The AKT1S1 Knockout HT29 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal knockout cell population generated from the HT29 human colorectal adenocarcinoma line. This product disrupts the AKT1S1 gene (encoding PRAS40), a constitutive negative regulator of mTORC1. The polyclonal nature preserves heterogeneous gene-editing events, enabling pooled studies of mTORC1 signaling, cancer biology, and autophagy. By eliminating PRAS40, tonic mTORC1 inhibition is relieved, facilitating investigation of downstream anabolic processes in a colorectal cancer context.
The HT29 cell line, a well-characterized epithelial model of human colorectal adenocarcinoma, serves as an ideal host for studying oncogenic signaling pathways. Originating from a primary colon tumor, HT29 cells retain key genetic features of colorectal cancer, including mutations in APC, TP53, and PI3K pathway components, which synergize with AKT1S1 loss to drive malignant phenotypes. This cellular context provides a relevant platform for examining tumor cell proliferation, survival, and metabolic reprogramming, and it is widely employed in drug discovery and functional genomics screens targeting colorectal malignancies.
AKT1S1 (PRAS40) is a substrate and inhibitor of mTORC1 that integrates insulin/PI3K/AKT and AMPK signals. It binds the mTOR-Raptor complex to suppress kinase activity; AKT phosphorylation at Thr246 promotes 14-3-3 binding and dissociation, relieving inhibition. Without PRAS40, mTORC1 hyperactivates, phosphorylating S6K and 4E-BP1 to boost protein synthesis, while inhibiting autophagy via ULK1 and ATG13. Key interactors include mTOR, Raptor, Rheb, and 14-3-3, placing AKT1S1 at a critical signaling node.
In the HT29 colorectal adenocarcinoma context, loss of AKT1S1 amplifies mTORC1-driven oncogenic processes. Constitutive mTORC1 activation promotes unchecked cell cycle progression, proliferation, and metabolic anabolism, while suppressing autophagy. This model enables dissection of mTORC1-dependent mechanisms in colorectal cancer, including resistance to PI3K/AKT-targeted therapies and metabolic rewiring. The polyclonal knockout population facilitates exploration of clonal heterogeneity in signaling responses and drug sensitivity, providing a more representative system than single-clone knockouts.
Researchers can use these cells for western blotting of mTORC1 substrates, proliferation assays (MTT, BrdU), and autophagy flux (LC3-II). Additional applications include RT-qPCR, high-throughput drug screening, colony formation, and migration/invasion assays. This model is valuable for identifying mTORC1 vulnerabilities in colorectal cancer and evaluating inhibitors of S6K, 4E-BP1, or upstream regulators like AKT and PI3K. For further technical specifications or assistance with assay setup, please contact Ascent Research.
