Aurora-A Kinase Activity Research Articles

LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr288 and subsequently impaired p53 phosphorylation at Ser315 which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy.

Abstract 4456: Aurora-A is a downstream target of RAS and forms a positive feedback regulation loop with NF-κB in non-small cell lung cancer

Abstract Previous studies have shown that inhibition of Aurora-A reduces mutant RAS oncogenic activity and that both Aurora-A and RAS activate NF-κB pathway through phosphorylation of IkBa and upregulation of GSKa, respectively. Here we show that Aurora-A expression and kinase activity are regulated by activating mutation of KRAS. Blockage of NF-κB by IκB-S32/36A abrogated KRAS-induced Aurora-A expression and kinase activity. Furthermore, we demonstrated that NF-κB directly bound to Aurora-A promoter and induces Aurora-A transcription. In addition, depletion of Aurora-A by siRNA and pharmacological inhibitor MLN8237 suppresses RAS-induced NF-κB activity and selectively induces cell death in KRAS mutant NSCLC cells. We also show that Aurora-A is induced by nicotine and is frequently overexpressed in NSCLCs. Overexpression of Aurora-A is associated with tobacco smoke and poor prognosis in lung adenocarcinoma but not squamous lung cancer. These data suggest that Aurora-A is regulated by RAS through NF-κB and that the feedback regulation loop between Aurora-A and NF-κB could play a pivotal role in NSCLC. Citation Format: Donghwa Kim, Masayuki Kanai, Xiangqian Zheng, Dali Zheng, Domenico Coppola, Jin Q. Cheng. Aurora-A is a downstream target of RAS and forms a positive feedback regulation loop with NF-κB in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2014-4456

NEDD9 Regulates Actin Dynamics through Cortactin Deacetylation in an AURKA/HDAC6–Dependent Manner

The prometastatic protein NEDD9 (neural precursor cell expressed, developmentally downregulated 9) is highly expressed in many cancers and is required for mesenchymal individual cell migration and progression to the invasive stage. Nevertheless, the molecular mechanisms of NEDD9-driven migration and the downstream targets effecting metastasis are not well defined. In the current study, knockdown of NEDD9 in highly metastatic tumor cells drastically reduces their migratory capacity due to disruption of actin dynamics at the leading edge. Specifically, NEDD9 deficiency leads to a decrease in the persistence and stability of lamellipodial protrusions similar to knockdown of cortactin (CTTN). Mechanistically, it was shown that NEDD9 binds to and regulates acetylation of CTTN in an Aurora A kinase (AURKA)/HDAC6-dependent manner. The knockdown of NEDD9 or AURKA results in an increase in the amount of acetylated CTTN and a decrease in the binding of CTTN to F-actin. Overexpression of the deacetylation mimicking (9KR) mutant of CTTN is sufficient to restore actin dynamics at the leading edge and migration proficiency of the tumor cells. Inhibition of AURKA and HDAC6 activity by alisertib and Tubastatin A in xenograft models of breast cancer leads to a decrease in the number of pulmonary metastases. Collectively, these findings identify CTTN as the key downstream component of NEDD9-driven migration and metastatic phenotypes. This study provides a mechanistic platform for therapeutic interventions based on AURKA and HDAC6 inhibition for patients with metastatic breast cancer to prevent and/or eradicate metastases.

A novel mechanism for activation of Aurora-A kinase by Ajuba

Aurora-A is a centrosome-localized serine/threonine kinase, which plays a critical role in mitotic and meiotic cell division processes. However, the regulation of Aurora-A is still not fully understood. Previously, we have found an intramolecular inhibitory regulation mechanism of Aurora-A: the N-terminal regulatory domain (aa 1-128, Nt) can interact with the C-terminal catalytic domain (aa 129-403, Cd) and inhibit the kinase activity of Aurora-A. In this study, we found that the PreLIM domain of Ajuba, another important activator of Aurora-A, induces the autophosphorylation of the C-terminal kinase domain of Aurora-A, and is phosphorylated by the C-terminal. Moreover, the LIM domain of Ajuba can competitively bind to the N-terminal of Aurora-A, and inhibited the interaction between N-terminal and C-terminal of Aurora A. Taken together, these results suggest a novel mechanism for regulation of Aurora-A by Ajuba.

