Non-Canonical Senescence Phenotype in Resistance to CDK4/6 Inhibitors in ER-Positive Breast Cancer

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have transformed the treatment landscape for estrogen receptor-positive (ER+) breast cancer, yet resistance remains a major clinical challenge. Although CDK4/6i induce G1 arrest and therapy-induced senescence (TIS), the exact nature of this senescent state and its contribution to resistance are not well understood. To explore this, we developed palbociclib- (2PR, 9PR, TPR) and abemaciclib- (2AR, 9AR, TAR) resistant ER+ breast cancer sublines through prolonged drug exposure over six months. Resistant cells demonstrated distinct phenotypic alterations, including cellular senescence, reduced mitochondrial membrane potential, and impaired glycolytic activity. Cytokine profiling and enzyme-linked immunosorbent assay (ELISA) validation revealed a non-canonical senescence-associated secretory phenotype (SASP) characterized by elevated growth/differentiation factor 15 (GDF-15) and serpin E1 (plasminogen activator inhibitor-1, PAI-1) and absence of classical pro-inflammatory interleukins, including IL-1α and IL-6. IL-8 levels were significantly elevated, but no association with epithelial–mesenchymal transition (EMT) was observed. Resistant cells preserved their epithelial morphology, showed no upregulation of EMT markers, and lacked aldehyde dehydrogenase 1-positive (ALDH1+) stem-like populations. Additionally, Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) was strongly upregulated in palbociclib-resistant cells. Together, these findings identify a distinct, non-canonical senescence phenotype associated with CDK4/6i resistance and may provide a foundation for identifying new vulnerabilities in resistant ER+ breast cancers through targeting SASP-related signaling.