bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3778: Inhibition of NF-κB translocation induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer
Authors: Nagaraju Ganji Purnachandra , Olatunji Alese , Gupta Sandhya , Shoji Mamoru , El-Rayes Bassel F.
Published: August 2015

Purpose Objectives: Cell cycle progression and DNA synthesis are essential steps in cancer cell growth and resistance. Thymidylate synthase (TS) is a therapeutic target for 5FU. Curcumin is a potent inhibitor of NF-κB. We tested the hypothesis that NF-κB translocation inhibition by curcumin and its analogs can inhibit cell cycle progression and downregulate TS levels in colorectal cell lines.

Methods: Two colorectal cancer cell lines were usedHCT-116 and HT-29. Cells were either untreated (control) or treated with curcumin (20μM), EF31 (7.5μM for HCT-116 and 2.5μM for HT-29) and UBS109 (620nM for HCT-116 and 1.25μM for HT-29) for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of curcumin and its analogues EF31 and UBS109 on different signal molecules involved in cell cycle arrest and DNA synthesis. Electromobility shift assay (EMSA) was performed in colon cancer cells to evaluate the effects of curcumin and its analogues on NF-κB activity. A subcutaneous xenograft mouse model was used to evaluate the in vivo effects of curcumin analogues.

Results: Treatment with curcumin and its analogues inhibited cyclin D1, pRb and E2F-1 at the transcriptional and translational level and induced p21, leading to G0/G1 cell cycle arrest. NF-κB over-expression induced E2F-1 and TS protein and mRNA levels in both cell lines. Arrest in the G0/G1 phase was associated with downregulation of the transcription factor E2F-1 and its target gene TS. Similar effects were observed in tumors from animals treated with EF31 and UBS109. Further, EF31 and UBS109 treatment significantly decreased the tumor growth compared to untreated tumors.

Conclusion: In this study, we present in vitro and in vivo data to support the use of curcumin analogs in colorectal cancer based on their ability to reduce expression of proteins important for DNA repair and cell cycle progression leading to cell cycle arrest and tumor shrinkage. Accordingly, combining curcumin analogs with chemotherapy is a rational approach for future drug development in colorectal cancer.

Citation Format: Ganji Purnachandra Nagaraju, Alese Olatunji, Sandhya Gupta, Mamoru Shoji, Bassel F. El-Rayes. Inhibition of NF-κB translocation induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3778. doi:10.1158/1538-7445.AM2015-3778
Abstract 3779: Mutational analysis of MCPH1 C-terminal tandem BRCT domain reveals residues essential for cell cycle arrest
Authors: Golubeva Volha A. , Woods Nicholas T. , Monteiro Alvaro N.A.
Published: August 2015

In this study, the C-terminal tandem BRCT domain of human microcephalin 1 protein (MCPH1) was mutagenized in Saccharomyces cerevisiae yeast to identify residues critical for the cell cycle arrest. Deleterious variants in MCPH1 causing the loss of the protein function are associated with primary microcephaly, characterized by decreased cerebral cortex size and mild mental retardation. The tandem BRCT domain of MCPH1 (amino acids 640-833) recognizes phospho-serine/threonine motifs, including □-H2AX, necessary for DNA damage response and cell cycle regulation. Expression of this region of human MCPH1 tethered to DNA in yeast through the Gal4 DNA binding domain (and not through the interaction with the Gal4 activation domain), results in growth inhibition and a small colony phenotype. To detect the essential residues of the MCPH1 tandem BRCT, mutations were randomly introduced into this region using error-prone polymerase chain reaction. The mutants were then transformed into yeast and transformants showing abrogation of the small colony phenotype were isolated and the specific mutations were assessed by sequencing. Screening revealed 34 missense, 4 nonsense, and 8 frameshift variants, and one large deletion of 72 base pairs. The 34 missense variants were further analyzed in silico by alignment with the BRCA1 tandem BRCT. Out of 34 missense variants, four (T694A, L755P, F756V, and S819P) were identified in two or more colonies. One of the mutated residues (T653) is involved in direct hydrogen bonding with □-H2AX, which is likely disrupted by the amino acid change induced during mutagenesis (T653I). Interestingly, when aligned with BRCA1, 16 (47%) of MCPH1 missense variants matched to a corresponding site in BRCA1 previously reported as a missense variant with a functional impact. MCPH1 residues 653, 756, and 819 all corresponded to amino acids in BRCA1 known for their likely pathogenicity. Thus, this study identifies functional determinants in MCPH1 C-terminal domain potentially involved in cell cycle regulation. Taken together, our results suggest that variants in BRCT domain containing proteins, can be clinically annotated by making comparative alignments with the conserved residues of BRCA1 BRCT. Structural inference using BRCT paralogs might aid in the clinical annotation for variants lacking genetic data.

