Seize the engine: Emerging cell cycle targets in breast cancer.

Breast cancer arises from a series of molecular alterations that disrupt cell cycle checkpoints, leading to aberrant cell proliferation and genomic instability. Targeted pharmacological inhibition of cell cycle regulators has long been considered a promising anti-cancer strategy. Initial attempts to drug critical cell cycle drivers were hampered by poor selectivity, modest efficacy and haematological toxicity. Advances in our understanding of the molecular basis of cell cycle disruption and the mechanisms of resistance to CDK4/6 inhibitors have reignited interest in blocking specific components of the cell cycle machinery, such as CDK2, CDK4, CDK7, PLK4, WEE1, PKMYT1, AURKA and TTK. These targets play critical roles in regulating quiescence, DNA replication and chromosome segregation. Extensive preclinical data support their potential to overcome CDK4/6 inhibitor resistance, induce synthetic lethality or sensitise tumours to immune checkpoint inhibitors. This review provides a biological and drug development perspective on emerging cell cycle targets and novel inhibitors, many of which exhibit favourable safety profiles and promising activity in clinical trials.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Mitotic kinases as drivers of the epithelial-to-mesenchymal transition and as therapeutic targets against breast cancers.

Biological therapies against breast cancer patients with tumors positive for the estrogen and progesterone hormone receptors and Her2 amplification have greatly improved their survival. However, to date, there are no effective biological therapies against breast cancers that lack these three receptors or triple-negative breast cancers (TNBC). TNBC correlates with poor survival, in part because they relapse following chemo- and radio-therapies. TNBC is intrinsically aggressive since they have high mitotic indexes and tend to metastasize to the central nervous system. TNBCs are more likely to display centrosome amplification, an abnormal phenotype that results in defective mitotic spindles and abnormal cytokinesis, which culminate in aneuploidy and chromosome instability (known causes of tumor initiation and chemo-resistance). Besides their known role in cell cycle control, mitotic kinases have been also studied in different types of cancer including breast, especially in the context of epithelial-to-mesenchymal transition (EMT). EMT is a cellular process characterized by the loss of cell polarity, reorganization of the cytoskeleton, and signaling reprogramming (upregulation of mesenchymal genes and downregulation of epithelial genes). Previously, we and others have shown the effects of mitotic kinases like Nek2 and Mps1 (TTK) on EMT. In this review, we focus on Aurora A, Aurora B, Bub1, and highly expressed in cancer (Hec1) as novel targets for therapeutic interventions in breast cancer and their effects on EMT. We highlight the established relationships and interactions of these and other mitotic kinases, clinical trial studies involving mitotic kinases, and the importance that represents to develop drugs against these proteins as potential targets in the primary care therapy for TNBC.

