Role of p27kip1 in CDK4/6 inhibitor-induced senescence in Breast Cancer

Hormone receptor positive breast cancer (HR+ BC) is the most frequent breast cancer subtype and relies on hormonal therapy and CDK4/6 inhibitors (CDK4/6i) as common treatments. Although initially effective, resistance to CDK4/6i frequently emerges and the comprehension of the mechanisms underlying this resistance represents an urgent clinical need. A known effect of the exposure to CDK4/6i is the induction of senescence, a non-proliferative yet metabolically active state, associated with secretion of inflammatory cytokines and chemokines collectively known as senescence-associated secretory phenotype (SASP). The SASP profoundly influences the tumor microenvironment and may promote residual tumor cell proliferation and therapeutic resistance. p27kip1 (p27) is a cell-cycle inhibitor that directly suppresses CDK4/6 activity. Notably, p27 is frequently downregulated in HR+ BC, where CDKN1B acts as a driver gene. Recent structural studies have proposed a role of p27 in modulating the response to palbociclib. We hypothesized that p27 downmodulation in HR+ BC cells could alter long-term responses to CDK4/6i and influence the induction of senescence. To test this hypothesis, we set up an experimental approach using T47D HR+ BC cells, either wild type (p27WT) or knockout for p27 (p27KO). Cells were treated with palbociclib for 72 hours, then released and replated in drug-free conditions and analyzed at days 1, 10, 20 post-drug washout to assess the induction of senescence and cell cycle re-entry. To further understand the adaptive behavior of p27KO cells exposed to palbociclib, at days 10 and 20 post washout, we performed functional assays, such as colony formation, soft agar, and mammosphere assays to evaluate proliferation and stem-like properties. Our results indicated that p27KO cells displayed a more pronounced senescent phenotype than p27WT cells, as evidenced by increased SA--Gal activity, a hallmark of senescence, and SASP secretion. Interestingly, we observed a concomitant increase in cell-cycle regulators, such as cyclin D1 and cyclin E, suggesting that p27KO cells could at least in part bypass palbociclib-induced cell cycle arrest. Functional assays supported this notion: despite exhibiting a stronger senescent phenotype, p27KO cells retained a greater capacity for growing in colony and soft-agar assays and displayed enhanced stem-like features compared with p27WT cells. Mechanistically, p27KO cells following palbociclib exposure exhibited hyperactivation of mTOR pathway and reduced sensitivity to mTORC1 inhibitor rapamycin. This suggests that mTOR signaling activation may confer p27KO cells the ability to bypass the palbociclib-induced cell cycle arrest, promoting cell survival and growth. Given the known function of mTOR in autophagy and cell metabolism, we explored whether these processes were also affected. While autophagy appeared unchanged, metabolomic profiling revealed a metabolic shift towards a OXPHOS-dependent phenotype with respect to glycolysis in p27KO cells. To explore the translational relevance of our in vitro findings, we took advantage of an HR+ BC PDX model with acquired resistance to palbociclib developed in our lab. Whole transcriptome RNA sequencing of the palbociclib-resistant cohort revealed enrichment of proliferation-associated pathways, including mTORC1 signaling, and of p27 degradation pathway, supporting a pivotal role of p27 downmodulation in the development of CDK4/6i resistance in HR+ BC. Overall, our data identify p27 expression as a critical determinant of CDK4/6i efficacy in HR+ BC. Our evidences indicate that p27 loss promotes CDK4/6i resistance through mTOR pathway activation, leading to a senescent-like state that fosters tumor adaptation and aggressiveness, potentially via SASP-mediated effects on neighboring cells. These findings highlight a possible role for p27 as a biomarker of therapeutic response and could reveal new therapeutic vulnerabilities to overcome CDK4/6i resistance.

Hormone receptor positive breast cancer (HR+ BC) is the most frequent breast cancer subtype and relies on hormonal therapy and CDK4/6 inhibitors (CDK4/6i) as common treatments. Although initially effective, resistance to CDK4/6i frequently emerges and the comprehension of the mechanisms underlying this resistance represents an urgent clinical need. A known effect of the exposure to CDK4/6i is the induction of senescence, a non-proliferative yet metabolically active state, associated with secretion of inflammatory cytokines and chemokines collectively known as senescence-associated secretory phenotype (SASP). The SASP profoundly influences the tumor microenvironment and may promote residual tumor cell proliferation and therapeutic resistance. p27kip1 (p27) is a cell-cycle inhibitor that directly suppresses CDK4/6 activity. Notably, p27 is frequently downregulated in HR+ BC, where CDKN1B acts as a driver gene. Recent structural studies have proposed a role of p27 in modulating the response to palbociclib. We hypothesized that p27 downmodulation in HR+ BC cells could alter long-term responses to CDK4/6i and influence the induction of senescence. To test this hypothesis, we set up an experimental approach using T47D HR+ BC cells, either wild type (p27WT) or knockout for p27 (p27KO). Cells were treated with palbociclib for 72 hours, then released and replated in drug-free conditions and analyzed at days 1, 10, 20 post-drug washout to assess the induction of senescence and cell cycle re-entry. To further understand the adaptive behavior of p27KO cells exposed to palbociclib, at days 10 and 20 post washout, we performed functional assays, such as colony formation, soft agar, and mammosphere assays to evaluate proliferation and stem-like properties. Our results indicated that p27KO cells displayed a more pronounced senescent phenotype than p27WT cells, as evidenced by increased SA--Gal activity, a hallmark of senescence, and SASP secretion. Interestingly, we observed a concomitant increase in cell-cycle regulators, such as cyclin D1 and cyclin E, suggesting that p27KO cells could at least in part bypass palbociclib-induced cell cycle arrest. Functional assays supported this notion: despite exhibiting a stronger senescent phenotype, p27KO cells retained a greater capacity for growing in colony and soft-agar assays and displayed enhanced stem-like features compared with p27WT cells. Mechanistically, p27KO cells following palbociclib exposure exhibited hyperactivation of mTOR pathway and reduced sensitivity to mTORC1 inhibitor rapamycin. This suggests that mTOR signaling activation may confer p27KO cells the ability to bypass the palbociclib-induced cell cycle arrest, promoting cell survival and growth. Given the known function of mTOR in autophagy and cell metabolism, we explored whether these processes were also affected. While autophagy appeared unchanged, metabolomic profiling revealed a metabolic shift towards a OXPHOS-dependent phenotype with respect to glycolysis in p27KO cells. To explore the translational relevance of our in vitro findings, we took advantage of an HR+ BC PDX model with acquired resistance to palbociclib developed in our lab. Whole transcriptome RNA sequencing of the palbociclib-resistant cohort revealed enrichment of proliferation-associated pathways, including mTORC1 signaling, and of p27 degradation pathway, supporting a pivotal role of p27 downmodulation in the development of CDK4/6i resistance in HR+ BC. Overall, our data identify p27 expression as a critical determinant of CDK4/6i efficacy in HR+ BC. Our evidences indicate that p27 loss promotes CDK4/6i resistance through mTOR pathway activation, leading to a senescent-like state that fosters tumor adaptation and aggressiveness, potentially via SASP-mediated effects on neighboring cells. These findings highlight a possible role for p27 as a biomarker of therapeutic response and could reveal new therapeutic vulnerabilities to overcome CDK4/6i resistance.