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

## TL;DR

This study shows that HES1 protein oscillations control dormancy and reactivation in breast cancer cells, offering a new way to prevent cancer recurrence.

## Contribution

The study reveals that HES1 oscillations are essential for cell cycle reentry in ER+ breast cancer cells.

## Key findings

- HES1 oscillations change during dormancy and reactivation in ER+ breast cancer cells.
- Disrupting HES1 oscillations prevents cell cycle reentry and induces cell death.
- Maintaining HES1 levels during reentry upregulates p21 and impairs cell cycle progression.

## Abstract

Breast cancer can recur years after initial treatment due to reactivation of dormant tumor cells. Understanding how these cells exit dormancy is crucial for preventing relapse. We investigated HES1, a transcription factor with rhythmic protein oscillations, and its role in regulating quiescence in estrogen receptor-positive (ER+) breast cancer cells. Using live-cell imaging and a reversible cell cycle arrest model, we show that HES1 dynamics change during dormancy and reactivation, and that disrupting these oscillations prevents cell cycle reentry and induces cell death. These findings reveal HES1 protein dynamics as a potential therapeutic vulnerability and highlight a promising strategy to target dormant cancer cells to prevent their reactivation.

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.