Characterization and risk association of polymorphisms in Aurora kinases A, B and C with genetic susceptibility to gastric cancer development.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in genes encoding mitotic kinases could influence development and progression of gastric cancer (GC). METHODS: Case-control study of nine SNPs in mitotic genes was conducted using qPCR. The study included 116 GC patients and 203 controls. In silico analysis was performed to evaluate the effects of polymorphisms on transcription factors binding sites. RESULTS: The AURKA rs1047972 genotypes (CT vs. CC: OR, 1.96; 95% CI, 1.05-3.65; p = 0.033; CC + TT vs. CT: OR, 1.94; 95% CI, 1.04-3.60; p = 0.036) and rs911160 (CC vs. GG: OR, 5.56; 95% CI, 1.24-24.81; p = 0.025; GG + CG vs. CC: OR, 5.26; 95% CI, 1.19-23.22; p = 0.028), were associated with increased GC risk, whereas certain rs8173 genotypes (CG vs. CC: OR, 0.60; 95% CI, 0.36-0.99; p = 0.049; GG vs. CC: OR, 0.38; 95% CI, 0.18-0.79; p = 0.010; CC + CG vs. GG: OR, 0.49; 95% CI, 0.25-0.98; p = 0.043) were protective. Association with increased GC risk was demonstrated for AURKB rs2241909 (GG + AG vs. AA: OR, 1.61; 95% CI, 1.01-2.56; p = 0.041) and rs2289590 (AC vs. AA: OR, 2.41; 95% CI, 1.47-3.98; p = 0.001; CC vs. AA: OR, 6.77; 95% CI, 2.24-20.47; p = 0.001; AA+AC vs. CC: OR, 4.23; 95% CI, 1.44-12.40; p = 0.009). Furthermore, AURKC rs11084490 (GG + CG vs. CC: OR, 1.71; 95% CI, 1.04-2.81; p = 0.033) was associated with increased GC risk. A combined analysis of five SNPs, associated with an increased GC risk, detected polymorphism profiles where all the combinations contribute to the higher GC risk, with an OR increased 1.51-fold for the rs1047972(CT)/rs11084490(CG + GG) to 2.29-fold for the rs1047972(CT)/rs911160(CC) combinations. In silico analysis for rs911160 and rs2289590 demonstrated that different transcription factors preferentially bind to polymorphic sites, indicating that AURKA and AURKB could be regulated differently depending on the presence of particular allele. CONCLUSIONS: Our results revealed that AURKA (rs1047972 and rs911160), AURKB (rs2241909 and rs2289590) and AURKC (rs11084490) are associated with a higher risk of GC susceptibility. Our findings also showed that the combined effect of these SNPs may influence GC risk, thus indicating the significance of assessing multiple polymorphisms, jointly. The study was conducted on a less numerous but ethnically homogeneous Bosnian population, therefore further investigations in larger and multiethnic groups and the assessment of functional impact of the results are needed to strengthen the findings.

Small molecule tools in mitosis research.

Mitosis belongs to the most appealing cellular processes. Yet, the highly dynamic and complex nature of mitosis represents a major challenge when it comes to the functional dissection of mitotic proteins. Due to their fast and often reversible mode of action, small molecules have proven themselves as invaluable tools to dissect mitotic processes. In this chapter, we provide a broad overview of available compounds affecting mitosis. We discuss the different application fields of small molecules and important aspects that have to be considered when using them. Finally, we provide two detailed protocols for the application of small molecules to study mitosis in tissue culture cells.

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation.

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases.

Anti-Melanoma Activities of Haspin Inhibitor CHR-6494 Deployed as a Single Agent or in a Synergistic Combination with MEK Inhibitor.

Background: Melanoma is a heterogeneous malignancy that presents an immense challenge in therapeutic development. Recent approaches targeting the oncogenic MAP kinase pathways have shown tremendous improvement in the overall survival of patients with advanced melanoma. However, there is still an urgent need for identification of new strategies to overcome drug resistances and to improve therapeutic efficacy. Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) belongs to a selected group of mitotic kinases and is required for normal mitosis progression. In contrast to inhibitors of other mitotic kinases, anti-tumor potential of haspin inhibitors has not been well explored. Herein, we aim to examine effects of CHR-6494, a small molecule inhibitor of haspin, in melanoma cells. Methods: Anti-tumor activities of the haspin inhibitor CHR-6494 were tested in a number of melanoma cell lines either as a single agent or in combination with the MEK inhibitor Trametinib (GSK1120212). Experiments are based on: 1) Cell viability determined by the crystal violet staining assay; 2) apoptotic responses measured by the caspase 3/7 activity assay and western blot analysis for the level of cleaved PARP (Poly ADP-Ribose Polymerase); 3) cell cycle analysis conducted using flow cytometry; and 4) cell migratory ability assessed by the scratch assay and the transwell migration assay. Results: We have found that CHR-6494 alone elicits a dose dependent inhibitory effect on the viability of several melanoma cell lines. This growth inhibition is accompanied by an increase in apoptotic responses. More importantly, CHR-6494 appears to synergize with the MEK inhibitor Trametinib in suppressing cell growth and enhancing apoptosis in both wild type and BRAFV600E mutant melanoma cell lines. Administering of these two small molecules as a combination is also capable of suppressing cell migration to a greater extent than the individual agent. Conclusion: These results suggest that haspin can be considered as a viable anti-melanoma target, and that concomitant inhibition of haspin and MEK activities with small molecules could represent a novel therapeutic strategy with improved efficacy for treatment of melanoma.

