Nuclear Aurora kinase A triggers programmed death-ligand 1-mediated immune suppression by activating MYC transcription in triple-negative breast cancer

Background:Increasing studies have reported that oncogenes regulate components of the immune system,suggesting that this is a mechanism for tumorigenesis.Aurora kinase A(AURKA),a serine/threonine kinase,is involved in cell mitosis and is essential for tumor cell proliferation,metastasis,and drug resistance.However,the mechanism by which AURKA is involved in immune response regulation is unclear.Therefore,this study aimed to investigate the role of AURKA in immune regulation in triple-negative breast cancer(TNBC).Methods:Peripheral blood mononuclear cells(PBMCs)were co-cultured with TNBC cells.The xCELLigence Real-Time Cell Analyzer-MP system was used to detect the killing efficiency of immune cells on TNBC cells.The expression of immune effector molecules was tested by quantitative real-time polymerase chain reaction(qRT-PCR)to evaluate immune function.Furthermore,to validate AURKA-regulated immune response in vivo,4T1 murine breast cancer cell line with AURKA overexpression or downregulation was engrafted into BALB/c mice.The distribution and proportion of immune cells in tumors were further evaluated by immunohistochemistry and flow cytometry.Results:Downregulation of AURKA in TNBC cells increased immune response by activating CD8^(+)T cell proliferation and activity.Nuclear rather than cytoplasmic AURKA-derived programmed death-ligand 1(PD-L1)expression was independent of its kinase activity.Mechanistic investigations showed that nuclear AURKA increased PD-L1 expression via an MYC-dependent pathway.PD-L1 overexpression mostly reversed AURKA silencing-induced expression of immune effector molecules,including interleukin-(IL-2),interferon-γ(IFN-γ),and perforin.Moreover,AURKA expression was negatively correlated with the enrichment and activity of tumor-infiltrating CD8^(+)T cells in 4T1 engrafted BALB/c mouse model.Conclusions:Nuclear AURKA elevated PD-L1 expression via an MYCdependent pathway and contributed to immune evasion in TNBC.Therapies targeting nuclear AURKA may restore immune responses against tumors.

MK615 inhibits pancreatic cancer cell growth by dual inhibition of Aurora A and B kinases

AIM:To investigate the anti-neoplastic effect of MK615, an anti-neoplastic compound isolated from Japanese apricot, against human pancreatic cancer cells in vitro. METHODS: Three human pancreatic cancer cell lines PANC-1, PK-1, and PK45H were cultured with MK615 at concentrations of 600, 300, 150, and 0 μg/mL. Growth inhibition was evaluated by cell proliferation assay, and killing activity was determined by lactate dehydrogenase (LDH) assay. Expression of Aurora A and B kinases was detected by real-time polymerase chain reaction (PCR) and Western blotting. Cell cycle stages were evaluated by flow cytometry. RESULTS: The growth inhibitory rates of MK615 at 150, 300, and 600 μg/mL were 2.3% ± 0.9%, 8.9% ± 3.2% and 67.1% ± 8.1% on PANC1 cells, 1.3% ± 0.3%, 8.7% ± 4.1% and 45.7 ± 7.6% on PK1 cells, and 1.2 ± 0.8%, 9.1% ± 2.1% and 52.1% ± 5.5% on PK45H cells, respectively (P <0.05). The percentage cytotoxicities of MK615 at 0, 150, 300, and 600 μg/mL were 19.6% ± 1.3%, 26.7% ± 1.8%, 25.5% ± 0.9% and 26.4% ± 0.9% in PANC1 cells, 19.7% ± 1.3%, 24.7% ± 0.8%, 25.9% ± 0.9% and 29.9% ± 1.1% in PK1 cells, and 28.0% ± 0.9%, 31.2% ± 0.9%, 30.4% ± 1.1% and 35.3 ± 1.0% in PK45H cells, respectively (P < 0.05). Real-time PCR and Western blotting showed that MK615 dually inhibited the expression of Aurora A and B kinases. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. CONCLUSION: MK615 exerts an anti-neoplastic effect on human pancreatic cancer cells in vitro by dual inhibition of Aurora A and B kinases.

The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

Objective： Aurora A kinase representing a family of evolutionadly conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A contributes to the carcinogenesis, chromosomal instability （CIN）, and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide （ASODN） technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods： Aurora AASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot respectively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results： The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours＇ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased （P 〈 0.05）, along with the induction of accumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora AASODN and paclitaxel was higher significantly than paclitaxel （P 〈 0.05） or Aurora AASODN alone （P 〈 0.05）. Conclusion： Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.

