bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 602: Ascorbic acid combined with a cytostatic inhibitor of glycolysis synergistically induces apoptosis in non-small lung cancer cells.
Authors: Vuyyuri Saleha B. , Rinkinen Jacob , Worden Erin , Davis Keith R.
Published: August 2014

Purpose An exceptional characteristic of many cancer cells is increased glucose uptake and elevated glycolysis with a concomitant reduction in oxidative respiration, also known as the Warburg effect. The increase in basal levels of reactive oxygen species (primarily, H2O2) and their increased dependence on glycolysis for their survival make tumor cells more susceptible than normal cells to treatment with a combination of pro-oxidant agents and chemotherapy. This common feature of tumor cells has been targeted for the development of new cancer therapeutics. Ascorbic acid (AA) has been shown to have cancer curative potential, at clinically achievable doses that have minimal or no toxic effects on normal cells. We have previously shown that some cancer cell lines are significantly more sensitive to treatment with ascorbic acid at concentrations where it may function as a pro-oxidant. In the present case, we investigated the effects of AA alone or in combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small lung cancer (NSCLC) cell lines vs immortalized lung epithelial cell line.

Results Cell viability assays showed that combined treatment of AA and 3-PO caused a synergistic increase in cell death in all the NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone, while no synergistic cytotoxic effect was observed in immortalized lung epithelial cells. Key markers such as DNA fragmentation, PARP cleavage, annexin-V binding, and caspase activity indicated that AA-induced cell death is caused via the activation of apoptosis. Metabolomic analysis revealed a decrease in NAD/NADH in the combination treatment that correlated well with the synergistic cell death observed in the viability results. The importance of NAD/NADH depletion in the synergistic response was confirmed by rescue experiments showing addition of NAD to the culture protected cells from the combination treatment. The major significance of these studies is that, we show for the first time that AA selectively synergizes with a glycolytic inhibitor in killing NSCLC cells and that pharmacologic AA treatment (either alone or combined with any other chemotherapeutic agent or glycolytic inhibitors used in the treatment of cancer) may be a promising therapy for non-small lung cancer and other challenging to-treat cancers.

Citation Format: Saleha B. Vuyyuri, Jacob Rinkinen, Erin Worden, Keith R. Davis. Ascorbic acid combined with a cytostatic inhibitor of glycolysis synergistically induces apoptosis in non-small lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2013-602
Abstract 614: Biological clues to potent DNA-damaging activities in food and flavoring.
Authors: Hossain Mohammad Zulfiquer , Gilbert Samuel F. , Patel Kalpesh , Ghosh Soma , Bhunia Anil K. , Kern Scott E.
Published: August 2014

Population differences in age-related diseases and cancer could stem from differences in diet. To characterize DNA strand-breaking activities in selected foods/beverages, flavorings, and some of their constituent chemicals, we used p53R cells, a cellular assay sensitive to such breaks. Substances testing positive included reference chemicals: quinacrine (peak response, 51X) and etoposide (33X); flavonoids: EGCG (19X), curcumin (12X), apigenin (9X), and quercetin (7X); beverages: chamomile (11X), green (21X), and black tea (26X) and coffee (3 to 29X); and liquid smoke (4 to 28X). Damage occurred at dietary concentrations: etoposide near 5 μg/ml produced responses similar to a 1:1000 dilution of liquid smoke, a 1:20 dilution of coffee, and a 1:5 dilution of tea. Pyrogallol-related chemicals and tannins are present in dietary sources and individually produced strong activity: pyrogallol (30X), 3-methoxycatechol (25X), gallic acid (21X), and 1,2,4-benzenetriol (21X). From structure-activity relationships, high activities depended on specific orientations of hydroxyls on the benzene ring. Responses accompanied cellular signals characteristic of DNA breaks such as H2AX phosphorylation. Breaks were also directly detected by comet assay. Cellular toxicological effects of foods and flavorings could guide epidemiologic and experimental studies of potential disease risks from DNA strand-breaking chemicals in diets.