Mitotic read-out genes confer poor outcome in luminal A breast cancer tumors.

Luminal breast tumors have been classified into A and B subgroups, with the luminal A being associated with a more favorable clinical outcome. Unfortunately, luminal A tumors do not have a universally good prognosis. We used transcriptomic analyses using public datasets to evaluate the differential expression between normal breast tissue and breast cancer, focusing on upregulated genes included in cell cycle function. Association of selected genes with relapse free survival (RFS) and overall survival (OS) was performed using the KM Plotter Online Tool (http://www.kmplot.com). Seventy-seven genes were differentially expressed between normal and malignant breast tissue. Only five genes were associated with poor RFS and OS. The mitosis-related genes GTSE1, CDCA3, FAM83D and SMC4 were associated with poor outcome specifically in Luminal A tumors. The combination of FAM83D and CDCA3 for RFS and GTSE1 alone for OS showed the better prediction for clinical outcome. CDCA3 was amplified in 3.4% of the tumors, and FAM83D and SMC4 in 2.3% and 2.2%, respectively. In conclusion, we describe a set of genes that predict detrimental outcome in Luminal A tumors. These genes may have utility for stratification in trials of antimitotic agents or cytotoxic chemotherapy, or as candidates for direct target inhibition.

In silico analyses identify gene-sets, associated with clinical outcome in ovarian cancer: role of mitotic kinases.

INTRODUCTION: Accurate assessment of prognosis in early stage ovarian cancer is challenging resulting in suboptimal selection of patients for adjuvant therapy. The identification of predictive markers for cytotoxic chemotherapy is therefore highly desirable. Protein kinases are important components in oncogenic transformation and those relating to cell cycle and mitosis control may allow for identification of high-risk early stage ovarian tumors. METHODS: Genes with differential expression in ovarian surface epithelia (OSE) and ovarian cancer epithelial cells (CEPIs) were identified from public datasets and analyzed with dChip software. Progression-free (PFS) and overall survival (OS) associated with these genes in stage I/II and late stage ovarian cancer was explored using the Kaplan Meier Plotter online tool. RESULTS: Of 2925 transcripts associated with modified expression in CEPIs compared to OSE, 66 genes coded for upregulated protein kinases. Expression of 9 of these genes (CDC28, CHK1, NIMA, Aurora kinase A, Aurora kinase B, BUB1, BUB1ßB, CDKN2A and TTK) was associated with worse PFS (HR:3.40, log rank p<0.001). The combined analyses of CHK1, CDKN2A, AURKA, AURKB, TTK and NEK2 showed the highest magnitude of association with PFS (HR:4.62, log rank p<0.001). Expression of AURKB predicted detrimental OS in stage I/II ovarian cancer better than all other combinations Conclusion: Genes linked to cell cycle control are associated with worse outcome in early stage ovarian cancer. Incorporation of these biomarkers in clinical studies may help in the identification of patients at high risk of relapse for whom optimizing adjuvant therapeutic strategies is needed.

Transcriptomic analyses identify association between mitotic kinases, PDZ-binding kinase and BUB1, and clinical outcome in breast cancer.

