In the developing vertebrate embryo, segmentation initiates through the formation of repeated segments, or somites, on either side of the posterior neural tube along the anterior to posterior axis. The periodicity of somitogenesis is regulated by a molecular oscillator, the segmentation clock, driving cyclic gene expression in the unsegmented paraxial mesoderm, from which somites derive. Three signaling pathways underlie the molecular mechanism of the oscillator: Wnt, FGF, and Notch. In particular, Notch has been demonstrated to be an essential piece in the intricate somitogenesis regulation puzzle. Notch is required to synchronize oscillations between neighboring cells, and is moreover necessary for somite formation and clock gene oscillations. Following ligand activation, the Notch receptor is cleaved to liberate the active intracellular domain (NICD) and during somitogenesis NICD itself is produced and degraded in a cyclical manner, requiring tightly regulated, and coordinated turnover. It was recently shown that the pace of the segmentation clock is exquisitely sensitive to levels/stability of NICD. In this review, we focus on what is known about the mechanisms regulating NICD turnover, crucial to the activity of the pathway in all developmental contexts. To date, the regulation of NICD stability has been attributed to phosphorylation of the PEST domain which serves to recruit the SCF/Sel10/FBXW7 E3 ubiquitin ligase complex involved in NICD turnover. We will describe the pathophysiological relevance of NICD-FBXW7 interaction, whose defects have been linked to leukemia and a variety of solid cancers.

Despite extraordinary advances that have been achieved in the last few decades, cancer continues to represent a leading cause of mortality worldwide. Lethal cancer types ultimately become refractory to standard of care approaches; thus, novel effective treatment options are desperately needed. Tumor Treating Fields (TTFields) are an innovative non-invasive regional anti-mitotic treatment modality with minimal systemic toxicity. TTFields are low intensity (1-3 V/cm), intermediate frequency (100-300 kHz) alternating electric fields delivered to cancer cells. In patients, TTFields are applied using FDA-approved transducer arrays, orthogonally positioned on the area surrounding the tumor region, with side effects mostly limited to the skin. The precise molecular mechanism of the anti-tumor effects of TTFields is not well-understood, but preclinical research on TTFields suggests it may act during two phases of mitosis: at metaphase, by disrupting the formation of the mitotic spindle, and at cytokinesis, by dielectrophoretic dislocation of intracellular organelles leading to cell death. This review describes the mechanism of action of TTFields and provides an overview of the most important in vitro studies that investigate the disruptive effects of TTFields in different cancer cells, focusing mainly on anti-mitotic roles. Lastly, we summarize completed and ongoing TTFields clinical trials on a variety of solid tumors.

All vertebrates share a segmented body axis. Segments form from the rostral end of the presomitic mesoderm (PSM) with a periodicity that is regulated by the segmentation clock. The segmentation clock is a molecular oscillator that exhibits dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanism by which NICD stability is regulated in this context is unclear. We identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle‐dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations and an increase in somite size.

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non-small cell lung cancer (NSCLC). Griseofulvin (GF; FDA approved treatment) inhibits CC, and combined with radiation therapy (RT) resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and RT resulted in a significant tumor growth delay. Both GF and RT treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased RT efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, RT and immunotherapy could be a promising strategy to treat NSCLC.

A striking example of coupling between growth and form occurs during the segmentation of the vertebrate embryo. During segmentation, pairs of segments, one on either side of the anterior-posterior axis, bud off from the presomitic mesoderm (PSM) at regular intervals in time. In the clock and wavefront model, a multicellular oscillator regulates the time at which the next pair of segments form whilst a wavefront regulates their spatial location. In most mathematical models of segmentation, it is assumed that cells in the PSM are oscillators with a constant characteristic frequency. Based on recent experimental findings, here we propose a model in which the natural oscillation frequency of each PSM cell is a function of its position in the cell cycle. Given adequate oscillator coupling and that cells in the PSM are randomly distributed in the cell cycle, we find that the emergent oscillator period is a weighted average of the constituent oscillator frequencies with the weightings dependent on the fraction of cells in a given cell cycle state. Here, we show that such a model can allow for coupling between pattern formation and growth rate in PSM tissue.

<div>Abstract<p>Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non–small cell lung cancer (NSCLC). Griseofulvin (GF; FDA-approved treatment) inhibits CC, and combined with radiation treatment (RT), resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability <i>in vitro. In vivo</i>, GF treatment was well tolerated by mice, and the combined therapy of GF and radiation treatment resulted in a significant tumor growth delay. Both GF and radiation treatment also induced the generation of micronuclei (MN) <i>in vitro</i> and <i>in vivo</i> and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased radiation treatment efficacy in lung cancer preclinical models <i>in vitro</i> and <i>in vivo</i>. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.</p></div>

<p>Figure S1 shows basal centrosome amplification in cell lines. Figure S2 shows the effect of GF on cell viability. Figure S3 shows HSET expression in cell lines. Figure S4 shows the effect of GF on clonogenic potential after RT. Figure S5 shows the induction of micronuclei after GF and/or RT. Figure S6 shows the expression of IFN-β after GF and/or RT.</p>

<p>Supplementary Materials & Methods</p>