Budding Uninhibited By Benzimidazoles 1 Research Articles

Bub1 suppresses inflammatory arthritis-associated bone loss in mice through inhibition of TNFα-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients' synovium and murine ankle tissue with arthritis. As CD45+CD11b+ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell-specific Bub1 conditional knockout (Bub1ΔLysM) mice were generated. Bub1ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum-transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi-quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated bone marrow-derived macrophages (BMMs) obtained from Bub1ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1β treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis-associated bone loss through inhibition of inflammation-mediated osteoclastogenesis.

Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization.

EGFR signaling initiates upon ligand binding which leads to activation and internalization of the receptor-ligand complex. Here, we evaluated if BUB1 impacted EGFR signaling by regulating EGFR receptor internalization and activation. BUB1 was ablated genomically (siRNA) or biochemically (2OH-BNPP1) in cells. EGF ligand was used to initiate EGFR signaling while disuccinimidyl suberate (DSS) was used for cross linking cellular proteins. EGFR signaling was measured by western immunoblotting and receptor internalization was evaluated by fluorescent microscopy (pEGFR (pY1068) colocalization with early endosome marker EEA1). siRNA mediated BUB1 depletion led to an overall increase in total EGFR levels and more phospho-EGFR (Y845, Y1092, and Y1173) dimers while the amount of total EGFR (non-phospho) dimers remained unchanged. BUB1 inhibitor (BUB1i) decreased EGF mediated EGFR signaling including pEGFR Y845, pAKT S473 and pERK1/2 in a time dependent manner. Additionally, BUB1i also reduced EGF mediated pEGFR (Y845) dimers (asymmetric dimers) without affecting total EGFR dimers (symmetric dimers) indicating that dimerization of inactive EGFR is not affected by BUB1. Furthermore, BUB1i blocked EGF mediated EGFR degradation (increase in EGFR half-life) without impacting half-lives of HER2 or c-MET. BUB1i also reduced co-localization of pEGFR with EEA1 positive endosomes suggesting that BUB1 might modulate EGFR endocytosis. Our data provide evidence that BUB1 protein and its kinase activity may regulate EGFR activation, endocytosis, degradation, and downstream signaling without affecting other members of the receptor tyrosine kinase family.

Budding uninhibited by benzimidazoles 1 promotes cell proliferation, invasion, and epithelial-mesenchymal transition via the Wnt/β-catenin signaling in glioblastoma

The pathogenesis and progression of GBM (glioblastoma), as one of the most frequently occurring malignancies of the central nervous system, are regulated by several genes. BUB1 (budding uninhibited by benzimidazoles 1) is a mitotic checkpoint that plays an important role in chromosome segregation as well as in various tumors. However, its role in glioma is unknown. The current study discovered prominently elevated BUB1 in glioma and a significant relationship between BUB1 expression, a high World Health Organization grade, and a poor prognosis in glioma patients. Moreover, BUB1 triggered EMT (epithelial-mesenchymal transition) apart from promoting glioma cell proliferation, migration, and infiltration. Besides, BUB1 promoted EMT by activating the Wnt/β-catenin axis. As implied by our study, BUB1 probably has the potential as a target for GBM management.

Budding uninhibited by benzimidazoles 1 might be a poor prognosis biomarker promoting the progression of papillary thyroid cancer.