Citation Format: Mohammad Zulfiquer Hossain, Samuel F. Gilbert, Kalpesh Patel, Soma Ghosh, Anil K. Bhunia, Scott E. Kern. Biological clues to potent DNA-damaging activities in food and flavoring. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 614. doi:10.1158/1538-7445.AM2013-614
Abstract 616: Insulin-like growth factor binding protein-3 (IGFBP-3) facilitates the response to DNA-damaging chemotherapy in triple-negative breast cancer cells.
Authors: Lin Mike Zhen , de Silva Hasanthi C. , Martin Janet Lee , Baxter Robert Charles
Published: August 2014

Introduction. Breast cancer cells frequently develop resistance to DNA-damaging therapy, and epidermal growth factor receptor (EGFR) may play an important role in this process. EGFR can modulate the repair of DNA double strand breaks (DSB) by non-homologous end-joining (NHEJ), by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). However many ER-negative breast tumors still respond poorly to DNA-damaging drugs even when combined with an EGFR inhibitor. IGFBP-3, an intracellular and secreted protein, is highly expressed in these tumors. Since we previously showed that IGFBP-3 can transactivate EGFR and translocate to the nucleus, the aim of this study was to determine whether IGFBP-3 forms part of the EGFR-DNA-PK complex that regulates the DNA damage response to DSB-inducing chemotherapeutic agents.

Results. In the triple-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, protein complex formation and nuclear localization of EGFR, DNA-PKcs and IGFBP-3 were enhanced by exposure to cytotoxic drugs etoposide or doxorubicin and reduced by IGFBP-3 knockdown or the EGFR kinase inhibitor gefitinib. Concomitantly, the co-location of EGFR-IGFBP-3 complexes with lipid rafts, visualized by proximity ligation assay (PLA) and confocal microscopy, decreased in response to drug treatment, consistent with the loss of this complex from the plasma membrane. Furthermore, the phosphorylation of DNA-PKcs at Ser2056 and its substrate Artemis nuclease at S516, and DNA repair capacity as measured by a non-homologous end-joining assay, were all decreased by IGFBP-3 silencing, while H2AX phosphorylation was increased, suggesting that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy.

Conclusion. The increased intranuclear interaction between EGFR and DNA-PKcs in response to DNA damage, and the subsequent stimulation of NHEJ activity, require the presence of IGFBP-3, which also forms part of the nuclear complex. This demonstrates a previously unrecognized “caretaker” role for IGFBP-3 in regulating DNA DSB repair by NHEJ. These novel findings suggests the possibility of a therapeutic approach for sensitizing ER-negative breast cancers to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3. (Supported by Australian Research Council grant DP0984232 to RCB).

Citation Format: Mike Zhen Lin, Hasanthi C. de Silva, Janet Lee Martin, Robert Charles Baxter. Insulin-like growth factor binding protein-3 (IGFBP-3) facilitates the response to DNA-damaging chemotherapy in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2013-616
Abstract 618: Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker.
Authors: Dizdaroglu Miral
Published: August 2014

Accumulated evidence points to DNA repair capacity as an important therapeutic factor in cancer in predicting patient response to DNA-damaging agents such as chemotherapeutic drugs and ionizing radiation. Recent findings suggest that some types of malignant tumors possess increased DNA repair capacity that may affect the therapy and outcome of cancer. Thus, the knowledge of the over-expression or under-expression levels of DNA repair proteins in tumors and disease-free tissues will help develop and guide treatment strategies that will likely lead to the best treatment results for patients. In this context, DNA repair proteins are becoming predictive, prognostic and therapeutic factors in cancer, and also promising drug targets for cancer treatment as DNA repair inhibitors are being developed to increase the efficacy of cancer therapy. We developed assays for the mass spectrometric measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. There is strong evidence for a critical role of NEIL1 in maintaining the genetic stability and in prevention of diseases such as cancer and metabolic syndrome. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), using the fully 15N-labeled analogue of hNEIL1 (15N-hNEIL1) as an internal standard, which we produced, purified and characterized. Both hNEIL1 and 15N-hNEIL1 were hydrolyzed with trypsin. Eighteen tryptic peptides of each protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1, and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides of both proteins were recorded and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and 15N-hNEIL1 on the basis of product ions. We also showed the detection of these proteins following their separation by gel electrophoresis and in-gel trypsin digestion. Our results suggest that the assays developed would be highly suitable for the positive identification and accurate quantification of hNEIL1 in tissues in vivo as a potential cancer biomarker.

