AURKA Expression Research Articles

Epigenetic silencing of miR-137 induces drug resistance and chromosomal instability by targeting AURKA in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Aberrant microRNAs (miRNAs) expression has been shown to be involved in the pathogenesis of MM. In this study, we further demonstrated that miR-137 was significantly downregulated in MM and negatively correlated with clinical prognosis. Moreover, we described the epigenetic regulation of miR-137 and its association with progression-free survival in MM patients. Furthermore, overexpression of miR-137 in MM cell line (miR-137 OE) increased its sensitivity to bortezomib and eprirubicin in vitro. Also, some high-risk genetic abnormalities in MM, including deletion of chromosome 1p22.2, 14q or 17p13, and gain of chromosome 1p22.2 were detected in NCI-H929 empty vector (NCI-H929 EV) treated cells but not in the NCI-H929 miR-137 overexpression (NCI-H929 miR-137 OE) cells. Luciferase reporter assays demonstrated that miR-137 targeted AURKA. Ectopic expression of miR-137 strongly reduced the expression of AURKA and p-ATM/Chk2 in MM cells, and increased the expression of p53, and p21. Importantly, miR-137 overexpression together with bortezomib treatment significantly inhibited tumor growth in MM xenograft model. Taken together, this study demonstrates that miR-137 is epigenetically silenced in MM, and overexpression of miR-137 could reduce drug resistance and overcome chromosomal instability of the MM cells via affecting the apoptosis and DNA damage pathways.

Abstract 1161: A new, therapeutically actionable target for the VHL E3 ubiquitin ligase in renal cell carcinoma

Abstract Background: Loss of function of the von Hippel Lindau (VHL) tumor suppressor is a nearly ubiquitous feature of clear cell renal cell carcinoma (ccRCC). The current therapeutic strategies in RCC were developed around knowledge that the VHL E3 ubiquitin ligase targets hypoxia inducible factors (HIFs) for degradation. AURKA is a well-established mitotic kinase and an emerging target in numerous cancers arising from its critical roles in tumor growth and survival. More recently AURKA was discovered to also have important non-mitotic functions in G1/G0 cells, where it activates HDAC6 to regulate microtubule stability. Here we report that AURKA is a novel target for the VHL E3 ligase, which plays an important role in modulating the non-mitotic activity of AURKA. Methods: In vitro and in vivo ubiquitination assays were performed to establish AURKA as a direct target of VHL. Immunoblot analyses were used to determine AURKA levels in human RCC patient material and VHL deficient cell lines. Immunofluorescence was utilized to assess the impact of VHL-mediated AURKA ubiquitination on microtubule targets using primary cilia. Results: In this study, we show that AURKA is a novel target for VHL's E3-ligase activity. VHL directly regulates AURKA expression in non-mitotic, quiescent cells by promoting AURKA degradation via the 26S proteasome. The ubiquitination assays showed enhanced AURKA ubiquitination in the presence of VHL. We found that VHL mediates AURKA degradation via a multi-monoubiquitin chain linkage, in contrast to the more traditional and abundant K48-linkage of proteins targeted for proteasome-mediated degradation. Biochemical studies revealed that unlike HIFα recognition by VHL, which requires proline hydroxylation, VHL interacted with, and degraded AURKA independent of its hydroxylation status, suggesting an alternate mechanism for recognition of AURKA. Importantly, using primary cilia as a biomarker for stabile microtubules and activity of AURKA/HDAC6, we showed that the loss of primary cilia observed in VHL-null cells was rescued by alisertib (AURKA inhibitor) and rocilinostat (HDAC6 inhibitor). Conclusion: In conclusion, the biochemical evidence presented here identifies AURKA as a novel target of VHL's E3 ubiquitin ligase activity. We demonstrate a previously unknown mechanism for VHL mediated multi-mono ubiquitination of AURKA in regulating its non-mitotic functions, important in enabling the G0/G1 transition of cells and maintaining the cilia-centrosome cycle. Importantly, as an alternative to the current HIF related anti-angiogenic therapies, identifying AURKA as a novel VHL target lays the foundation for new therapeutic avenues in RCC, targeted specifically at the epithelial defects associated with VHL-null renal cell carcinoma. Citation Format: Elshad Hasanov, Guang Chen, Pratim Chowdhury, Justin Weldon, Eric Jonasch, Subrata Sen, Cheryl Lyn Walker, Ruhee Dere. A new, therapeutically actionable target for the VHL E3 ubiquitin ligase in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1161.

Effect of AURKA Gene Expression Knockdown on Angiogenesis and Tumorigenesis of Human Ovarian Cancer Cell Lines.

