Mitotic Cell Cycle Arrest Research Articles

FBXO42 activity is required to prevent mitotic arrest, spindle assembly checkpoint activation and lethality in glioblastoma and other cancers.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. To identify genes differentially required for the viability of GBM stem-like cells (GSCs), we performed functional genomic lethality screens comparing GSCs and control human neural stem cells. Among top-scoring hits in a subset of GBM cells was the F-box-containing gene FBXO42, which was also predicted to be essential in ∼15% of cell lines derived from a broad range of cancers. Mechanistic studies revealed that, in sensitive cells, FBXO42 activity prevents chromosome alignment defects, mitotic cell cycle arrest and cell death. The cell cycle arrest, but not the cell death, triggered by FBXO42 inactivation could be suppressed by brief exposure to a chemical inhibitor of Mps1, a key spindle assembly checkpoint (SAC) kinase. FBXO42's cancer-essential function requires its F-box and Kelch domains, which are necessary for FBXO42's substrate recognition and targeting by SCF (SKP1-CUL1-F-box protein)ubiquitin ligase complex. However, none of FBXO42's previously proposed targets, including ING4, p53 and RBPJ, were responsible for the observed phenotypes. Instead, our results suggest that FBOX42 alters the activity of one or more proteins that perturb chromosome-microtubuledynamics in cancer cells, whichin turn leads to induction of the SAC and cell death.

Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids.

Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n=2× =18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin-proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids.

MicroRNA-508-3p regulates the proliferation of human lung cancer cells by targeting G1 to S phase transition 1 (GSPT1) protein.

Due to its crucial cancer regulatory role, microRNA-508-3p has been reported as a potential therapeutic anticancer molecular target. The present work encompassed the molecular characterization of microRNA-508-3p in lung cancer emphasizing on understanding the possible mechanism of its regulatory action. qRT-PCR was performed to estimate the relative gene expression of microRNA-508-p in the tissue samples. The proliferation of cancer cells was determined by cell counting kit-8. The colony formation from cancer cells was analyzed by clonogenic assay. Mitotic phase distribution was understood by employing the flow cytometric technique. Edu-Hoechst staining was used for the assessment of cell viability. In silico analysis and dual-luciferase assay were used for target identification of microRNA-508-3p in lung cancer. Immunofluorescence and western blotting studies were carried out for relative protein expression. The rat models were used for performing the in vivo experimental procedures. The study showed the significant down-regulation of microRNA-508-3p in lung cancer. The lower expression levels of microRNA-508-3p were shown to be associated with poor survival of lung cancer patients. The over-expression of microRNA-508-3p was found to decline the proliferation and viability of cancer cells together with the induction of mitotic cell cycle arrest at G1 by targeting G1 to S phase transition 1 (GSPT1) protein. MicroRNA-508-3p up-regulation inhibited the in vivo tumor growth in rat models. Our study identifies miR-508-3p as a pivotal regulator of lung cancer cell proliferation by targeting the GSPT1 protein. This highlights its potential as a tumor suppressor and a therapeutic target for lung cancer. Our findings offer mechanistic insights into miRNA-mediated cancer progression, prompting further research in this intricate regulatory network.

Effect of 13.56 MHz radiofrequency hyperthermia on mitotic cell cycle arrest in MCF7 breast cancer cell line and suggest a time interval for radiotherapy

After surgery, radiotherapy is the most common technique to treat breast cancer. Over the past decades, the thermal effects of radiofrequency-wave hyperthermia combined with radiotherapy have been used to increase radiosensitivity in cancer treatment. The cells have various radiation and thermal sensitivities at different stages of the mitotic cycle. Furthermore, ionizing radiation and the thermal effect of hyperthermia affect the cells' mitotic cycle and can partly induce cell cycle arrest. However, the time interval between hyperthermia and radiotherapy, as an essential factor influencing hyperthermia effect on cancer cells' cycle arrest, has not been studied before. In this study, we investigated the effect of hyperthermia on the MCF7 cancer cell cycle arrest in mitotic cycles at various selected time intervals after hyperthermia to find and propose appropriate time intervals between hyperthermia and radiotherapy. In this experimental study, we used the MCF7 breast cancer cell line to investigate the effect of 13.56 MHz hyperthermia (at a temperature of 43°C for a period of 20 min) on their cell cycle arrest. We performed the flowcytometry assay to assess the changes in the mitotic phases of the cell population at different time intervals (1, 6, 24, and 48 h) after hyperthermia. Our flowcytometry results indicated the 24-h time interval has the most significant effect on the cell population at S and G2/M phases. Therefore, the 24-h time interval can be proposed as the most appropriate time after hyperthermia for carrying out combinational radiotherapy procedure. Among various investigated time intervals examined in our research, the 24-h time interval can be proposed as the most appropriate time between hyperthermia and radiotherapy for combinational therapy of breast cancer cells.