Papillary thyroid carcinoma (PTC) is one of the most widespread malignant tumors of the endocrine system, with a high incidence. Budding uninhibited by benzimidazoles 1 (BUB1), one of the spindle assembly checkpoint (SAC) genes, is a multitask protein kinase required for eukaryotic chromosome segregation. Although BUB1 has been explored in several types of cancer, its biological role and molecular mechanisms in PTC remain unclear. In this study, we performed an examination of four public datasets along with local PTC cohorts and discovered that BUB1 was elevated in PTC compared to non-cancer tissues. High BUB1 expression was linked with the status of BRAFV600E , RAS, and TERT after statistical analysis. Clinically, BUB1 is associated with a variety of clinicopathological features in PTC patients. Interestingly, analysis of the TCGA database showed that BUB1 was closely associated with poor prognosis of PTC and significantly correlated with PFS. As determined by regression analysis, BUB1, and T stage were independent predictors of PTC and were related to BRAFV600E and lymph node metastatic status. By RT-qPCR, BUB1 was considerably overexpressed in PTC cell lines in comparison with normal thyroid epithelial cells. We confirmed that the knockdown of BUB1 in BCPAP and TPC1 cell lines significantly inhibited cell proliferation, cloning, and migration in vitro experiments. These results imply that BUB1 may be a significant oncogenic gene that is directly associated with the prognosis of PTC and may represent a future target for therapeutic intervention.

Abstract 2816: BUB1 inhibition radiosensitizes triple-negative breast cancer by targeting the DNA-damage repair pathways

Abstract Triple-negative breast cancer (TNBC) is the most lethal type of breast cancer (BC). TNBC has a substantial risk of locoregional recurrence and is difficult to cure due to absence of molecular targets or intrinsic heterogeneity. As radiotherapy (RT) is crucial in the treatment of BC, there is a need to identify novel molecular targets for increasing the efficacy of radiation therapy. BUB1 (Budding uninhibited by benzimidazoles 1) is a serine/threonine kinase implicated in chromosomal segregation during mitosis. However, it is still uncertain how BUB1 contributes to radioresistance in TNBC. By performing expression analysis (Oncomine), we observed that BUB1 is overexpressed in BC and even more in TNBC, indicating that this overexpression could be a prognostic factor for TNBC. MTT assay in several TNBC cell lines demonstrated that pharmacological (BAY1816032) or genomic (CRISPR) ablation of BUB1 was cytotoxic. By performing clonogenic survival assays, we observed that BUB1 ablation led to clinically meaningful radiosensitization in TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468) with radiation enhancement ratios ranging from 1.1 - 1.38. In addition, we showed that inhibition of BUB1 kinase function is essential for the radiosensitization phenotype using siRNAs and CRISPR knockouts by depleting endogenous BUB1 and reintroducing wild-type (WT) or kinase-dead (KD) BUB1. We observed significant increase in tumor doubling time in SUM159 xenografts (mammary fat pads in CB17/SCID mice) when BUB1i was combined with RT (DT=29.6 vs 7.9 days). A reduction in Ki67 was observed in these tumors further confirming the effectiveness of combination treatment. The effect of BUB1 depletion on DNA double strand break repair was assessed using γ-H2AX foci formation assay, which revealed sustained increase in γ-H2AX foci following radiation in BUB1i-treated cells. Further, BLRR assays confirmed that BUB1i reduces non-homologous end joining (NHEJ) efficiency. Biochemical analyses showed that BUB1i increases and stabilizes radiation-induced DNAPKcs phosphorylation (S2056). To evaluate the recruitment of DDR proteins to the chromatin following irradiation (IR), soluble nuclear and the chromatin-enriched fractionation was performed which demonstrated that cells pretreated with BUB1i had increased recruitment of DDR proteins to chromatin suggesting that BUB1 may play a role in the initial recruitment of NHEJ proteins to DSBs. Our findings demonstrate that both genetic and pharmacological inhibition of BUB1 sensitizes TNBC cells to RT and that BUB1 mediated radiosensitization may occur through NHEJ pathway. Our results suggest that BUB1 may represent a novel molecular target for radiosensitization in women with TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Pin Li, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. BUB1 inhibition radiosensitizes triple-negative breast cancer by targeting the DNA-damage repair pathways [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 2816.

Identification and Validation of a Potential Stemness-Associated Biomarker in Hepatocellular Carcinoma.