Ovarian cancer is one of the most common malignant gynecological cancers. Higher expression of AURKA has been found in immortalized human ovarian epithelial cells in previous studies, implying the relationship between AURKA and ovarian cancer pathogenesis. We investigated the effect of AURKA on angiogenesis and tumorigenesis of human ovarian cancer cells. Firstly, the expression of AURKA in HO8910 and SKOV3 ovarian cancer cell lines was knocked down using a vector expressing a short hairpin small interfering RNA (shRNA). Next, the effect of knockdown of AURKA on cell angiogenesis, proliferation, migration, and invasion was determined by microtubule formation assay, proliferation assay, transwell migration, and invasion assays. In addition, the effect of AURKA knockdown on angiogenesis and tumorigenesis was also determined in a chicken chorioallantoic membrane (CAM) model and in nude mice. The results of the microtubule formation assay indicated that knockdown of AURKA significantly inhibited ovarian cancer cell-induced angiogenesis of endothelial cells compared to its control (P < 0.001). Knockdown of AURKA also significantly inhibited cell proliferation, migration, and invasion of HO8910 and SKOV3 cells in vitro. Furthermore, the Matrigel plug assay showed that knockdown of AURKA significantly repressed ovarian cancer cell-induced angiogenesis in nude mice (P < 0.05), and the CAMs model also showed that AURKA knockdown significantly attenuated the angiogenesis (P < 0.001) and tumorigenesis (P < 0.001) of HO8910 cells compared to the control. Finally, the tumorigenicity assay in vivo further indicated that AURKA shRNA reduced tumorigenesis in nude mice inoculated with ovarian cancer cells (P < 0.001). These results suggest the potential role of AURKA in angiogenesis and tumorigenesis of ovarian cancer, which may provide a potential therapeutic target for the disease.

AurkA controls self-renewal of breast cancer-initiating cells promoting wnt3a stabilization through suppression of miR-128.

AurkA overexpression was previously found in breast cancer and associated to its ability in controlling chromosome segregation during mitosis, however whether it may affect breast cancer cells, endorsed with stem properties (BCICs), is still unclear. Surprisingly, a strong correlation between AurkA expression and β-catenin localization in breast cancer tissues suggested a link between AurkA and Wnt signaling. In our study, AurkA knock-down reduced wnt3a mRNA and suppressed metastatic signature of MDA-MB-231 cells. As a consequence, the amount of BCICs and their migratory capability dramatically decreased. Conversely, wnt3a mRNA stabilization and increased CD44+/CD24low/− subpopulation was found in AurkA-overexpressing MCF7 cells. In vivo, AurkA-overexpressing primary breast cancer cells showed higher tumorigenic properties. Interestingly, we found that AurkA suppressed the expression of miR-128, inhibitor of wnt3a mRNA stabilization. Namely, miR-128 suppression realized after AurkA binding to Snail. Remarkably, a strong correlation between AurkA and miR-128 expression in breast cancer tissues confirmed our findings. This study provides novel insights into an undisclosed role for the kinase AurkA in self-renewal and migration of BCICs affecting response to cancer therapies, metastatic spread and recurrence. In addition, it suggests a new therapeutic strategy taking advantage of miR-128 to suppress AurkA-Wnt3a signaling.

Abstract P2-02-05: Circulating tumor cells in triple-negative and non-triple negative breast cancer patients show different genetic profiles

