The conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.

Severe, irreversible salivary gland disease and oral dryness is experienced by sufferers of Sjögren's syndrome and those treated with irradiation for head and neck cancer. Therefore, major efforts have been made in the last decade to unravel key molecular signals that can drive salivary gland (SG) regeneration and functional restoration. However, the earliest molecular determinants that accompany SG regeneration remain incompletely defined. The present study examined the initial mitogenic events marking the regenerative response of the murine submandibular gland (SMG), following innate immune-mediated injury. Local intraductal administration of the synthetic double stranded (ds) RNA polyinosinic-polycytidylic acid (poly (I:C)) widely, but transiently, depleted the acinar and progenitor cells, 24 hours post poly (I:C) introduction. While the progenitor and duct cells started to proliferate and expand at 72 hours, the Mist1-positve acinar cells did not re-appear until 96 hours post poly (I:C) injury. The cellular replenishment during regeneration involved significant upregulation of the cell cycle promoter Aurora kinase B (AURKB). AURKB, which is expressed in healthy proliferating and cancerous cells, is a serine/threonine protein kinase, well known to orchestrate key events in cell division and cytokinesis. However, the expression and role of AURKB in regeneration of post mitotic salivary gland cells has not been previously explored. In vivo inhibition of AURKB using the selective inhibitor Barasertib (AZD1152-HQPA) interfered with SMG recovery from the transient, but severe poly (I:C)-mediated injury and cellular depletion. AURKB deficiency during regeneration of the injured tissues: disrupted cell cycle progression, repressed renewal of Mist1-positive acinar cells and prevented recovery of salivary secretion. The knowledge gained in this study may be utilized in the development of therapeutic targets for irreversible salivary gland disease.

Recessive mutations in WD repeat domain 62 (WDR62) cause microcephaly and a wide spectrum of severe brain malformations. Disruption of the mouse ortholog results in microcephaly underlain by reduced proliferation of neocortical progenitors during late neurogenesis, abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. Spindle pole localization of WDR62 and mitotic progression are defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Expression of WDR62 is closely correlated with components of the chromosome passenger complex (CPC), a key regulator of mitosis. Wild type WDR62, but not disease-associated mutant forms, interacts with the CPC core enzyme Aurora kinase B and staining of CPC components at centromeres is altered in patient-derived fibroblasts. Our findings demonstrate critical and diverse functions of WDR62 in neocortical development and provide insight into the mechanisms by which its disruption leads to a plethora of structural abnormalities.

Aurora kinase B plays an important role in the cell cycle to orchestrate the mitotic process. The amplification and overexpression of this kinase have been implicated in several human malignancies. Therefore, Aurora kinase B is a potential drug target for anticancer therapies. Here, we combine atom-based 3D-QSAR analysis and pharmacophore model generation to identify the principal structural features of acylureidoindolin derivatives that could potentially be responsible for the inhibition of Aurora kinase B. The selected CoMFA and CoMSIA model showed significant results with cross-validation values (q2) of 0.68, 0.641 and linear regression values (r2) of 0.971, 0.933 respectively. These values support the statistical reliability of our model. A pharmacophore model was also generated, incorporating features of reported crystal complex structures of Aurora kinase B. The pharmacophore model was used to screen commercial databases to retrieve potential lead candidates. The resulting hits were analyzed at each stage for diversity based on the pharmacophore model, followed by molecular docking and filtering based on their interaction with active site residues and 3D-QSAR predictions. Subsequently, MD simulations and binding free energy calculations were performed to test the predictions and to characterize interactions at the molecular level. The results suggested that the identified compounds retained the interactions with binding residues. Binding energy decomposition identified residues Glu155, Trp156 and Ala157 of site B and Leu83 and Leu207 of site C as major contributors to binding affinity, complementary to 3D-QSAR results. To best of our knowledge, this is the first comparison of WaterSwap field and 3D-QSAR maps. Overall, this integrated strategy provides a basis for the development of new and potential AK-B inhibitors and is applicable to other protein targets.