Citation Format: Volha A. Golubeva, Nicholas T. Woods, Alvaro N.A. Monteiro. Mutational analysis of MCPH1 C-terminal tandem BRCT domain reveals residues essential for cell cycle arrest. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3779. doi:10.1158/1538-7445.AM2015-3779
Abstract 3781: Effects of a novel microtubule inhibitor, AK301 on Apc heterozygote colon cells
Authors: Chopra Avijeet , Anderson Amy , Rosenberg Daniel , Giardina Charles
Published: August 2015

Over 80% of the sporadic colon cancers harbor somatic mutations of APC, predominantly resulting in APC protein lacking its C-terminal end. The basic region within the C-terminus of APC interacts with microtubules to promote polymerization, a process essential for establishment of cell polarity and faithful segregation of chromosomes during mitosis. Truncation mutations of Apc have been proposed to act in a dominant negative manner to slow microtubule growth and anchoring. We recently identified a novel microtubule inhibitor, 4-(3-chlorophenyl)piperazin-1-yl) (2-ethoxyphenyl)methanone, referred to as AK301. Our in vitro tubulin polymerization assay indicates that AK301 disrupts microtubule polymerization, but does not completely abrogate microtubule growth. In addition, in silico molecular docking predicts a novel and unique binding orientation of AK301 to the colchicine-binding domain of β-tubulin. We examined the effects of AK301 on microtubule dynamics in Apc+/+ and ApcMin/+ cells. Here we show that ApcMin/+ cells normally maintain shorter microtubules under normal growth conditions. Following treatment with AK301, mutant cells show more extensive cytoskeletal and mitotic spindle abnormalities. Moreover, AK301-treated ApcMin/+ cells showed centrosome declustering, evident from multiple Aurora A kinase foci; bipolar assembly was observed in the corresponding untreated ApcMin/+ cells or treated Apc+/+ cells. Overall, AK301 and its derivatives may help illuminate the regulation of microtubule dynamics in Apc mutant cells and provide insight into the consequences of targeting their microtubule assembly defect.

Note: This abstract was not presented at the meeting.

Citation Format: Avijeet Chopra, Amy Anderson, Daniel Rosenberg, Charles Giardina. Effects of a novel microtubule inhibitor, AK301 on Apc heterozygote colon cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2015-3781
Abstract 3785: Cdk1 coordinates telomere replication by regulating the temporal recruitment of telomerase and the CST complex
Authors: Gopalakrishnan Veena , Liu Chang-Ching , Poon Lai Fong , Yan Tingdong , Li Shang
Published: August 2015

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. They ensure genomic stability and complete replication of the genome. The enzyme telomerase mediates telomere elongation. Considering that telomerase is up-regulated in about 90% of human cancers and telomerase-mediated telomere elongation is significantly deregulated in cancers, insights from our study can be used to target the telomere replication machinery for therapeutic intervention in cancer. In our study, we use budding yeast to understand the mechanism of telomere maintenance. In budding yeast, Cdk1 coordinates the cell cycle-dependent elongation of telomere by telomerase. Cdk1 phosphorylates the telomere-specific ssDNA binding protein, Cdc13, which promotes the recruitment of telomerase to the telomere and thereby, telomere elongation. Cdc13 is also an integral part of the CST (Cdc13-Stn1-Ten1) complex, which provides the protective cap at the telomere and inhibits telomere elongation by telomerase. Therefore, the telomere length homeostasis is balanced between these two telomerase-extendable and CST-unextendable states. However, the cellular signaling that regulates the switch between these two states is largely unknown. Here we show that Cdk1 phosphorylates Stn1 sequentially to that of Cdc13. Cdk1-dependant phosphorylation of Stn1 is essential for telomere maintenance in vivo, CST stability and the inhibition of telomerase at the telomeres. By controlling the timing of Cdc13 and Stn1 phosphorylation during cell cycle progression, Cdk1 coordinates the recruitment of telomerase holoenzyme and CST complex to the telomeres respectively and hence the switch between the telomerase-extendable and CST-unextendable states of the telomeres in budding yeast. With increasing evidence of human CST complex being involved in telomere length maintenance, the results from my study will provide insights into telomere homeostasis in humans and its disruption in cancers.

Citation Format: Veena Gopalakrishnan, Chang-Ching Liu, Lai Fong Poon, Tingdong Yan, Shang Li. Cdk1 coordinates telomere replication by regulating the temporal recruitment of telomerase and the CST complex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2015-3785
Abstract 3787: EphrinB1 is a novel microtubule associated protein whose expression may predict sensitivity to taxane
Authors: Vermeer Paola D. , Colbert Paul L. , Vermeer Daniel W. , Wieking Bryant G. , Lee John H.
Published: August 2015