Abstract Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior, poor prognosis and still remains as a difficult disease since treatment options are limited. Despite some success in PARP inhibition in BRCA gene mutation patients or platinating agents that may offer superior outcomes in a subset of TNBC patients (pts), currently, there are no targeted therapies for TNBC available. Specific biomarkers are urgently needed for developing effective treatments to predict which patients will respond to the given therapy. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. Since TNBC is closely related to epithelial-mesenchymal transition (EMT), a stem cell phenotype and, in addition, androgen receptor (AR) expression has been detected in up to a third of TNBC pts, we here established a multi-marker gene panel for the characterization of CTCs in TNBC pts and compared these findings with CTC characteristics in non-TNBC pts. Methods: 2x5 ml blood of 30 TNBC pts before and/or after neoadjuvant therapy and 30 non- TNBC pts (E+/PR+: n=23; ER+/PR-: n=4; HER2+: n=1; HER2+/ER+: n=1; HER2+/ER+/PR+: n=1) before therapy were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was gene specifically pre-amplified using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, Aurka, HER2, HER3, EGFR, ALK, AR (androgene receptor), BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taken the false positive rate in healthy donors into account and defined as Ct(cutoff)-Ct(sample)-[Ct(CD45cutoff)-Ct(CD45sample)]. Results: In general, the distribution of the markers across all patients was highly variable. However, different expression patterns were found when CTCs of TNBC pts were compared with those of non-TNBC pts. In TNBC pts, SRC1 was the gene that was predominantly expressed, followed by c-Kit, HER3, BRCA1 and AURKA expression, before as well as after therapy. Interestingly, AKT2, EGFR, ERCC1 and PARP1 expression could not be detected at any time point studied. In addition, ALK, AR, c-Met, HER2 and KRT5 were only detected before but not after therapy. All other genes were expressed below 15%. In contrast, in non-TNBC pts, AKT2 was the gene that was predominantly expressed, followed by c-MET, HER3 and PI3K whereas c-KIT, ERCC1, mTOR and NOTCH1 were never found. All other genes were expressed below 10%. Conclusion: We successfully established a gene panel for the detection of the heterogeneous CTC population and demonstrated that CTCs in TNBC pts and non-TNBC pts show different genetic profiles. Although these data have to be confirmed in a bigger patient cohort, the knowledge about the individual target gene expression profile might efficiently help to predict a personalized targeted therapy for these pts in the future. Citation Format: Bittner A-K, Hoffmann O, Hauch S, Kimmig R, Kasimir-Bauer S. Circulating tumor cells in triple-negative and non-triple negative breast cancer patients show different genetic profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-05.

Aurora kinase targeting in lung cancer reduces KRAS-induced transformation.

BackgroundActivating mutations in KRAS are prevalent in lung cancer and have been causally linked to the oncogenic process. However, therapies targeted to oncogenic RAS have been ineffective to date and identification of KRAS targets that impinge on the oncogenic phenotype is warranted. Based on published studies showing that mitotic kinases Aurora A (AURKA) and B (AURKB) cooperate with oncogenic RAS to promote malignant transformation and that AURKA phosphorylates RAS effector pathway components, the aim of this study was to investigate whether AURKA and AURKB are KRAS targets in lung cancer and whether targeting these kinases might be therapeutically beneficial.MethodsIn order to determine whether oncogenic KRAS induces Aurora kinase expression, we used qPCR and western blotting in three different lung cell-based models of gain- or loss-of-function of KRAS. In order to determine the functional role of these kinases in KRAS-induced transformation, we generated KRAS-positive A549 and H358 cells with stable and inducible shRNA-mediated knockdown of AURKA or AURKB and evaluated transformation in vitro and tumor growth in vivo. In order to validate AURKA and/or AURKB as therapeutically relevant KRAS targets in lung cancer, we treated A549 and H358 cells, as well as two different lung cell based models of gain-of-function of KRAS with a dual Aurora kinase inhibitor and performed functional in vitro assays.ResultsWe determined that KRAS positively regulates AURKA and AURKB expression. Furthermore, in KRAS-positive H358 and A549 cell lines, inducible knockdown of AURKA or AURKB, as well as treatment with a dual AURKA/AURKB inhibitor, decreased growth, viability, proliferation, transformation, and induced apoptosis in vitro. In addition, inducible shRNA-mediated knockdown of AURKA in A549 cells decreased tumor growth in vivo. More importantly, dual pharmacological inhibiton of AURKA and AURKB reduced growth, viability, transformation, and induced apoptosis in vitro in an oncogenic KRAS-dependent manner, indicating that Aurora kinase inhibition therapy can specifically target KRAS-transformed cells.ConclusionsOur results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0494-6) contains supplementary material, which is available to authorized users.

Phase 1 Study of AMG 900, an Orally Administered Pan-Aurora Kinase Inhibitor, in Adult Patients (Pts) with Acute Myeloid Leukemia (AML)