Curcumin-induced Aurora-A suppression not only causes mitotic defect and cell cycle arrest but also alters chemosensitivity to anticancer drugs

Overexpression of oncoprotein Aurora-A increases drug resistance and promotes lung metastasis of breast cancer cells. Curcumin is an active anticancer compound in turmeric and curry. Here we observed that Aurora-A protein and kinase activity were reduced in curcumin-treated human breast chemoresistant nonmetastatic MCF-7 and highly metastatic cancer MDA-MB-231 cells. Curcumin acts in a similar manner to Aurora-A small interfering RNA (siRNA), resulting in monopolar spindle formation, S and G2/M arrest, and cell division reduction. Ectopic Aurora-A extinguished the curcumin effects. The anticancer effects of curcumin were enhanced by Aurora-A siRNA and produced additivity and synergism effects in cell division and monopolar phenotype, respectively. Combination treatment with curcumin overrode the chemoresistance to four Food and Drug Administration (FDA)-approved anticancer drugs (ixabepilone, cisplatin, vinorelbine, or everolimus) in MDA-MB-231 cells, which was characterized by a decrease in cell viability and the occurrence of an additivity or synergy effect. Ectopic expression of Aurora-A attenuated curcumin-enhanced chemosensitivity to these four tested drugs. A similar benefit of curcumin was observed in MCF-7 cells treated with ixabepilone, the primary systemic therapy to patients with invasive breast cancer (stages IIA–IIIB) before surgery. Antagonism effect was observed when MCF-7 cells were treated with curcumin plus cisplatin, vinorelbine or everolimus. Curcumin-induced enhancement in chemosensitivity was paralleled by significant increases (additivity or synergy effect) in apoptosis and cell cycle arrest at S and G2/M phases, the consequences of Aurora-A inhibition. These results suggest that a combination of curcumin with FDA-approved anticancer drugs warrants further assessment with a view to developing a novel clinical treatment for breast cancer.

A non-mitotic role for Aurora kinase A as a direct activator of cell migration upon interaction with PLD, FAK and Src.

ABSTRACTTimely activation of Aurora kinase A (AURA, also known as AURKA) is vital for centrosome formation and the progression of mitosis. Nonetheless, it is still unclear if and when other cellular functions are activated by AURA. We report here that Src phosphorylates and activates AURA at T288, and AURA also activates focal adhesion kinase (FAK, also known as PTK2), leading to initiation of cell movement. An additional and new way by which AURA is regulated, is by phospholipase D2 (PLD2), which causes AURA activation. In addition, AURA phosphorylates PLD, so both proteins engage in a positive reinforcement loop. AURA and PLD2 form a protein–protein complex and colocalize to cytoplasmic regions in cells. The reason why PLD activates AURA is because of the production of phosphatidic acid by the lipase, which binds directly to AURA, with the region E171–E211 projected to be a phosphatidic-acid-binding pocket. Furthermore, this direct interaction with phosphatidic acid enhances tubulin polymerization and cooperates synergistically with AURA, FAK and Src in yielding a fully effectual cellular migration. Thus, Src and FAK, and PLD and phosphatidic acid are new upstream regulators of AURA that mediate its role in the non-mitotic cellular function of cell migration.

Go6976, a Potent FLT3 Kinase Inhibitor, Exerts Antiproliferative Activity Against Acute Myeloid Leukemia Via Inhibition Of Survivin and Mcl-1 In The Presence Of Human Serum

The FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis, and thus recent studies are directed at developing and testing novel FLT3 inhibitors for the treatment of AML. Go6976 is an indolocarbazole with a simillar structural backbone to staurosporine. In the present study, we demonstrated that Go6976 displays a potent inhibitory activity against recombinant FLT3 using in vitro kinase assay. Its IC50 value is 0.7nM. We also tested the effect of Go6976 on several kinds of other kinases. Go6976 inhibited the kinase activity of Aurora-A, Aurora-B and JAK2 with IC50 values of 118.2 nM, 77.7 nM and 92.7 nM, respectively. Go6976 did not show the inhibitory activity against the FGFR3 even at 1 microM. These data indicated Go6976 preferentially and potently inhibit the FLT3. Go6976 significantly inhibited the proliferation of human leukemia cells having FLT3-ITD. The IC50 values of Go6976 against MV4-11 and MOLM-13 were 0.044 and 0.008 microM, respectively. In contrast, human leukemia HL-60 and U937 cells which lack FLT3-ITD showed strong resistance to Go6976 treatment. Furthermore, we observed that Go6976 shows minimal toxicity for purified human normal CD34(+) cells. Go6976 suppressed the phosphorylation of FLT3 in MV4-11 and MOLM13 cells. Consistent with FLT3 inhibition, Go6976 potently inhibited phosphorylation of constitutively activated STAT3/5, Erk1/2, and Akt. Western blotting analysis revealed that MV4-11 and MOLM13 cells possess abundant survivin and Mcl-1 protein. We hypothesized that the expression of survivin and Mcl-1 may be regulated by constitutive activation of FLT3. In order to test this hypothesis, the effect of siRNA for FLT3-ITD was examined. Indeed, we observed that siRNA-induced down-regulation of FLT3 decreased survivin and Mcl-1 expression in MOLM13 cells, suggesting that up-regulated survivin and Mcl-1 may be closely associated with FLT3 signaling. Interestingly, we found that both survivin mRNA and protein were rapidly downregulated by Go6976 treatment in MOLM13 and MV4-11 cells. It was also observed that Go6976 significantly suppressed Mcl-1 mRNA and protein. It has been reported that STAT-3 and STAT-5 signaling play a pivotal role in the regulation of survivin and Mcl-1, respectively. Thus, inhibitory effects of Go6976 on the expression Survivin and Mcl-1 may be a consequence of the suppression of phosphorylation of STAT-3 and STAT-5 by Go6976 in FLT3-ITD cells. This inhibition of anti-apoptotic proteins by Go6976 may be critical for its antiproliferative effect in FLT3-ITD cells. It has been known that previous FLT3 inhibitors such as PKC412 and CEP-701 bind to the human plasma protein, alpha1-acid glycoprotein, resulting in significantly diminished inhibitory activity against FLT3. Indeed, inhibitory effect of PKC412 on FLT3 was significantly decreased, when MOLM13 cells were treated with PKC412 in the presence of human serum. In contrast, we found that Go6976 potently inhibits phosphorylation of FLT3 and exerts cytotoxicity even in the presence of human serum or human alpha1-acid glycoprotein. In conclusion, our data indicate that Go6976 may have a unique therapeutic potential for FLT3-driven acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Insights into Aurora-A kinase activation using unnatural amino acids incorporated by chemical modification.

Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated and supported substrate phosphorylation. Aurora-A activity is enhanced by phosphorylation at position 287 alone but is suppressed when position 288 is also phosphorylated. This is rationalized by competition between phosphorylated T287 and T288 for a binding site composed of arginines, based on a structure of Aurora-A in which phospho-T287 occupies this site. This is, to our knowledge, the first example of a Ser/Thr kinase whose activity is controlled by the phosphorylation state of adjacent residues in its activation loop. Overall we demonstrate an approach that combines mutagenesis and selective chemical modification of selected cysteine residues to investigate otherwise impenetrable aspects of kinase regulation.

SMAD5 expression and inhibition of the mitotic kinase aurora-A on sensitivity of breast cancer cells to chemotherapy.

e13516 Background: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A will lead to increased sensitivity to chemotherapy. Methods: The hypothesis was tested in vitro. Cellular proliferation was tested using MTT assay and protein levels were determined on Western blots. Results: In ER+ cell line, MCF7, constitutive activation of Raf-1/MAPK signaling pathway leads to aberrant activation of Aurora-A kinase activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to parental MCF7 cells. This effect is even more pronounced with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by novel inhibitor, Alisertib. This was further explored in triple negative cells (MDA-MB-231) and cells expressing Her-2/neu (BT474) which express higher levels of Aurora-A. Combination of Alisertib and paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are investigating a role of Aurora-A in developing chemoresistance through activating SMAD5. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. Conclusions: Inhibition of Aurora-A by Alisertib resulted in decreased SMAD5 nuclear phosphorylation and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be extremely useful in developing more effective treatments for breast cancer patients both in neo-adjuvant, adjuvant and metastatic settings.