Mitosis is a complex sequence of highly regulated events that ensures the proper segregation of sister chromatids into daughter cells. At the heart of the mitotic machinery lie the microtubules. Microtubules (MTs) are components of the cytoskeleton made up of polymerized alpha and beta tubulin dimers. MT structure and function must be maintained throughout the cell cycle to ensure proper execution of mitosis and cellular homeostasis. It has previously been reported that the protein tyrosine phosphatase, PTPN13, localizes at centrosomes from interphase, through metaphase and dramatically shifts to the spindle midzone during anaphase. During telophase, PTPN13 accumulates at the midzone, becoming concentrated at the center of the midbody during cytokinesis. We have previously demonstrated that the HPV16 E6 oncoprotein binds PTPN13 and leads to its degradation. Given PTPN13's localization to distinct compartments during mitosis/cytokinesis, we speculated that HPV infection may affect the process of mitosis. In addition, the newly appreciated roles of the PTPN13 phosphatase substrate, EphrinB1, in oncogenesis, further prompted us to define its role in mitosis. Here we report that EphrinB1 co-localizes with MTs during all phases of the cell cycle. Specifically, unphosphorylated EphrinB1 directly binds tubulin, while its phosphorylated form lacks MT binding. These characteristics are hallmarks of microtubule associated proteins (MAPs) and suggest that EphrinB1 is a novel MAP. Importantly, we demonstrate that expression of PTPN13 correlates with sensitivity to taxanes while its absence may confer resistance. In addition, we show that EphrinB1 expression affects taxane response in vitro and in vivo. We speculate that this reflects PTPN13's modulation of EphrinB1 phosphorylation and suggest that EphrinB1 is an important contributor to taxane sensitivity/resistance phenotypes in epithelial cancers. HPV infection or functional mutations of PTPN13 in non-viral cancers may predict taxane sensitivity.

Note: This abstract was not presented at the meeting.

Citation Format: Paola D. Vermeer, Paul L. Colbert, Daniel W. Vermeer, Bryant G. Wieking, John H. Lee. EphrinB1 is a novel microtubule associated protein whose expression may predict sensitivity to taxane. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2015-3787
Abstract 3792: SKP2 supports cell proliferation and is regulated by Notch signaling in myoblasts and embryonal rhabdomyosarcoma
Authors: Rota Rossella , Adesso Laura , Conti Beatrice , Ciarapica Roberta , De Raimondi Lavinia , Salvo Maria , Rodriguez Sonia , Carlesso Nadia , Miele Lucio , Locatelli Franco
Published: August 2015

Background:

Rhabdomyosarcoma (RMS) in a pediatric tumor of myogenic origin. It includes two subtypes: embryonal and alveolar. Embryonal RMS (ERMS) cells express key myogenic factors such as MyoD and Myogenin, but proliferate indefinitely and have lost the ability to terminally differentiate into skeletal myofibers. Differently from the alveolar tumors bearing specific chromosomal translocations, ERMS has cytogenetic aberrations and molecular deregulations of pathways regulating senescence, proliferation and differentiation. It has been shown that SKP2, an F-box protein and a component of the ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box), is over-expressed in RMS primary samples and correlates with a dismal outcome. Therefore, we sought here to investigate the regulation of SKP2 and its role in ERMS.

Methods:

We modulated SKP2 expression through silencing, by using a siRNA validated in the literature, and forcing its expression through retroviral infection. In parallel, we investigate the effect of Notch signaling modulation on SKP2 expression.

Results:

SKP2 silencing resulted in cell cycle slowdown in both normal myoblasts and ERMS cells. Down-regulation of Notch1 led to SKP2 reduction while that of Notch3 supported SKP2 expression. Cosilencing SKP2 and Notch3 gave raise to myoblast-like structure formation in ERMS and facilitated myoblasts fusion. Finally, using a SKP2 inhibitor ERMS cell proliferation was completely blocked.

Conclusion:

Altogether, these preliminary experiments suggest that SKP2 could be regulated by Notch signaling in ERMS and that its inhibition hampers tumor cell proliferative capability.

Note: This abstract was not presented at the meeting.

Citation Format: Rossella Rota, Laura Adesso, Beatrice Conti, Roberta Ciarapica, Lavinia Raimondi, Maria De Salvo, Sonia Rodriguez, Nadia Carlesso, Lucio Miele, Franco Locatelli. SKP2 supports cell proliferation and is regulated by Notch signaling in myoblasts and embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2015-3792
Abstract 3793: Overexpression of A20 and activated-Src result in TRAIL resistance in oral cancer cells
Authors: Hino Satoshi , Hamakawa Tomohiro , Nakashiro Koh-ichi , Hamakawa Hiroyuki
Published: August 2015

The 5-year survival rate for oral cancer patients is around 50%, and has remained unchanged for the last several decades. This poor survival rate is due to the delay in diagnosis, the high incidence of recurrence, and the difficulty of controlling metastases. It is considered that the acquisition of resistance to anoikis and apoptosis is an essential event to successfully metastasize. We have to withdraw these resistance of cancer cells to improve the survival ratio.