Anticancer effect of 7-hydroxycoumarin in cisplatin-resistant ovarian cancer cell is mediated via apoptosis induction, caspase activation and cell cycle arrest at G2M phase


 
 
 
 Purpose: To investigate the anticancer effects of 7-hydroxycoumarin against cisplatin-resistant ovarian cancer cell line, and the underlying mechanism(s).
 Methods: Cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The 4’,6-diamidino-2-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) dual staining methods were used for measuring cell apoptosis in terms of DNA damage. Flow cytometry was used for analysis of mitosis of cancer cells, while protein expression levels were assayed with western blotting.
 Results: The 7-hydroxycoumarin preferentially inhibited the proliferation of the ovarian cancer cells, but had significantly less prominent effects on normal cells (p < 0.05). The decrease in cell proliferation was due to induction of cell apoptosis via caspase-linked apoptotic pathway. Treatment with 7- hdoxycoumarin further led to the arrest of cancer cell cycle at G2/M stage (p < 0.05) via down-regulation of the expressions of regulatory proteins that promote mitotic entry.
 Conclusion: 7-Hydroxycoumarin exerts significant anticancer effect against cisplatin-resistant ovarian cancer cells via decrease in cell proliferation, induction of apoptosis and mitotic cell cycle arrest. Thus, the compound could emerge as a vital lead molecule in the treatment of cisplatin-resistant type of human ovarian cancer.
 
 
 

Using Budding Yeast to Identify Molecules That Block Cancer Cell 'Mitotic Slippage' Only in the Presence of Mitotic Poisons.

Research on the budding yeast Saccharomyces cerevisiae has yielded fundamental discoveries on highly conserved biological pathways and yeast remains the best-studied eukaryotic cell in the world. Studies on the mitotic cell cycle and the discovery of cell cycle checkpoints in budding yeast has led to a detailed, although incomplete, understanding of eukaryotic cell cycle progression. In multicellular eukaryotic organisms, uncontrolled aberrant cell division is the defining feature of cancer. Some of the most successful classes of anti-cancer chemotherapeutic agents are mitotic poisons. Mitotic poisons are thought to function by inducing a mitotic spindle checkpoint-dependent cell cycle arrest, via the assembly of the highly conserved mitotic checkpoint complex (MCC), leading to apoptosis. Even in the presence of mitotic poisons, some cancer cells continue cell division via ‘mitotic slippage’, which may correlate with a cancer becoming refractory to mitotic poison chemotherapeutic treatments. In this review, knowledge about budding yeast cell cycle control is explored to suggest novel potential drug targets, namely, specific regions in the highly conserved anaphase-promoting complex/cyclosome (APC/C) subunits Apc1 and/or Apc5, and in a specific N-terminal region in the APC/C co-factor cell division cycle 20 (Cdc20), which may yield molecules which block ‘mitotic slippage’ only in the presence of mitotic poisons.

Antibody\u2013drug nanoparticle induces synergistic treatment efficacies in HER2 positive breast cancer cells