Background Cancer stem cells (CSCs) are typically related to metastasis, recurrence, and drug resistance in malignant tumors. However, the biomarker and mechanism of CSCs need further exploration. This study is aimed at comprehensively depicting the stemness characteristics and identify a potential stemness-associated biomarker in hepatocellular carcinoma (HCC). Methods The data of HCC patients from The Cancer Genome Atlas (TCGA) were collected and divided based on the mRNA expression-based stemness index (mRNAsi) in this study. Weighted gene coexpression network analysis (WGCNA) and the protein-protein interaction (PPI) network were performed, and the genes were screened through the Cytoscape software. Then, we constructed a prognostic expression signature using the multivariable Cox analysis and verified using the GEO and ICGC databases. Even more importantly, we used the three-dimensional (3D) fibrin gel to enrich the tumor-repopulating cells (TRCs) to validate the expression of the signature in CSCs by quantitative RT-PCR. Results mRNAsi was significantly elevated in tumor and high-mRNAsi score was associated with poor overall survival in HCC. The positive stemness-associated (blue) module with 737 genes were screened based on WGCNA, and Budding uninhibited by benzimidazoles 1 (BUB1) was identified as the hub gene highly related to stemness in HCC. Then, the prognostic value and stemness characteristics were well validated in the ICGC and GSE14520 cohorts. Further analysis showed the expression of BUB1 was elevated in TRCs. Conclusion BUB1, as a potential stemness-associated biomarker, could serve as a therapeutic CSCs-target and predicted the clinical outcomes of patients with HCC.

BUB1 predicts poor prognosis and immune status in liver hepatocellular carcinoma.

Accurate assessment of the tumour immune microenvironment promotes individualized immunotherapy regimens and screens dominant populations suitable for immunotherapy. Therefore, potential molecular markers were investigated to make an overall assessment of the immune microenvironment status of liver hepatocellular carcinoma (LIHC). In this study, a total of 121 differentially expressed genes (DEGs) were identified, and DEGs were enriched in the epithelial-mesenchymal transition, hypoxia, myogenesis, and p53 pathways. A total of 20 hub genes were selected and a strong correlation was identified between these hub genes and prognosis. The expression of budding uninhibited by benzimidazoles 1 (BUB1) was found to be upregulated in LIHC and was strongly related to immune cells and immune checkpoint molecule expression. Immunohistochemistry (IHC) indicated that BUB1 expression was higher in LIHC tissues than in normal liver tissues. BUB1 knockdown resulted in reduced proliferation and vertical migration ability of LIHC cells, and reduced the expression of phospho-SMAD family member 2 and phospho-SMAD family member 3 proteins. IHC showed that BUB1 expression was accompanied by immune cell infiltration into LIHC tissues. These results suggest that BUB1 may serve as a potential prognostic biomarker for LIHC and as an indicator of its immune status.

BUBs Are New Biomarkers of Promoting Tumorigenesis and Affecting Prognosis in Breast Cancer.

Background A tumor occurs because of abnormal cell multiplication caused by many variables like a significant disturbance in the regulation of cell growth and the instability of chromosome mitosis. Budding uninhibited by benzimidazoles 1 (BUB1), BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B), and budding uninhibited by benzimidazoles 3 (BUB3) are key regulators of mitosis, and their abnormal expression is highly correlated with breast cancer (BrCa), sarcoma, hepatic carcinoma, and other malignant tumors. However, the occurrence of BUBs (BUB1, BUB1B, and BUB3) and the development of BrCa have not been systematically explained. Methods Find out the target gene by looking up literature on PubMed and CNKI. Using the R software, TCGA, GEO, Kaplan-Meier Plotter, TIMER, and other databases, we studied the level of transcription, genetic changes, and physiological functions of BUBs in BrCa patients and their relationship with the origin, development, prognosis, immunity, and drug resistance of BrCa patients. Findings. We found that the high expression level of BUBs in BrCa tissues proposed a poor prognosis. The multivariate Cox regression analysis suggested that BUB1B and BUB3 might be independent prognostic factors of BrCa. In addition, the Metascape functional enrichment analysis showed that BUBs may be involved in the composition of the spindle, chromosome, and other structures and play a role in mitosis, sister chromatid separation, and other processes. Pathway enrichment suggests that BUBs may affect the cell cycle and lead to abnormal proliferation. Meanwhile, we also found that BUB3 can negatively regulate B lymphocytes, and BUB1 and BUB1B inhibit immune responses by promoting the secretion level of checkpoint molecules of the immune system, leading to immune escape of tumor cells. Conclusion We speculate that BUB1, BUB1B, and BUB3 may be therapeutic targets for BrCa patients and also provide new therapeutic strategies for BrCa treatment.