Wild-type p53 is a stress-responsive transcription factor and potent tumor suppressor. P53 activates or represses genes involved in cell cycle progression or apoptosis in order to arrest the cell cycle or induce cell death. Transcription repression by p53 is indirect and requires repressive members of the RB-family (RB1, RBL1, RBL2) and formation of repressor complexes of RB1-E2F and RBL1/RBL2-DREAM. Many aurora kinase A/B (AURKA/B) pathway genes are repressed in a p53-DREAM-dependent manner. We found heightened expression of RBL2 and reduced expression of AURKA/B pathway genes is associated with improved outcomes in p53 wild-type but not p53 mutant non-small cell lung cancer (NSCLC) patients. Knockdown of p53, RBL2, or the DREAM component LIN37 increased AURKA/B pathway gene expression and reduced paclitaxel and radiation toxicity in NSCLC cells. In contrast, pharmacologic inhibition of AURKA/B or knockdown of AURKA/B pathway components increased paclitaxel and IR sensitivity. The results support a model in which p53-RBL2-DREAM-mediated repression of the AURKA/B pathway contributes to tumor suppression, improved tumor therapy responses, and better outcomes in p53 wild-type NSCLCs.

The Ku heterodimer (Ku70/Ku80) is the central DNA binding component of the classical non-homologous end joining (NHEJ) pathway that repairs DNA double-stranded breaks (DSBs), serving as the scaffold for the formation of the NHEJ complex. Here we show that Ku70 is phosphorylated on Serine 155 in response to DNA damage. Expression of Ku70 bearing a S155 phosphomimetic substitution (Ku70 S155D) in Ku70-deficient mouse embryonic fibroblasts (MEFs) triggered cell cycle arrest at multiple checkpoints and altered expression of several cell cycle regulators in absence of DNA damage. Cells expressing Ku70 S155D exhibited a constitutive DNA damage response, including ATM activation, H2AX phosphorylation and 53BP1 foci formation. Ku70 S155D was found to interact with Aurora B and to have an inhibitory effect on Aurora B kinase activity. Lastly, we demonstrate that Ku and Aurora B interact following ionizing radiation treatment and that Aurora B inhibition in response to DNA damage is dependent upon Ku70 S155 phosphorylation. This uncovers a new pathway where Ku may relay signaling to Aurora B to enforce cell cycle arrest in response to DNA damage.

Hyperactivation of Wnt and Ras-MAPK signalling are common events in development of colorectal adenomas. Further progression from adenoma-to-carcinoma is frequently associated with 20q gain and overexpression of Aurora kinase A (AURKA). Interestingly, AURKA has been shown to further enhance Wnt and Ras-MAPK signalling. However, the molecular details of these interactions in driving colorectal carcinogenesis remain poorly understood. Here we first performed differential expression analysis (DEA) of AURKA knockdown in two colorectal cancer (CRC) cell lines with 20q gain and AURKA overexpression. Next, using an exact algorithm, Heinz, we computed the largest connected protein-protein interaction (PPI) network module of significantly deregulated genes in the two CRC cell lines. The DEA and the Heinz analyses suggest 20 Wnt and Ras-MAPK signalling genes being deregulated by AURKA, whereof β-catenin and KRAS occurred in both cell lines. Finally, shortest path analysis over the PPI network revealed eight 'connecting genes' between AURKA and these Wnt and Ras-MAPK signalling genes, of which UBE2D1, DICER1, CDK6 and RACGAP1 occurred in both cell lines. This study, first, confirms that AURKA influences deregulation of Wnt and Ras-MAPK signalling genes, and second, suggests mechanisms in CRC cell lines describing these interactions.