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Citation Format: Miral Dizdaroglu. Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2013-618
Abstract 62: Detection and comparison of EGFR mutations in matched tumor tissue, cell block, pleural effusion and serum with PNA clamping and direct sequencing in NSCLC with malignant pleural effusion.
Authors: Park Chan Kwon , Dong Chang , Jin Woo Jin Woo , Kim Kwan Hyoung , Ha Jick Hwan , Rhee Chin Kook , Kim Young Kyoon , Lee Sang Haak , Park Mi Sun , Yim Hyeon Woo , Kim Seung Joon
Published: August 2014

Background: Peptide nucleic acid (PNA)-mediated real-time PCR clamping was recently used to detect mutations because it has higher sensitivity than conventional direct sequencing. Pleural effusion and serum samples could be good sources of detecting the epidermal growth factor receptor (EGFR) mutational status of non-small cell lung cancer (NSCLC) patients. Methods: We studied 37 NSCLC patients with malignant pleural effusion. In each patient, EGFR mutational status was measured from the samples of tumor tissue, cell block, pleural effusion and serum using both PNA clamping and direct sequencing. The concordance between PNA clamping and direct sequencing, and the diagnostic performance of pleural effusion was investigated. Results: The detection rate of EGFR mutation in the pleural effusion and the serum was 27% and 2.8%, respectively by both PNA clamping and direct sequencing. The κ coefficient between the two methods were 0.68 (p-value = 0.0007), 0.91 (p-value < 0.0001), 0.75 (p-value < 0.0001) and -0.01 (p-value = 0.8639) in tissue, cell block, pleural effusion and serum, respectively. The diagnostic performance of pleural effusion compared with combined tumor tissue plus cell block showed sensitivity 89%, specificity 100%, positive predictive value 100% and negative predictive value 95% with PNA clamping, and sensitivity 67%, specificity 90%, positive predictive value 75% and negative predictive value 86% with directing sequencing. Interestingly, a patient who was confirmed EGFR mutation only with the PNA clamping showed significant response to EGFR TKI (tyrosine kinase inhibitor). Conclusion: Despite the limited role of serum samples, pleural effusion had a diagnostic performance comparable with tumor tissue and cell block to detect EGFR mutational status in NSCLC. Although the diagnostic performance of PNA clamping was similar to that of direct sequencing, additional benefit is expected considering the response to the EGFR TKI.

Citation Format: Chan Kwon Park, Chang Dong, Jin Woo Jin Woo, Kwan Hyoung Kim, Jick Hwan Ha, Chin Kook Rhee, Young Kyoon Kim, Sang Haak Lee, Mi Sun Park, Hyeon Woo Yim, Seung Joon Kim. Detection and comparison of EGFR mutations in matched tumor tissue, cell block, pleural effusion and serum with PNA clamping and direct sequencing in NSCLC with malignant pleural effusion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2013-62
Abstract 623: The PARP3- and ATM-dependent phosphorylation of APLF facilitates DNA double-strand break repair.
Authors: Koch Christine A. , Fenton Amanda L. , Shirodkar Purnata , Meng Li
Published: August 2014