Protein kinases are important components in oncogenic transformation of breast cancer. Evaluation of upregulated genes that codify for protein kinases could be used as biomarkers to predict clinical outcome. Gene expression and functional analyses using public datasets were performed to identify differential gene expression and functions in basal-like tumors compared with normal breast tissue. Overall survival (OS) associated with upregulated genes was explored using the KM Plotter online tool. The prognostic influence of these genes in luminal tumors and systemically untreated patients was also assessed. Of the 426 transcripts identified in basal-like tumors, 11 genes that coded for components of protein kinases were upregulated with more than a fourfold change. Regulation of cell cycle was an enriched function containing 10 of these 11 identified genes. Among them, expression of four genes, BUB1ß, CDC28, NIMA, and PDZ binding kinase, were all associated with improved OS when using at least one probe in the basal-like subtype. Two genes, BUB1ß and PDZ binding kinase, showed consistent association with improved OS irrespective of the gene probe used for the analysis. No association was observed for these genes with relapse-free survival. In contrast, both BUB1ß and PDZ binding kinase showed worse OS in luminal tumors and in a cohort of systemically untreated patients. BUB1ß and PDZ binding kinase are associated with improved OS in basal-like tumors and worse OS in luminal and untreated patients. The association with a better outcome in basal-like tumors could be due to a more favorable response to chemotherapy.

Mitotic kinase cascades orchestrating timely disjunction and movement of centrosomes maintain chromosomal stability and prevent cancer.

Centrosomes are microtubule-organizing centers that duplicate in S phase to form bipolar spindles that separate duplicated chromosomes faithfully into two daughter cells during cell division. Recent studies show that proper timing of centrosome dynamics, the disjunction and movement of centrosomes, is tightly linked to spindle symmetry, correct microtubule-kinetochore attachment, and chromosome segregation. Here, we review mechanisms that regulate centrosome dynamics, with emphasis on the roles of key mitotic kinases in the proper timing of centrosome dynamics and how aberrancies in these processes may cause chromosomal instability and cancer.

Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis.

Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel-Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell-cell adhesion and apical-basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the "non-mitotic" unconventional functions of these kinases in renal tumorigenesis.

FBXW7 and USP7 regulate CCDC6 turnover during the cell cycle and affect cancer drugs susceptibility in NSCLC.

CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet.We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

Molecular insight into the regulation and function of MCAK.

Chromosome stability is ensured by precisely fine-tuned dynamics of mitotic spindles, which are controlled by a network of various microtubule-associated and interacting proteins including the kinesin-13 family. The best characterized member of this family is the mitotic centromere-associated kinesin (MCAK). By efficiently depolymerizing microtubules, MCAK influences various key events during mitosis. MCAK itself is regulated by its interaction partners, its intrinsic conformation switch and the phosphorylation of mitotic kinases like Aurora A/B, cyclin-dependent kinase 1 and Polo-like kinase 1. Perturbing its regulation alters MCAK's conformation, catalytic activity, subcellular localization and stability, leading further to mitotic defects in spindle formation and chromosome movement. Indeed, MCAK is aberrantly regulated in various cancer types, which is linked to increased invasiveness, metastasis and drug resistance. In the current review, we summarize recently published data concerning MCAK, correlate its conformation changes with its depolymerization activity and function, propose a model of its regulation by multiple mitotic kinases and highlight its potential involvement in oncogenesis and drug resistance.

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals.

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

Targeting prostate cancer cell lines with polo-like kinase 1 inhibitors as a single agent and in combination with histone deacetylase inhibitors.

Combinations of anticancer therapies with high efficacy and low toxicities are highly sought after. Therefore, we studied the effect of polo-like kinase 1 (Plk1) inhibitors on prostate cancer cells as a single agent and in combination with histone deacetylase (HDAC) inhibitors valproic acid and vorinostat. IC50s of Plk1 inhibitors BI 2536 and BI 6727 were determined in prostate cancer cells by MTS assays. Morphological and molecular changes were assessed by immunoblotting, immunofluorescence, flow cytometry, real-time RT-PCR, and pulldown assays. Efficacy of combination therapy was assessed by MTS and clonogenic assays. IC50 values in DU145, LNCaP, and PC3 cells were 50, 75, and 175 nM, respectively, for BI 2536 and 2.5, 5, and 600 nM, respectively, for BI 6727. Human prostate fibroblasts and normal prostate epithelial cells were unaffected at these concentrations. While DU145 and LNCaP cells were solely arrested in mitosis on treatment, PC3 cells accumulated in G2 phase and mitosis, suggesting a weak spindle assembly checkpoint. Combining Plk1 inhibitors with HDAC inhibitors had synergistic antitumor effects in vitro. DMSO-treated prostate cancer cells were used as controls to study the effect of Plk1 and HDAC inhibition. Plk1 inhibitors decreased proliferation and clonogenic potential of prostate cancer cells. Hence, Plk1 may serve as an important molecular target for inhibiting prostate cancer. Combining HDAC inhibitors with BI 2536 or BI 6727 may be an effective treatment strategy against prostate cancer.