Background: Aurora kinases form a family of highly conserved serine/threonine protein kinases that regulate key steps in mitosis. Aurora kinases A and B are amplified and/or overexpressed in many malignancies, including various types of leukemia, and are associated with high proliferation rates, poor prognosis, and therapeutic resistance. AMG 900 is an investigational, orally administered, highly potent, selective, small-molecule pan-aurora kinase inhibitor that has shown single-agent activity in heavily pretreated pts with chemotherapy-resistant/refractory solid tumors. This open-label, multicenter, sequential dose escalation study (NCT01380756) assessed AMG 900 in adult pts with AML.Methods: The primary objectives of this study were to evaluate the safety and tolerability of AMG 900, to evaluate pharmacokinetics after multiple oral administrations, to determine the optimal dose schedule, and to determine the maximum tolerated dose (MTD). A secondary objective was to evaluate antitumor activity in pts with AML. Key inclusion criteria included definitively diagnosed AML that had failed standard treatments or for which no standard therapy was available, Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2, and life expectancy > 3 months. Dose escalation included parallel evaluation of 2 schedules in separate groups. In group 1, AMG 900 was administered daily 4 days on/10 days off at doses of 15, 25, 40, 60, 80, 100, 125, and 150 mg. In group 2, AMG 900 was administered daily 7 days on/7 days off at doses of 30, 40, 50, 60, and 75 mg. Dose escalation was conducted using a 3+3+3 design; intrapatient dose escalation was not allowed. The relationship between gene expression at baseline and clinical response was an exploratory objective. RNA levels for preselected genes were measured by microarray in mononuclear cells obtained from bone marrow (BM) aspirates.Results: A total of 35 pts were enrolled: 22 in group 1 and 13 in group 2. Twenty-three pts (65.7%) were male, 27 (77.1%) were white, and mean (SD) age was 66.1 (12.2) years. ECOG status was 0 in 7 pts (20.0%), 1 in 22 pts (62.9%), and 2 in 6 pts (17.1%). Pts received a median (min, max) of 14 (4, 49) doses of AMG 900. Mean maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC) increased with increasing dose. Thirty pts (85.7%) had treatment-related adverse events (AEs). The most common AEs (in ≥ 10% of pts overall) were nausea (31.4%), diarrhea (28.6%), febrile neutropenia (28.6%), fatigue (22.9%), vomiting (17.1%), alopecia (14.3%), dyspnea (11.4%), epistaxis (11.4%), mucosal inflammation (11.4%), and rash (11.4%). Nine pts (25.7%) died during the study from lung infection and respiratory failure (2 pts each) and acute respiratory failure, cardiorespiratory arrest, respiratory distress, sepsis, and septic shock (1 pt each). Only respiratory failure and septic shock (1 pt each) were considered potentially related to AMG 900 by the investigators. All 35 pts discontinued treatment. Reasons for discontinuation were disease progression (65.7%), AEs (11.4%), death (8.6%), withdrawal of consent (5.7%), other reasons (5.7%), and requirement for alternative therapy (2.9%). Two pts (5.7%) experienced dose-limiting toxicities: 1 pt from group 1 (40 mg) had grade 3 pancytopenia, and 1 pt from group 2 (50 mg) had febrile neutropenia and grade 3 abdominal pain. The MTD of AMG 900 was not formally reached in either dose schedule. Three pts (8.6%) from group 1 (40 mg [2 pts] and 60 mg [1 pt]) had a best response of complete response with incomplete count recovery (CRi). Overall, the objective response rate for CRi was 8.6% (80% confidence interval: 3%, 18%). Higher gene expression of BIRC5, AURKB, AURKA, TTK, and CCNB1 was associated with objective response in univariate logistic regression models (P < .02), and was still significant after adjusting for average dose and percentage of blasts in the BM in a multivariate model (P < .01). Expression profiles of responders were clustered together in a principal component analysis.Conclusions: AMG 900 had an acceptable safety profile in this grievously ill population of adult pts with recurrent/refractory AML. Prolonged cytopenias hampered further dose escalation in this single-agent treatment setting. Combination of low doses of AMG 900 with other anticancer agents should be evaluated in future studies. DisclosuresSchuster:Amgen Inc.: Equity Ownership, Honoraria, Speakers Bureau. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Gamelin:Amgen Inc.: Employment, Equity Ownership. Rasmussen:Amgen Inc.: Employment, Equity Ownership. Juan:Amgen Inc.: Employment, Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Chow:Amgen Inc.: Employment, Equity Ownership. Friberg:Amgen Inc.: Employment, Equity Ownership. Vogl:Amgen Inc.: Employment, Equity Ownership.

Identification of genes responsible for RelA-dependent proliferation arrest in human mammary epithelial cells conditionally expressing RelA.

