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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 in vitro. In vivo, 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) 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 radiation treatment 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, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

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.

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 in vitro. In vivo, 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) 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 radiation treatment 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, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

Supplementary Materials & Methods

Mitosis is a delicate event that must be executed with high fidelity to ensure genomic stability. Recent work has provided insight into how mitotic errors shape cancer genomes by driving both numerical and structural alterations in chromosomes that contribute to tumor initiation and progression. Here, we review the sources of mitotic errors in human tumors and their effect on cell fitness and transformation. We discuss new findings that suggest that chromosome missegregation can produce a proinflammatory environment and impact tumor responsiveness to immunotherapy. Finally, we survey the vulnerabilities exposed by cell division errors and how they can be exploited therapeutically.

International audience; Multiciliated cells (MCCs) amplify large numbers of centrioles that convert into basal bodies, which are required for producing multiple motile cilia. Most centrioles amplified by MCCs grow on the surface of organelles called deuterosomes, whereas a smaller number grow through the centriolar pathway in association with the two parent centrioles. Here, we show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. Therefore, deuterosomes may have evolved to relieve, rather than supplement, the centriolar pathway during multiciliogenesis. Remarkably, MCCs lacking parent centrioles and deuterosomes also amplify the appropriate number of centrioles inside a cloud of pericentriolar and fibrogranular material. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize.

Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional genetic defects. We show that increasing centrosome number elevated tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive aneuploidy and the development of spontaneous tumors in multiple tissues. Tumors arising from centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship among centrosome amplification, genomic instability, and tumor development.

Multiciliated cells (MCCs) are a specialized population of postmitotic cells that are decorated with tens to hundreds of hairlike protrusions, termed motile cilia, that beat back and forth to direct fluid flow across an epithelium ( 1 ). MCCs line the respiratory tract, brain ventricles, and reproductive tracts of vertebrates and play a crucial role in tissue homeostasis; defects in the formation or movement of motile cilia can cause fertility defects, chronic respiratory infections, and/or a buildup of fluid in the brain. Despite their importance to human health, the pathways controlling the production of motile cilia in differentiating MCCs remain poorly understood. On page 803 of this issue, Al Jord et al. ( 2 ) shed light on this question by showing that multiciliated progenitor cells implement components of the mitotic cell cycle machinery to coordinate events that are required for motile ciliation and cellular differentiation, while avoiding cell division (mitosis).

The centrosome is a microtubule-organizing center that plays an important role during M-phase and cell division where it organizes the two poles of the bipolar microtubule spindle apparatus from which chromosomes are segregated. In contrast to most normal cells, the vast majority of lung cancers contain extra copies of centrosomes, and a large body of circumstantial evidence links extra centrosomes to the development of cancer. Cells with supernumerary centrosomes form multipolar mitotic spindles, which, if not corrected, lead to lethal multipolar divisions or “mitotic catastrophe.” To overcome this, lung cancer cells cluster their centrosomes into two spindle poles, enabling tumor cells to survive. Thus, inhibition of centrosome clustering (CCi) can force cancer cells into multipolar divisions, lead to chromosomal instability, make cancer cells more sensitive to radiation treatment, and eventually result in the selective killing of cancer cells. At the same time, this approach should spare healthy tissues with normal centrosome numbers, resulting in a favorable therapeutic index. To examine the relationship between CCi and radiation-treatment sensitivity, we first treated three NSCLC cell lines (A549, H460 and H358) and one immortalized primary human bronchial epithelial cell (HBEC) with griseofulvin—a centrosomal clustering inhibitor. Immunofluorescent staining for pericentrin and alpha-tubulin, we observed a significant increase in the number of cells with multipolar spindles in NSCLC cells compared to HBECs (40-80% vs. 15%). Next, we examined the ability of griseofulvin to radiosensitize these cells to radiation treatment. By using clonogenic assays, we saw significantly less clonogenic potential of NSCLC cells following radiation treatment after exposure to 30 uM of griseofulvin, but griseofulvin did not radiosensitize HBECs. To further confirm our results, we genetically targeted KIFC1, a member of kinesin-14 family of motor proteins that have been shown to be essential for clustering centrosomes in cancer cells, but are not required for division in normal cells. In our experiments, knockdown of KIFC1 expression with siRNA also significantly sensitized NSCLC cells to radiation treatment. Finally, when we treated the NSCLC cells with griseofulvin, we saw a significant increase in the formation of micronuclei in cancer cells. Recently, it has been shown that micronuclei are important sources of immunostimulatory DNA via activation of the cGAS-STING pathway. Therefore, inhibition of CC could potentially augment immunogenicity and increase efficiency of immunotherapy (IMT). In conclusion, inhibition of CC resulted in the formation of multipolar spindle in NSCLCs and further sensitized NSCLCs to radiation treatment. CC is not typically required for normal cells, and thus CCi may be a more specific and perhaps less toxic therapy for NSCLCs. The potential of CCi to enhance IMT is under investigation. Citation Format: Hailun Wang, Katriana Nugent, Matthew Ballew, Ghali Lemtiri-Chlieh, Natasha Raman, Michelle Levine, Andrew Holland, Phuoc Tran. Centrosome clustering inhibition as a novel strategy to sensitize non-small cell lung cancer to radiation treatment and immunotherapy [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B12.

Plk4 family kinases control centriole assembly. Plk4s target to mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or asterless/Cep152. Here, we report a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular β-sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a new crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over asterless-derived acidic regions. Overall, our findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly.