Abstract The PI3K pathway plays a pivotal role in regulating cell proliferation and survival of cancer cells including breast cancer. This pathway is frequently hyperactivated in cancer due to genetic mutations, amplifications, or functional alterations in key components such as PIK3CA, PTEN, and AKT. Over the years, several PI3K inhibitors have been developed and approved for the treatment of breast cancer. However, drug tolerance and adaptive responses often emerge, leading to reactivation of the PI3K pathway and ultimately limiting the clinical efficacy of these inhibitors. This study aims to elucidate the mechanism underlying resistance to PI3K inhibitors, and to understand how tumor cells evade PI3K inhibition. At this purpose, we developed a model of acquired resistant to taselisib, a selective PI3KCA inhibitor, by generating resistant subline (MCF7 Tas-r) from MCF7 human breast cancer cells through stepwise exposure of MCF7 parental cells (MCF7-Par) to increasing concentrations of taselisib (ranging from 50 nM to 500 nM). Then, since phosphorylation is one of the major posttranslational modification of proteins that regulates several signaling pathways, including PI3K signaling, we performed phosphoproteomic profiling followed by functional enrichment analysis in order to identify the molecular alterations associated with acquired resistance. Our analysis identified 253 differentially phosphorylated proteins (196 upregulated and 57 downregulated) in resistant cells compared to parental cells. Functional enrichment analysis revealed significant enrichment of pathways related to autophagy regulation, as “mTOR signaling,” and “AMPK signaling,” highlighting autophagy as a key process altered in resistant cells. Notably, SQSTM1/p62, a critical cargo adaptor in autophagy, emerged as a central hub, together with AMPK, reinforcing the hypothesis that autophagy activation contributes to taselisib resistance. These findings were validated by Western blot analysis, which confirmed the upregulation of key proteins involved in PI3K and autophagy signaling in MCF7 Tas-r cells compared to parental cells, including p-ULK1, SQSTM1/p62, ATG5, and Beclin-1. Furthermore, we observed increased levels of phosphorylated AMPK (p-AMPK) along with downregulation of phosphorylated mTOR (p-mTOR), suggesting that AMPK activation may contribute to mTOR suppression and promote autophagy induction in resistant cells. Given the central role of autophagy in resistance, we tested the therapeutic potential of chloroquine (CQ), an anti-malarial agent known to block autophagy by inhibiting autophagosome-lysosome fusion. CQ enhanced the antitumor activity of taselisib in vitro, as demonstrated by cell viability and clonogenic assays. Taken together, our findings suggest that autophagy activation serves as a key adaptive mechanism driving acquired resistance to PI3K inhibitors such as taselisib in breast cancer. Targeting autophagy-related pathways may represent a promising therapeutic strategy to overcome or delay resistance and enhance the clinical efficacy of PI3K-targeted treatments. Citation Format: B. Pucci, R. Lombardi, T. Moccia, R. Migliorino, C. Ciardiello, E. Di Gennaro, A. Budillon, M. De Laurentiis, A. Leone, S. Cocco. Mechanisms of Acquired Resistance to PI3K Inhibitors in Breast Cancer: The Central Role of Autophagy abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-11-11.
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