Abstract LB-214: Common genomic alterations in malignant peripheral nerve sheath tumors augment Aurora A activity and sensitize tumors to aurora kinase inhibitors.

Abstract Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary, cancers associated with mutations in the neurofibromin 1 gene 1. MPNSTs are often resistant to chemotherapies and have high rates of disease recurrence, highlighting the lack of effective treatment options for this cancer. Aurora kinase A inhibitors (AKIs) have shown promise against MPNST cell lines 2. We expanded this study by testing AKI in human MPNST xenotransplant mice models. Treatment resulted in stabilized disease with tumor cells undergoing senescence and endoreduplication. Aurora kinase A (AURKA) is an emerging target in cancer, however, targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumor response. Therefore, we utilized three MPNST cell lines and profiled them for the expression and activity of AURKA as well as their responses to AKIs. The most proliferative lines, S462 and 2884, express equivalent levels of AURKA, however, S462 cells were more sensitive to kinase inhibition. Both cell lines experienced apoptosis, senescence and endoreduplication in response to AKI treatment. AURKA activity is regulated by a co-activator, the Targeting Protein for XKlp2 (TPX2) and a molecular brake, the Receptor for Hyaluronan Mediated Motility (RHAMM)3. Interestingly, published analysis of copy number variation has identified hemizygous loss of the RHAMM gene in half of the examined high-grade MPNST, but not in benign or low grade tumors 4. We proposed that MPNSTs with RHAMM deletions are oncogene addicted to AURKA activity and are therefore, particularly susceptible to AKI. We profiled our MPNST lines for RHAMM and TPX2 expression and found that S462 cells express significantly more TPX2 and less RHAMM compared to 2884 cells. Furthermore, S462 cells had increased kinase. To determine whether levels of these molecular regulators could affect AKI efficacy we depleted RHAMM and TPX2 in 2884 and S462 cells respectively. While cells with reduced TPX2 have unchanged responses to AKIs, RHAMM depleted cells have a 2 fold reduction in IC-50s. We also looked at the effect of AKI against a population of MPNST tumor-initiating cells (TICs) from the S462 line. Compared to adherent cells, S462 TICs have elevated AURKA activity and their ability to self-renew in vitro is arrested by AKI. Indeed, the altered levels of kinase activity in the RHAMM and TPX2 depletion lines correlated with their ability to form and maintain sphere culture. In addition, we find that AKI treated S462 TICs differentiated into terminal neurons. All in all, these data indicate AURKA as a rational therapy for aggressive MPNSTs with RHAMM serving as a biomarker for AKI efficacy. Citation Format: Pooja Mohan, Joan Castellsague, Jihong Jiang, Kristi Allen, Helen Chen, Oksana Nemirovsky, Melanie Spyra, Kaiji Hu, Lan Kluwe, Miguel Pujana, Alberto Villanueva, Victor Mautner, Sandra Dunn, Jonathan Keats, Conxi Lazaro, Christopher Maxwell. Common genomic alterations in malignant peripheral nerve sheath tumors augment Aurora A activity and sensitize tumors to aurora kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2013-LB-214

AEG-1 overexpression is essential for maintenance of malignant state in human AML cells via up-regulation of Akt1 mediated by AURKA activation

