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Centrosome amplification primes ovarian cancer cells for apoptosis and potentiates the response to chemotherapy [1]

['Frances Edwards', 'Biology Of Centrosomes', 'Genetic Instability', 'Institut Curie', 'Psl Research University', 'Cnrs Umr', 'Paris', 'Giulia Fantozzi', 'Anthony Y. Simon', 'Jean-Philippe Morretton']

Date: 2024-09

Centrosome amplification is a feature of cancer cells associated with chromosome instability and invasiveness. Enhancing chromosome instability and subsequent cancer cell death via centrosome unclustering and multipolar divisions is an aimed-for therapeutic approach. Here, we show that centrosome amplification potentiates responses to conventional chemotherapy in addition to its effect on multipolar divisions and chromosome instability. We perform single-cell live imaging of chemotherapy responses in epithelial ovarian cancer cell lines and observe increased cell death when centrosome amplification is induced. By correlating cell fate with mitotic behaviors, we show that enhanced cell death can occur independently of chromosome instability. We identify that cells with centrosome amplification are primed for apoptosis. We show they are dependent on the apoptotic inhibitor BCL-XL and that this is not a consequence of mitotic stresses associated with centrosome amplification. Given the multiple mechanisms that promote chemotherapy responses in cells with centrosome amplification, we assess such a relationship in an epithelial ovarian cancer patient cohort. We show that high centrosome numbers associate with improved treatment responses and longer overall survival. Our work identifies apoptotic priming as a clinically relevant consequence of centrosome amplification, expanding our understanding of this pleiotropic cancer cell feature.

Data Availability: All relevant data are within the paper and its Supporting Information file. Data underlying this study are from the Biological Resource Center (BRC) of Institut Curie (certification number: 2009/33837.4; AFNOR NF S 96 90). The access to the tumor cohort is restricted and subjected to the ethical evaluation by the Institut Curie. To inquiry about this cohort please consult https://institut-curie.org/ , or contact the head of the pathology department at Institut Curie: Dr Anne Salomon: [email protected] .

Here, we chose to study how centrosome amplification influences the response to combined Carboplatin and Paclitaxel in the context of epithelial ovarian cancer, a disease with poor clinical outcome related to late diagnosis, and frequent relapse [ 36 ]. Centrosome amplification is observed in ovarian cancer cell lines, and we recently also characterized its occurrence in situ in patient samples [ 5 ]. We use an inducible PLK4 overexpression system in ovarian cancer cell lines to induce centrosome amplification in isogenic backgrounds. We perform single-cell live-imaging of cells to assess the correlations between mitotic behaviors and cell fate during chemotherapy. We show that centrosome amplification potentiates the response to combined Carboplatin and Paclitaxel via multiple mechanisms. Beyond multipolar divisions associated with Paclitaxel exposure, we found that centrosome amplification also enhances cell death independently of mitotic behaviors. We show that centrosome amplification, although well tolerated by ovarian cancer cells, leads to mitochondria outer membrane permeabilization (MOMP) priming. We assess the level of centrosome amplification in a previously characterized ovarian cancer patient cohort and observe an association between high centrosome numbers and the patient time to relapse as well as their overall survival. Together, our work shows for the first time that centrosome amplification can synergize with combined chemotherapy, advancing our understanding of its consequences in cancer.

Carboplatin and Paclitaxel are cytotoxic agents used in treatment of various solid cancers, and their combination is the standard of care for treatment of advanced stage epithelial ovarian cancer patients [ 26 ]. Carboplatin induces DNA damage and Paclitaxel stabilizes microtubules leading to cell death via mitotic catastrophe which is defined as death during or following abnormal mitosis [ 27 , 28 ]. Despite the central role of centrosomes in spindle assembly, how centrosome amplification influences the response to combined Carboplatin and Paclitaxel remains unexplored. Paclitaxel has been shown to induce multipolar divisions [ 29 ] and this can be increased by centrosome amplification [ 30 ]. The impact of centrosome amplification on the response to DNA damaging agents has however not been explored despite centrosomes regulating multiple signaling pathways that could influence chemotherapy responses [ 31 – 35 ]. Multiple consequences of centrosome amplification could therefore synergize with combined Carboplatin and Paclitaxel to induce efficient cancer cell elimination.

Centrosomes are the major microtubule organizing centers (MTOCs) in proliferating animal cells, whose structure and number are tightly regulated during the cell cycle [ 1 ]. The centrosome is duplicated during S-phase in a PLK4 kinase-dependent manner and the 2 centrosomes contribute to the timely and functional assembly of a bipolar spindle during mitosis. In cancer cell lines, centrosome structural and numerical defects are common [ 2 ] and in particular centrosome amplification—more than 2 centrosomes per cell—has also been observed in situ in tumor samples [ 3 – 5 ]. Cells with centrosome amplification perform bipolar mitosis via centrosome clustering mechanisms during spindle assembly [ 6 – 10 ]. Centrosome amplification is nevertheless associated with chromosome instability [ 11 , 12 ] and increased invasive behaviors [ 13 – 15 ]. As first postulated by T. Boveri [ 16 ], centrosome amplification can drive tumorigenesis in vivo [ 7 , 17 – 19 ]. Defects in centrosome clustering capacity are associated with lethal multipolar divisions [ 8 , 11 , 20 ], motivating the search for inhibitors that limit centrosome clustering and eliminate cells with centrosome amplification [ 20 – 25 ].

