Accumulation Of Mitotic Cells Research Articles

Spectroscopic, DFT, molecular docking and anti-cancer studies of regioselectively designed 1-aryl-indenopyrazoles

L-Proline catalyzed condensation of 1-indanones with DMF-DMA (N,N-dimethylformamide dimethyl acetal) resulted in (Z)-2-((dimethylamino)methylene-2,3-dihydro-1H-inden-1-ones 2 instead of their E-isomers 3, which is further regioselectively transformed into 1-aryl-2,4-dihydroindeno[1,2-c]pyrazole 4 rather than 2-aryl-2,4-dihydroindeno[1,2-c]pyrazoles 5 by condensing with aryl hydrazines. Spectrochemical characterizations and single crystal X-ray diffraction studies were used to resolve syntactic ambiguity between the possible geometric isomers of enaminones (E & Z) and regioisomers 1H-indenopyrazole and 2H-indenopyrazoles. The experimental findings were correlated with computed values and have shown good correlation with the proposed structures. The synthesized indenopyrazoles 4a–f were tested for their ability to inhibit the proliferation of A549 human lung cancer cell lines, and the compound 4b have showed IC50 value 13.77 µM comparable to the reference medication erlotinib (10.26 µM). The complete cell cycle analysis have revealed the accumulation of mitotic cells in G1/S phase. The flow cytometric analysis also revealed the increase in apoptotic cells indicating tumor suppression by the derivative 4b. To get insight into the interactions of the compound and EGFR tyrosine kinase protein molecular docking was performed. Also, pharmacokinetic profiling revealed about the oral compatibility of the synthesized compounds.

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, β-Catenin, and TRK pathways.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC50 value of 1.04nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC50 value of 1.46, 1.53, and 1.47nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.

Abstract LB-288: CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics

Abstract DNA replication is a tightly regulated process required for faithful duplication of the genome. Previous research in lower eukaryotes has demonstrated a requisite role for the CDC7 protein kinase in DNA replication origin firing through phosphorylation of the MCM2-7 helicase complex. Owing to its overexpression in various neoplasms, CDC7 has emerged as an attractive target for cancer treatment. We previously reported that SRA141, a potent and selective CDC7 inhibitor, is cytotoxic to multiple tumor cell lines in vitro and demonstrates robust anti-tumor efficacy in colorectal and leukemia xenograft models. Here, we explored the effects of SRA141 on DNA replication and cell cycle dynamics and how these effects relate to the mechanism of SRA141-induced cell death in colorectal cancer cell lines. SRA141 treatment caused a complete inhibition of MCM2 phosphorylation and reduced overall cellular DNA synthesis within 3 hours, with the greatest effects observed in late S-phase. Unexpectedly, DNA combing experiments indicated that SRA141 caused a 70-90% increase in the rate of replication fork progression. These findings suggest that SRA141 may indeed limit origin firing, but this effect is compensated for in part by increased replication fork rates. Despite delay, nucleotide analog pulse-chase experiments indicated that SRA141 treated cells ultimately finished S phase and transitioned into mitosis. In contrast to other reports, no evidence of replication fork collapse was observed, indicating that the main mechanism of SRA141 cytotoxicity is not mediated through replication fork collapse. Intriguingly, some of the most pronounced effects of SRA141 treatment were on progression through M phase, as evidenced by an accumulation of mitotic cells and increased Cyclin B levels following treatment. Moreover, inhibition of Aurora B kinase, which regulates mitotic progression, strongly synergized with SRA141 in caspase-dependent cancer cell killing. Taken together, our findings indicate that the mechanism of cytotoxicity of CDC7 inhibitors is distinct from agents that cause replication fork collapse or inhibit CDKs, and thus may define a new class of cancer therapeutic agents that synergize with drugs that target certain mitotic pathways. Citation Format: Veena Jagannathan, Snezana Milutinovic, Ryan Hansen, Bryan Strouse, Christian Hassig, Eric Brown. CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-288.