The molecular mechanisms responsible for opposing oncogenic and tumor-suppressor activities of NF-kB are obscure. Semi-quantitative immunohistochemistry of primary breast tumors using antibodies to RelA, the pleiotropic NF-kB factor, and Ki67 revealed a negative correlation between RelA levels and Ki67-index among ER +/HER2 − tumors [1]. Similarly, expression of AURKA, a marker for proliferation, negatively correlates with expression of NFKBIA, a surrogate for RelA expression and activity, in ER +/HER2 − tumors analyzed by The Cancer Genome Atlas [2], [3], [4]. Furthermore, conditional expression of RelA using a Tetracycline-inducible system in Human Mammary Epithelial Cells (HRA cells) caused proliferation arrest while withdrawal of Doxycycline (Dox) and suppression of RelA expression in arrested cells restored cell cycle progression [1]. To identify genes responsible for the negative relationship between RelA levels and proliferation, we performed genome-wide gene expression analysis of HRA cells under the following conditions: RelA un-induced, No Dox (ND); Dox induced for 24 h; Dox induced for 72 h; Dox induced for 24 h then Dox withdrawn for 48 h. The expression data was submitted to Gene Expression Ominibus (GEO) and the accession number is GSE65040. Analysis of the data identified cross-talk between basal RelA activity and the Interferon pathway mediated by IRF1, a target of RelA [5]. Activation of the Interferon pathway lead to down-regulation of CDK4 expression resulting in RB1 hypo-phosphorylation and suppression of cell cycle progression. The tumor-suppressor activity of NF-kB, specifically RelA, may stem from cross-talk with the Interferon pathway.

Quantitative Proteomics Reveals That the Inhibition of Na(+)/K(+)-ATPase Activity Affects S-Phase Progression Leading to a Chromosome Segregation Disorder by Attenuating the Aurora A Function in Hepatocellular Carcinoma Cells.

Many studies have shown the Na(+)/K(+)-ATPase (NKA) might be a potential target for anticancer therapy. Cardiac glycosides (CGs), as a family of naturally compounds, inhibited the NKA activity. The present study investigates the antitumor effect of ouabain and elucidates the pharmacological mechanisms of CG activity in liver cancer HepG2 cell using SILAC coupled to LC-MS/MS method. Bioinformatics analysis of 330 proteins that were changed in cells under treatment with 0.5 μmol/L ouabain showed that the biological processes are associated with an acute inflammatory response, cell cycle, oxidation reduction, chromosome segregation, and DNA metabolism. We confirmed that ouabain induced chromosome segregation disorder and S-cell cycle block by decreasing the expression of AURKA, SMC2, Cyclin D, and p-CDK1 as well as increasing the expression of p53. We found that the overexpression or inhibition of AURKA significantly reduced or enhanced the ouabain-mediated the anticancer effects. Our findings suggest that AURKA is involved in the anticancer mechanisms of ouabain in HepG2 cells.

MiR-137 contributes to drug susceptibility and chromosomal instability of multiple myeloma through targeting AURKA

Background: MicroRNAs are non-coding small RNAs that modulate protein expression and implicated in the pathogenesis of much kind of cancers, including multiple myeloma (MM). Previous study revealed that mic-137 was significant down regulated in MM patients compared with health donors. The purpose of this study is to investigate how miR-137 involved in pathogenesis and drug resistance in MM and its potential as prognostic biomarker. Materials and Methods: Real-time RT-PCR was performed to identify the expression of miR-137 in 6 MM cell lines and CD138+ cell sorted from 21 MM patients and 10 healthy donors. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing, methylation specific polymerase chain reaction （MSP）and bisulfite sequencing PCR (BSP). The functional roles of miR-137 in MM were characterized by CCK-8 assay, soft agar clonogenic- formation, standard apoptosis assay and CGH array (CytoScanHD Array, Affymetrix). Effect of miR-137 on MM progression in vivo was assessed in the NOD/SCID mice models. In addition, to further identify miR-137 targets, we used bioinformatics analysis and confirmed by luciferase reporter assay. In addition, a total of 19 paired sequential samples with GEP and clinical data, obtained from published studies, were analyzed. Results: The expression of miR-137 was down regulated in all 6 MM cell lines and MM patients (p=0.0017). Further study revealed that methylation of the miR-137 CpG islands was frequently observed in MM cell lines (p<0.0001) and patients (p=0.004) compared with healthy donor by MSP and BSP. Functional study of Cck-8 assay, soft agar clonogenic assay and standard apoptosis assay showed miR-137-OE NCI-H929 significantly inhibited cell proliferation and increased cell drug sensitivity compared with NCI-H929-EV in vitro. Meanwhile, the tumorigenicity of miR-137 overexpressed NCI-H929 reduces tumor volume in xenograft models at day 20 and prolonged the survival of NOD-SCID mice following tail vein injection of miR-137-OE NCI-H929 compared with control (p=0.0198). Overexpression of miR-137 inactivates both AKT and MAPK/ERK Signaling pathway by up-regulating p53 and down-regulating pAKT. Interestingly, CGH-array analysis indicated miR-137 overexpression could decrease chromosomal instability in NCI-H929, such as gain at 1q21, loss at 12p13.31 and 14q22.2 etc. To explore the mechanism of miR-137 involved in chromosome instability, our results demonstrated miR-137 mainly through up regulated AURKA expression and luciferase assay confirmed miR-137 targeted at position 374-380 of AURKA 3’UTR. Conclusion: Our data suggested that miR-137 was epigenetic silenced and targeted AURKA expression to contribute to drug susceptibility and chromosomal Instability in MM. Future studies will be performed on how miR-137 targets AURKA linked to the p53 pathway and evaluating miR-137 as prognostic biomarkers and targets for treatment in MM. Disclosures No relevant conflicts of interest to declare.