Inhibition of BUB1 suppresses tumorigenesis of osteosarcoma via blocking of PI3K/Akt and ERK pathways.

Osteosarcoma (OS) is a primary malignant bone tumour that mainly affects teenagers, with patients displaying poor prognosis. Budding uninhibited by benzimidazoles 1 (BUB1), a type of serine/threonine kinase that is linked to pro‐tumorigenic phenomena, has not been well studied in OS. Hence, this study aimed to explore the role of BUB1 in OS. The expression of BUB1 in OS specimens and cell lines was assessed using immunohistochemistry and Western blot analysis. Univariate and multivariate analyses were applied to evaluate the impact of BUB1 on patient survival. Cell counting kit‐8, wound‐healing and Transwell assays, as well as flow cytometry, were used to investigate the influence of BUB1 inhibition on OS in vitro. Moreover, a tumour xenograft model was established to investigate the in vivo effect of BUB1 inhibition on OS tumour growth. Results showed that BUB1 was overexpressed in OS specimens and cell lines. Furthermore, BUB1 overexpression was closely associated with the poor clinical outcomes of patients with OS. Inhibition of BUB1 markedly suppressed cell proliferation and tumour growth, cell migration, invasion and induced cell apoptosis of OS by blocking the PI3K/Akt and ERK signalling pathways. Thus, our study suggested that overexpression of BUB1 protein contributed to poor survival of OS patients and that inhibition of BUB1 resulted in considerable anti‐tumour activity associated with proliferation, migration, invasion and apoptosis of OS.

KIF4A knockdown suppresses ovarian cancer cell proliferation and induces apoptosis by downregulating BUB1 expression.

Ovarian cancer is one of the most common lethal gynecological malignancies worldwide. Abnormal kinesin family member 4A (KIF4A) expression has been implicated in ovarian cancer progression; however, the potential mechanism underlying KIF4A in ovarian cancer is not completely understood. The present study aimed to clarify the molecular basis of KIF4A in ovarian cancer. KIF4A and budding uninhibited by benzimidazoles 1 (BUB1) expression levels were detected via reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8, colony formation, wound healing, TUNEL and flow cytometry assays were performed to assess cell proliferation, migration, apoptosis and cell cycle distribution, respectively. Ki67 expression levels were detected by conducting immunofluorescence assays. The expression levels of migration- and apoptosis-related proteins were measured via western blotting. A co-immunoprecipitation assay was conducted to determine the association between KIF4A and BUB1. The results demonstrated that KIF4A was expressed at significantly higher levels in ovarian cancer cell lines compared with IOSE-80 cells. Compared with the short hairpin RNA-negative control group, KIF4A knockdown significantly inhibited cell viability, colony formation and migration, and markedly induced cell apoptosis. The results indicated that KIF4A could bind to BUB1 and regulate BUB1 expression. BUB1 overexpression weakened KIF4A knockdown-mediated effects on cell viability, colony formation, migration and apoptosis. Overall, the present study demonstrated that KIF4A knockdown suppressed ovarian cancer progression by regulating BUB1, and suggested the potential value of KIF4A and BUB1 as therapeutic targets for ovarian cancer.

Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma.

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

Bioinformatics analysis shows that TOP2A functions as a key candidate gene in the progression of cervical cancer.