APLF is a forkhead associated (FHA)-containing protein with poly(ADP-ribose)-binding zinc finger (PBZ) domains, which undergoes ionizing radiaiton (IR)-induced and ATM-dependent phosphorylation at serine-116 (Ser116). Here we demonstrate that the phosphorylation of APLF at Ser116 in human U2OS cells by ATM is dependent on PARP3 levels and the APLF PBZ domains. The interaction of APLF at sites of DNA damage was diminished by the single substitution of APLF Ser116 to alanine, and the cellular depletion or chemical inhibition of ATM or PARP3 also altered the retention of APLF at sites of laser-induced DNA damage, and impaired the accumulation of Ser116-phosphorylated APLF at IR-induced γH2AX foci in human cells. The data further suggest that ATM and PARP3 participate in a common signaling pathway to facilitate APLF-Ser116 phosphorylation, which, in turn, appears to be required for efficient DNA double-strand break (DSB) repair kinetics and cell survival following IR. Collectively, these findings provide a more detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage, and suggest that Ser116-APLF phosphorylation facilitates APLF-dependent DSB repair.

Citation Format: Christine A. Koch, Amanda L. Fenton, Purnata Shirodkar, Li Meng. The PARP3- and ATM-dependent phosphorylation of APLF facilitates DNA double-strand break repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 623. doi:10.1158/1538-7445.AM2013-623
Abstract 635: PARP inhibition in HPV positive head and neck cancers.
Authors: Cooper Tiffiny , Rodriguez Marcela , Trummell Hoa Q. , Weaver Alice , Bonner James A. , Yang Eddy S.
Published: August 2014

Purpose: The human papilloma virus (HPV) has recently become a prevalent cause of human head and neck cancer (HNC), which often exhibits dysregulated epidermal growth factor receptor (EGFR) signaling. We previously reported that in non-HPV-associated HNC, inhibition of EGFR (EGFRi) attenuates DNA double stranded break (DSB) repair and subsequently sensitizes tumors to inhibitors of poly (ADP-ribose) polymerase (PARP), which target DNA repair deficient tumors. Because patients with HPV-associated HNC have been shown to exhibit increased sensitivity to DNA damage, we evaluated the response of HPV (+) HNC cells to combination EGFRi and PARPi compared to HPV (-) HNC cells.

Methods: HPV (-) UM-SCC1 and UM-SCC6, as well as HPV (+) SCC-47 and SCC-90 were utilized for in vitro studies. Cell growth and survival fraction were evaluated via cell proliferation and colony formation assays, respectively. DNA damage studies were performed using standard γ-H2AX and Rad51 foci. Lastly, in vivo studies were conducted in mice bearing HNC tumor xenografts as well as a human tumor explant from a lymph node metastasis of a HPV(+) HNC patient.

Results: Interestingly, HPV (+) HNCs exhibited the greatest sensitivity both in vitro and in vivo to PARP inhibition alone. This correlated with a blunted DNA damage response. In contrast, HPV (-) HNCs were most sensitive to combination EGFRi/PARPi, which correlated with persistent DNA damage.

Conclusions: HPV(+) head and neck cancers may be sensitive to PARP inhibition alone. Our in vitro and in vivo results warrant further testing of PARP inhibitors as part of therapy for HNCs.

Citation Format: Tiffiny Cooper, Marcela Rodriguez, Hoa Q. Trummell, Alice Weaver, James A. Bonner, Eddy S. Yang. PARP inhibition in HPV positive head and neck cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 635. doi:10.1158/1538-7445.AM2013-635
Abstract 641: Correlation of methylation status of deep surgical margins with the local reccurence of head and neck squamous cell carcinoma.
Authors: Hayashi Masamichi , Wu Gaosong , Chang Xiaofei , Li Xiufeng , Ahn Julie , Khan Zubair , Akpeng Belinda , Roh Jong–Lyel , Koch Wayne
Published: August 2014

Background: Locoregional recurrence occurs in as many as 40% of patients undergoing surgery for head and neck squamous cell carcinoma (HNSCC). We have been focused some molecular alterations exsiting even in histologically negative surgical margins. However, in head and neck cancer resection, surgical margins are hard to evaluate because the most of them usually consists of complicated muscular margins. In order to evaluate these deep surgical margins, before collecting some margin tissues, we often conduct tissue imprinting by using one layer nitrocellulose sheet which can collect proteins without hurting surgical samples.