Design, synthesis, in silico pharmacokinetics prediction and biological evaluation of 1,4-dihydroindeno[1,2-c]pyrazole chalcone as EGFR /Akt pathway inhibitors

In an attempt to develop potent and selective anticancer agents, a series of 15 conjugates of 1,4-dihydroindeno[1,2-c]pyrazole chalcone (12a-o) were designed, synthesized and evaluated for their antiproliferative activity against MCF7, A549, MDA-MB-231, HCT116 and SKBR3 human cancer cell lines. Among them, 12h, 12l and 12m showed IC50 values: 3.82, 5.33 and 4.21 μM, respectively, on A549 cell with respect to the positive control, Erlotinib (IC50 value: 10.26 μM). Detailed biological assays showed accumulation of mitotic cells in G2/M phase. In addition, Western blot analysis and immunofluorescence study revealed inhibition of EGFR and Akt pathways. In silico computational studies were also carried out to predict the binding modes and pharmacokinetic parameters of these conjugates.

CREPT/RPRD1B associates with Aurora B to regulate Cyclin B1 expression for accelerating the G2/M transition in gastric cancer

Gastric cancer, like most of other cancers, has an uncontrolled cell cycle regulated by cyclins and cyclin-dependent kinases (CDKs). In this study, we reported that gastric cancer cells showed an accelerated G2/M transition promoted by CREPT/RPRD1B and Aurora kinase B (Aurora B). We found that CREPT/RPRD1B and Aurora B were coordinately expressed during the cell cycle in gastric cancer cells. Deletion of CREPT/RPRD1B disturbed the cell progression and extended the length of cell cycle, leading to a significant accumulation of mitotic cells. Mechanistically, we revealed that CREPT/RPRD1B interacted with Aurora B to regulate the expression of Cyclin B1 in gastric cancer cells. Interestingly, Aurora B phosphorylates S145 in a well-conserved motif of CREPT/RPRD1B. We proposed that phosphorylation of CREPT/RPRD1B by Aurora B is required for promoting the transcription of Cyclin B1, which is critical for the regulation of gastric tumorigenesis. Our study provides a mechanism by which gastric tumor cells maintain their high proliferation rate via coordination of Aurora B and CREPT/RPRD1B on the expression of Cyclin B1. Targeting the interaction of Aurora B and CREPT/RPRD1B might be a strategy for anti-gastric cancer therapy in the future.

Loss-of-function mutations in KIF14 cause severe microcephaly and kidney development defects in humans and zebrafish.

Mutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development.

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent ApcMin/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Design, synthesis and biological evaluation of 1, 4-dihydro indeno[1,2-c] pyrazole linked oxindole analogues as potential anticancer agents targeting tubulin and inducing p53 dependent apoptosis

A series of 1, 4-dihydroindeno-[1,2-c] pyrazole linked oxindole conjugates have been synthesized by using Knoevenagel condensation method and further evaluated for their antiproliferative activity against HeLa, A549 and MDA-MB-231 human cancer cell lines along with HEK-293 (normal human embryonic kidney cells). Among the derivatives, compounds 12a, 12b, and 12d showed excellent cytotoxicity with IC50 values ranging between 1.33 to 4.33 μM. Furthermore, detailed biological assays showed that there was accumulation of mitotic cells in G2/M phase, disruption of microtubule network and increase in the G2/M checkpoint proteins (Cyclin B1 and CDK1). Moreover, compound 12d with IC50 value of 1.33 μM showed significant upregulation of tumor suppressor proteins like p53, p21 and pro-apoptotic Bax. The molecular docking analysis demonstrated that these congeners occupy the colchicine binding pocket of the tubulin.

Loss of the RNA helicase SKIV2L2 impairs mitotic progression and replication-dependent histone mRNA turnover in murine cell lines.

RNA surveillance via the nuclear exosome requires cofactors such as the helicase SKIV2L2 to process and degrade certain noncoding RNAs. This research aimed to characterize the phenotype associated with RNAi knockdown of Skiv2l2 in two murine cancer cell lines: Neuro2A and P19. SKIV2L2 depletion in Neuro2A and P19 cells induced changes in gene expression indicative of cell differentiation and reduced cellular proliferation by 30%. Propidium iodide-based cell-cycle analysis of Skiv2l2 knockdown cells revealed defective progression through the G2/M phase and an accumulation of mitotic cells, suggesting SKIV2L2 contributes to mitotic progression. Since SKIV2L2 targets RNAs to the nuclear exosome for processing and degradation, we identified RNA targets elevated in cells depleted of SKIV2L2 that could account for the observed twofold increase in mitotic cells. Skiv2l2 knockdown cells accumulated replication-dependent histone mRNAs, among other RNAs, that could impede mitotic progression and indirectly trigger differentiation.

Stabilization of 4E-BP1 by PI3K kinase and its involvement in CHK2 phosphorylation in the cellular response to radiation.