The effects of AURKA overexpression associated with poor clinical outcomes have been attributed to increased cell cycle progression and the development of genomic instability with aneuploidy. We used RNA interference to examine the effects of AURKA overexpression in human bladder cancer cells. Knockdown had minimal effects on cell proliferation but blocked tumor cell invasion. Whole genome mRNA expression profiling identified nicotinamide N-methyltransferase (NNMT) as a downstream target that was repressed by AURKA. Chromatin immunoprecipitation and NNMT promoter luciferase assays revealed that AURKA's effects on NNMT were caused by PAX3-mediated transcriptional repression and overexpression of NNMT blocked tumor cell invasion in vitro. Overexpression of AURKA and activation of its downstream pathway was enriched in the basal subtype in primary human tumors and was associated with poor clinical outcomes. We also show that the FISH test for the AURKA gene copy number in urine yielded a specificity of 79.7% (95% confidence interval [CI] = 74.2% to 84.1%), and a sensitivity of 79.6% (95% CI = 74.2% to 84.1%) with an AUC of 0.901 (95% CI = 0.872 to 0.928; P < 0.001). These results implicate AURKA as an effective biomarker for bladder cancer detection as well as therapeutic target especially for its basal type.

The emergence of resistance to imatinib mediated by mutations in the BCR-ABL has become a major challenge in the treatment of chronic myeloid leukemia (CML). Alternative therapeutic strategies to override imatinib-resistant CML are urgently needed. In this study, we investigated the effect of AKI603, a novel small molecule inhibitor of Aurora kinase A (AurA) to overcome resistance mediated by BCR-ABL-T315I mutation. Our results showed that AKI603 exhibited strong anti-proliferative activity in leukemic cells. AKI603 inhibited cell proliferation and colony formation capacities in imatinib-resistant CML cells by inducing cell cycle arrest with polyploidy accumulation. Surprisingly, inhibition of AurA by AKI603 induced leukemia cell senescence in both BCR-ABL wild type and T315I mutation cells. Furthermore, the induction of senescence was associated with enhancing reactive oxygen species (ROS) level. Moreover, the anti-tumor effect of AKI603 was proved in the BALB/c nude mice KBM5-T315I xenograft model. Taken together, our data demonstrate that the small molecule AurA inhibitor AKI603 may be used to overcome drug resistance induced by BCR-ABL-T315I mutation in CML.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator for an accurate kinetochore-microtubule attachment. However, the regulatory mechanism underlying precise MCAK depolymerase activity control during mitosis remains elusive. Here, we describe a novel pathway involving an Aurora B-PLK1 axis for regulation of MCAK activity in mitosis. Aurora B phosphorylates PLK1 on Thr210 to activate its kinase activity at the kinetochores during mitosis. Aurora B-orchestrated PLK1 kinase activity was examined in real-time mitosis using a fluorescence resonance energy transfer-based reporter and quantitative analysis of native PLK1 substrate phosphorylation. Active PLK1, in turn, phosphorylates MCAK at Ser715 which promotes its microtubule depolymerase activity essential for faithful chromosome segregation. Importantly, inhibition of PLK1 kinase activity or expression of a non-phosphorylatable MCAK mutant prevents correct kinetochore-microtubule attachment, resulting in abnormal anaphase with chromosome bridges. We reason that the Aurora B-PLK1 signaling at the kinetochore orchestrates MCAK activity, which is essential for timely correction of aberrant kinetochore attachment to ensure accurate chromosome segregation during mitosis.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder caused by ATTCT expansion in the ATXN10 gene. Previous investigations have identified that depletion of Ataxin-10, the gene product, leads to cellular apoptosis and cytokinesis failure. Herein we identify the mitotic kinase Aurora B as an Ataxin-10 interacting partner. Aurora B interacts with and phosphorylates Ataxin-10 at S12, as evidenced by in vitro kinase and mass spectrometry analysis. Both endogenous and S12-phosphorylated Ataxin-10 localizes to the midbody during cytokinesis, and cytokinetic defects induced by inhibition of ATXN10 expression is not rescued by the S12A mutant. Inhibition of Aurora B or expression of the S12A mutant renders reduced interaction between Ataxin-10 and polo-like kinase 1 (Plk1), a kinase previously identified to regulate Ataxin-10 in cytokinesis. Taken together, we propose a model that Aurora B phosphorylates Ataxin-10 at S12 to promote the interaction between Ataxin-10 and Plk1 in cytokinesis. These findings identify an Aurora B-dependent mechanism that implicates Ataxin-10 in cytokinesis.