Method: Both 161 deep margin tissues and 166 deep margin imprints were collected from 74 histologically margin negative resections of primary HNSCC from May, 2009 to September, 2012. Bisulfited DNA from samples were amplified using quantitive methylation-specific polymerase chain reaction (QMSP) for 6 genes (DCC, EDNRB, HOXA9, KIF1A, NID2 and NR2B). The cut off value was calculated using QMSP data of tumors and normal muscles, and was applied to deep margin samples. The correlation between the QMSP status of deep margin samples and clinical outcome was evaluated.

Results: The average QMSP of tumor tissues, that of normal muscles and the cut off value are 21.0, 0.6, 2.1 in DCC, 23.3, 0.6, 2.2 in EDNRB, 32.0, 0.7, 2.3 in HOXA9, 20.3, 0.4, 1.3 in KIF1A, 36.0, 3.0, 9.4 in NID2 and 10.7, 0.1, 0.4 in NR2B. Those who has the higher QMSP values of deep margin tissues than the cut off value tends to have a poor recurrence free survival (P=0.073 in DCC, P=0.018 in HOXA9, P=0.005 in KIF1A, P=0.011 in NID2) and a poor overall survival (P=0.076 in HOXA9, P=0.059 in NID2). Also, those who has the higher QMSP values of deep margin imprints than the cut off value tends to have a poor recurrence free survival (P=0.051 in HOXA9) and a poor overall survival (P=0.075 in HOXA9).

Conclusion: The methylation status of HNSCC deep surgical margin samples is correlated with the local recurrence. Especially, hyper-methylated HOXA9 gene of both deep margin tissues and imprints is a significant biomarker of poor recurrence free survival and overall survival.

Citation Format: Masamichi Hayashi, Gaosong Wu, Xiaofei Chang, Xiufeng Li, Julie Ahn, Zubair Khan, Belinda Akpeng, Jong–Lyel Roh, Wayne Koch. Correlation of methylation status of deep surgical margins with the local reccurence of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2013-641
Abstract 647: Novel promoter hypermethylation marker for prognostic in cervicouterine cancinogenesis.
Authors: Brebi Priscilla , Andana Alejandra , Hoffstetter Rene , Ili Carmen , Viscarra Tamara , Silva Ramon , Garcia Patricia , Leal Pamela , Weber Helga , Roa Juan C.
Published: August 2014

New biomarkers are needed to improve cervical cancer screening technologies, which are mostly based on cytological examination and HPV detection. However, PAP-smear has a low-sensitivity to detect low grade squamous intraepithelial lesions and not all HPV infected women will develop preneoplastic or neoplastic lesions. Previous results of our group showed that genes Gen Z (patent pending), CDH1 and MEGF9 could be hypermethylated in cervical cancer and not in normal epithelia. The aim of this study was to determinate if promoter methylation status of genes (Gen Z, CDH1 and MEGF9) are related with progression or diagnosis of cervical carcinogenesis.

For this study, 107 citobrush, urine and blood samples were collected from women who attended to gynecological care in a public health center in Temuco, Chile. Citobrush DNA from 50 normal, 40 low grade squamous intraepithelial lesion (LSIL), 40 high grade squamous intraepithelial lesion (HSIL) and 17 squamous cervical cancer were bisulfite converted for methylation specific PCR (MSP). Bisulfite conversion was confirmed by amplification of a 133-bp fragment of the β-actin. MSP primers were specifically design for CpG island of promotor region of each gene.

Gen Z was found 100% methylated in SCC samples, 65% in HSIL, 43% in LSIL and in normal samples only a 26%. MEGF9 and CDH1 genes were found methylated in 36% and 48% of normal samples, 45% and 55% of LSIL, 70% and 77% of HSIL and 47% and 71% of SCC, respectively.

