HES1 oscillations are required for cell cycle progression and re-entry in oestrogen receptor positive breast cancer cells

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumour cells (DTCs) and remains a major clinical challenge, particularly in oestrogen receptor positive (ER⁺) tumours. This process is underpinned by regulation of the cell cycle machinery that controls quiescence maintenance and exit. HES1, a Notch pathway transcription factor, regulates key cell cycle genes and has been shown to demonstrate protein expression oscillations which are crucial to its function. Here, we sought to establish whether HES1 oscillations may regulate ER+ cancer cell quiescence and reactivation. To investigate this, we developed a fundamental in vitro model of cell cycle arrest and re-entry based on reversible CDK4/6 inhibition (CDK4/6i), compatible with quantitative single-cell live-imaging of an endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited ~24-hour protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to G1/S. During CDK4/6i-mediated arrest the ~24h HES1 oscillation was lost, HES1 levels were maintained at a moderately higher level and HES1 exhibited smaller dips. Similar changes were observed in unperturbed, spontaneously quiescent cells. Following release from CDK4/6i and cell cycle re-entry, these alterations were reversed and the characteristic G1/S HES1 dip was observed. Preventing this dip at the point of release, by inducibly sustaining HES1 with a Tet-On system, impeded cell cycle re-entry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumour cells.