Cervical cancer (CC) is one of the most prevalent types of cancer affecting females worldwide. However, the molecular mechanisms underlying the development and progression of CC remains to be elucidated. Taking the high incidence and mortality rates amongst women into consideration, the identification of novel biomarkers to prevent CC is of great significance and required to improve diagnosis. Using three raw microarray datasets from the Gene Expression Omnibus database, 188 differentially expressed genes (DEGs) were identified. Gene Ontology and pathway analyses were performed on the DEGs. Through protein-protein interaction network construction and module analysis, eight hub genes [cell division cycle 6, cyclin-dependent kinase 1 (CDK1), cell division control protein 45, budding uninhibited by benzimidazoles 1 (BUB1), DNA topoisomerase II α (TOP2A) and minichromosome maintenance complex component 4, CCNB2 and CCNB1] were identified, but only TOP2A was considered a prognostic factor in survival analysis. There were strong positive correlations between TOP2A and BUB1 (P<0.0001, rs=0.635), CDK1 (P<0.0001, rs=0.511), centromere protein F (CENPF) (P<0.0001, rs=0.677), Rac GTPase activating protein 1 (RACGAP1) (P<0.0001, rs=0.612), F-box protein 5 (FBXO5) (P<0.0001, rs=0.585) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) (P<0.0001, rs=0.584). Additionally, BUB1, CDK1, CENPF, RACGAP1, FBXO5 and BUB1B are all potentially suitable candidate targets for the diagnosis and treatment of CC. In conclusion, the present study identified TOP2A as a potential tumor oncogene and a biomarker for the prognosis of CC.

BUB1 promotes proliferation of liver cancer cells by activating SMAD2 phosphorylation.

Budding uninhibited by benzimidazoles 1 (BUB1) is a mitotic checkpoint serine/threonine kinase that has been reported as an oncogene or tumor suppressor gene in various types of cancer, including breast cancer, pancreatic ductal adenocarcinoma, prostate and gastric cancers. However, its role in liver cancer remains unclear. The present study aimed to explore the biological function of BUB1 in liver cancer. The present study demonstrated that BUB1 mRNA expression levels and the intensity of immunohistochemical staining were significantly increased in liver cancer tissues compared with normal tissues. The role of BUB1 in cell proliferation was also determined. Overexpression of BUB1 significantly promoted cell proliferation, whereas knockdown of BUB1 expression inhibited the proliferation of liver cancer cell lines. In experiments investigating the underlying mechanism, overexpression of BUB1 increased the levels of SMAD2 phosphorylation, whereas knockdown of BUB1 reduced the levels of SMAD2 phosphorylation. Therefore, BUB1 may promote proliferation of liver cancer cells by activating phosphorylation of SMAD2, and BUB1 may serve as a potential target in the diagnosis and/or treatment of liver cancer.

TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling

BUB1 (budding uninhibited by benzimidazoles-1) is required for efficient TGF-β signaling, through its role in stabilizing the TGFBR1 and TGFBR2 complex. Here we demonstrate that TGFBR2 phosphorylates BUB1 at Serine-318, which is conserved in primates. S318 phosphorylation abrogates the interaction of BUB1 with TGFBR1 and SMAD2. Using BUB1 truncation domains (1–241, 241–482 and 482–723), we demonstrate that multiple contact points exist between BUB1 and TGF-β signaling components and that these interactions are independent of the BUB1 tetratricopeptide repeat (TPR) domain. Moreover, substitutions in the middle domain (241–482) encompassing S318 reveals that efficient interaction with TGFBR2 occurs only in its dephosphorylated state (241–482 S318A). In contrast, the phospho-mimicking mutant (241–482 S318D) exhibits efficient binding with SMAD2 and its over-expression results in a decrease in TGFBR1-TGFBR2 and TGFBR1-SMAD2 interactions. These findings suggest that TGFBR2 mediated BUB1 phosphorylation at S318 may serve as a switch for the dissociation of the SMAD2-TGFBR complex, and therefore represents a regulatory event for TGF-β signaling. Finally, we provide evidence that the BUB1-TGF-β signaling axis may mediate aggressive phenotypes in a variety of cancers.

