREG1 in ER Stress Response

RETREG1 (FAM134B) localizes to ER sheets and mediates their degradation during ER stress. Studies suggest RETREG1 may link ER stress to autophagic pathways, but its function in ACD—a non-apoptotic cell death mode relevant to GBM therapy—requires further exploration .

3. Therapeutic Gap in GBM

Current GBM treatments have limited efficacy, and resistance to apoptosis-inducing therapies is common. Targeting ACD via reticulophagy regulators like RETREG1 could offer a novel therapeutic strategy for refractory GBM .

1. Generation of RETREG1 KO Cell Lines

Method: CRISPR/Cas9 genome editing was used to disrupt RETREG1 in human GBM cell line MZ-54 and mouse embryonic fibroblasts (MEFs). KO was confirmed by Western blotting and qPCR, showing complete loss of RETREG1 protein and reduced mRNA expression .

Validation: RETREG1 KO cells exhibited impaired ER stress-induced reticulophagy, as measured by reduced ER fragment engulfment in autophagosomes .

2. Functional Assays in RETREG1 KO Cells

ER Stress Response: Treatment with loperamide (LOP), an ACD-inducing compound, induced ER stress markers (ATF4, HSPA5/BiP) in both WT and RETREG1 KO cells, but RETREG1 KO cells showed reduced LC3B lipidation and autophagosome formation .

Reticulophagy Impairment: RETREG1 KO cells displayed decreased degradation of ER sheet marker CKAP4 and reduced formation of ssRFP+ GFP-KDEL− puncta (indicative of reticulophagic flux) compared to WT cells .

ACD Sensitivity: RETREG1 KO significantly reduced LOP-induced ACD, as measured by propidium iodide (PI) uptake and clonogenic survival assays. Double KO of RETREG1 and TEX264 further attenuated ACD, indicating redundant roles of reticulophagy receptors .

3. Mechanistic Studies

RETREG1-ATF4 Axis: LOP-induced ATF4 upregulation was unaffected in RETREG1 KO cells, but ATF4-dependent autophagy gene transcription (e.g., ATG13, WIPI1) was partially impaired, linking RETREG1 to ATF4-mediated autophagic signaling .

ER Ultrastructure: Electron microscopy revealed fewer ER fragments within autophagosomes in RETREG1 KO cells, confirming RETREG1’s role in ER sheet engulfment .

1. Mechanistic Insights

RETREG1 regulates glioblastoma cell survival through two key mechanisms:

ER Homeostasis Maintenance: RETREG1 mediates selective degradation of ER sheets during ER stress, preventing excessive lipid peroxidation and cell death .

ATF4-Driven Autophagy: RETREG1 supports ATF4-induced autophagy gene expression, enabling efficient autophagosome formation and ACD execution .

2. Translational Significance

Biomarker Potential: Low RETREG1 expression in glioblastoma tissues correlates with poor prognosis, making RETREG1 a candidate prognostic biomarker .

Therapeutic Target: Inhibiting RETREG1 (e.g., via siRNA or small molecules) sensitizes glioblastoma cells to ER stress-inducing drugs (e.g., LOP) and immunotherapies, offering a novel combination strategy for treatment-resistant tumors .