Chemotherapeutic drugs suffer from non-specific binding, undesired toxicity, and poor blood circulation which contribute to poor therapeutic efficacy. In this study, antibody–drug nanoparticles (ADNs) are engineered by synthesizing pure anti-cancer drug nanorods (NRs) in the core of nanoparticles with a therapeutic monoclonal antibody, Trastuzumab on the surface of NRs for specific targeting and synergistic treatments of human epidermal growth factor receptor 2 (HER2) positive breast cancer cells. ADNs were designed by first synthesizing ~ 95 nm diameter × ~ 500 nm long paclitaxel (PTX) NRs using the nanoprecipitation method. The surface of PTXNRs was functionalized at 2′ OH nucleophilic site using carbonyldiimidazole and conjugated to TTZ through the lysine residue interaction forming PTXNR-TTZ conjugates (ADNs). The size, shape, and surface charge of ADNs were characterized using scanning electron microscopy (SEM), SEM, and zeta potential, respectively. Using fluorophore labeling and response surface analysis, the percentage conjugation efficiency was found > 95% with a PTX to TTZ mass ratio of 4 (molar ratio ≈ 682). In vitro therapeutic efficiency of PTXNR-TTZ was evaluated in two HER2 positive breast cancer cell lines: BT-474 and SK-BR-3, and a HER2 negative MDA-MB-231 breast cancer cell using MTT assay. PTXNR-TTZ inhibited > 80% of BT-474 and SK-BR-3 cells at a higher efficiency than individual PTX and TTZ treatments alone after 72 h. A combination index analysis indicated a synergistic combination of PTXNR-TTZ compared with the doses of single-drug treatment. Relatively lower cytotoxicity was observed in MCF-10A human breast epithelial cell control. The molecular mechanisms of PTXNR-TTZ were investigated using cell cycle and Western blot analyses. The cell cycle analysis showed PTXNR-TTZ arrested > 80% of BT-474 breast cancer cells in the G2/M phase, while > 70% of untreated cells were found in the G0/G1 phase indicating that G2/M arrest induced apoptosis. A similar percentage of G2/M arrested cells was found to induce caspase-dependent apoptosis in PTXNR-TTZ treated BT-474 cells as revealed using Western blot analysis. PTXNR-TTZ treated BT-474 cells showed ~ 1.3, 1.4, and 1.6-fold higher expressions of cleaved caspase-9, cytochrome C, and cleaved caspase-3, respectively than untreated cells, indicating up-regulation of caspase-dependent activation of apoptotic pathways. The PTXNR-TTZ ADN represents a novel nanoparticle design that holds promise for targeted and efficient anti-cancer therapy by selective targeting and cancer cell death via apoptosis and mitotic cell cycle arrest.

Abstract 5817: Anti-tropomyosin drugs prevent the rescue of vincristine-induced mitotic spindle defects

Abstract Drugs targeting a major component of the actin filaments of cancer cells, tropomyosin Tpm3.1, synergize with anti-microtubule drugs in neuroblastoma and lung cancer models both in vitro and in vivo and a wide range of other cancer types in vitro. We have determined the mechanism of synergy in HeLa cells to gain insight into the potential interaction of actin filaments and microtubules in the survival and proliferation of cancer cells. HeLa cells exhibit a strong synergistic response to the combined treatment of vincristine (VCR) and anti-Tpm3.1 compounds, marked by an enhanced reduction in cell viability, apoptosis induction and mitotic cell cycle arrest. Tpm3.1 localizes to the cell cortex during mitosis, potentially associating with the microtubule network, particularly the dynein/dynactin complexes responsible for mediating cortical pulling forces during spindle assembly. VCR alone causes supernumerary NuMA organized acentrosomal microtubule organizing centers upon nuclear envelope breakdown, which can be resolved via a clustering mechanism to achieve bipolar cell division. The addition of anti-Tpm3.1 compounds inhibits NuMA-associated clustering in VCR-treated cells, leading to irreparable defects during spindle assembly and thus a largely increased number of cells with multi-polar spindles undergoing mitotic delay and catastrophe. We conclude that actin/Tpm3.1 filaments contribute to the formation of the bipolar spindle and play a critical role in the clustering of acentrosomal microtubule asters. Citation Format: Peter Gunning, Yao Wang, Jeff H. Stear, Ashleigh Swain, Xing Xu, Nicole Bryce, Irina B. Alieva, Vera B. Dugina, Timothy Cripe, Justine Stehn, Edna C. Hardeman. Anti-tropomyosin drugs prevent the rescue of vincristine-induced mitotic spindle defects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5817.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.