In this study, we have used oral squamous cell carcinoma cell lines with and without distinct metastatic ability, and revealed the relationship between their anoikis and apoptosis sensitivity and the metastatic ability. At first we examined their sensitivity to anoikis by using the low attachment culture condition. There was no apparent relation between their metastatic potential and the sensitivity to anoikis. SAS cells which have no metastatic potency showed modest sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), whereas SAS-T5 cells and SAS-L1 cells, a clone with high metastatic potential showed robust resistance. Fas ligand (Anti-Fas/CD95 monoclonal antibody) showed the similar effects for apoptosis induction as well as TRAIL, but TNF-α had no effect in these cells. We confirmed that the expression levels and the localization of TRAIL receptors were not correlated with TRAIL sensitivity. We found that the mechanisms underlying the differences in these resistance to TRAIL was an activity of Src kinase and the basal levels of A20. Phosphorylated Src (Tyr 416), the active form, was upregulated in SAS-T5 and SAS-L1 cells compared to parental SAS cells. Src inhibitor PP2 suppressed cell proliferation in response to dependence on Src signaling. Downregulation of Src or A20 with siRNA made metastatic cells dissolve the resistance to TRAIL-induced apoptosis. These results suggest that combination therapy with TRAIL and some drug inhibiting Src kinase and /or A20 expression may be a treatment strategy for oral squamous cell carcinoma.

Citation Format: Satoshi Hino, Tomohiro Hamakawa, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Overexpression of A20 and activated-Src result in TRAIL resistance in oral cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3793. doi:10.1158/1538-7445.AM2015-3793
Abstract 3794: Mechanistic studies on the anti-tumor effect of jacaric acid on murine macrophage-like leukemia PU5-1.8 cells
Authors: Liu Wai Nam , Leung Kwok Nam
Published: August 2015

This study aims at demonstrating the in vitro and in vivo anti-tumor effect and action mechanisms of jacaric acid, a conjugated linolenic acid isomer which is present in jacaranda seed oil, on murine macrophage-like leukemia PU5-1.8 cells. Our results demonstrated the growth-inhibitory effect of jacaric acid on PU5-1.8 cells in a time- and concentration-dependent manner, as measured by MTT reduction and fluorometric cell proliferation assays, while it exerted minimal cytotoxicity on normal murine cells. Pre-treatment of PU5-1.8 cells with jacaric acid significantly decreased the leukemic cell growth in syngeneic BALB/c mice, while consumption of jacaric acid is apparently non-toxic to the mice. Flow cytometric analysis revealed the induction of G0/G1 cell cycle arrest, accompanied by a decrease in cyclin E and CDK2 proteins as demonstrated by Western blotting. Jacaric acid also triggered apoptosis in PU5-1.8 cells as reflected by induction of DNA fragmentation, phosphatidylserine externalization and mitochondrial membrane depolarization, which was quantified by ELISA, Annexin V assay and JC-1 dye staining method, respectively. Up-regulation of Bax and down-regulation of Bcl-2 and Bcl-xL proteins might account for the occurrence of apoptotic events. Collectively, our results suggest that the growth-inhibitory effect of jacaric acid on PU5-1.8 cells might be mediated through inducing cell cycle arrest and apoptosis of the leukemia cells. Therefore, jacaric acid represents a naturally-occurring potential candidate for the treatment of some forms of myeloid leukemia with minimal toxicity and fewer side effects.

Citation Format: Wai Nam Liu, Kwok Nam Leung. Mechanistic studies on the anti-tumor effect of jacaric acid on murine macrophage-like leukemia PU5-1.8 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2015-3794
Abstract 3802: Selective cytotoxicty and mechanism of Myrothamnus flabellifolius, an edible medicinal plant, on acute myeloid leukemia cells
Authors: Badiab Abeer , Dhillon Jasjit , Nabbie Fizal , Peethambaran Bela
Published: August 2015

Current therapies for acute myeloid leukemia involve chemotherapy and radiation, which cause damage to normal and cancerous cells. There is a dire need to discover novel drugs that target only cancer cells. Myrothamnus flabellifolius is a South African resurrection plant known for its anti-viral, anti-microbial and anti-inflammatory properties. Human myeloid leukemia (HL-60) cells and normal (TK-6) lymphocytes were treated with the methanolic extract of M. flabellifolius. Cancer cells treated with extract depicted a significant reduction in viability as compared to normal cells. The first part of this project involved fractionation of the methanolic extract using HPLC and testing each fraction on the normal and leukemic cell lines to find a fraction that selectively killed the cancer cells. A phytochemical analysis was also conducted for characterization of the anticancer compounds. In terms of mechanism, the loss in HL-60 viability was accompanied by the induction of caspase dependent apoptosis by way of caspase-7 cleavage. Heat shock proteins are molecular chaperones that aid in protein reassembly under stressful conditions and protect cells from apoptosis. TK-6 cells express heat shock protein 70 at a high level while HL-60 cells do not express the protein under normal conditions. The expression of HSP70 exhibits a positive correlation with the viability of cells post treatment with extract, indicating that this vital protein plays an important role in the survival of cells post treatment with M. flabellifolius extract.