Acute myeloid leukemia (AML) remains highly fatal, highlighting the need for improved understanding of signal pathways that can lead to the development of new therapeutic regimens targeting common molecular pathways shared across different AML subtypes. Here we demonstrate that astrocyte elevated gene-1 (AEG-1) is one of such pathways, involving in cell cycle and apoptosis regulation and contributing to enhanced proliferation and chemoresistance in HL-60 and U937 AML cells. The pleiotropic effects of AEG-1 on AML were found to correlate with two novel target genes, Aurora kinase A (AURKA) and Akt1. Down-regulation of AEG-1 by short-hairpin RNA (shRNA) could not only decrease AURKA expression both on mRNA and protein levels but also decrease the levels of pAkt473 and pAkt308 (the active forms of phosphorylated Akt), similar effect as using AURKA inhibitor Tozasertib (VX680). Furthermore, the AEG-1 shRNA-induced malignant phenotype changes could be mitigated by forced overexpression of AURKA through increased Akt1 activation and phosphorylation in AML cells. On the other hand, although exogenous expression of AEG-1 could increase both AURKA and Akt expression levels the simultaneous use of AURKA inhibitor Tozasertib blocked AEG-1's role of up-regulation of Akt expression in ECV304 cells, suggesting that AURKA might be a key mediator of AEG-1 in regulating Akt activation, and a key effector of AEG-1 in maintaining the malignant state of AML. Moreover, knockdown AEG-1 expression also changed the expression levels of PTEN, survivin and stathmin, the genes that have been reported to be involved in the development of several other malignant tumors. Our results provide evidence for AEG-1's carcinogenesis role in AML and reveal a novel functional link between AEG-1 and AURKA on Akt1 activation. AEG-1 can be an important candidate as a drug design target within AURKA signal pathway for more specific killing of AML cells while sparing normal cells.

Inhibition of Cdk2 activity decreases Aurora-A kinase centrosomal localization and prevents centrosome amplification in breast cancer cells

Centrosome amplification plays a key role in the origin of chromosomal instability (CIN) during cancer development and progression. In this study, MCF-7 breast cancer cell lines harboring abrogated p53 function (vMCF-7DNp53) were employed to investigate the relationship between induction of genotoxic stress, activation of cyclin-A/Cdk2 and Aurora-A oncogenic signalings and development of centrosome amplification. Introduction of genotoxic stress in the vMCF-7DNp53 cell line by treatment with hydroxyurea (HU) induced centrosome amplification that was mechanistically linked to Aurora-A kinase activity. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, methotrexate. Importantly, we demonstrated that Aurora-A kinase-induced centrosome amplification was mediated by Cdk2 kinase since molecular inhibition of Cdk2 activity by SU9516 suppressed Aurora-A centrosomal localization and consequent centrosome amplification. In addition, we employed vMCF-7DRaf-1 cells that display high levels of endogenous cyclin-A and demonstrated that molecular targeting of Aurora-A by Alisertib reduces cyclin-A expression. Taken together, these findings demonstrate a novel positive feed-back loop between cyclin-A/Cdk2 and Aurora-A pathways in the development of centrosome amplification in breast cancer cells. They also provide the translational rationale for targeting ‘druggable cell cycle regulators’ as an innovative therapeutic strategy to inhibit centrosome amplification and CIN in breast tumors resistant to conventional chemotherapeutic drugs.

Phosphorylation of NuMA by Aurora‐A kinase in PC‐3 prostate cancer cells affects proliferation, survival, and interphase NuMA localization

Aurora-A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora-A are up-regulated in multiple malignancies. Aurora-A is a key regulator of mitosis that localizes to the centrosome from the G2 phase through mitotic exit and regulates mitotic spindle formation as well as centrosome separation. Overexpression of Aurora-A in multiple malignancies has been linked to higher tumor grade and poor prognosis through mechanisms that remain to be defined. Using an unbiased proteomics approach, we identified the protein nuclear mitotic apparatus (NuMA) as a robust substrate of Aurora-A kinase. Using a small molecule Aurora-A inhibitor in conjunction with a reverse in-gel kinase assay (RIKA), we demonstrate that NuMA becomes hypo-phosphorylated in vivo upon Aurora-A inhibition. Using an alanine substitution strategy, we identified multiple Aurora-A phospho-acceptor sites in the C-terminal tail of NuMA. Functional analyses demonstrate that mutation of three of these phospho-acceptor sites significantly diminished cell proliferation. In addition, alanine mutation at these sites significantly increased the rate of apoptosis. Using confocal immunofluorescence microscopy, we show that the NuMA T1804A mutant mis-localizes to the cytoplasm in interphase nuclei in a punctate pattern. The identification of Aurora-A phosphorylation sites in NuMA that are important for cell cycle progression and apoptosis provides new insights into Aurora-A function.