Potent antitumor property of Allium bakhtiaricum extracts

BackgroundAllium species are magnificently nutritious and are commonly used as a part of the diet in Iran. They have health enhancing benefits including anticancer properties due to the presence of numerous bioactive compounds. Herein, we investigated in vitro and in vivo anticancer properties of Allium bakhtiaricum extracts.MethodsAnti-growth activity of different fractions was explored in vitro on different cancerous cells using MTT assay, Annexin V/PI and SA-β-gal staining, Western blotting, flowcytometric and immunofluorescence microscopic evaluations. In vivo antitumor activity was investigated in BALB/c mice bearing 4 T1 mammary carcinoma cells.ResultsWe demonstrated that chloroformic and ethyl acetate fractions exert cytotoxic activity toward MDA-MB-231 cells, the most sensitive cell line, after 72 h of treatment with IC50 values of 0.005 and 0.006 mg/ml, respectively. Incubation of MDA-MB-231 cells with ¼ and ½ IC50-72h concentrations of each fraction resulted in a significant G2/M cell cycle arrest. ¼ IC50-72h concentration of the chloroform fraction led to the disruption of polymerization in mitotic microtubules. Exposure of human breast cancer cells to different concentrations of the extracts at different incubation times did not induce apoptosis, autophagy or senescence. Our in vivo study revealed that administration of the chloroform extract at a dose of 1 mg/kg/day strongly suppressed mammary tumor progression and decreased the number of proliferative cells in the lung tissues indicating its anti-metastatic effect.ConclusionOur findings imply that the chloroform fraction of Allium bakhtiaricum possesses the suppressive action on breast cancer through mitotic cell cycle arrest suggesting a mechanism associated with disturbing microtubule polymerization.

Microarray meta-analysis identifies candidate genes for human spermatogenic arrest.

Male infertility affects approximately half of couples who have difficulty becoming pregnant, and its prevalence is continuously rising. Many studies have been performed using animal testes to reveal the mechanisms of male infertility, but few studies have investigated human testes due to various limitations. The aim of this study was to investigate the gene expression profile of impaired human testes through a meta-analysis of microarray data sets, which was accomplished by using 178 human testis samples and 7 microarray data sets. Impaired testes were categorised into four pathological phenotypes or the normal phenotype based on their Johnsen score. Then, a meta-analysis was performed to screen out the differentially expressed genes (DEGs) in each phenotype. The DEGs were used in a subsequent bioinformatics analysis. Our results identified several novel hub genes and pathways and suggested that G1 mitotic cell cycle arrest was a remarkable feature in pre-meiotic arrest. Furthermore, fifteen p53-interacting proteins, such as ABL1 and HDAC2, whose roles in spermatogenesis have not been well characterised, were selected from the DEGs through a strict screening procedure.

Mouse models of hypoplastic left heart syndrome (HLHS) reveal new targets for potential intervention

Hypoplastic left heart syndrome (HLHS) is one of the most lethal congenital heart disease (CHD), with a 30% 5‐year mortality rate after surgical palliation. Patients can succumb to heart failure and among surviving patients, neurodevelopmental delay and neurobehavioral and neurocognitive disabilities are observed at a high prevalence. While hypoxia and hemodynamic disturbance related to the structural heart defect and from cardiopulmonary bypass surgical intervention are suggested as factors driving these poor outcomes, studies over the past decade indicate they are largely attributable to as yet unidentified patient intrinsic factors. The recent recovery of mouse models of HLHS has provided some insights into the nature of such intrinsic factors. Analysis of the HLHS mutant mice have revealed HLHS has a complex genetic architecture and is profoundly genetically heterogeneous. Detailed investigation of the Ohia HLHS mutant mouse line showed HLHS arises combinatorially from mutations in two genes, Sap130 – a chromatin modifying protein in the Sin3A HDAC repressor complex, and PchdhA9 ‐ a protocadherin mediating cell‐cell adhesion. While Sap130 drives the LV hypoplasia, Pcdha9 drives the aortic valve defects and can cause bicuspid aortic valve. These findings have been validated with the production and analysis of CRISPR gene targeted mice and zebrafish models.Analyses of CHD defects in Ohia mutant mice and the emergence of HLHS during embryonic/fetal development indicated the LV hypoplasia is not hemodynamically driven, but instead is associated with cell intrinsic defects involving mitotic cell cycle arrest. Interestingly, the Ohia mutant mice also showed forebrain abnormalities involving hypoplasia of the cerebral cortex, and dysplasia of the hippocampus, corpus callosum and cerebellum, brain structures also observed to be impacted in HLHS patients. Further analysis of a floxed Sap130 allele with Emx1‐Cre targeted forebrain‐specific deletion of Sap130 yielded viable adult mice with normal heart structure but with brain abnormalities that included microcephaly, similar to findings in the Ohia mutant fetuses. This was associated with cell intrinsic defects involving loss of neural progenitors. Brain MRI and neuronal fiber tractography with diffusion tensor imaging revealed extensive alterations in neural network connectivity. Behavioral assessments further showed learning and memory deficits. Together these findings suggest a shared genetic etiology for the heart and brain defects in HLHS. This likely is mediated by cell intrinsic defects, suggesting the need to shift the focus of therapeutic intervention from hemodynamic modulation to targeting the cell intrinsic deficits associated with HLHS. Only then will it be possible to achieve substantive improvement in cardiac and neurodevelopmental outcome for HLHS.Support or Funding InformationFunded by NIH Grants HL142788 and HL132024This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential anti-tumour effects of MTH1 inhibitors in patient-derived 3D colorectal cancer cultures