Abstract 3430: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling

Abstract The Ser/Thr kinase BUB1 (budding uninhibited by benzimidazoles-1) is required for efficient signaling by the TGF-β receptor. Recruitment of BUB1 to the activated heteromeric TGF-β-receptor complex including SMAD2 and SMAD3, is required for ligand mediated canonical and non-canonical downstream cascades (Nyati et. al. 2015). In an effort to delineate the structure-function basis for this, we utilized in vitro kinase reactions followed by mass spectrometry to demonstrate that TGFBR2 phosphorylates BUB1 at Serine 318. To elucidate the functional significance of TGFBR2 mediated phosphorylation of BUB1, S318 was substituted with alanine (S318A) or aspartic acid (S318D). Co-immunoprecipitation studies of these phospho-deficient or phospho-mimicking BUB1 mutants with TGFBR1, and SMAD2 revealed that the S318A mutant interacted more efficiently to TGFBR1 and SMAD2 compared to the S318D BUB1. To investigate if the Ser318 residue was involved in mediating BUB1 interaction with TGF-β signaling components, we utilized deletion mutants of BUB1. Our findings reveal that the N-terminal (1-241) and C-terminal (482-723) domains of BUB1 independently interact with SMAD2, SMAD3, TGFBR1, or TGFBR2. The middle domain (241-482) having a S318A mutation interacted efficiently with TGFBR2 while the S318D mutant did not. Additionally, the 241-482 S318D mutant exhibited an increased interaction with SMAD2. Over-expression of 241-482 S318D resulted in a decrease in TGFBR1-TGFBR2 interaction as well as TGFBR1-SMAD2 interaction, suggesting that TGFBR2 mediated BUB1 phosphorylation at S318 maybe the trigger for the dissociation of the activated receptor complex, and therefore a key regulator of TGF-β signaling. Taken together, we show that TGFBR2 phosphorylates BUB1 at Ser318 and propose a model with the following steps: (i) BUB1 is recruited to TGFBR1-TGFBR2 complex in response to ligand, (ii) BUB1 participates in the recruitment of SMAD2/3 to the receptor, (iii) TGFBR2 phosphorylates BUB1 at S318, which triggers the disassembly of the activated complex. Citation Format: Shyam Nyati, Brandon Gregg, Jiaqi Xu, Grant Young, Lauren Kimmel, Nyati Mukesh, Dipankar Ray, Hongtao Yu, Alnawaz Rehemtulla. TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3430.

Insights into mitotic checkpoint by integrating CRISPR and RNAi

ABSTRACTOur recent study of the mitotic checkpoint protein BUB1 (budding uninhibited by benzimidazoles 1) revealed several apparent BUB1 knock-out cell lines expressing low levels of BUB1 protein sufficient to support spindle assembly checkpoint activity. This rings alarm bells on the application of CRISPR technology.

HNRNPL Restrains miR-155 Targeting of BUB1 to Stabilize Aberrant Karyotypes of Transformed Cells in Chronic Lymphocytic Leukemia.

Aneuploidy and overexpression of hsa-miR-155-5p (miR-155) characterize most solid and hematological malignancies. We recently demonstrated that miR-155 sustains aneuploidy at early stages of in vitro cellular transformation. During in vitro transformation of normal human fibroblast, upregulation of miR-155 downregulates spindle checkpoint proteins as the mitotic checkpoint serine/threonine kinase budding uninhibited by benzimidazoles 1 (BUB1), the centromere protein F (CENPF) and the zw10 kinetochore protein (ZW10), compromising the chromosome alignment at the metaphase plate and leading to aneuploidy in daughter cells. Here we show that the heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to the polymorphic marker D2S1888 at the 3′UTR of BUB1 gene, impairs the miR-155 targeting, and restores BUB1 expression in chronic lymphocytic leukemia. This mechanism occurs at advanced passages of cell transformation and allows the expansion of more favorable clones. Our findings have revealed, at least in part, the molecular mechanisms behind the chromosomal stabilization of cell lines and the concept that, to survive, tumor cells cannot continuously change their genetic heritage but need to stabilize the most suitable karyotype.