The mitotic kinase Aurora-A promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα+ breast cancer cells

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα(+) breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα(+) breast cancer patients resistant to conventional endocrine therapy.

Genomic imbalance ofHMMR/RHAMMregulates the sensitivity and response of malignant peripheral nerve sheath tumour cells to aurora kinase inhibition

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM.

Melanoma-associated mutations in protein phosphatase 6 cause chromosome instability and DNA damage due to dysregulated Aurora-A

Mutations in the PPP6C catalytic subunit of protein phosphatase 6 (PP6) are drivers for the development of melanoma. Here, we analyse a panel of melanoma-associated mutations in PPP6C and find that these generally compromise assembly of the PP6 holoenzyme and catalytic activity towards a model substrate. Detailed analysis of one mutant, PPP6C-H114Y, in both primary melanoma and engineered cell lines reveals it is destabilized and undergoes increased proteasome-mediated turnover. Global analysis of phosphatase substrates by mass spectrometry identifies the oncogenic kinase Aurora-A as the major PP6 substrate that is dysregulated under these conditions. Accordingly, cells lacking PPP6C or carrying the PPP6C-H114Y allele have elevated Aurora-A kinase activity and display chromosome instability with associated Aurora-A-dependent micronucleation. Chromosomes mis-segregated to these micronuclei are preferentially stained by the DNA damage marker γ-H2AX, suggesting that loss of PPP6C promotes both chromosome instability and DNA damage. These findings support the view that formation of micronuclei rather than chromosome instability alone explains how loss of PPP6C, and more generally mitotic spindle and centrosome defects, can act as drivers for genome instability in melanoma and other cancers.

Abstract P2-07-01: BRCA1 regulates RHAMM function in breast cancer

Abstract Objectives: The receptor for hyaluronan-mediated motility (RHAMM, intracellular hyaluronan binding protein [IHABP], CD168), whose over-expression in tumors is associated with poor prognosis and early age at diagnosis, localizes to the centrosome, interacts with microtubules, functions in the maintenance of spindle pole stability and is required for centrosomal targeting. A previous study has shown that genetic variation at the HMMR locus (encoding for RHAMM) modifies breast cancer risk among BRCA1 mutation carriers and interplay between BRCA1, AURKA (aurora kinase A), RHAMM and phosphorylated RHAMM (pRHAMM) plays an important role in epithelial apicobasal polarization and breast carcinogenesis. Aim of this study was to investigate mechanisms by which BRCA1 regulates the function of RHAMM. Methods: Four pairs of shBRCA1 oligonucleotides containing target sequences against BRCA1 transcript were designed and synthesized. After annealing, these four double-strand shBRCA1 fragments were inserted into pLVTH vector and stably transfected into MCF-12A cells for knock-down of BRCA1 expression. The BRCA1 knock-out breast cancer cell line HCC1937 with homozygous BRCA1 c.5382insC mutation was transfected with the full length wt-BRCA1 to restore BRCA1 function. HCC1937 cells were also transfected with the BRCA1-delExon11 isoform that lacks BRCA1 exon 11 in its entirety. Western blotting analysis and immunohistochemical staining with anti-BRCA1, anti-RHAMM, anti-pRHAMM and anti-AURKA antibodies were performed. Real-time PCR was used to quantify RHAMM and AURKA expression in these different cell lines and transfectants. Results: Stable transfection of MCF-12A cells with the different shRNAs resulted in a significant decrease of BRCA1 transcript and protein levels. Real-time PCR showed increased RHAMM expression in MCF-12A cells transfected with shRNA BRCA1 knock-down constructs. Increased RHAMM gene expression was also determined in HCC1937 cells lacking BRCA1 function due to homozygous deleterious BRCA1 mutation c.5382insC. Restored wt-BRCA1 expression in HCC1937 cells transfected with the full length BRCA1 cDNA results in was significantly reduced RHAMM expression. Inhibition of RHAMM gene expression was even more pronounced in HCC1937 cells transfected with the BRCA1-delExon11variant lacking 1,143 amino acids of the BRCA1 protein. On the protein level loss of BRCA1 function affects nuclear localization of pT703-RHAMM the product of AURKA activity: pT703-RHAMM staining was revealed to be strong at the nuclear envelope in MCF12A BRCA1 knock-down transfectants compared to the homogenous and less intense but clearly nuclear pT703-RHAMM staining in MCF12A wt-BRCA1 cells. Conclusions: BRCA1 negatively regulates RHAMM gene expression and might be involved in the nuclear localization of pRHAMM. Interestingly on the transcription level the BRCA1delexon11 isoform of BRCA1 displayed a stronger RHAMM repressor function than BRCA1 wild type. Taken together, our data provide insight into new functions of BRCA1 regulating RHAMM expression and mediating pRHAMM function. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-07-01.

