Role In Spindle Assembly Checkpoint Research Articles

Abstract 2413: Glioblastoma stem-like cells are resistant to the negative effects of increased aneuploidy on in vitro survival and radiosensitivity

Abstract Glioblastomas (GBM) have long been classified as radioresistant tumors. Glioblastoma stem-like cells (GSCs) are a portion of GBM’s heterogenous cell population and are generally considered to be crucial drivers of tumorigenesis and radioresistance. As for most tumor cells, GSCs display a moderate level of chromosomal instability and aneuploidy. However, whether aneuploidy promotes or inhibits GSC survival and its effects on radiosensitivity remain unclear. In this study we compared the radiation response of GSC lines (NSC11 and NSC20) and the standard glioma cell line U251 after an increase in aneuploidy. This increase was induced by treatment with CFI-402257, a selective inhibitor of the mitotic kinase TTK, which plays a key role in spindle-assembly checkpoint (SAC) regulation. After an initial drug exposure of 24h, the drug was removed and cells were allowed to grow for 5 days. In both GSC lines and U251, as demonstrated by interphase chromosome FISH, this protocol resulted in approximately 150%, 50%, and 125% increases in aneuploidy of chromosomes 2, 7 and 10 respectively. After these increases in aneuploidy, the clonogenicity (in vitro survival) of U251 cells was reduced by 80%, whereas it was reduced by 50% and 30% in NSC11 and NSC20 respectively. Moreover, the increase in aneuploidy significantly enhanced the radiosensitivity of U251 cells, whereas it had no effect on the radiosensitivity of the GSCs. These changes could not be attributed to residual inhibition of TTK or deficiencies in the SAC: at 5 days after CFI inhibition no changes in nocodazole-induced SAC arrest in mitosis were detected. Additionally, after aneuploid induction no changes in the expression of the stem cell marker CD133 were detected in the GSC lines, indicating no change in differentiation status. To begin to investigate the mechanism behind these differences in radiosensitivity, γH2AX foci dispersal, a surrogate marker for DNA double strand break (DSB) repair was evaluated. After the increase in aneuploidy, cells were irradiated (2Gy) and collected 1-24h after for analysis of γH2AX. In U251 cells, the increase in aneuploidy resulted in slower foci dispersal, indicative of a decrease in DSB repair; whereas in GSCs the increased aneuploidy had no effect on foci dispersal as compared to control. Mitotic catastrophe, the primary mechanism of solid tumor cell death after irradiation, was also evaluated. The increase in aneuploidy significantly increased the frequency of radiation-induced mitotic catastrophe in U251 cells, but no changes in either GSC line were detected. Overall, these results suggest that GSCs have an enhanced ability to tolerate the negative consequences of aneuploidy on survival as well as on radiosensitivity. Such aneuploid tolerance may provide a mechanism through which GBMs exploit karyotype diversity to survive under harsh environmental conditions and after treatment. Citation Format: Ian C. Sutton, Charlotte M. Degorre, Komal Rawal, Philip J. Tofilon. Glioblastoma stem-like cells are resistant to the negative effects of increased aneuploidy on in vitro survival and radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2413.

Spindle assembly checkpoint gene BUB1B is essential in breast cancer cell survival.

The study aimed to investigate the role of spindle assembly checkpoint (SAC) in cancer cells with compromised genomic integrity. Chromosomal instability (CIN) gives cancer cells an adaptive advantage. However, maintaining the balance of this instability is crucial for the survival of cancer cells as it could lead them to the mitotic catastrophe. Therefore, cancer cells adapt to the detrimental effects of CIN. We hypothesized that changes in SAC might be one such adaptation mechanism. The focus of the study was BUB1B, an integral part of the checkpoint. Clinical datasets were analyzed to compare expression levels of SAC genes in normal tissue vs. breast carcinoma. The effects of the reduction of BUB1B expression was examined utilizing RNA interference method with siRNAs. In vitro viability, clonogenicity, apoptosis, and SAC activity levels of a variety of breast cancer (BrCa) cell lines, as well as in vivo tumorigenicity of the triple-negative breast cancer (TNBC) cell line MDA-MB-468, were tested. Additionally, the chromosomal stability of these cells was tested by immunofluorescence staining and flow cytometry. In clinical breast cancer datasets, SAC genes were elevated in BrCa with BUB1B having the highest fold change. BUB1B overexpression was associated with a decreased probability of overall survival. The knockdown of BUB1B resulted in reduced viability and clonogenicity in BrCa cell lines and a significant increase in apoptosis and cell death. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. BUB1B knockdown also impaired chromosome alignment and resulted in acute chromosomal abnormalities. We also showed that BUB1B knockdown on the MDA-MB-468 cell line decreases tumor growth in mice. A functional spindle assembly checkpoint is essential for the survival of BrCa cells. BUB1B is a critical factor in SAC, and therefore breast cancer cell survival. Impairment of BUB1B has damaging effects on cancer cell viability and tumorigenicity, especially on the more aggressive variants of BrCa.

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.

Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis.

Chromosome instability usually leads to tumorigenesis. Bloom syndrome (BS) is a genetic disease associated with chromosome instability. The BS gene product, BLM, has been reported to function in the spindle assembly checkpoint (SAC) to prevent chromosome instability. BTR complex, composed of BLM, topoisomerase IIIα (Topo IIIα), RMI1 (RecQ-mediated genome instability protein 1, BLAP75) and RMI2 (RecQ-mediated genome instability protein 2, BLAP18), is crucial for maintaining genome stability. Recent work has demonstrated that RMI2 also plays critical role in SAC. However, little is know about RMI1 regulation during the cell cycle. Here we present that RMI1 protein level does not change through G1, S and G2 phases, but significantly increases in M phase. Moreover, phosphorylation of RMI1 occurs in mitosis. Upon microtubule-disturbing agent, RMI1 is phosphorylated primarily at the sites of Serine 284 and Serine 292, which does not interfere with the formation of BTR complex. Additionally, this phosphorylation is partially reversed by roscovitine treatment, implying cycling-dependent kinase 1 (CDK1) might be one of the upstream kinases.

DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

Bir1 Deletion Causes Malfunction of the Spindle Assembly Checkpoint and Apoptosis in Yeast

Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H2O2 treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.