All promoter regions studied showed a higher methylation frequency in LSIL, HSIL and SCC than normal samples. Significant statistical differences in Gen Z and CDH1 Methylation frequencies between normal and SCC samples were found (P<0.05). Methylation of Gen Z increased in a sequential and cumulative way as the lesion progress.

Our results suggest that the Gen Z could be useful tool for identifying women with a higher risk of progression to cervical cancer. Examination of these biomarkers in a larger, independent cohort is warranted. Grant Support: This investigation was financed by Proyect CORFO-INNOVA N°07CN13PBT-222 and Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141

Citation Format: Priscilla Brebi, Alejandra Andana, Rene Hoffstetter, Carmen Ili, Tamara Viscarra, Ramon Silva, Patricia Garcia, Pamela Leal, Helga Weber, Juan C. Roa. Novel promoter hypermethylation marker for prognostic in cervicouterine cancinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2013-647
Abstract 650: IGSF4 methylation is an independent marker of HPV positive OPSCC.
Authors: Chen Kang Mei , Stephen Josena K. , Mahan Meredith , Havard Shaleta , Divine George , Gardner Glendon , Schweitzer Vanessa P. , Worsham Maria J.
Published: August 2014

Human papilloma virus (HPV) is a known causative agent for oropharyngeal squamous cell cancer (OPSCC). The goal of our study was to determine the relationship of HPV type 16 (HPV16) infection and methylation status in an OPSCC cohort. HPV16 was detected using real-time quantitative PCR (qPCR) in a retrospective cohort of 111 primary OPSCC. A 24 tumor suppressor gene candidate panel was assessed for methylation using the methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) assay. Univariate associations with HPV and gene methylation, age, gender, race and smoking were analyzed using Fisher's exact tests and logistic regression. A final multiple logistic regression model, to include patient characteristics and genes significant on univariate analysis, was built using backwards selection of genes. Statistical significance was set at p<0.05 and all analyses were done using SAS 9.2. In univariate analyses, Caucasian American (CA) OPSCC were more likely to be HPV positive as compared to African American (AA) OPSCC (OR=3.01, 95% CI 1.28, 7.07, p=0.011). Also, smoking (never vs current) [OR=3.60 (1.06, 12.24), p=0.04], and TIMP3, DAPK1, ESR1, and IGSF4 were associated with HPV positive status (OR=5.03, p=0.021; OR=3.71, p=0.04; OR=5.03, p=0.021; OR=7.68, p=0.012, respectively). The final model, after controlling for patient characteristics and after backward elimination of genes, indicated IGSF4 methylation is an independent predictor of HPV positive status [OR=7.07 (1.22, 40.79), p=0.029]. HPV infection status is independently associated with methylation of IGSF4, indicating interplay of DNA methylation and HPV in OPSCC pathogenesis.

Citation Format: Kang Mei Chen, Josena K. Stephen, Meredith Mahan, Shaleta Havard, George Divine, Glendon Gardner, Vanessa P. Schweitzer, Maria J. Worsham. IGSF4 methylation is an independent marker of HPV positive OPSCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 650. doi:10.1158/1538-7445.AM2013-650
Abstract 651: The clinical significance of tissue miR-124 methylation in sporadic and ulcerative colitis-associated colorectal cancer.
Authors: Toiyama Yuji , Hur Keun , Tanaka Koji , Inoue Yasuhiro , Nagasaka Takeshi , Kusunoki Masato , Boland C. Richard , Goel Ajay
Published: August 2014