The AURKA/TPX2 axis drives colon tumorigenesis cooperatively with MYC

The MYC oncogene has long been established as a central driver in many types of human cancers including colorectal cancer. However, the realization of MYC-targeting therapies remains elusive; as a result, synthetic lethal therapeutic approaches are alternatively being explored. A synthetic lethal therapeutic approach aims to kill MYC-driven tumors by targeting a certain co-regulator on the MYC pathway. We analyzed copy number and expression profiles from 130 colorectal cancer tumors together with publicly available datasets to identify co-regulators on the MYC pathway. Candidates were functionally tested by in vitro assays using colorectal cancer and normal fibroblast cell lines. Additionally, survival analyses were carried out on another 159 colorectal cancer patients and public datasets. Our in silico screening identified two MYC co-regulator candidates, AURKA and TPX2, which are interacting mitotic regulators located on chromosome 20q. We found the two candidates showed frequent co-amplification with the MYC locus while expression levels of MYC and the two genes were positively correlated with those of MYC downstream target genes across multiple cancer types. In vitro, the aberrant expression of MYC, AURKA and TPX2 resulted in more aggressive anchorage-independent growth in normal fibroblast cells. Furthermore, knockdown of AURKA or TPX2, or treatment with an AURKA-specific inhibitor effectively suppressed the proliferation of MYC-expressing colorectal cancer cells. Additionally, combined high expression of MYC, AURKA and TPX2 proved to be a poor prognostic indicator of colorectal cancer patient survival. Through bioinformatic analyses and experiments, we proposed TPX2 and AURKA as novel co-regulators on the MYC pathway. Inhibiting the AURKA/TPX2 axis would be a novel synthetic lethal therapeutic approach for MYC-driven cancers.

Assessing associations between the AURKA-HMMR-TPX2-TUBG1 functional module and breast cancer risk in BRCA1/2 mutation carriers.

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04 – 1.15, p = 1.9 x 10−4 (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03 – 1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted pinteraction values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients’ survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers.

Expression of proliferation markers Ki67, cyclin A, geminin and aurora-kinase A in primary breast carcinomas and corresponding distant metastases

AimsTo assess the expression of the following cell cycle regulatory proteins in primary metastatic breast carcinomas (MBCs) and on availability in matched distant metastases (DMs): Ki67, cyclin A, geminin and...

AURKA is a predictor of chemotherapy response and prognosis for patients with advanced oral squamous cell carcinoma.

Increasing evidence proposes the benefits of cisplatin-based chemotherapy in a subpopulation of patients with oral squamous cell carcinoma (OSCC), yet reliable indicators for this subpopulation are poorly explored. AURKA, also known as aurora kinase A, playing important functions in cell mitosis and making cells resistant to cisplatin through dysregulation of DNA damage repair networks, has been reported to be upregulated in OSCC, making AURKA a promising indicator. In this study, we recruited 78 patients with advanced OSCC to examine the expression of AURKA and the correlation with chemotherapy response and clinical outcomes. We found that AURKA was strongly expressed in 31 (39.74 %) of the 78 advanced OSCC samples and its expression was significantly associated with cisplatin resistance (P = 0.023), clinical recurrence (P = 0.021), and 5-year survival (P = 0.019). Chemotherapy increased AURKA expression in post-chemotherapy samples, yet with no significance (P = 0.101). Multivariate Cox proportional hazards regression model analysis demonstrated that lymph node samples with positive, strong AURKA staining, and poor chemotherapy response were independently associated with the 5-year survival and disease-free survival. Inhibiting AURKA expression in OSCC cell lines remarkably increased their sensitivity to cisplatin treatment by 2.5-fold difference. Our results imply that the overexpression of AURKA in advanced OSCC not only plays a role in the disease course but also shows an involvement in cisplatin treatment response. AURKA level may be a valuable predictor for patients with advanced OSCC, with downregulation of AURKA being a promising adjuvant therapy in this patient population.