Kinases are essential regulators of a variety of cellular signaling processes, including neurite formation-a foundational step in neurodevelopment. Aberrant axonal sprouting and failed regeneration of injured axons are associated with conditions like traumatic injury, neurodegenerative disease, and seizures. Investigating the mechanisms underlying neurite formation will allow for identification of potential therapeutics. We used a kinase inhibitor library to screen 493 kinase inhibitors and observed that 45% impacted neuritogenesis in Neuro2a (N-2a) cells. Based on the screening, we further investigated the roles of Aurora kinases A, B, and C and Nuak kinases 1 and 2. The roles of Aurora and Nuak kinases have not been thoroughly studied in the nervous system. Inhibition or overexpression of Aurora and Nuak kinases in primary cortical neurons resulted in various neuromorphological defects, with Aurora A regulating neurite initiation, Aurora B and C regulating neurite initiation and elongation, all Aurora kinases regulating arborization, and all Nuak kinases regulating neurite initiation and elongation and arborization. Our high-throughput screening and analysis of Aurora and Nuak kinases revealed their functions and may contribute to the identification of therapeutics.

The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the Km value of the phosphorylation is increased from 10 to 45 μM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177-197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the kcat value from about 40 to 7 min-1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.

During mitosis, spatiotemporal regulation of phosphorylation at the kinetochore is essential for accurate chromosome alignment and proper chromosome segregation. Aurora B kinase phosphorylates kinetochore substrates to correct improper kinetochore-microtubule (KT-MT) attachments, whereas tension across the centromeres inactivates Aurora B kinase, and PP2A phosphatase dephosphorylates the kinetochore proteins to stabilize the attachments. However, the molecular entity of the tension sensing mechanism remains elusive. In a previous report, we showed that centromeric SET/TAF1 on Sgo2 up-regulates Aurora B kinase activity via PP2A inhibition in prometaphase. Here we show that Aurora B and Bub1 at the centromere/kinetochore regulate both kinase activities one another in an inter-kinetochore distance-dependent manner, indicating a positive feedback loop. We further show that the centromeric pool of SET on Sgo2 depends on Bub1 kinase activity, and the centromeric localization of SET decreases in a distance-dependent manner, thereby inactivating Aurora B in metaphase. Consistently, ectopic targeting of SET to the kinetochores during metaphase hyperactivates Aurora B via PP2A inhibition, and thereby rescues the feedback loop. Thus, we propose that SET, Aurora B and Bub1 form a distance-dependent positive feedback loop, which spatiotemporally may act as a tension sensor at centromeres.

Tubulin-binding agents (TBAs) are designed to target microtubule (MT) dynamics, resulting in compromised mitotic spindles and an unsatisfied spindle assembly checkpoint. The activity of Aurora B kinase is indispensable for TBA-induced mitotic arrest, and its inhibition causes mitotic slippage and postmitotic endoreduplication. However, the precise phenomenon underlying mitotic slippage, which is caused by treatment with both Aurora B inhibitors and TBAs, and the cell fate after postmitotic slippage are not completely understood. Here, we found that HeLa and breast cancer cells treated with the different types of TBAs, such as paclitaxel and eribulin (MT-stabilizing and MT-destabilizing agents, respectively), exhibited distinct behaviors of mitotic slippage on inhibition of Aurora B. In such conditions, the cell fates after postmitotic slippage vastly differed with respect to cell morphology, cell proliferation, and cytotoxicity in short-term culture; that is, the effects of inhibition of Aurora B were beneficial for cytotoxicity enhancement in eribulin treatment but not in paclitaxel. However, in long-term culture, the cells that survived after mitotic slippage underwent endoreduplication and became giant cells in both cases, resulting in cellular senescence. We propose that MT-destabilizing agents may be more appropriate than MT-stabilizing agents for treating cancer cells with a weakened Aurora B kinase activity.