Background: Growing evidence indicates that the expression of several miRNAs can be epigenetically regulated through methylation of their promoter CpG sequences. MiR-124 is one such tumor suppressive miRNA, which is expressed at lower levels in various human cancers due to methylation of its promoter region. The present study aimed to determine miR-124 methylation status and determine its clinical significance in sporadic and ulcerative colitis-associated colorectal cancer (CRC). Materials & Methods: We analyzed 579 colorectal tissues, which included 176 sporadic CRC and the corresponding adjacent normal colonic mucosa (NC), 57 colorectal adenomas and 20 normal colonic mucosae from non-cancer patients (NN). In addition, we also examined 135 normal mucosae (N-UC), 12 dysplasia(D-UC) and 12 colitic cancers (C-UC) from UC patients. We performed quantitative bisulfite pyrosequencing analysis to determine miR-124 promoter methylation levels. Results: MiR-124 methylation rate was significantly higher in CRCs and adenomas compared to NC or NN (P<0.0001, CRC vs. NC; P<0.0001, CRC vs. NN). Receiver operating characteristic (ROC) analysis revealed that methylation levels of miR-124 can robustly discriminate patients with CRC (AUC=0.977) and adenomas (AUC=0.985) from healthy controls. Kaplan-Meier survival analysis showed that patients with high miR-124 methylation demonstrated poor overall survival (p=0.001), and high miR-124 methylation was an independent prognostic factor of sporadic CRC (p=0.028). Similarly, miR-124 methylation was significantly higher in C-UC and D-UC compared to N-UC (p<0.0001). ROC analysis showed that miR-124 methylation can successfully discriminate C-UC (AUC=0.978) and D-UC (AUC=0.884) from N-UC. Interestingly, high levels of miR-124 methylation in rectal specimens were an independent predictor for the development of UC-associated CRC. Conclusions: miR-124 methylation is an early event in sporadic, as well as ulcerative colitis-associated CRC. Since miR-124 methylation could robustly identify patients with colorectal adenomas and cancers, this miRNA has the potential to serve as a diagnostic marker in colorectal neoplasia.

Citation Format: Yuji Toiyama, Keun Hur, Koji Tanaka, Yasuhiro Inoue, Takeshi Nagasaka, Masato Kusunoki, C. Richard Boland, Ajay Goel. The clinical significance of tissue miR-124 methylation in sporadic and ulcerative colitis-associated colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 651. doi:10.1158/1538-7445.AM2013-651
Abstract 672: Histone deacetylase inhibition has potential to suppresse growth of androgen dependent and independent prostate cancer in vitro and in vivo.
Authors: Sato Shinya , Suzuki Shugo , Naiki-Ito Aya , Yamashita Yoriko , Asamoto Makoto , Shirai Tomoyuki , Takahashi Satoru
Published: August 2014

Histone acetylation plays an important role in chromatin remodeling and gene expression. Histone deacetylase (HDAC) inhibitors regulate gene expressions through the induction of histone acetylation and have been reported to suppress growth of several malignancies including lymphoma. In this study, we examined the effects of one example on androgen dependent and independent prostate cancer growth in vivo and in vitro. In in vitro study, androgen dependent and independent prostate cancer cell lines (LNCaP and VCaP) were used. The HDAC inhibitor, OBP-801 (Generously donated by Dr. Urata, Oncolys Biopharma), significantly suppressed cell proliferation with induction of G2/M arrest, and decreased expression of cdc2, androgen receptor (AR) and PSA protein in both cell lines. OBP-801 also significantly induced apoptosis with activation of caspase 3 in LNCaP. Next, we performed two in vivo experiments. First, we transplanted an androgen independent rat prostate cancer (PLS-P) into the femurs and backs of male F344 rats, and OBP-801 was administered intravenously at doses of 0, 2.5 and 5mg/kg every other day for 2 weeks. OBP-801 significantly reduced the volume of the transplanted tumor, while inducing cleaved caspase 3 protein expression. OBP-801 also suppressed bone destruction and induction of osteoclasts in xenografts transplanted to femurs. Second, we prepared male Transgenic Rat for Adenocarcinoma of Prostate (TRAP) rats which spontaneously develop androgen dependent rat prostate cancers, and OBP-801 was given intravenously at doses of 0, 0.5 and 2mg/kg once a week for 8 weeks. As a result, body weight loss was observed in treated groups, and numbers of carcinoma acini in the lateral and ventral prostates were significantly decreased compared with the control group values. In conclusion, the obtained data suggests that OBP-801 may be a candidate therapeutic or preventing agent for androgen dependent and independent prostate cancer, although side effects such as body weight loss may occur. Elucidation of mechanisms of HDAC underlying these effects is needed.