MiR-137 Contributes to Drug Susceptibility and Chromosomal Instability of Multiple Myeloma through Targeting AURKA

Background: MicroRNAs are non-coding small RNAs that modulate protein expression and implicated in the pathogenesis of much kind of cancers, including multiple myeloma (MM). Previous study revealed that mic-137 was significant down regulated in MM patients compared with health donors. The purpose of this study is to investigate how miR-137 involved in pathogenesis and drug resistance in MM and its potential as prognostic biomarker.Materials and Methods: Real-time RT-PCR was performed to identify the expression of miR-137 in 6 MM cell lines and CD138+ cell sorted from 21 MM patients and 10 healthy donors. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing, methylation specific polymerase chain reaction （MSP）and bisulfite sequencing PCR (BSP). The functional roles of miR-137 in MM were characterized by CCK-8 assay, soft agar clonogenic- formation, standard apoptosis assay and CGH array (CytoScan"HD Array, Affymetrix). Effect of miR-137 on MM progression in vivo was assessed in the NOD/SCID mice models. In addition, to further identify miR-137 targets, we used bioinformatics analysis and confirmed by luciferase reporter assay. In addition, a total of 19 paired sequential samples with GEP and clinical data, obtained from published studies, were analyzed.Results: The expression of miR-137 was down regulated in all 6 MM cell lines and MM patients (p=0.0017). Further study revealed that methylation of the miR-137 CpG islands was frequently observed in MM cell lines (p<0.0001) and patients (p=0.004) compared with healthy donor by MSP and BSP. Functional study of Cck-8 assay, soft agar clonogenic assay and standard apoptosis assay showed miR-137-OE NCI-H929 significantly inhibited cell proliferation and increased cell drug sensitivity compared with NCI-H929-EV in vitro. Meanwhile, the tumorigenicity of miR-137 overexpressed NCI-H929 reduces tumor volume in xenograft models at day 20 and prolonged the survival of NOD-SCID mice following tail vein injection of miR-137-OE NCI-H929 compared with control (p=0.0198). Overexpression of miR-137 inactivates both AKT and MAPK/ERK Signaling pathway by up-regulating p53 and down-regulating pAKT. Interestingly, CGH-array analysis indicated miR-137 overexpression could decrease chromosomal instability in NCI-H929, such as gain at 1q21, loss at 12p13.31 and 14q22.2 etc. To explore the mechanism of miR-137 involved in chromosome instability, our results demonstrated miR-137 mainly through up regulated AURKA expression and luciferase assay confirmed miR-137 targeted at position 374-380 of AURKA 3’UTR.Conclusion: Our data suggested that miR-137 was epigenetic silenced and targeted AURKA expression to contribute to drug susceptibility and chromosomal Instability in MM. Future studies will be performed on how miR-137 targets AURKA linked to the p53 pathway and evaluating miR-137 as prognostic biomarkers and targets for treatment in MM. DisclosuresNo relevant conflicts of interest to declare.

Aurora kinase A mediates c-Myc's oncogenic effects in hepatocellular carcinoma.

Dysregulation of c-Myc (Myc) has been shown to contribute to progression of hepatocellular carcinoma, however, the detailed molecular mechanism remains poorly understood. Here, we report that Myc binds to the Aurora kinase A (Aurka) promoter and induces expression of Aurka in HCC cells. Increased expression of Aurka correlates with that of Myc in HCC. Nuclear accumulation of Aurka was confirmed by subcellular protein fractionation and immunoblot experiments in HCC cells. Myc inhibition decreases the nuclear accumulation of Aurka in HCC cells. Also Aurka accumulating in the nucleus up-regulates Myc transcription by binding the Myc promoter containing the highly conserved CCCTCCCCA in the NHE region of the CpG islands. Inhibition of Myc or Aurka diminishes the malignant phenotypes of HCC cells by down-regulating some common target genes. Also Aurka and Myc mediates the effects of each other, at least partially, on proliferation, anchorage-independent soft agar growth, and ATP production. Blocking Aurka in an orthotopic model significantly impairs tumor growth in mice. These results identify a Myc-Aurka feedback loop in which Myc and Aurka regulate expression of each other at the transcriptional level and both play an important role in hepatocarcinogenesis.

Lineage-restricted function of the pluripotency factor NANOG in stratified epithelia.