Objectives: 4E-BP1 is a family member of eIF4E binding proteins (4E-BPs) which act as the suppressors of cap-dependent translation of RNA via competitively associating with cap-bound eIF4E. RNA translation regulation is an important manner to control the cellular responses to a series of stress conditions such as ionizing radiation (IR)-induced DNA damage response and cell cycle controlling. This study aimed to determine the mechanism of 4E-BP1 stabilization and its potential downstream target(s) in the response to IR.Methods: PI3Ks kinase inhibitors were used to determine the signaling control of 4E-BP1 phosphorylation and protein stability. shRNA strategy was employed to silence the expression of 4E-BP1 in HeLa and HepG2 cells, and determine its effect on the irradiation-induced CHK2 phosphorylation. The protein degradation/stability was investigated by western blotting on the condition of blocking novel protein synthesis by cycloheximide (CHX).Results: The phosphorylation of 4E-BP1 at Thr37/46 was significantly increased in both HepG2 and HeLa cells by ionizing radiation. Depression of 4E-BP1 by shRNA strategy resulted in an incomplete G2 arrest at the early stage of 2 hours post-irradiation, as well as a higher accumulation of mitotic cells at 10 and 12 hours post-irradiation as compared to the control cells. Consistently, the CHK2 phosphorylation at Thr68 induced by IR was also attenuated by silencing 4E-BP1 expression. Both PI3K and DNA-PKcs kinase inhibitors significantly decreased the protein level of 4E-BP1, which was associated with the accelerated degradation mediated by ubiquitination-proteasome pathway.Conclusion: PI3K kinase activity is necessary for maintaining 4E-BP1 stability. Our results also suggest 4E-BP1 a novel biological role of regulating cell cycle G2 checkpoint in responding to IR stress in association with controlling CHK2 phosphorylation.

The p90 ribosomal S6 kinase 2 specifically affects mitotic progression by regulating the basal level, distribution and stability of mitotic spindles.

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles.

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin.

The effect of the anticancer drug doxorubicin on cytomegalovirus-infected human fibroblasts

The anticancer antibiotic doxorubicin (DOX) is highly toxic and has life-threatening side effects. The effect of DOX on normal cells is not well studied. It has been proposed that DOX induces cancer cell death by apoptosis. However, information regarding the role of p73 protein, a member of the p53 tumor suppressor family, is sparse. Cytomegalovirus (CMV) triggers an antiapoptotic program, and the virus is able to carry out genome replication before the death of the target cell. Our goal was to examine the effect of DOX on normal cells, analyze the ability of the CMV-induced antiapoptotic program to reduce DOX toxicity, and examine the involvement of p73 protein and its isoforms in the regulation of death of CMV-infected and DOX-treated cells. Cell counting revealed that DOX killed about 70% of human embryonic lung fibroblasts (HELFs) in culture. Combined exposure to DOX and CMV reduced cell death. The cell number with DNA breaks found by TUNEL declined from 5.2 to 3.2% (p < 0.05) after DOX exposure of CMV-infected cells. Analysis of mitotic figures revealed that DOX caused accumulation of mitotic cells, which was not observed in CMV-infected and DOX-treated cells. PCR analysis of mRNA of two p73 protein isoforms (TAp73 and dNp73) showed that, in uninfected cells, TAp73 isoform expression was low, while in CMV-infected cells the level of TAp73 was significantly higher and for the first time dNp73 expression was registered. Infected cells treated with DOX did not exhibit mitosis block. The cells displayed activation of caspases 8, 9, and 3 accompanied by the cells’ death; however, this was not as large-scale as in cultures exposed only to DOX. From these findings, it can be concluded that CMV reduces DOX-related damage in normal cells. This suggests that TAp73 and dNp73 induction provides conditions that diminish DOX-produced DNA damage and death of normal cells.

New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines

Abstract Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines. Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC50 values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism. Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines. Disclosures No relevant conflicts of interest to declare.