Reactive oxygen species (ROS) function as second messengers in signal transduction, but high ROS levels can also cause cell death. MTH1 dephosphorylates oxidized nucleotides, thereby preventing their incorporation into DNA and protecting tumour cells from oxidative DNA damage. Inhibitors of MTH1 (TH588 and (S)-crizotinib) were shown to reduce cancer cell viability. However, the MTH1-dependency of the anti-cancer effects of these drugs has recently been questioned. Here, we have assessed anti-tumour effects of TH588 and (S)-crizotinib in patient-derived 3D colorectal cancer cultures. Hypoxia and reoxygenation – conditions that increase intracellular ROS levels – increased sensitivity to (S)-crizotinib, but not to TH588. (S)-crizotinib reduced tyrosine phosphorylation of c-MET and ErbB3 whereas TH588 induced a mitotic cell cycle arrest, which was not affected by adding ROS-modulating compounds. Furthermore, we show that both compounds induced DNA damage that could not be prevented by adding the ROS inhibitor N-acetyl-L-cysteine. Moreover, adding ROS-modulating compounds did not alter the reduction in viability in response to TH588 and (S)-crizotinib. We conclude that TH588 and (S)-crizotinib have very clear and distinct anti-tumour effects in 3D colorectal cancer cultures, but that these effects most likely occur through distinct and ROS-independent mechanisms.

Chelidonine and Homochelidonine Induce Cell Death through Cell Cycle Checkpoints and MAP Kinase Pathways

This study focuses on the comparative in vitro cytotoxicity of chelidonine and homochelidonine on human cancer and non-cancer cells. Both alkaloids produced a decrease in cellular growth in a dose-dependent manner exhibiting greater potency in cancer cells. The growth inhibitory effect was evidenced in both ovarian carcinoma A2780 and lung fibroblast MRC-5 cells by inducing G2 and mitotic phase cell cycle arrest. Results indicated that the extent of apoptosis induced by chelidonine and homochelidonine was correlated to sensitivity to the antiproliferative activity of the evaluated compounds. Western blotting suggested that the cellular toxicological mechanism of chelidonine is related to the differential upregulation of phospho-Chk2, p21Cip1/Waf1, phospho-ERK1/2 and phospho-p38 in various cell types, leading to alternations in the suppression of proliferation and either induction or prevention of apoptosis. Chelidonine showed the more potent effects and also affected the cell cycle checkpoints and MAPK signaling pathways within cells.

Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

Endogenous circadian clocks regulate 24-h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse-chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA-transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.-Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

Abstract 5440: miRNAs as novel therapeutic adjuvants for improving the efficacy of vincristine and radiation therapy in treating medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite recent improvements in the overall survival, only a modest percentage of patients survive Myc-driven high-risk MB. The quality of life for surviving patients is substantially reduced due to the devastating and often irreversible side effects of radiation and chemotherapy. Recently, in a large unbiased genomic screen, we uncovered a group of microRNAs (miRNAs) capable of meditating drug sensitivity in c-myc amplified high-risk MB. Our functional screen of ~1900 miRNAs identified miR-584-5p as a potent candidate that uniquely sensitizes high-risk MB to radiation as well as vincristine (VCR) (20 to 25-fold dose reduction), an anti-mitotic agent routinely administered alongside radiation and in combination with other chemotherapeutic drugs to treat medulloblastoma patients. Our studies revealed that miR-584 might act as a potent tumor suppressor as it inhibited MB growth in vivo as well as migration and invasion of c-myc amplified MB. We show that miR-584 overexpression results in defective mitosis leading to mitotic catastrophe, apoptosis, DNA damage and G2-M cell-cycle arrest in high-risk MB cells without affecting normal neural stem cell growth. Notably, we discovered that miR-584 directly regulates the expression and activity of genes including histone deacetylase 1 (HDAC1), and eukaryotic translation initiation factor 4e family member 3 (EIF4E3) that are known to play important roles in microtubule dynamics, metaphase-anaphase transition and radio-resistance. Moreover, silencing either of these two target genes resulted in significant inhibition of MB growth and enhanced sensitivity to VCR and ionizing radiation. Overexpressing miR-584 or silencing either HDAC1 or EIF4E3 also inhibited the MB stem cell proliferation and self-renewal. We report that while miR-584-5p is predominantly expressed in normal brain and cerebellum, its expression is significantly reduced in MB patient derived xenografts (PDXs). In contrast to miR-584, EIF4E3 and HDAC1 were found to be overexpressed in medulloblastoma patients. These findings are highly significant, unexpected and innovative as this miRNA and its target genes are the first to be shown to affect the therapeutic efficacy of VCR and radiation in c-myc amplified high-risk medulloblastoma. Citation Format: Nourhan Abdelfattah, Subapriya Rajamanickam, Panneerdoss Subbarayalu, Santosh Timilsina, Benjamin Onyeagucha, Yidong Chen, Manjeet Rao. miRNAs as novel therapeutic adjuvants for improving the efficacy of vincristine and radiation therapy in treating medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5440. doi:10.1158/1538-7445.AM2017-5440

