Evaluation of novel and established inhibitors of the PI3K/AKT pathway in PTEN-Null breast cancer cell lines.

Abstract

Introduction: The PI3K/AKT pathway is a critical signalling cascade involved in regulating cell survival, proliferation, and metabolism, with its dysregulation often implicated in cancer initiation and progression. PTEN-null or PI3K-mutant cancers exhibit hyperactivation of AKT, presenting a therapeutic challenge. This study aimed to evaluate the effects of novel PIP analogues, specifically PIP-Dec, on the PI3K/AKT pathway and compare their efficacy to established PI3K/AKT inhibitors. Through cytotoxicity assays, molecular analyses, and kinase activity profiling, the research assessed their impact on key components of the pathway and their potential therapeutic relevance. Methods: Cytotoxicity of PIP analogues was assessed using SRB assays on PTEN-null (MDA-MB-468, HCC70) and PTEN-wild-type (MDA-MB-231) breast cancer cell lines. Western blotting and TR-FRET were used to investigate the inhibition of AKT phosphorylation, while immunofluorescence studies analysed AKT localization to the cell membrane. High-throughput PamGene kinase profiling was employed to determine the broader impact of PIP-Dec on kinase activity, comparing its effects to those of AZD-5363 and Pictilisib. Statistical analysis and PCA clustering were used to evaluate consistency across assays. Results: PIP-Dec demonstrated limited selectivity for PTEN-null cell lines, exhibiting enhanced cytotoxicity compared to PTEN wild-type lines, albeit requiring higher concentrations than established PI3K/AKT inhibitors. Western blot and TR-FRET confirmed its ability to inhibit AKT phosphorylation, while immunofluorescence showed its disruption of AKT localization to the cell membrane. PamGene profiling revealed distinct kinase modulation by PIP-Dec, including activation of PRKACA and PRKG2 and inhibition of NUAK1 and COQ8A. Compared to Pictilisib’s broader kinase effects through upstream PI3K inhibition, AZD-5363 showed greater similarity to PIP-Dec’s downstream actions, as highlighted by PCA clustering. Conclusion: This study underscores the potential of PIP-Dec as a novel modulator of the PI3K/AKT pathway, demonstrating effects comparable to established inhibitors in specific contexts. The results provide valuable insights into the activity of PIP analogues, highlighting their therapeutic relevance in PTEN-null cancers, such as TNBC. Future research should aim to explore the cytotoxic effects of PIP analogues further by employing assays such as flow cytometry and caspase activity measurements. Additionally, extending timepoints, validating kinase interactions, and investigating combinatorial therapies will be crucial for optimizing these molecules towards potential clinical utility.

Date of Award	14 Mar 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sam Butterworth (Main Supervisor) & Katie Finegan (Co Supervisor)