CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

A cell cycle centric view of tumour dormancy.

Tumour dormancy and recurrent metastatic cancer remain the greatest clinical challenge for cancer patients. Dormant tumour cells can evade treatment and detection, while retaining proliferative potential, often for years, before relapsing to tumour outgrowth. Cellular quiescence is one mechanism that promotes and maintains tumour dormancy due to its central role in reducing proliferation, elevating cyto-protective mechanisms, and retaining proliferative potential. Quiescence/proliferation decisions are dictated by intrinsic and extrinsic signals, which regulate the activity of cyclin-dependent kinases (CDKs) to modulate cell cycle gene expression. By clarifying the pathways regulating CDK activity and the signals which activate them, we can better understand how cancer cells enter, maintain, and escape from quiescence throughout the progression of dormancy and metastatic disease. Here we review how CDK activity is regulated to modulate cellular quiescence in the context of tumour dormancy and highlight the therapeutic challenges and opportunities it presents.

An image-based screen for secreted proteins involved in breast cancer G0 cell cycle arrest.

Secreted proteins regulate the balance between cellular proliferation and G0 arrest and therefore play important roles in tumour dormancy. Tumour dormancy presents a significant clinical challenge for breast cancer patients, where non-proliferating, G0-arrested cancer cells remain at metastatic sites, below the level of clinical detection, some of which can re-enter proliferation and drive tumour relapse. Knowing which secreted proteins can regulate entry into and exit from G0 allows us to manipulate their signalling to prevent tumour relapse. To identify novel secreted proteins that can promote breast cancer G0 arrest, we performed a secretome-wide, image-based screen for proteins that increase the fraction of cells in G0 arrest. From a secretome library of 1282 purified proteins, we identified 29 candidates that promote G0 arrest in non-transformed and transformed breast epithelial cells. The assay we have developed can be adapted for use in other perturbation screens in other cell types. All datasets have been made available for re-analysis and our candidate proteins are presented for alternative bioinformatic refinement or further experimental follow up.