Citation Format: Abeer Badiab, Jasjit Dhillon, Fizal Nabbie, Bela Peethambaran. Selective cytotoxicty and mechanism of Myrothamnus flabellifolius, an edible medicinal plant, on acute myeloid leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3802. doi:10.1158/1538-7445.AM2015-3802
Abstract 3803: Transcriptome analysis of MCF7 breast cancer cells with different expression profile of PAR-4 (prostate apoptosis response-4) in the presence and absence of docetaxel
Authors: de Bessa Garcia Simone A. , Pavanelli Ana C. , Melo Natalia C. , Nagai Maria A.
Published: August 2015

The ability of cancer cells to evade apoptosis is one of the essential alterations in cell physiology that dictate tumor growth and impact chemo- and radio-resistance. Experimental evidence indicates that protein kinase C apoptosis WT1 regulator (PAWR) gene, also known as prostate apoptosis response-4 (PAR-4), plays an important role in tumor cell survival, and may be considered a candidate for tumor cell selective therapy. This selective ability of Par-4 to directly cause apoptosis in most cancer cells is associated with its nuclear translocation and its phosphorylation state. PAR-4 acts in both intrinsic and extrinsic apoptotic pathways. In the present study, we sought to evaluate the effects of PAR-4 on the expression profile of MCF7 breast cancer cells in the presence and absence of docetaxel. MCF7 breast cancer cells were transfected with the expression vector pCMV6-PAR-4 or pCMV6-Neo. Real time PCR, western blot and immunofluorescence were used to characterize the clones over-expressing PAR-4. Using cDNA microarray analysis, we evaluated the transcriptome changes in the comparison between MCF7 cells with increased expression of PAR-4 and the control cells before and after docetaxel treatment. Several differentially expressed genes were identified as potentially involved in PAR-4-mediated chemosensitivity to docetaxel. Using the Ingenuity program, several gene networks were generated from the differentially expressed genes, including many genes direct and indirectly involved in the canonical WNT (Wingless-Type MMTV Integration 1) signaling pathway. The Wnt Signaling Pathway Human RT2 Profiler PCR Array was used to validate the differentially expressed genes involved in the Wnt pathway identified in MCF-7 cells with differences of Par-4 expression before and after exposure to docetaxel. The validation results confirm the data observed by cDNA microarray showing that the increase in PAR-4 expression modulates the expression of many genes involved in the Wnt signaling pathway in MCF7 breast cells. The results generated could be of particular interest for the understanding the action of PAR-4 in chemosensitivity, especially to increase the specificity and effectiveness of drug treatment and overcoming resistance to chemotherapy.

Supported by FAPESP and CNPq.

Citation Format: Simone A. de Bessa Garcia, Ana C. Pavanelli, Natalia C. Melo, Maria A. Nagai. Transcriptome analysis of MCF7 breast cancer cells with different expression profile of PAR-4 (prostate apoptosis response-4) in the presence and absence of docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3803. doi:10.1158/1538-7445.AM2015-3803
Abstract 3804: Disruption of spindle microtubules by TTFields result in abnormal chromosomes segregation and subsequent caspase-dependent cell death
Authors: Voloshin Sela Tali , Schneiderman Rosa S. , Giladi Moshe , Porat Yaara , Munster Mijal , Blat Roni , Sherbo Shay , Bomzon Zeev , Urman Noa , Kirson Eilon D. , Weinberg Uri , Palti Yoram
Published: August 2015

Tumor Treating Fields (TTFields) are low intensity, intermediate frequency alternating electric fields. TTFields efficacy was demonstrated in vitro, in vivo and in clinical trials. The Optune device, designated to deliver TTFields to the tumor region, was approved by the FDA for the treatment of recurrent Glioblastoma multiforme (GBM). While there is a growing body of evidence demonstrating TTFields effect on mitotic cells, little is known about the mechanisms by which TTFields act to induce cancer cell death. The aim of the present study was to provide a mechanistic insight into the mode of action of TTFields.

Cancerous cell lines were treated with TTFields using the inovitro system. Disruption of spindle microtubules was demonstrated by confocal microscopy. Divergence from normal chromosome number was evaluated using SKY analysis and chromosome spreads. Induction of apoptosis was examined using flow cytometry based on Annexin V and Propidium Iodide staining. Detection of caspase activity was carried using poly caspase assay kit. Broad-spectrum caspase inhibitor was used in combination with TTFields to evaluate to what extent caspase dependent apoptosis was accountable for treatment efficacy.

Our results demonstrate that TTFields application prevents proper mitotic spindle assembly by interfering with microtubule polymerization. We also show that the outcomes of mitosis under TTFields application include abnormal chromosome segregation and caspase dependent apoptosis of daughter cells. Addition of caspase inhibitor completely abrogated apoptosis confirming that TTFields induced cell death is caspase dependent manner.

Taken together, our results demonstrate that TTFields treatment interfere with normal mitotic spindle formation thereby promoting mitotic catastrophe which result in caspase dependent apoptosis.

Citation Format: Tali Voloshin Sela, Rosa S. Schneiderman, Moshe Giladi, Yaara Porat, Mijal Munster, Roni Blat, Shay Sherbo, Zeev Bomzon, Noa Urman, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Disruption of spindle microtubules by TTFields result in abnormal chromosomes segregation and subsequent caspase-dependent cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2015-3804
Abstract 3805: Effect of Imatinib mesylate in gastric cancer cell progression
Authors: Kim Jung Lim , Kim Bo Ram , Na Yoo Jin , Park Seong Hye , Jeong Yoon A , Oh Sang Cheul
Published: August 2015

Imatinib is a powerful tyrosine kinase inhibitor that specifically targets BCR-ABL, KIT, and PDGFR kinases that is used in the treatment of chronic myelogenous leukemia (CML), gastrointestinal stromal tumors (GIST), and other cancers. But, imatinib mesylate has not yet been reported about anti-cancer effect in gastric cancer. This study was undertaken to evaluate the in vitro effect of imatinib mesylate in gastric cancer cells and to define molecular mechanism underlying these effects.