Abstract 2730: RNAi knockdown or chemical inhibition of anaphase-promoting complex components is synthetic lethal with HSP90 inhibition

Abstract The molecular chaperone heat shock protein 90 (HSP90) maintains the conformation, stability and function of oncogenic client proteins, many of which are mutated or overexpressed. Therefore, HSP90 is an important cancer therapeutic target. To further increase the efficacy of HSP90 inhibitors, combinatorial therapeutic strategies may be beneficial. Here we report an unbiased global screening approach to identify genes that encode potentially druggable proteins that modulate cellular responses to HSP90 inhibition. From the 7,593 genes that were tested, three components of the anaphase-promoting complex (APC/C) were among the genes which when silenced caused significant HSP90 inhibitor sensitisation. siRNA knockdown of ANAPC1, -8 or -4 produced up to three-fold sensitisation to HSP90 inhibitors 17-AAG and AUY922 in human colon cancer cells. Intriguingly, we show that combinatorial siRNA-mediated ANAPC1 knockdown and HSP90 inhibition induced an accumulation of mitotic cells exhibiting a tri-polar spindle formation. A marked increase in cyclin B1 and aurora A protein levels was observed in response to combinatorial ANAPC1 knockdown and HSP90 inhibition. These effects may have been responsible for the observed formation of aberrations in the mitotic spindle architecture. Furthermore, after a period of prolonged mitotic arrest, cancer cells treated with combinational ANAPC1 knockdown and HSP90 inhibition were committed to apoptosis. Mimicking siRNA knockdown, treatment with the APC/C inhibitor proTAME synergistically sensitised human colon carcinoma cells to HSP90 inhibition. These exciting results provide a mechanistic explanation underlying the HSP90 inhibitor sensitisation phenotype, and suggest that the APC/C may represent an interesting therapeutic target to exploit in combination with HSP90 inhibitors. Furthermore, therapeutic approaches may be developed which exploit the vulnerability of mitotic cancer cells in the context of HSP90 inhibition. Citation Format: Jennifer Howes, Bing-Feng Lu, Marissa Powers, Costas Mitsopoulos, Bissan Al-Lazikani, Spiros Linardopoulos, Paul Clarke, Paul Workman. RNAi knockdown or chemical inhibition of anaphase-promoting complex components is synthetic lethal with HSP90 inhibition. [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 2730. doi:10.1158/1538-7445.AM2014-2730

Depletion of RNA-binding protein RBM8A (Y14) causes cell cycle deficiency and apoptosis in human cells

RBM8A (Y14) contains an RNA-binding motif and forms a tight heterodimer with Magoh. The heterodimer is known to be a member of the exon junction complex that forms on mRNA before export and it is required for mRNA metabolism processes such as splicing, mRNA export and nonsense-mediated mRNA decay. Recently, deficient cellular proliferation has been observed in RBM8A- or Magoh-depleted cells. These results prompted us to study the role of RBM8A in cell cycle progression of human tumour cells. The depletion of RBM8A in A549 cells resulted in poor cell survival and the accumulation of mitotic cells. After release from G1/S arrest induced by a double thymidine block, the RBM8A-silenced cells could not proceed to the next G1 phase beyond G2/M phase. Finally, the sub-G1 population increased and the apoptosis markers caspases 3/7 were activated. Silenced cells exhibited an increased frequency of multipolar or monopolar centrosomes, which may have caused the observed deficiency in cell cycle progression. Finally, silencing of either RBM8A or Magoh resulted in mutual downregulation of the other protein. These results illustrate that the RBM8A-Magoh mRNA binding complex is required for M phase progression and both proteins may be novel targets for anticancer therapy.

SB225002 Promotes Mitotic Catastrophe in Chemo-Sensitive and -Resistant Ovarian Cancer Cells Independent of p53 Status In Vitro

Recent evidence indicates that CXCR2 signaling is crucial for cancer progression, and its antagonist SB225002 induces apoptosis in Wilms’ tumor cells. Here, we investigated the effect of SB225002 on cell cycle progression and apoptosis induction in vitro, using CDDP-sensitive and -resistant OVCA cell lines with different p53 status (wild type, mutant or null). Adenovirus infection of wild-type p53 or transfection of p53 siRNA was used to over-express or knock-down p53. Cell cycle and apoptosis were determined by flow cytometry or Hoechst staining and observation of nuclear morphology. Our data demonstrated that SB225002 induced apoptosis in both wild-type and p53-deficient ovarian cancer (OVCA) cells through alternative mechanisms. SB225002 promoted mitotic catastrophe, as evidenced by the accumulation of mitotic cells with spindle abnormalities, chromosome mis-segregation, multi-polar cell division, multiple nuclei, aneuploidy/polyploidy and subsequent extensive apoptosis. SB225002-induced mitotic catastrophe appeared to be mediated by down-regulation of checkpoint kinase Chk1 and Cdk1-cyclin B activation. In cells expressing wild-type p53 (OV2008 and C13*), SB225002 increased total and phospho-Ser p53 levels, and p53 knock-down decreased SB225002-induced apoptosis, without affecting premature mitosis. These results suggest that SB225002 induces p53-dependent apoptosis, and provokes mitotic catastrophe in p53-independent manner in p53 wild-type cells. Reconstitution with wild-type P53 in P53-null SKOV3 cell attenuated SB225002-induced mitotic catastrophe, suggesting p53 prevented mitotic catastrophe induced by SB225002 in p53-deficient OVCA cells. Finally, the effect of SB225002 could not be prevented by pretreatment with CXCR2 ligand or its neutralizing antibody. The present studies demonstrate for the first time that SB225002 has dual actions in OVCA cells, inducing classic apoptosis through p53 activation and provoking mitotic catastrophe in both p53 wild-type and deficient cells by Chk1 inhibition and Cdk activation. These findings raise the possibility of SB225002 as a new candidate molecule for OVCA therapy independent of the p53 status.

