Abstract 6050: Phosphorylated p27 transcriptionally co-regulates p65 and STAT3 to activate pro-oncogenic tumor inflammation

Abstract Breast cancer is the leading cause of cancer-related mortality in women. Breast cancer stem cells (CSCs) contribute to therapy resistance in part by altering the tumor microenvironment (TME) to drive cancer promoting inflammation and immune evasion. Our prior work revealed that the cell cycle inhibitor, p27 promotes CSC expansion when phosphorylated at T157 and T198 (p27pTpT) by PI3K-activated kinases. This occurs through p27/STAT3 recruitment to promoter sites to induce CSC driver genes. p27pTpT activates stem cell driver genes and also drives inflammatory signaling. New work shows p27pTpT promotes p65 (NFκB subunit) activation. Here we investigated the hypothesize that p27pTpT drives cancer-promoting inflammation through NF-κB activation, contributing to a pro-tumor inflammatory TME that supports immune evasion and therapy resistance. We used triple negative BC models MDA-MB-231 and 1833 with low and high metastatic ability, respectively. Cyclin-CDK-defective phosphomimetic mutant of p27 (p27CK-DD) was overexpressed in 231 cells and p27 was also knocked down in 1833 cells. Combined ATAC-seq, RNA-seq, and p27 ChIP-seq showed p27 increases chromatin accessibility to upregulate genes associated with immune-related pathways, including NFκB, TGF-β, SMAD, and interleukin signaling. This p27pTpT-dependent increase in chromatin accessibility leads to upregulation of pro-inflammatory genes and mediators of immune evasion including CCL2 and PDL1. ChIP-qPCR validated that p27 promotes p65 and STAT3 recruitment to these promoter sites in p27-activated 231p27CK-DD and 1833. p27/p65/STAT3 chromatin recruitment fell with p27 depletion and by PI3K inhibition in 1833. To investigate how p27 regulates chromatin accessibility and immune-related gene expression, its potential interaction with the chromatin remodeling regulator BAF complex was assayed. p27CK-DD expression in 231 and the high levels of p27pTpT in 1833 cells upregulate BAF complex subunits (ARID1A, BRG1, SMARCC1) at RNA and protein levels. This effect was reduced by p27 knockdown and by PI3K inhibition. ChIP-qPCR confirmed p27/p65/STAT3 recruitment to ARID1A promoter, indicating a functional interplay between p27 and the BAF complex gene regulation. In addition to its recruitment to the promotes of BAF complex subunits and induction of their expression, p27 also associates with BAF complex. p27 immunoprecipitates contain BAF complex proteins with greater association seen in 231CK-DD and 1833 cells. p27/BAF complexes decreased upon PI3K inhibition. These findings support a model in which p27 enhances chromatin accessibility through upregulation of genes encoding BAF components and potentially by binding to and altering the BAF complex composition or stability. p27 also acts as a transcriptional coregulator with p65 (NF-κB) and STAT3 to drive pro-inflammatory genes and to induce the PDL1 mediator of immune evasion. Citation Format: Seyedeh Fatemeh Razavipour, Amir Bagheri, Jiaxiang Tao, Michael B. Atkins, Samir N. Khleif, Joyce M. Slingerland. Phosphorylated p27 transcriptionally co-regulates p65 and STAT3 to activate pro-oncogenic tumor inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6050.