Identification of hub genes and pathways in glioblastoma by bioinformatics analysis.

Glioblastoma (GBM) is the most common type of malignant brain tumor, and is associated with poor patient prognosis. A comprehensive understanding of the molecular mechanism underlying GBM may help to guide the identification of novel diagnoses and treatment targets. The gene expression profile of the GSE4290 GBM dataset was analyzed in order to identify differentially expressed genes (DEGs). Enriched pathways were identified through Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analyses. A protein-protein interaction network was constructed in order to identify hub genes and for module analysis. Expression and survival analyses were conducted in order to screen and validate critical genes. A total of 1,801 DEGs were recorded, including 620 upregulated and 1,181 downregulated genes. Upregulated DEGs were enriched in the terms ‘mitotic cell cycle process’, ‘mitotic cell cycle’ and ‘cell cycle process’. Downregulated genes were enriched in ‘transsynaptic signaling’, ‘anterograde transsynaptic signaling’ and ‘synaptic signaling’. A total of 15 hub genes, which displayed a high degree of connectivity, were selected. These genes included vascular endothelial growth factor A, cyclin-dependent kinase 1 (CDK1), cell-division cycle protein 20 (CDC20), aurora kinase A (AURKA), and budding uninhibited by benzimidazoles 1 (BUB1). The identified DEGs and hub genes may help guide investigations on the mechanisms underlying the development and progression of GBM. CDK1, CDC20, AURKA and BUB1, which are involved in cell cycle pathways, may be potential targets in the diagnosis and therapy of GBM.

Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics.

Background & AimsA generalized human pacemaking syndrome, chronic atrial and intestinal dysrhythmia (CAID) (OMIM 616201), is caused by a homozygous SGO1 mutation (K23E), leading to chronic intestinal pseudo-obstruction and arrhythmias. Because CAID patients do not show phenotypes consistent with perturbation of known roles of SGO1, we hypothesized that noncanonical roles of SGO1 drive the clinical manifestations observed.MethodsTo identify a molecular signature for CAID syndrome, we achieved unbiased screens in cell lines and gut tissues from CAID patients vs wild-type controls. We performed RNA sequencing along with stable isotope labeling with amino acids in cell culture. In addition, we determined the genome-wide DNA methylation and chromatin accessibility signatures using reduced representative bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing. Functional studies included patch-clamp, quantitation of transforming growth factor-β (TGF-β) signaling, and immunohistochemistry in CAID patient gut biopsy specimens.ResultsProteome and transcriptome studies converge on cell-cycle regulation, cardiac conduction, and smooth muscle regulation as drivers of CAID syndrome. Specifically, the inward rectifier current, an important regulator of cellular function, was disrupted. Immunohistochemistry confirmed overexpression of Budding Uninhibited By Benzimidazoles 1 (BUB1) in patients, implicating the TGF-β pathway in CAID pathogenesis. Canonical TGF-β signaling was up-regulated and uncoupled from noncanonical signaling in CAID patients. Reduced representative bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing experiments showed significant changes of chromatin states in CAID, pointing to epigenetic regulation as a possible pathologic mechanism.ConclusionsOur findings point to impaired inward rectifier potassium current, dysregulation of canonical TGF-β signaling, and epigenetic regulation as potential drivers of intestinal and cardiac manifestations of CAID syndrome. Transcript profiling and genomics data are as follows: repository URL: https://www.ncbi.nlm.nih.gov/geo; SuperSeries GSE110612 was composed of the following subseries: GSE110309, GSE110576, and GSE110601.

Kinases Involved in Both Autophagy and Mitosis

Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.