BACKGROUND: Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes.
METHODS: In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model.
RESULTS: Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the \"blue\" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7.
CONCLUSIONS: We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Intraperitoneal (IP) chemotherapy with paclitaxel (PTX) for gastric cancer (GC) with peritoneal metastasis (PM) is considered a promising treatment approach, however, there are no useful biomarkers to predict the efficacy of IP therapy. We examined the association between intra-peritoneal exosomes, particularly exosomal micro-RNAs (exo-miRNAs), and IP-chemo sensitivity. MKN45 cells that were cultured with intra-peritoneal exosomes from patients who did not respond to IP therapy with PTX (IPnon-respond group) exhibited resistance to PTX compared with exosomes from responding patients (IPrespond group) (p = 0.002). A comprehensive search for exo-miRNAs indicated that miR-493 was significantly up-regulated in exosomes from the IPnon-respond group compared with those collected from the IPrespond group. The expression of miR-493 in PTX-resistant MKN45 cells (MKN45PTX-res) was higher compared with that in MKN45. In addition, MKN45PTX-res cells exhibited lower MAD2L1 gene and protein expression compared with MKN45. Finally, miR-493 enhancement by transfection of miR-493 mimics significantly down-regulated MAD2L1 expression in MKN45 cells and reduced PTX sensitivity. Our results suggest that intra-peritoneal exo-miR-493 is involved in chemoresistance to PTX by downregulating MAD2L1 in GC with PM. Exo-miR-493 may be a biomarker for chemoresistance and prognosis of GC patients with PM and may also be a promising therapeutic target.

Bladder cancer (BC) is the most common urinary tract malignancy. Aurora kinase B (AURKB), a component of the chromosomal passenger protein complex, affects chromosomal segregation during cell division. Mitotic arrest-deficient 2-like protein 2 (MAD2L2) interacts with various proteins and contributes to genomic integrity. Both AURKB and MAD2L2 are overexpressed in various human cancers and have synergistic oncogenic effects; therefore, they are regarded as emerging therapeutic targets for cancer. However, the relationship between these factors and the mechanisms underlying their oncogenic activity in BC remains largely unknown. The present study aimed to explore the interactions between AURKB and MAD2L2 and how they affect BC progression via the DNA damage response (DDR) pathway.
Bioinformatics was used to analyze the expression, prognostic value, and pro-tumoral function of AURKB in patients with BC. CCK-8 assay, colony-forming assay, flow cytometry, SA-β-gal staining, wound healing assay, and transwell chamber experiments were performed to test the viability, cell cycle progression, senescence, and migration and invasion abilities of BC cells in vitro. A nude mouse xenograft assay was performed to test the tumorigenesis ability of BC cells in vivo. The expression and interaction of proteins and the occurrence of the senescence-associated secretory phenotype were detected using western blot analysis, co-immunoprecipitation assay, and RT-qPCR.
AURKB was highly expressed and associated with prognosis in patients with BC. AURKB expression was positively correlated with MAD2L2 expression. We confirmed that AURKB interacts with, and modulates the expression of, MAD2L2 in BC cells. AURKB knockdown suppressed the proliferation, migration, and invasion abilities of, and cell cycle progression in, BC cells, inducing senescence in these cells. The effects of AURKB knockdown were rescued by MAD2L2 overexpression in vitro and in vivo. The effects of MAD2L2 knockdown were similar to those of AURKB knockdown. Furthermore, p53 ablation rescued the MAD2L2 knockdown-induced suppression of BC cell proliferation and cell cycle arrest and senescence in BC cells.
AURKB activates MAD2L2 expression to downregulate the p53 DDR pathway, thereby promoting BC progression. Thus, AURKB may serve as a potential molecular marker and a novel anticancer therapeutic target for BC.

The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.

Oocyte cryopreservation is offered to women of various age groups for both health and social reasons. Oocytes derived from either controlled ovarian stimulation or in vitro maturation (IVM) are cryopreserved via vitrification. As maternal age is a significant determinant of oocyte quality, there is limited data on the age-related susceptibility of oocytes to the vitrification-warming procedure alone or in conjunction with IVM. In the present study, metaphase II oocytes obtained from 2, 6, 9, and 12 month old Swiss albino mice either by superovulation or IVM were used. To understand the association between maternal age and oocyte cryotolerance, oocytes were subjected to vitrification-warming and compared to non vitrified sibling oocytes. Survived oocytes were evaluated for mitochondrial potential, spindle integrity, relative expression of spindle checkpoint protein transcripts, and DNA double-strand breaks. Maturation potential and vitrification-warming survival were significantly affected (p < 0.001 and p < 0.05, respectively) in ovulated oocytes from the advanced age group but not in IVM oocytes. Although vitrification-warming significantly increased spindle abnormalities in ovulated oocytes from advanced maternal age (p < 0.01), no significant changes were observed in IVM oocytes. Furthermore, Bub1 and Mad2 transcript levels were significantly higher in vitrified-warmed IVM oocytes (p < 0.05). In conclusion, advanced maternal age can have a negative impact on the cryosusceptibility of ovulated oocytes but not IVM oocytes in mice.