Abstract 5118: A novel polyamine sulfonamide with anti-leukemic activity

Abstract The greatest challenge in treating Acute Myeloid Leukemia (AML) is refractory disease. Although 60-80% of AML patients achieve complete remission after induction chemotherapy, majority of these patients relapse and die of progressive disease. New treatment options targeting the vulnerabilities of AML biology are highly critical for disease regression, in particular the chemo-resistant leukemia-initiating population, to ensure relapse-free survival in patients. Identifying novel druggable targets that are selective to AML despite their location in the vascular niche is thus highly warranted. We developed an in vitro co-culture model consisting of AML cells and Bone Marrow- derived Endothelial Cells (BMECs), mimicking the protective effect of the vascular niche in the bone marrow microenvironment. Using this unique model, and taking advantage of combinatorial chemistry, we performed high-throughput chemical-phenotypic screening of approximately 30 million novel compounds and identified a novel polyamine sulfonamide 2470-51 that can selectively kill AML cells by overcoming the protective effect of the BMECs. In addition to AML blasts, 2470-51 also exhibited highly selective activity against the leukemic stem and progenitor cell population while sparing normal hematopoietic stem and progenitor cells. In vivo studies using patient-derived xenograft models indicated significant regression in AML engraftment post 2470-51 treatment. Target identification experiments involving unbiased label-free shotgun proteomic analysis in combination with targeted Selected Ion Monitoring (SIM) revealed covalent drug binding targets of 2470-51. By performing differential protein expression analysis using ITRAQ analysis, we identified downstream mechanisms that led to mitotic cell cycle arrest and cell death. Collectively, our findings display the in vitro as well as in vivo efficacy of a novel polyamine sulfonamide in eliminating AML, including the leukemia initiating compartment. We also uncover a novel mechanism in AML that can be taken advantage of for selective toxicity. Furthermore, we establish the role of 2470-51 as a potential therapeutic agent in treating AML. Citation Format: Vindhya Vijay, Amy Meacham, Leylah Drusbosky, Elizabeth Wise, Christopher Cogle. A novel polyamine sulfonamide with anti-leukemic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2017-5118

The Zebrafish curly fry Is Required for Proper Centrosome and Mitotic Spindle Assembly.

The zebrafish curly fry (cfy) mutation leads to a dramatic increase in mitotic index and cell death starting during neural tube formation. The mutant phenotype is cell autonomous and does not result from defects in apical/basal polarity within the neuroepithelium. The increase in mitotic index could be due to increased proliferation or cell cycle arrest in mitosis. cfy embryos were analyzed to examine these two possibilities. By labeling embryos with a pulse of BrdU and anti-phospho-histone 3 and examining the DNA content by fluorescence activated cell sorting, we show that cfy mutants exhibit no increase in proliferation, but a significant increase in the number of cells arrested in mitosis. Furthermore, time-lapse microscopy in vivo confirmed that a great majority of dividing cells arrest during mitosis and that these mitotically arrested cells die in cfy embryos. Finally, immunostaining and confocal microscopy in cfy mutant embryos revealed that mitotic cells in mutants contain aberrant centrosomes and often exhibit monopolar spindles, thereby leading to mitotic cell cycle arrest. Our results suggest that the cfy gene is required for proper centrosome assembly and mitotic spindle formation, therefore critical for normal cell division.

