HES1 oscillations are required for cell cycle reentry in oestrogen receptor–positive breast cancer cells

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumor cells (DTCs) and remains a major clinical challenge, particularly in estrogen receptor-positive (ER⁺) tumors. 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. 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 reentry based on reversible CDK4/6 inhibition (CDK4/6i) with palbociclib, compatible with quantitative single-cell live-imaging of a knock-in endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited ~24 h protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to S-Phase. During CDK4/6i-mediated arrest, the ~24 h 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 reentry, 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, upregulated the cell cycle inhibitor p21, impeded cell cycle reentry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumor cells.