Kinasas y fosfatasas: el yin y el yan de la vida / Kinases and phosphatases: the life´s yin yan

Introducción: la existencia de tendencias opuestas en un proceso o fenómeno de la naturaleza es la fuente principal del desarrollo. Objetivo: el principal mecanismo de control de la mayoría de los procesos biológicos al nivel molecular son las modificaciones postraduccionales y dentro de ella los ciclos de fosforilación/desfosforilación. Las proteínas al ser fosforiladas o desfosforiladas experimentan un cambio conformacional que modifica su actividad, sus propiedades, su localización celular y las interacciones con otras moléculas. Resultados: las enzimas implicadas en este ciclo son las kinasas y las fosfatasas, cuyas acciones coordinadas y opuestas dan el soporte mecanístico necesario para el desarrollo de múltiples funciones celulares. Conclusiones: en este trabajo se pretende demostrar, mediante el estudio de la participación de kinasas y fosfatasas en la división celular, que estas enzimas representan la expresión molecular de los contrarios, yin y yan, de la antigua Filosofía China(AU)

Introduction: opposite forces in the inside of a process are the main sources for development. Objective: the principal mechanism to control most biological processes is the postranslational modifications of proteins. Among these the phosphorylation/ ephosphorylation cycle is the most extended in nature. Phosphorylation or dephosphorylation of proteins causes a conformational change that modifies their activities, properties, celular localization and interactions with other molecules. Results: kinases and phosphatese are the main enzymes implied in this cycle which coordinated and opposite actions give the mechanistic support for several cellular functions. Conclusions: by mean of an study about the involvement of kinases and phosphatases in cell division, this paper tray to show that these enzymes represent the molecular expression of the yin and yan, the opposite element on ancient Chinese philosophy(AU)

Kinasas y fosfatasas: el yin y el yan de la vida / Kinases and phosphatases: the life´s yin yan

Introducción: la existencia de tendencias opuestas en un proceso o fenómeno de la naturaleza es la fuente principal del desarrollo. Objetivo: el principal mecanismo de control de la mayoría de los procesos biológicos al nivel molecular son las modificaciones postraduccionales y dentro de ella los ciclos de fosforilación/desfosforilación. Las proteínas al ser fosforiladas o desfosforiladas experimentan un cambio conformacional que modifica su actividad, sus propiedades, su localización celular y las interacciones con otras moléculas. Resultados: las enzimas implicadas en este ciclo son las kinasas y las fosfatasas, cuyas acciones coordinadas y opuestas dan el soporte mecanístico necesario para el desarrollo de múltiples funciones celulares. Conclusiones: en este trabajo se pretende demostrar, mediante el estudio de la participación de kinasas y fosfatasas en la división celular, que estas enzimas representan la expresión molecular de los contrarios, yin y yan, de la antigua Filosofía China.

Introduction: opposite forces in the inside of a process are the main sources for development. Objective: the principal mechanism to control most biological processes is the postranslational modifications of proteins. Among these the phosphorylation/ ephosphorylation cycle is the most extended in nature. Phosphorylation or dephosphorylation of proteins causes a conformational change that modifies their activities, properties, celular localization and interactions with other molecules. Results: kinases and phosphatese are the main enzymes implied in this cycle which coordinated and opposite actions give the mechanistic support for several cellular functions. Conclusions: by mean of an study about the involvement of kinases and phosphatases in cell division, this paper tray to show that these enzymes represent the molecular expression of the yin and yan, the opposite element on ancient Chinese philosophy.