The AKT3 Knockout CAL-27 Polyclonal Cells are a CRISPR/Cas9-edited pooled population of human tongue squamous cell carcinoma cells with targeted disruption of the AKT3 gene. This polyclonal knockout cell product is generated by lentiviral delivery of Cas9 and an AKT3-specific guide RNA, followed by selection to enrich for cells harboring gene-disrupting edits. The resulting population provides a heterogeneous loss-of-function model that reflects the genetic diversity inherent in pooled CRISPR screens, making it suitable for studying AKT3-dependent phenotypes without clonal bias. Researchers can use this tool to interrogate AKT3 function in an oral cancer context, leveraging the preserved oncogenic landscape of the parental CAL-27 line while ablating a critical node in the PI3K signaling network.
The parental CAL-27 cell line is a well-characterized model of human tongue squamous cell carcinoma derived from a primary lesion of a 56-year-old male patient. These cells exhibit adherent epithelial morphology and harbor a homozygous TP53 mutation (p.R175H), which abrogates wild-type p53 tumor suppressor function. CAL-27 cells display robust activation of the PI3K/AKT/mTOR axis, making them particularly dependent on AKT signaling for survival and proliferation. Their tumorigenic properties, including anchorage-independent growth and invasive potential, render them a valuable system for investigating head and neck squamous cell carcinoma biology and for preclinical evaluation of targeted therapeutics.
AKT3 (PKB??) is a member of the AGC family of serine/threonine kinases and serves as a principal effector of phosphoinositide 3-kinase (PI3K) signaling. Activated by upstream regulators such as EGFR, IGFR, and HER2, signal transduction proceeds through PI3K-mediated generation of PIP3, which recruits AKT3 to the plasma membrane where it is phosphorylated at Thr305 by PDK1 and at Ser472 by mTORC2. Active AKT3 phosphorylates numerous downstream targets including GSK3??, FoxO1/3a, TSC2, PRAS40, and BAD, thereby promoting cell survival, proliferation, and metabolism while inhibiting apoptosis. It also forms complexes with regulatory partners like HSP90, PP2A, and TCL1, which modulate its stability and activity. In parallel, negative regulators such as PTEN and PHLPP counteract AKT3 signaling by dephosphorylating PIP3 and the kinase itself, respectively, establishing a tightly controlled node that integrates growth factor inputs with metabolic and survival outputs.
Disruption of AKT3 in the CAL-27 background is expected to attenuate oncogenic signaling cascades that drive oral cancer progression. Given the mutant p53 status of these cells, which disables one of the major apoptotic barriers, AKT3 knockout may further impair survival signals transduced through MDM2 and NF-??B, potentially sensitizing cells to genotoxic stress or targeted agents. The model enables dissection of AKT isoform-specific contributions, as AKT1 and AKT2 remain intact, allowing researchers to evaluate functional redundancy and isoform-selective dependencies. By uncoupling the PI3K/AKT/mTOR axis at the level of AKT3, this system can reveal adaptive feedback mechanisms and compensatory pathway activation, providing insights into resistance mechanisms encountered with pan-AKT or PI3K inhibitors.
This polyclonal knockout cell product is suited for a broad range of applications in oncology and signal transduction research. It can be employed in isoform-specific functional studies using western blotting for phospho-AKT (Ser473), phospho-S6, and phospho-GSK3?? to monitor pathway activity. Cell viability assays (MTT or CellTiter-Glo) and soft agar colony formation assays enable assessment of anchorage-dependent and -independent growth. Migration and invasion transwell assays facilitate analysis of metastatic potential, while xenograft tumor models in immunocompromised mice permit evaluation of in vivo tumorigenicity and drug responses. Additionally, these cells serve as an isogenic platform for AKT inhibitor sensitivity profiling with agents such as MK-2206 or ipatasertib, and for genome-wide CRISPR screens to identify synthetic lethal partners. For additional information or technical support, please contact Ascent Research.