Ovarian cancer (OVCA), a prevalent gynecological malignancy, ranks as the fourth most common cancer among women. Mitotic Arrest Deficient 2 Like 2 (MAD2L2), a chromatin-binding protein and a component of DNA polymerase ζ, has been previously identified as an inhibitor of tumor growth in colorectal cancer. However, the roles of MAD2L2 in OVCA, including its expression, impact, and prognostic significance, remain unclear. We employed bioinformatics tools, Cox Regression analysis, and in vitro cell experiments to investigate its biological functions. Our findings reveal that MAD2L2 typically undergoes genomic alterations, such as amplifications and deep deletions. Moreover, we observed an overexpression of MAD2L2 mRNA in OVCA patients, correlating with reduced survival rates, particularly in those with Grade IV tumors. Furthermore, analysis of mRNA biofunctions indicated that MAD2L2 is predominantly localized in the organellar ribosome, engaging mainly in NADH dehydrogenase activity. This was deduced from the results of gene ontology enrichment analysis, which also identified its role as a structural constituent in mitochondrial translation elongation. These findings were corroborated by KEGG pathway analysis, further revealing MAD2L2\'s involvement in tumor metabolism and the cell death process. Notably, MAD2L2 protein expression showed significant associations with various immune cells, including CD4+T cells, CD8+T cells, B cells, natural killer cells, and Myeloid dendritic cells. Additionally, elevated levels of MAD2L2 were found to enhance cell proliferation and migration in OVCA cells. The upregulation of MAD2L2 also appears to inhibit the ferroptosis process, coinciding with increased mTOR signaling activity in these cells. Our study identifies MAD2L2 as a novel regulator in ovarian tumor progression and offers new insights for treating OVCA.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major health concern, necessitating a deeper understanding of its prognosis and underlying mechanisms. This study aimed to investigate the mechanism and prognostic value of CD8+ T Cell exhaustion (CD8+ TEX)-related genes in HCC and construct a survival prognosis prediction model for patients with HCC.
METHODS: CD8+ TEX-related genes associated with HCC prognosis were analysed and identified, and a prognostic prediction model was constructed using the \'least absolute shrinkage and selection operator\' Cox regression model. Immunohistochemistry was used to verify the expression of the model genes in HCC tissues. A nomogram was constructed based on risk scores and clinical features, and its predictive efficacy was verified. The expression of STAM, ANXA5, and MAD2L2 in HCC cell lines was detected by western blotting; subsequently, these genes were knocked down in HCC cell lines by small interfering RNA, and their effects on the proliferation and migration of HCC cell lines were detected by colony formation assay, cck8, wound healing, and transwell assays.
RESULTS: Six genes related to CD8+ TEX were included in the risk-prediction model. The prognosis of patients with HCC in the low-risk group was significantly better than that of those in the high-risk group. Cox regression analysis revealed that the risk score was an independent risk factor for the prognosis of patients with HCC. The differentially expressed genes in patients with high-risk HCC were mainly enriched in the nucleotide-binding oligomerization domain-containing protein-like receptor, hypoxia-inducible factor-1, and tumour programmed cell death protein (PD)-1/PD-L1 immune checkpoint pathways. The CD8+ TEX-related genes STAM, ANXA5, and MAD2L2 were knocked down in HCC cell lines to significantly inhibit cell proliferation and migration. The prediction results of the nomogram based on the risk score showed a good fit and application value.
CONCLUSIONS: The prediction model based on CD8+ TEX-related genes can predict the prognosis of HCC and provide a theoretical basis for the early identification of patients with poor HCC prognosis.

Glioblastoma, the most common primary malignant tumor of the brain, is associated with poor prognosis. Glioblastoma cells exhibit high proliferative and invasive properties, and glioblastoma stem cells (GSCs) have been shown to play a crucial role in the malignant behavior of glioblastoma cells. This study aims to investigate the molecular mechanisms involved in GSCs maintenance and malignant progression.
Bioinformatics analysis was performed based on data from public databases to explore the expression profile of Mitotic arrest deficient 2 like 2 (MAD2L2) and its potential function in glioma. The impact of MAD2L2 on glioblastoma cell behaviors was assessed through cell viability assays (CCK8), colony formation assays, 5-Ethynyl-2\'-deoxyuridine (EDU) incorporation assays, scratch assays, and transwell migration/invasion assays. The findings from in vitro experiments were further validated in vivo using xenograft tumor model. GSCs were isolated from the U87 and LN229 cell lines through flow cytometry and the stemness characteristics were verified by immunofluorescence staining. The sphere-forming ability of GSCs was examined using the stem cell sphere formation assay. Bioinformatics methods were conducted to identified the potential downstream target genes of MAD2L2, followed by in vitro experimental validation. Furthermore, potential upstream transcription factors that regulate MAD2L2 expression were confirmed through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.
The MAD2L2 exhibited high expression in glioblastoma samples and showed significant correlation with patient prognosis. In vitro and in vivo experiments confirmed that silencing of MAD2L2 led to decreased proliferation, invasion, and migration capabilities of glioblastoma cells, while decreasing stemness characteristics of glioblastoma stem cells. Conversely, overexpression of MAD2L2 enhanced these malignant behaviors. Further investigation revealed that MYC proto-oncogene (c-MYC) mediated the functional role of MAD2L2 in glioblastoma, which was further validated through a rescue experiment. Moreover, using dual-luciferase reporter gene assays and ChIP assays determined that the upstream transcription factor E2F-1 regulated the expression of MAD2L2.
Our study elucidated the role of MAD2L2 in maintaining glioblastoma stemness and promoting malignant behaviors through the regulation of c-MYC, suggesting its potential as a therapeutic target.