The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi Sensitivity in Triple-Negative Breast Cancers.

The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.

Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair.

Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viability.

Effect of Emi1 gene silencing on the proliferation and invasion of human breast cancer cells.

Breast cancer is the most common malignant tumour in women. The early silk-splitting inhibitor protein 1 Emi1 is responsible for mediating ubiquitin protein degradation. The present study investigated the effects of the decreased expression of the Emil gene on the proliferation and invasion of breast cancer cells. The interference efficiency of small interfering ribonucleic acid (siRNA) was quantitatively verified using fluorescence real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, and the effect of Emi1 gene silencing on cell vitality and invasion was determined using MTT and Transwell assays, respectively. The expression of the proliferation genes programmed cell death receptor 4 (PDCD-4), fatty acid synthase ligand (FasL), PTEN and RhoB, along with the invasive genes Maspin, TIMP3 and RECK, was measured using fluorescence RT-qPCR. In breast cancer cells, siRNA successfully reduced the expression of the Emi1 gene, and the expression level of the cell proliferation genes PDCD-4, FasL, PTEN and RhoB, along with invasive genes Maspin, TIMP3 and RECK, decreased significantly (P < 0.05). Furthermore, Emi1 gene silencing reduced the proliferation and invasion abilities of MDA-MB-231 and SUM149PT cells by reducing the expression of proliferative and invasive genes.

Expression significance of Emi1, UBCH10 and CyclinB1 in esophageal squamous cell carcinoma.

Despite significant advances in the diagnosis and treatment of esophageal squamous cell carcinoma (ESCC), esophageal cancer is still a heavy social and medical burden due to its high incidence. Uncontrolled division and proliferation is one of the characteristics of tumor cells, which will promote rapid tumor growth and metastasis. Early mitotic inhibitor 1 (Emi1), ubiquitin-conjugating enzyme 10 (UBCH10) and CyclinB1 are important proteins involved in the regulation of cell cycle. In this study, the expression of Emi1, UBCH10 and CyclinB1 in ESCC tissues and adjacent normal tissues will be analyzed by immunohistochemistry and in-situ hybridization techniques, and their relationship with tumor proliferation and apoptosis will be analyzed. The results showed that Emi1, UBCH10 and CyclinB1 genes and proteins were highly expressed in tumor tissues, which were correlated with tumor grade, lymph node metastasis and pathological stage, and positively correlated with tumor proliferation. Emi1, UBCH10 and CyclinB1 are also positively correlated. It is speculated that Emi1, UBCH10 and CyclinB1 genes synergically promote tumor proliferation and inhibit apoptosis, which may be potential diagnostic and therapeutic targets for ESCC.

Afu-Emi1 Contributes to Stress Adaptation and Voriconazole Susceptibility in Aspergillus fumigatus.

Invasive aspergillosis (IA) is the second most common invasive fungal disease and is associated with high mortality rates. Aspergillus fumigatus is the predominant causal agent of this life-threatening infection. Triazoles are still the cornerstone of antifungal treatment, and voriconazole remains the first-line choice. However, voriconazole resistance has been increasingly reported, which results in significantly higher mortality rates for IA and is particularly problematic. In the present study, we report the identification and functional study of a protein with previously unknown function that is encoded by the gene designated Afu-emi1 (AFUA_1G07360). High-throughput gene replacement technology was applied to construct the knockout ΔAfu-emi1 strain and a revertant strain. The MICs for azoles, including posaconazole, itraconazole, and voriconazole, were evaluated via the broth microdilution method and E-tests, which revealed that disruption of Afu-emi1 resulted in 4-fold increased susceptibility to voriconazole. Colony growth in the presence of oxidants, namely, H2O2 and menadione, and osmotic pressure-altering agents, namely, NaCl and d-sorbitol, was measured. The Afu-emi1 mutant strain exhibited a significant growth defect under oxidative and osmotic stress. The reactive oxygen species (ROS) production levels with or without voriconazole pretreatment were determined, and the Afu-emi1 mutant strain exhibited significantly lower ROS production levels. The effects of Afu-emi1 disruption on voriconazole susceptibility, growth under stress, and ROS production were restored in the revertant strain. In addition, the expression of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b in the ΔAfu-emi1 strain was significantly reduced. In conclusion, deletion of the gene Afu-emi1 resulted in increased voriconazole susceptibility, attenuated ability for oxidative and osmotic stress adaptation, decreased ROS production, and downregulation of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. IMPORTANCE Voriconazole is the first-line choice for IA, a life-threatening disease. Therefore, voriconazole resistance has become particularly problematic. Disruption of Afu-emi1 resulted in increased susceptibility to voriconazole, a significant growth defect under oxidative and osmotic stress, and downregulation of target enzyme Cyp51A and efflux pump expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. Targeting Afu-Emi1 might help to enhance azole therapeutic efficacy and impede azole resistance.