Low Concentration of Arsenic-Induced Aberrant Mitosis in Keratinocytes Through E2F1 Transcriptionally Regulated Aurora-A

Chronic exposure to low-concentration arsenic promotes cell proliferation and carcinogenesis both in vitro and in vivo. Centrosome amplification, the major cause of chromosome instability, occurs frequently in cancers. Aurora-A is a mitotic kinase and causes centrosome amplification and chromosome instability when overexpressed. Our previous study revealed that low-concentration arsenic induces Aurora-A overexpression in immortalized bladder cells. In this study, we hypothesized that low-concentration arsenic induces aberrant mitosis in keratinocytes due to Aurora-A overexpression. The specimen of Bowen's disease (BD) and squamous cell carcinoma obtained from arseniasis-endemic areas in Taiwan showed Aurora-A overexpression. The mRNA/protein levels and kinase activity of Aurora-A were increased in immortalized keratinocyte HaCaT cells after arsenic treatment at low concentration (< 1µM). Aberrant spindles, multiple centrosomes, and multinucleated cells were detected under fluorescent microscopy in HaCaT cells after arsenic treatment. These findings were associated with increased expression of Aurora-A. We further revealed that Aurora-A was regulated by arsenic-induced transcriptional factor E2F1 as demonstrated by chromosome immunoprecipitation, promoter activity, and small interfering RNA assays. Finally, in arsenic-treated HaCaT cells and in BD, a significant increase of dysfunctional p53 was found, and this event correlated with the increase in expression of Aurora-A. Altogether, our data suggest that low concentration of arsenic induces activation of E2F1-Aurora-A axis and results in aberrant mitosis of keratinocytes. Overexpression of Aurora-A and dysfunctional p53 may act synergistically to trigger skin tumor formation. Our findings suggest that Aurora-A may be a potential target for the prevention and treatment of arsenic-related cancers.

Aurora Kinase A Drives MTOC Biogenesis but Does Not Trigger Resumption of Meiosis in Mouse Oocytes Matured In Vivo1

Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.

Calmodulin activation of Aurora-A kinase (AURKA) is required during ciliary disassembly and in mitosis

The centrosomal Aurora-A kinase (AURKA) regulates mitotic progression, and overexpression and hyperactivation of AURKA commonly promotes genomic instability in many tumors. Although most studies of AURKA focus on its role in mitosis, some recent work identified unexpected nonmitotic activities of AURKA. Among these, a role for basal body-localized AURKA in regulating ciliary disassembly in interphase cells has highlighted a role in regulating cellular responsiveness to growth factors and mechanical cues. The mechanism of AURKA activation involves interactions with multiple partner proteins and is not well understood, particularly in interphase cells. We show here that AURKA activation at the basal body in ciliary disassembly requires interactions with Ca(2+) and calmodulin (CaM) and that Ca(2+)/CaM are important mediators of the ciliary disassembly process. We also show that Ca(2+)/CaM binding is required for AURKA activation in mitosis and that inhibition of CaM activity reduces interaction between AURKA and its activator, NEDD9. Finally, mutated derivatives of AURKA impaired for CaM binding and/or CaM-dependent activation cause defects in mitotic progression, cytokinesis, and ciliary resorption. These results define Ca(2+)/CaM as important regulators of AURKA activation in mitotic and nonmitotic signaling.