The GRAMD1A Knockout NCI-H1975 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population targeting the human GRAMD1A gene in the NCI-H1975 lung adenocarcinoma cell line. This heterogeneous loss-of-function model enables studies of GRAMD1A-mediated non-vesicular cholesterol transport without the clonal biases of single-cell-derived lines.
NCI-H1975 is a human lung adenocarcinoma epithelial cell line derived from a 62-year-old female nonsmoker. It harbors activating EGFR mutations (L858R, T790M) and serves as a key model for EGFR-mutant non-small cell lung cancer with acquired tyrosine kinase inhibitor resistance. The oncogenic EGFR signaling and altered metabolic state provide a relevant context for investigating cholesterol homeostasis?Ccancer biology links.
GRAMD1A localizes to ER?Cplasma membrane contact sites, where its GRAM domain senses cholesterol and phosphatidylserine enrichment. It engages VAPA and VAPB on the ER to mediate non-vesicular cholesterol transfer, regulating ER cholesterol levels and the SREBF2?CSCAP?CInsig-1 axis. This controls proteolytic activation of SREBP2 and transcription of cholesterol synthesis (HMGCR, LDLR) and uptake genes. Downstream effects include modulation of PLIN2, ACAT, and lipid droplet formation. GRAMD1A thus integrates lipid sensing with SREBP signaling and broader lipid homeostasis.
Disruption of GRAMD1A in NCI-H1975 cells is predicted to impair cholesterol delivery to the ER, causing constitutive SREBP2 activation and upregulation of cholesterol synthesis and uptake. This may exacerbate ER stress and alter autophagy, potentially sensitizing EGFR-mutant cancer cells to metabolic stress. The model allows dissection of cholesterol-dependent survival mechanisms crucial for NSCLC membrane biogenesis and signaling.
These polyclonal knockout cells can be used in proximity ligation assays (PLA) for ER?CPM contacts, Filipin or Amplex Red cholesterol measurements, and Western blotting of SREBP2 cleavage. RNA-seq profiling, cell viability, colony formation, and BODIPY lipid droplet staining further evaluate metabolic and phenotypic consequences. Applications include functional genomics, cholesterol transport inhibitor validation, and mechanistic studies of metabolic reprogramming. For more information, please contact Ascent Research.