A FOXO-dependent replication checkpoint restricts proliferation of damaged cells.

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Substantial accumulation of single-stranded DNA during DNA replication activates the DNA replication stress checkpoint response that slows progression from S/G2 to M phase to protect genomic integrity. Whether and how mild replication stress restricts proliferation remains controversial. Here, we identify a cell cycle exit mechanism that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the anaphase promoting complex/cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead box O (FOXO)-mediated inhibition of its transcription factor E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible and thereby play an important role in maintaining genomic stability.

EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells.

BACKGROUND: The Ecotropic viral integration site 5 (EVI5), an important protein in regulating cell cycle, cytokinesis and cellular membrane traffic, functions as a stabilizing factor maintaining anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 in S/G2 phase. However, the mechanism by which EVI5 promotes malignant transformation of non-small cell lung cancer (NSCLC) remains unknown. In the present study, we addressed the role of EVI5 in NSCLC by regulating tumor growth, migration and invasion. METHODS: The expression levels of EVI5 and miR-486-5p in NSCLC tissues and cells were measured by real-time PCR. Meanwhile, EVI5 and its associated protein expression were analyzed by western blot and co-immunoprecipitation assay. Flow cytometry was performed to determine cell proliferation and apoptosis. CCK-8 and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of EVI5 in vivo, lung carcinoma xenograft mouse model was applied.. RESULTS: EVI5 was upregulated in NSCLC tissues and cell lines when compared with that in normal tissues and cell line. Knockdown of EVI5 in vitro inhibited tumor cell proliferation, migration and invasion in NSCLC cells. Further, inoculation of EVI5-deficient tumor cells into nude mice suppressed tumor proliferation and metastasis compared to control mice inoculated with unmanipulated tumor cells. These data indicated that EVI5 promote the proliferation of NSCLC cells which was consistent with our previous results. Additionally, we showed that EVI5 was directly regulated by miR-486-5p, and miR-486-5p-EVI5 axis affected the NSCLC migration and invasion through TGF-ß/Smad signaling pathway by interacting with TGF-ß receptor II and TGF-ß receptor I. CONCLUSIONS: Based on these results, we demonstrated a new post-transcriptional mechanism of EVI5 regulation via miR-486-5p and the protumoral function of EVI5 in NSCLC by interacting with Emi1 and/or TGF-ß receptors, which provides a new insight into the targeted therapy of NSCLC.

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage.

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects.

Regulation of mRNA translation during mitosis.

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

Human papillomavirus type 16 E7 oncoprotein inhibits the anaphase promoting complex/cyclosome activity by dysregulating EMI1 expression in mitosis.

The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase complex that orchestrates mitotic progression by targeting key mitotic regulators for proteasomal degradation. APC/C dysfunction is a frequent event during cancer development and can give rise to genomic instability. Here we report that the HPV16 E7 oncoprotein interferes with the degradation of APC/C substrates and that the APC/C inhibitor, EMI1, is expressed at higher levels in HPV16 E7-expressing mitotic cells. HPV16 E7 expression causes increased EMI1 mRNA expression and also inhibits EMI1 degradation. The resulting abnormally high EMI1 levels in HPV16 E7-expressing mitotic cells may inhibit degradation of APC/C substrates and cause the prometaphase delay that we have previously observed in such cells.