Expression level is a key determinant of E2F1-mediated cell fate

The Rb/E2F network has a critical role in regulating cell cycle progression and cell fate decisions. It is dysfunctional in virtually all human cancers, because of genetic lesions that cause overexpression of activators, inactivation of repressors, or both. Paradoxically, the downstream target of this network, E2F1, is rarely strongly overexpressed in cancer. E2F1 can induce both proliferation and apoptosis but the factors governing these critical cell fate decisions remain unclear. Previous studies have focused on qualitative mechanisms such as differential cofactors, posttranslational modification or state of other signaling pathways as modifiers of the cell fate decisions downstream of E2F1 activation. In contrast, the importance of the expression levels of E2F1 itself in dictating the downstream phenotypes has not been rigorously studied, partly due to the limited resolution of traditional population-level measurements. Here, through single-cell quantitative analysis, we demonstrate that E2F1 expression levels have a critical role in determining the fate of individual cells. Low levels of exogenous E2F1 promote proliferation, moderate levels induce G1, G2 and mitotic cell cycle arrest, and very high levels promote apoptosis. These multiple anti-proliferative mechanisms result in a strong selection pressure leading to rapid elimination of E2F1-overexpressing cells from the population. RNA-sequencing and RT-PCR revealed that low levels of E2F1 are sufficient to induce numerous cell cycle-promoting genes, intermediate levels induce growth arrest genes (i.e., p18, p19 and p27), whereas higher levels are necessary to induce key apoptotic E2F1 targets APAF1, PUMA, HRK and BIM. Finally, treatment of a lung cancer cell line with a proteasome inhibitor, MLN2238, resulted in an E2F1-dependent mitotic arrest and apoptosis, confirming the role of endogenous E2F1 levels in these phenotypes. The strong anti-proliferative activity of moderately overexpressed E2F1 in multiple cancer types suggests that targeting E2F1 for upregulation may represent an attractive therapeutic strategy in cancer.

P2.01-081 CDCA3 is a Novel Prognostic Cell Cycle Protein and Target for Therapy in Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related mortality worldwide with a 5 year survival rate of 15%. Non-small cell lung cancer (NSCLC) is the most commonly diagnosed form of lung cancer. Cisplatin-based regimens are currently the most effective chemotherapy for NSCLC, however, chemoresistance poses a major therapeutic problem. New and reliable strategies are required to avoid drug resistance in NSCLC. Cell division cycle associated 3 (CDCA3) is a key regulator of the cell cycle. CDCA3 modulates this process by enabling cell entry into mitosis through degradation of the mitosis-inhibitory factor WEE1. CDCA3 itself is also degraded in G1 yet re-expressed in G2/M phase, to allow successful progression through the cell cycle. Herein, we describe CDCA3 as a novel prognostic factor in NSCLC and target to delay or prevent cisplatin resistance in NSCLC. CDCA3 expression was investigated in squamous and non-squamous NSCLC using several approaches including bioinformatic analysis of publicly available datasets, immunohistochemistry of a tissue microarray and western blot analysis of matched tumor and normal tissue and NSCLC cell lines. CDCA3 function in NSCLC was determined using several in vitro assays by siRNA depleting CDCA3 in a panel of three immortalized bronchial epithelial cell lines (HBEC) and seven NSCLC cell lines. CDCA3 transcripts and protein levels are elevated in NSCLC patient tissue and highly expressed in tumor cells relative to proximal normal cells. High mRNA levels are associated with poor survival in resected NSCLC. Depletion of CDCA3 in vitro markedly impairs proliferation in seven NSCLC cell lines by inducing a mitotic cell cycle arrest, ultimately resulting in p21-dependent cellular senescence. Importantly, silencing of CDCA3 also greatly sensitizes NSCLC cell lines to cisplatin. In line with these in vitro data, NSCLC patients that have elevated levels of CDCA3 and are treated with cisplatin have a poorer outcome than patients with reduced levels of the protein. To improve patient response to cisplatin, we are exploring novel strategies to suppress CDCA3 expression in tumor cells. Our data highlight CDCA3 as a novel factor in mediating NSCLC. We propose that evaluating novel strategies to target CDCA3 may prove a useful strategy is enhancing the anti-tumor activity of platinum-based chemotherapy and may ultimately benefit patient outcomes by preventing cisplatin resistance.