Differential phase register of Hes1 oscillations with mitoses underlies cell-cycle heterogeneity in ER+ breast cancer cells

Abstract

Here, we study the dynamical expression of endogenously labelled Hes1, a transcriptional repressor implicated in controlling cell proliferation, to understand how cell-cycle length heterogeneity is generated in ER+ breast cancer cells. We find that Hes1 shows oscillatory expression and during the cell-cycle has a variable peak in G1, a trough around G1-S transition and a less variable second peak in G2/M. Compared to other subpopulations, the cell-cycle in CD44HighCD24Low cancer stem cells is longest and most variable. Most cells divide around the peak of the Hes1 expression wave but mitoses in slow dividing CD44HighCD24Low cells appear phase-shifted, resulting in a late-onset Hes1 peak in G1. The position, duration and shape of this peak, rather than the Hes1 expression levels, are good predictors of cell-cycle length. Diminishing Hes1 oscillations impairs proliferation, indicating their functional importance for efficient cell-cycle progression. We propose that the position of mitosis in relation to the Hes1 wave underlies cell-cycle length heterogeneity in cancer cell subpopulations.Significance statementTumours exhibit heterogeneities that are not due to mutations, including Cancer Stem Cells with different potencies. We show that the cancer stem cell state predisposed to dormancy in vivo has a highly variable and long cell-cycle. Using single-cell live-imaging for the transcriptional repressor Hes1 (a key molecule in cancer), we show a new type of oscillatory expression of Hes1 in all cells in the population. The most potent cancer stem cells tend to divide around the trough of the Hes1 oscillatory wave, a feature predictive of a long cell-cycle. A novel concept proposed here is that the position that a cell is with respect to the Hes1 wave when it divides is predictive of its prospective cell-cycle length and characteristic of its cellular sub-state.Abstract in picture

A spatialised agent-based model of NOTCH signalling pathway in Endothelial Cells predicts emergent heterogeneity due to continual dynamic phenotypic adjustments

“…Assuming that NOTCH signalling is sufficient to generate the heterogeneous maps we observed in vitro and in vivo, then NSH could emerge either due to generation of multiple stable phenotypes or alternatively instable, oscillating ones as reported previously (Hirata et al, 2002; Marinopoulou et al, 2021; Sabherwal et al, 2021; Shimojo et al, 2008; Yoshioka-Kobayashi et al, 2020). To evaluate these hypotheses in the context of EC monolayers we built a spatialised multiscale cellular model of NSP including Lateral Inhibition (LIb) and Lateral Induction (LId) as described in previous work (Boareto et al, 2016; Sprinzak et al, 2010).…”

“…Our previous results show that HES1 is repressed in few cells within a confluent EC monolayer suggesting that these cells could be licensed for proliferation (Chesnais et al, 2022). Furthermore, these results suggest a non-stable NSP dynamics in EC monolayers paralleling results from angiogenic EC (Ubezio et al, 2016) and breast cancer cell lines (Sabherwal et al, 2021).…”

“…We have previously demonstrated the value of measuring images of cultured EC to assess phenotypical and intracellular signalling heterogeneity in endothelial monolayers using our EC profiling tool (ECPT)(Chesnais et al, 2022). Our work using ECPT has established that quiescent EC within the same monolayer in vitro display heterogeneous levels of NICD (second messenger of NOTCH Signalling Pathway, NSP) and HES1 (NSP target gene), echoing results obtained in cancer cells (Sabherwal et al, 2021).…”

“…The current hypothesis is that mechanisms of lateral induction (LId) and lateral inhibition (LIb, trans-interactions) as well as ligand-receptors interactions in the same cell (cis-interactions, CI) (Boareto et al, 2016; Chesnais et al, 2022) coexist to induce differential signalling levels (e.g., levels of HES1) and phenotypes. Beyond the well-known stable alternating spatial patterns in NSP observed in several contexts (e.g., developing mouse retina neuroepithelium) (Formosa-Jordan et al, 2013), temporal oscillations in NSP (levels of NICD or NSP target genes) have been predicted in silico and observed experimentally (Marinopoulou et al, 2021; Sabherwal et al, 2021; Ubezio et al, 2016) The current mechanistic hypothesis is that NSP levels oscillations may result from cell autonomous negative autoregulation of HES1 transcription (Hirata et al, 2002). HES1 is a well-established NSP target gene in EC which differential expression regulates functions such as cell proliferation and inter-cellular junctional stability (Bentley et al, 2014; Fang et al, 2017; Fernández-Martín et al, 2012).…”

Dynamic switching of lateral inhibition spatial patterns

“…It is important to consider that perturbations could reasonably come from sources other than altered signalling in differentiating cells. Processes such as the extension and retraction of signalling protrusions, cell cycle variations in HES5/coupling strength, interkinetic nuclear migration and pulsatile Dll1 signalling are all reasonable candidates in contributing to the switching behaviour [25,28,50–52]. The DBP algorithm is general enough that it could reasonably be adapted to any of the listed alternatives, by altering the magnitude and duration of the perturbation, as well as the parameter it is applied to.…”