Targeting the cell cycle in esophageal adenocarcinoma:An adjunct to anticancer treatment

Esophageal adenocarcinoma is a major cause of cancer death in men in the developed world.Continuing poor outcomes with conventional therapies that predominantly target apoptosis pathways have lead to increasing interest in treatments that target the cell cycle.A large international effort has led to the development of a large number of inhibitors,which target cell cycle kinases,including cyclin-dependent kinases,Aurora kinases and polo-like kinase.Initial phase Ⅰ/Ⅱ trials in solid tumors have often demonstrated only modest clinical benefits of monotherapy.This may relate in part to a failure to identify the patient populations that will gain the most clinical benefit.Newer compounds lacking the side effect profile of first-generation compounds may show utility as adjunctive treatments targeted to an individual's predicted response to treatment.

Aurora B facilitates cholangiocarcinoma progression by stabilizing c-Myc

Background: Cholangiocarcinoma(CCA), a malignancy that arises from biliary epithelial cells, has a dismal prognosis, and few targeted therapies are available. Aurora B, a key mitotic regulator, has been reported to be involved in the progression of various tumors, yet its role in CCA is still unclarified.Methods: Human CCA tissues and murine spontaneous CCA models were used to assess Aurora B expression in CCA. A loss-of-function model was constructed in CCA cells to determine the role of Aurora B in CCA progression. Subcutaneous and liver orthotopic xenograft models were used to assess the therapeutic potential of Aurora B inhibitors in CCA.Results: In murine spontaneous CCA models, Aurora B was significantly upregulated. Elevated Aurora B expression was also observed in 62.3% of human specimens in our validation cohort(143 CCA specimens), and high Aurora B expression was positively correlated with pathological parameters of tumors and poor survival. Knockdown of Aurora B by siRNA and heteroduplex oligonucleotide(HDO)or an Aurora B kinase inhibitor(AZD1152) significantly suppressed CCA progression via G2/M arrest induction. An interaction between Aurora B and c-Myc was found in CCA cells. Targeting Aurora B significantly reduced this interaction and accelerated the proteasomal degradation of c-Myc, suggesting that Aurora B promoted the malignant properties of CCA by stabilizing c-Myc. Furthermore, sequential application of AZD1152 or Aurora B HDO drastically improved the efficacy of gemcitabine in CCA.Conclusions: Aurora B plays an essential role in CCA progression by modulating c-Myc stability and represents a new target for treatment and chemosensitization in CCA.

Mutations of the aurora kinase C gene causing macrozoospermia are the most frequent genetic cause of male infertility in Algerian men

Klinefelter syndrome and Y-chromosomal microdeletion analyses were once the only two genetic tests offered to infertile men. Analyses of aurora kinase C （AURKCj and DPY19L2 are now recommended for patients presenting macrozoospermia and globozoospermia, respectively, two rare forms of teratozoospermia particularly frequent among North African men. We carried out genetic analyses on Algerian patients, to evaluate the prevalence of these syndromes in this population and to compare it with the expected frequency of Klinefelter syndrome and Y-microdeletions. We carried out a retrospective study on 599 consecutive patients consulting for couple infertility at the assisted reproduction unit of the Ibn Rochd Clinique, Constantine, Algeria. Abnormal sperm parameters were observed in 404 men. Fourteen and seven men had typical macrozoospermia and globozoospermia profiles, respectively. Molecular diagnosis was carried out for these patients, for the AURKC and DPY19L2 genes. Eleven men with macrozoospermia had a homozygous AURKC mutation （79%）, corresponding to 2.7% of all patients with abnormal spermograms. All the men with globozoospermia studied （n = 5）, corresponding to 1.2% of all infertile men, presented a homozygous DPY19L2deletion. By comparison, we would expect 1.6% of the patients in this cohort to have Klinefelter syndrome and 0.23% to have Y-microdeletion. Our findings thus indicate that AURKCmutations are more frequent than Klinefelter syndrome and constitute the leading genetic cause of infertility in North African men. Furthermore, we estimate that AURKCand DPY19L2 molecular defects are 10 and 5 times more frequent, respectively, than Y-microdeletions.

The Pharmacophore Hypothesis of Novel Inhibitors for Aurora A Kinase

A three-dimensional pharmacophore model was developed from a series of inhibitors of Aurora A kinase to discover new potent anti-cancer agents using the HypoGen module in the Catalyst software. The pharmacophore model was developed based on the structure of 20 currently available inhibitors, which were carefully selected from the literature. The best hypothesis （Hypo 1） was defined by four features： one hydrogen-bond donor and three hy- drophobic points, with the best correlation coefficient of 0.909, the lowest rms deviation of 1.563, and the highest cost difference of 99.075. The Hypo 1 was then validated by a test set consisting of 24 compounds and by a cross-validation of 95% confidence level through randomizing the data using the CatScramble program, which suggested that a predictive pharmacophore model had been successfully obtained.

Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos

Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B （AurkB） is ubiquitously expressed while Aurora kinase C （AurkC） is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 （H3S10P） and regulate metaphase timing. Using an Oct4-photoactivat- able GFP fusion protein （Oct4-paGFP） and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.

Spatio-Temporal Expression Patterns of Aurora Kinases A, B in Mouse Zygotes during the First Mitosis

Objective To investigate the expression and localization of Aurora kinase A （A URKA ） and Aurora kinase B （A URKB） in mouse zygotes during the process of the first mitosis. Methods Quantitative real-time RT-PCR and Western blotting were performed to analyze the expression of AURKA and AURKB. The subcellular location of AURKA and A URKB was studied by confocal microscopy. Results A URKA and A URKB were increasingly expressed from phase G1 and peaked at phase M. After the entrance into mitosis A URKB became the predominant form both in mRNA and protein levels. The proteins of A URKA and A URKB both distributed in the cytoplasm and were associated with nucleus during the first mitosis of mouse zygotes, with some details in different. Conclusion The expression and localization of Aurora kinases A and B was in a cell- cycle regulated manner during the process of the cleavage of mouse zygotes. This discovery will aid in future investigations on their specific roles and molecular mecha- nisms in the regulation of mammalian early embryonic development.

Matrine and CYC116 Synergistically Inhibit Growth and Induce Apoptosis in Multiple Myeloma Cells

Objective： To investigate whether CYC116 can potentiate matdne-dependent growth inhibition and apoptosis in multiple myeloma （MM） cells. Methods： The dose response relationship of matrine to dexamethasone-resistant and dexamethasone-sensitive MM cells was first established. Myeloma RPMI8226 cells were treated with matrine alone or combined with CYC116 for 24 h. Cell proliferation was measured using an M＇I-F assay and apoptosis induction was evaluated by flow cytometry. Activation ot the caspase pathway and expression of apoptosis regulator proteins were detected by Western blotting. Results： Matrine significantly induced growth arrest and apoptosis in both drug-resistant and drug-sensitive MM cells. Treatment with the combination of matrine and CYC116 had a stronger cytotoxic effect on MM cells than did single drug treatments. Enhanced apoptosis observed following the combined treatment of matrine and CYC116 was associated with higher levels of activation of caspase-9, caspase-3, and poly adenosine diphosphate ribose polymerase （PARP） and down-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1 and the signaling proteins p-Akt and nuclear factor K B （NF-κB）. Conclusions： CYC116 enhances the growth inhibitory and apoptotic effects of matrine on MM cells.

Comprehensive analysis identifies as a critical prognostic prediction gene in breast cancer

Background:Aurora kinases(AURKs)family plays a vital role not only in cell division but also in tumorigenesis.However,there are still rare systematic analyses of the diverse expression patterns and prognostic value of the AURKs family in breast cancer(BC).Systematic bioinformatics analysis was conducted to explore the biological role,prognostic value,and immunologic function of AURKs family in BC.Methods:The expression,prognostic value,and clinical functions of AURKs family in BC were evaluated with several bioinformatics web portals:ONCOMINE Gene Expression Profiling Interactive Analysis,Kaplan-Meier plotter,cBioPortal,Metascape,GeneMANIA,and LinkedOmics;and the result was verified using human tissues.Results:The expression of AURKA and AURKB were upregulated in BC in subgroup analyses based on tumor stage(all P<0.05).BC patients with high AURKA and AURKB expression had a worse overall survival,relapse-free survival,and distant metastasisfree survival(all P<0.05).Verification experiment revealed that AURKA and AURKB were upregulated in BC(P<0.05).AURKA and AURKB were specifically associated with several tumor-associated kinases(polo-like kinase 1 and cyclin-dependent kinase 1),miRNAs(miR-507 and miR-381),and E2F transcription factor 1.Moreover,AURKA and AURKB were correlated with immune cell infiltration.Functional enrichment analysis revealed that AURKA and AURKB were involved in the cell cycle signaling pathway,platinum drug resistance signaling pathway,ErbB signaling pathway,Hippo signaling pathway,and nucleotide-binding and oligomerization domain-like receptor signaling pathway.Conclusions:Aurora kinases AURKA and AURKB could be employed as novel prognostic biomarkers or promising therapeutic targets for BC.