This study was to determine the expression of PDGFR molecules in gastric cancer cells (AGS, MKN28, MKN45, SNU638) by semi-quantitative polimerase chain reaction (PCR). The effects of imatinib mesylate on gastric cancer cells were examined in four human gastric cancer cell lines. The results showed expression of PDGFR in all four gastric cancer cells. We measured the sensitivity of gastric cancer cell line (AGS, MKN28, MKN45, SNU638) to imatinib mesylate using MTT assay. Cell viability of cell treated with imatinib mesylate was decreased in high dose. But cell viability was not change in high dose. Next, the results showed expression of PDGFR and suppression of its phosphorylation by imatinib mesylate in all four gastric cancer cells. Cell cycle analysis for sub-G1 and G2/M fraction also detected the increased cell death in the cells treated with imatinib mesylates. Results by Western blotting indicated that imatinib mesylates promotes apoptosis of gastric cancer cells by both activating caspase-3 and -9 and inducing PARP cleavage. And expression of p-AKT level was decreased. Cell migration and invasion that imatinib treated cells is decreased in low dose. Cell viability assay revealed that the combination of imatinib mesylate and chemo-reagent synergistically inhibited cell growth compared to cells treated with any of the agent alone.

In conclusion, inhibition the PDGFR by imatinib mesylates lead to apoptosis of gastric cancer cell lines. And Imatinib suppressed motility and invasion of gastric cancer cells. The results suggest that imatinib mesylate may be useful in the treatment of gastric cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Jung Lim Kim, Bo Ram Kim, Yoo Jin Na, Seong Hye Park, Yoon A Jeong, Sang Cheul Oh. Effect of Imatinib mesylate in gastric cancer cell progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3805. doi:10.1158/1538-7445.AM2015-3805
Abstract 3811: Arsenic trioxide induces cell cycle arrest, apoptosis through interaction of DAXX and degradation of MDM2 in acute leukemia cells
Authors: Kumar Sanjay , Tchounwou Paul B.
Published: August 2015

Arsenic trioxide (ATO) alone or combination with all trans retinoic acid (ATRA) has been successfully used in the treatment of all age group of acute promyelocytic leukemia (APL) patients. Recent human clinical trails result of ATO and their combination with ATRA shown complete remission in both de novo and relapsed APL patients. However, the detailed molecular mechanisms of its anti-leukemic action through cell cycle arrest and apoptosis are poorly known. We have used APL cell line and transgenic mice model to elucidate the anti-cancer properties of arsenic trioxide. We hypothesized that ATO arrests cell cycle and apoptosis through activation of p53 and interaction of death domain-associated protein (DAXX), which disrupted the MDM2-DAXX-HAUSP interactions. It also promoted MDM2 self-ubiquitination and degradation. To test the hypothesis, we used western blotting, confocal imaging and other molecular techniques to identify detailed cellular and molecular mechanisms of ATO action in APL cells and transgenic mice hepatocytes as well as bone marrow cells. We found that the expression levels of p53 and p21 increased significantly, whereas MDM2, DAXX and HAUSP decreased dose dependent manner. Our immunoprecipitation (IP) studies shown that they are well associated each other in cells and ATO disrupts associations. After 21 days treatment of ATO different doses (1.25, 2.5, 5.0 and 7.5 mg/kg body wt) in transgenic mice, we have collected liver tissue and isolated hepatocytes and bone marrow. In liver tissue, we found p53 nicely activated dose dependent ATO treated mice. On the basis of these findings, we conclude that ATO induces cell cycle arrest and apoptosis in APL cell line as well as mice hepatocytes through p53 activation, MDM2 self-ubiquitination and disruption of MDM2-DAXX-HAUSP interaction. It is novel target for treatment of APL patients through designing of new anti-leukemic drugs.

Keywords: Arsenic trioxide, HL-60 cells, cell cycle arrest, apoptosis, mitochondrial pathway, Mice hepatocytes and bone marrow.

Acknowledgements: This research was financially supported by National Institutes of Health NCRR Grant No. 5G12RR013459 and MIMHD Grant No. 8G12MD007581, through the RCMI-Center for Environmental Health at Jackson State University.