NANOG is a pluripotency transcription factor in embryonic stem cells; however, its role in adult tissues remains largely unexplored. Here we show that mouse NANOG is selectively expressed in stratified epithelia, most notably in the oesophagus where the Nanog promoter is hypomethylated. Interestingly, inducible ubiquitous overexpression of NANOG in mice causes hyperplasia selectively in the oesophagus, in association with increased cell proliferation. NANOG transcriptionally activates the mitotic programme, including Aurora A kinase (Aurka), in stratified epithelia, and endogenous NANOG directly binds to the Aurka promoter in primary keratinocytes. Interestingly, overexpression of Nanog or Aurka in mice increased proliferation and aneuploidy in the oesophageal basal epithelium. Finally, inactivation of NANOG in cell lines from oesophageal or head and neck squamous cell carcinomas (ESCCs or HNSCCs, respectively) results in lower levels of AURKA and decreased proliferation, and NANOG and AURKA expression are positively correlated in HNSCCs. Together, these results indicate that NANOG has a lineage-restricted mitogenic function in stratified epithelia.

AURKA regulates JAK2–STAT3 activity in human gastric and esophageal cancers

Aurora kinase A is a frequently amplified and overexpressed gene in upper gastrointestinal adenocarcinomas (UGCs). Using in vitro cell models of UGCs, we investigated whether AURKA can regulate Signal Transducer and Activator of Transcription 3 (STAT3). Our data indicate that overexpression of AURKA in FLO-1 and AGS cells increase STAT3 phosphorylation at the Tyr705 site, whereas AURKA genetic depletion by siRNA results in decreased phosphorylation levels of STAT3 in FLO-1 and MKN45 cells. Immunofluorescence analysis showed that AURKA overexpression enhanced STAT3 nuclear translocation while AURKA genetic knockdown reduced the nuclear translocation of STAT3 in AGS and FLO-1 cells, respectively. Using a luciferase reporter assay, we demonstrated that AURKA expression induces transcriptional activity of STAT3. Pharmacological inhibition of AURKA by MLN8237 reduced STAT3 phosphorylation along with down-regulation of STAT3 pro-survival targets, BCL2 and MCL1. Moreover, by using clonogenic cells survival assay, we showed that MLN8237 single dose treatment reduced the ability of FLO-1 and AGS cells to form colonies. Additional experiments utilizing cell models of overexpression and knockdown of AURKA indicated that STAT3 upstream non-receptor tyrosine kinase Janus kinase 2 (JAK2) is mediating the effect of AURKA on STAT3. The inhibition of JAK2 using JAK2-specific inhibitor AZD1480 or siRNA knockdown, in presence of AURKA overexpression, abrogated the AURKA-mediated STAT3 activation. These results confirm that the AURKA-JAK2 axis is the main mechanism by which AURKA regulates STAT3 activity. In conclusion, we report, for the first time, that AURKA promotes STAT3 activity through regulating the expression and phosphorylation levels of JAK2. This highlights the importance of targeting AURKA as a therapeutic approach to treat gastric and esophageal cancers.

Mantle cell lymphoma harboring Burkitt’s-like translocations presents differential expression of aurora kinase genes compared with others 8q abnormalities

We compared the levels of AURKA and AURKB in 24 (mantle cell lymphoma) MCL patients harboring 8q abnormalities and its relationship with MYCC gene status. Two distinct subgroups were observed, in terms of MYCC expression. Except for the patients with Burkitt's-like translocation, none of the patients harboring 8q abnormalities, including balanced translocations or duplications of MYCC band, identified both by G-banding and SKY, showed differential expression levels of MYCC. These previous findings also reflected in the differential expression of AURKA and AURKB genes. We found that AURKA and AURKB mRNA were expressed at significantly higher levels in MCL patients harboring Burkitt's-like translocation, when compared to patients with 8q rearrangements. The high expression of aurora kinase genes is reported to be associated with some parameters of clinical oncologic aggressiveness, such as high histological grade, invasion and increased rates of metastasis in several types of cancers. It is possible that in MCL patients expressing abnormal levels of MYCC together with a high expression of AURKA might offer some resistant to the conventional therapy purposes. Thus, aurora kinase inhibitors may also be considered for this specific subgroup on MCL, whose aggressive clinical course resembles high-grade lymphoma.

Relationship between expression of Aurka and clinicopathological characteristics in gastric cancer patients

The aim of the study was to clarify the role of Aurka in the process of formation and development of gastric cancer. The expression of Aurka in gastric cancer and corresponding non-cancerous gastric tissues of 52 gastric cancer patients was assessed with the real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry. We also analyzed the relationship between the expression level and clinicopathological characteristics. Aurka gene and protein expression in gastric cancer tissues in both cases were significantly higher than in corresponding non-cancerous gastric tissues (both P 0.05, respectively). Furthermore, Aurka gene expression was markedly higher in gastric cancer tissues of Helicobacter pylori (HP)-positive patients than negative ones (P