Phase I Assessment of New Mechanism-Based Pharmacodynamic Biomarkers for MLN8054, a Small-Molecule Inhibitor of Aurora A Kinase

The mitotic kinase Aurora A is an important therapeutic target for cancer therapy. This study evaluated new mechanism-based pharmacodynamic biomarkers in cancer patients in two phase I studies of MLN8054, a small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received MLN8054 orally for 7 consecutive days in escalating dose cohorts, with skin and tumor biopsies obtained before and after dosing. Skin biopsies were evaluated for increased mitotic cells within the basal epithelium. Tumor biopsies were assessed for accumulation of mitotic cells within proliferative tumor regions. Several patients in the highest dose cohorts showed marked increases in the skin mitotic index after dosing. Although some tumors exhibited increases in mitotic cells after dosing, others displayed decreases, a variable outcome consistent with dual mechanisms of mitotic arrest and mitotic slippage induced by antimitotics in tumors. To provide a clearer picture, mitotic cell chromosome alignment and spindle bipolarity, new biomarkers of Aurora A inhibition that act independently of mitotic arrest or slippage, were assessed in the tumor biopsies. Several patients, primarily in the highest dose cohorts, had marked decreases in the percentage of mitotic cells with aligned chromosomes and bipolar spindles after dosing. Evidence existed for an exposure-effect relationship for mitotic cells with defects in chromosome alignment and spindle bipolarity that indicated a biologically active dose range. Outcomes of pharmacodynamic assays from skin and tumor biopsies were concordant in several patients. Together, these new pharmacodynamic assays provide evidence for Aurora A inhibition by MLN8054 in patient skin and tumor tissues.

Suspension of Mitotic Activity in Dentate Gyrus of the Hibernating Ground Squirrel

Neurogenesis occurs in the adult mammalian hippocampus, a region of the brain important for learning and memory. Hibernation in Siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h (from 35-36°C to +4–6°C) permits accumulation of mitotic cells at different stages of the cell cycle. Histological methods used to study adult neurogenesis are limited largely to fixed tissue, and the mitotic state elucidated depends on the specific phase of mitosis at the time of day. However, using an immunohistochemical study of doublecortin (DCX) and BrdU-labelled neurons, we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity. Our data suggest that doublecortin-labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus.

Proteomic Analysis of Annexin A2 Phosphorylation Induced by Microtubule Interfering Agents and Kinesin Spindle Protein Inhibitors

Microtubule interfering agents (MIAs) are antitumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause the accumulation of mitotic cells and subsequently cell death. We used two-dimensional gel electrophoresis (2DE) followed by MALDI-MS analysis to demonstrate that the MIAs vinblastine (Velban) and paclitaxel (Taxol), as well as the KSP inhibitor S-tritil-L-cysteine, induce the phosphorylation of annexin A2 in human lung carcinoma A549 cells. Further tandem mass spectrometry analysis using a combination of peptide fragmentation methods (CID and ETD) and multiple reaction monitoring (MRM) analysis determined that this modification occurs mainly at threonine 19. We show that MIAs and KSP inhibitors only induce this phosphorylation in cells capable of reaching the M phase. Furthermore, we demonstrate that CDK activity is required for the phosphorylation of annexin A2 induced by MIAs and KSP inhibitors. Finally, we have used double thymidine block synchronization to demonstrate that annexin A2 is not phosphorylated during a normal mitosis, indicating that this phosphorylation of annexin A2 is a specific response to these drugs.