Citation Format: Sanjay Kumar, Paul B. Tchounwou. Arsenic trioxide induces cell cycle arrest, apoptosis through interaction of DAXX and degradation of MDM2 in acute leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3811. doi:10.1158/1538-7445.AM2015-3811
Abstract 3814: Metformin induces ER stress-dependent apoptosis through miR-708-5p/NNAT pathway in prostate cancer
Authors: Yang Jian , Wei Juncheng , Wu Yushi , Wang Zhilin , Guo Yuqi , Li Xin
Published: August 2015

Previous studies showed that metformin is associated with lower risk of tumorigenesis for several cancer types including prostate cancer. However, the molecular basis of anti-tumor effect induced by this biguanide agent is still unclear. In this study, we identified miR-708-5p as a novel downstream effector of metformin in prostate cancer. By increasing the expression of miR-708-5p, metformin suppresses the expression of endoplasmic reticulum (ER) membrane protein neuronatin (NNAT) and subsequently induces apoptosis of prostate cancer cells through ER stress pathway. Notably, down-regulated NNAT is associated with down-regulated intracellular calcium level and induces malformation of endoplasmic reticulum-ribosome structure which is revealed by electronic microscopy. Furthermore, western blot shows that the unfolded-protein response (UPR) proteins including CHOP, p-eIF2α, Calreticulin, GRP78 and ATP2A1, all of which are also considered as the ER stress markers, were up-regulated by metformin and miR-708-5p. Importantly, an increased apoptosis of prostate cancer cells is also observed when miR-708-5p mimic or NNAT siRNA was introduced. In summary, our findings clearly reveals that metformin stimulates miR-708-5p to target on NNAT mediated response to ER stress and apoptosis. This novel regulatory mechanism of metformin in prostate cancer cells not only advances our knowledge on the molecular mechanism of metformin in cancer progression, but also provide a promising therapeutic target for prostate cancer treatment.

Citation Format: Jian Yang, Juncheng Wei, Yushi Wu, Zhilin Wang, Yuqi Guo, Xin Li. Metformin induces ER stress-dependent apoptosis through miR-708-5p/NNAT pathway in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3814. doi:10.1158/1538-7445.AM2015-3814
Abstract 3820: Ultrasensitive DNA methylation detection using sputum and plasma for early detection of NSCLC
Authors: Chen Chen , Hulbert Alicia , Stark Alejandro , Rodgers Kristen , Lee Beverly , Brock Malcolm , Herman James
Published: August 2015

Purpose:

The National Lung Cancer Screening Trial provides hope that early detection can result in a reduction of mortality from lung cancer. This study was to determine if molecular alterations of sputum and plasma in patients with CT detected pulmonary nodules could predict patients with malignancy from those without.

Materials and Methods: We conducted a prospective case-control study obtaining cases and controls from the Lung Cancer Spore Trial at Johns Hopkins Hospital. Plasma, sputum and pre-operative CT scans were obtained from all patients. We processed the plasma, sputum and tumor by using promoter Methylation on beads followed by quantitative methylation specific real-time PCR to investigate promoter methylation status of six cancer-related genes, CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZFP42.

Results:

223 patients were analyzed, including 162 patients with NSCLC and 61 patients with benign lesions. The sensitivity and specificity values for lung cancer detection in plasma, sputum or in both are shown. Plasma methylation seems to be more specific than sputum methylation. Both concomitant methylation in both plasma and sputum yielded the highest specificity for all gene combinations. Promoter methylation of CDO1, TAC1, HOXA9 and SOX17 has a high specificity but low sensitivity to detect lung cancer.

Sensitivity and specificity for lung cancer detection using methylation changes in plasma and sputumPlasmaPlasmaSputumSputumPlasma & SputumPlasma & SputumGeneSpecificitySensitivitySpecificitySensitivitySpecificitySensitivityCDO180%45%67%63%93%26%TAC167%52%40%65%80%31%HOXA7100%11%80%27%100%6%HOXA960%66%7%90%67%60%SOX1767%56%73%52%93%27%ZFP4247%69%33%74%60%52%4 or more genes67%50%67%63%97%26%CDO1, TAC1, HOXA9, SOX1787%39%80%40%93%15%

Conclusion: Our gene panel of promoter methylation has a high enough specificity to be clinical useful but its sensitivity is low. The integration of this panel with other biomarkers would be needed to create a platform of high enough specificity and sensitivity to be clinically applicable.

Citation Format: Chen Chen, Alicia Hulbert, Alejandro Stark, Kristen Rodgers, Beverly Lee, Malcolm Brock, James Herman. Ultrasensitive DNA methylation detection using sputum and plasma for early detection of NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2015-3820
Abstract 3823: Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma
Authors: Mitsui Yozo , Hirata Hiroshi , Arichi Naoko , Hiraki Miho , Yasumono Hiroaki , Chang Inik , Fukuhara Shinichiro , Yamamura Soichiro , Shahryari Varahram , Deng Guoren , Saini Sharanjot , Majid Shahana , Tanaka Yuichiro , Dahiya Rajvir , Shiina Hiroaki
Published: August 2015

Introduction and Objective

Bone morphogenetic protein-2 (BMP-2) has been shown to function as a tumor suppressor with several different cancers, while its expression level is down-regulated by CpG hypermethylation. We hypothesized that impaired regulation of BMP-2 through epigenetic pathways is associated with the pathogenesis of renal cell carcinoma (RCC).

Methods

To test our hypothesis, CpG methylation of the BMP-2 gene was analyzed using 2 RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells.