Results

Centrosome amplification primes for MOMP independently of chromosome instability or lengthened mitosis Centrosome amplification induces an increase in chromosome instability and a spindle-assembly checkpoint-dependent extension of mitosis duration [7,8,11], both of which we observed in PLK4OE OVCAR8 cells (S1E and S1G Fig). Apoptotic priming and in particular sensitization to BCL-XL inhibition has previously been linked to mitotic defects. In particular chromosome instability, micronuclei formation and cGAS/STING signaling can drive a transcriptional response that drives apoptosis or priming [43,44]. Alternatively, extended mitotic duration can lead to the proteosomal degradation of anti-apoptotic BCL2 family proteins, leading to BCL-XL sensitization [45–48]. We were therefore interested in determining if apoptotic priming observed in response to centrosome amplification is caused by cumulated mitotic stress in these cells. First, we aimed to better characterize the mitotic stress induced by centrosome amplification in the already chromosomally instable OVCAR8 cell line (S1G Fig). We used single-cell DNA-sequencing to assess karyotype heterogeneity and observed scores of 0,119 in PLK4Ctl, 0,137 in PLK4OE cells, and 0,283 in PLK4Ctl cells treated with 1 μm of the MPS1 inhibitor AZ3146 as a positive control of chromosome mis-segregation (S6A Fig). PLK4OE therefore only mildly increased aneuploidy, in line with levels of chromosome-mis-segregation observed by time-lapse imaging (S6B Fig). We also assessed the extent of mitotic lengthening induced in PLK4OE cells (S6C Fig) and observed it was mild (median = 60 min in PLK4OE and median = 35 min in PLK4Ctl) compared to that induced by low doses of the CENP-E inhibitor GSK923295 (median = 100 min at 30 nM and 175 min at 35 nM), despite levels of chromosome mis-segregation being similar (S6B Fig). Mitotic stress is therefore mild in PLK4OE compared to the other perturbations we tested, but we were nevertheless interested in determining if it contributes to apoptotic priming. We were not able to reduce mitotic duration in PLK4OE cells via spindle assembly checkpoint inhibition without inducing a strong increase in multipolar divisions, so we used MPS1 and CENP-E inhibition to mimic mitotic stress observed in PLK4OE. We pretreated PLK4Ctl cells with inhibitors during 72 h before adding WEHI-539 for an additional 24 h. In PLK4OE cells, this lead to 33% Annexin V positive cells, whereas it only induces 9% in response to MPS1 inhibition (S6D Fig), making it unlikely that priming occurs in response to chromosome instability in PLK4OE. In CENP-E inhibition pretreated cells at 30 nM and 35 nM, WEHI-539 induces 12% and 25% Annexin V cells, respectively, in line with mitotic lengthening inducing priming [48]. Importantly however, 35 nM CENP-E inhibition pretreatment already induces 12% Annexin V positive cells which is considerable compared to the 4% observed in PLK4OE (S6D Fig), and most likely is explained by the extensive mitotic lengthening observed in response to 35 nM CENP-E inhibition (S6C Fig). This results in the ratio of cell death induced by WEHI-539 relative to the basal observed level of cell death to be comparable in PLK4Ctl cells and CENP-E inhibition pretreated cells (around 2-fold). In contrast, the ratio of cell death was much higher and close to 8-fold increase upon PLK4OE (Fig 4I). Therefore, the priming induced in PLK4OE stands out from that induced by other sources of mitotic stress in that PLK4OE cells are viable but strongly dependent on BCL-XL. These results also suggest that the combination of chromosome instability and mitotic lengthening is not the major contributor to MOMP priming upon centrosome amplification in OVCAR8 cells. To identify transcriptomic signatures that may influence cell death responses in cells with extra centrosomes, we used bulk RNAseq comparing PLK4OE and PLK4Ctl OVCAR8 cells. A strong inflammatory signature in PLK4OE (S6E Fig) was identified, and we also observed STING phosphorylation (S6F Fig), suggesting that the cGAS/STING pathway may shape the transcriptional response to centrosome amplification. We were therefore interested in directly testing if cGAS/STING signaling might contribute to priming, although this seemed unlikely as CENP-E and MPS1 inhibition also activate STING (S6F Fig) but are not associated with priming. We used a bulk LentiCRISPR knock-out approach of STING, but observed no influence on PLK4OE cells sensitivity to WEHI-539 (S6G and S6H Fig). We therefore identify that centrosome amplification in OVCAR8 leads to MOMP priming which is revealed by a selective sensitization to the BCL-XL inhibitor WEHI-539. Comparing with other mitotic perturbations, we conclude that the centrosome amplification associated priming is independent of mitotic lengthening and chromosome instability.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002759

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