Aurora A is a serine/threonine kinase whose role in cell cycle progression and tumour generation has been widely studied. Recent work has revealed an unexpected function for Aurora A during CD4+ T cell activation and, also, in graft versus host disease development. However, it remains unknown whether Aurora A is involved in CD8+ T cell effector function and in cytotoxic T lymphocyte-mediated antiviral response. Here, we show that Aurora A chemical inhibition leads to an impairment of both the peptide-specific cytotoxicity and the degranulation activity of CD8+ T cells. This finding was similarly proven for both mice and human CD8+ CTL activity. As a result of Aurora A blockade, we detected a reduction in the expression induced by T cell activation of genes classically related to the effector function of cytotoxic T lymphocytes such as granzyme B or perforin1. Finally, we have found that Aurora A is necessary for CD8+ T cell-mediated antiviral response, in an in vivo model of vaccinia virus infection. Thus, we can conclude that Aurora A activity is, indeed, needed for the proper effector function of cytotoxic T lymphocytes and for their activity against viral threats.

Glioblastoma multiforme (GBM) is the most frequent type of primary astrocytomas. We examined the association between single nucleotide polymorphisms (SNPs) in Aurora kinase A (AURKA), Aurora kinase B (AURKB), Aurora kinase C (AURKC) and Polo-like kinase 1 (PLK1) mitotic checkpoint genes and GBM risk by qPCR genotyping. In silico analysis was performed to evaluate effects of polymorphic biological sequences on protein binding motifs. Chi-square and Fisher statistics revealed a significant difference in genotypes frequencies between GBM patients and controls for AURKB rs2289590 variant (p = 0.038). Association with decreased GBM risk was demonstrated for AURKB rs2289590 AC genotype (OR = 0.54; 95% CI = 0.33-0.88; p = 0.015). Furthermore, AURKC rs11084490 CG genotype was associated with lower GBM risk (OR = 0.57; 95% CI = 0.34-0.95; p = 0.031). Bioinformatic analysis of rs2289590 polymorphic region identified additional binding site for the Yin-Yang 1 (YY1) transcription factor in the presence of C allele. Our results indicated that rs2289590 in AURKB and rs11084490 in AURKC were associated with a reduced GBM risk. The present study was performed on a less numerous but ethnically homogeneous population. Hence, future investigations in larger and multiethnic groups are needed to strengthen these results.

Effective experimental prophylactic vaccines against viral pathogens such as herpes simplex virus type 1 (HSV-1) have been shown to protect the host through T and/or B lymphocyte-driven responses. Previously, we found a live-attenuated HSV-1 mutant, 0ΔNLS used as a prophylactic vaccine, provided significant protection against subsequent ocular HSV-1 challenge aligned with a robust neutralizing antibody response. Yet, how the virus mutant elicited the humoral immune response relative to parental virus was unknown. Herein, we present the characterization of B cell subsets in vaccinated mice at times after primary vaccination and following boost compared to the parental virus, termed GFP105. We found that 0∆NLS-vaccinated mice possessed more CD4+ follicular helper T (TFH) cells, germinal B cells and class-switched B cells within the first 7 days post-vaccination. Moreover, 0∆NLS vaccination resulted in an increase in plasmablasts and plasma cells expressing amino-acid transporter CD98 along with an elevated titer of HSV-1-specific antibody compared to GFP105-vaccinated animals. Furthermore, O∆NLS-vaccine-induced CD4+ (TFH) cells produced significantly more IL-21 compared to mice immunized with the parental HSV-1 strain. In contrast, there were no differences in the number of regulatory B cells comparing the two groups of immunized mice. In comparing sera recognition of HSV-1-encoded proteins, it was noted antiserum from GFP105-vaccinated mice immunoprecipitated HSV-1 thymidine kinase (TK) and glycoprotein M (gM) whereas sera from 0∆NLS-immunized mice did not even though both groups of vaccinated mice displayed similar neutralizing antibody titers to HSV-1 and were highly resistant to ocular HSV-1 challenge. Collectively, the results suggest (1) the live-attenuated HSV-1 mutant 0∆NLS elicits a robust B cell response that drives select B cell responses greater than the parental HSV-1 and (2) HSV-1 TK and gM are likely expendable components in efficacy of a humoral response to ocular HSV-1 infection.

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.