Results

BMP-2 mRNA expression was significantly enhanced in the RCC cells by 5-aza-2′-deoxycitidie treatment. The prevalence of BMP-2 promoter methylation was significantly higher, while BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in the tumors. There was also a significant association of BMP-2 mRNA expression with tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after a radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression effectively induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells.

Conclusion

This is the first study to show inactivation of the BMP-2 gene via epigenetic pathways in RCC. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be useful as a molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

Citation Format: Yozo Mitsui, Hiroshi Hirata, Naoko Arichi, Miho Hiraki, Hiroaki Yasumono, Inik Chang, Shinichiro Fukuhara, Soichiro Yamamura, Varahram Shahryari, Guoren Deng, Sharanjot Saini, Shahana Majid, Yuichiro Tanaka, Rajvir Dahiya, Hiroaki Shiina. Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3823. doi:10.1158/1538-7445.AM2015-3823
Abstract 3834: Validation of potential methylation biomarkers in HPV-associated HNSCC
Authors: Chen Kang Mei , Stephen Josena K. , Divine George , Chitale Dhananjay , Ghanem Tamer , Chang Steven , Worsham Maria J.
Published: August 2015

Epidemiological and laboratory evidence indicate the human papilloma virus (HPV) as a causative agent for some squamous head and neck cancer (HNSCC). The biologic significance of HPV as independent risk factor is also underscored by the improved prognosis for patients with HPV positive HNSCC relative to HPV negative HNSCC, due in part to a better therapeutic response to chemoradiotherapy. The mechanism for this improved prognosis remains underexplored.

To assess the potential of HPV specific methylation biomarkers, we evaluated the status of 10 genes (CDH8, CRMP1, HTRE1, ELMO1, MEI1, MSX2, PCDH10, PCDHB11, C14orf162, and PITX2) in a cohort of 18 HPV positive and 14 HPV negative HNSCC using targeted sequencing (Illumina MiSeq). The genes were previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC using the Infinium HumanMethylation450 BeadChip (Lechner et al., 2013).

In our sample, of the 10 genes, targeted sequencing confirmed significant differential methylation between HPV positive and negative for C14orf162 (p = 0.007), MEI1(p = 0.004), MSX2 (p = 0.002), and PCDHB11(p = 0.002). PCDHB11 and MSX2 were hypermethylated; MEI1 and C14orf162 were hypomethylated, concordant with the Lechner et al., study.

In this independent sample set, confirmation of 4 of the 10 genes as differentially methylated in HPV positive vs HPV negative HNSCC with concordance of methylation direction (hypo- vs hypermethylated), validates their potential for differentiating HPV positive and HPV negative HNSCC. Cadherins are implicated in tumor progression and metastasis and are targets of the Polycomb group (PcG) proteins, which form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Our study further supports cadherin PCDHB11, together with C14orf162, MEI1, and MSX2 as likely biomarkers in HPV-associated HNSCC.

Support: Komen KG110218

Citation Format: Kang Mei Chen, Josena K. Stephen, George Divine, Dhananjay Chitale, Tamer Ghanem, Steven Chang, Maria J. Worsham. Validation of potential methylation biomarkers in HPV-associated HNSCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3834. doi:10.1158/1538-7445.AM2015-3834
Abstract 3835: DNA methylation levels in mononuclear DNA in breast cancer from the New York site of the BCFR
Authors: Wu Hui-Chen , Wang Qiao , Santella Regina M. , Terry Mary B.
Published: August 2015

Alterations in DNA methylation both at specific loci and overall, have been associated with breast cancer. These alterations have primarily been observed at the tissue level, although emerging studies are evaluating white blood cell (WBC) DNA methylation.

To examine whether DNA methylation change measured prospectively in a family-based cohort are associated with breast cancer risk, we measured methylation levels of two repetitive elements (LINE-1, and Sat2) and three genes in mononuclear cell DNA (BLNK, CUEDC1, and IBSP) previously identified as differentially methylated in breast cancer in a genome-wide association study. After an average of 8 years of followup, we observed 81 invasive incident breast cancers and compared their methylation levels measured with baseline bloods with the remaining 1,508 women not affected with breast cancer in the New York site of the Breast Cancer Family Registry (BCFR). We found that the overall percent methylation in CUEDC1 was statistically significantly higher in breast cancer cases than in controls (78.0% versus 76.8%, respectively, p = 0.02). In a multivariable Cox Proportional Hazards model adjusting for age at blood draw, with each 1 unit increase in CUEDC1 methylation level, the OR for breast cancer increased by 1.07 (95% Confidence Interval (CI) = 1.00-1.14). We did not find the other methylation markers to be related to risk. The area under the curve was 67.6% (95% CI = 61.4% to 73.8%) for the 5 methylation markers and Gail risk score, compared with 63.3% for the Gail score only.

Our finding for CUEDC1 replicates the finding from the Sisters Study supporting that selected markers selected markers of epigenetic changes measured in WBC may be potential biomarkers of breast cancer risk.

Citation Format: Hui-Chen Wu, Qiao Wang, Regina M. Santella, Mary B. Terry. DNA methylation levels in mononuclear DNA in breast cancer from the New York site of the BCFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2015-3835