Citation Format: Shinya Sato, Shugo Suzuki, Aya Naiki-Ito, Yoriko Yamashita, Makoto Asamoto, Tomoyuki Shirai, Satoru Takahashi. Histone deacetylase inhibition has potential to suppresse growth of androgen dependent and independent prostate cancer in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 672. doi:10.1158/1538-7445.AM2013-672
Abstract 674: The lysine demethylase KDM1 is a novel therapeutic target for the treatment of gliomas.
Authors: Sareddy Gangadhara R. , Saran Sandeep , Nair Binoj C. , Krishnan Samaya R. , Gonugunta Vijay K. , Brenner Andrew J. , Vadlamudi Ratna K.
Published: August 2014

Glioma development is a multistep process, involving alterations in genetic and epigenetic mechanisms. Understanding the mechanisms and enzymes that promote epigenetic changes in gliomas are urgently needed to identify novel therapeutic targets. In the present study we explored the significance of histone demethylase KDM1 in glioma progression. In order to know the status of expression of KDM1, we utilized glioma tissue microarrays which consist of different grades of astrocytomas, oligodendrogliomas, ependymomas and normal brain tissues. Immunohistochemical analysis showed that KDM1 expression is overexpressed in the gliomas compared to normal brain, and KDM1 expression levels were positively correlated with histological malignancy. KDM1 expression was also found to be elevated in various established glioma cell lines. To study the functional significance of KDM1 in gliomas, KDM1 expression was silenced using siRNA or its pharmacological inhibition using pargyline or NCL-1. Silencing of KDM1 or inhibition of its enzyme activity significantly reduced the proliferation and colony formation of glioma cells. Mechanistic studies showed that inhibition of KDM1 promoted the acetylation of p53 and up regulation of its target genes p21 and PUMA. Patient-derived primary GBM cells expressed high levels of KDM1 and pharmacological inhibition of KDM1 decreased their proliferation. Further, KDM1 inhibition reduced the expression of stemness markers CD133 and nestin in GBM cells. Mouse xenograft assays revealed that inhibition of KDM1 using pargyline significantly reduced U87 glioma xenograft tumor growth. Inhibition of KDM1 increased levels of H3K4-me2 and H3K9-Ac histone modifications, reduced H3K9-me2 modification and promoted expression of p53 target genes (p21 and PUMA), leading to apoptosis of glioma xenograft tumors. Our results suggest that KDM1 is overexpressed in gliomas and could be a potential therapeutic target for the treatment of gliomas.

Citation Format: Gangadhara R. Sareddy, Sandeep Saran, Binoj C. Nair, Samaya R. Krishnan, Vijay K. Gonugunta, Andrew J. Brenner, Ratna K. Vadlamudi. The lysine demethylase KDM1 is a novel therapeutic target for the treatment of gliomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2013-674
Abstract 682: Synergistic effects of CG200745 combining with Gemcitabine and/or Erotinib in pancreatic cancer.
Authors: Ro Seonggu , Hong Jang-Won , Kwon Sool-Ki , Cha Hyunju , Jung Dong Sub , Chang Ho-Jin , Cho Joong Myung
Published: August 2014

CG200745 is a HDAC inhibitor at the final stage of phase I clinical trial. In the current human studies with patient with terminal solid tumors, CG200745 is showing highly desirable PK and safety profiles. Actually, the AUC/dose of CG200745 is the highest among all the clinical stage HDAC inhibitors. CG200745 is more distributed in tumor tissue that in plasma. Moreover, SD's (stable disease) were observed even in single dose study. Currently, we are planning phase II studies using regimens combining CG200745 and existing therapy in patients with solid tumors. As a choice of indications, pancreatic cancer was considered based on biological studies and observation in phase I clinical studies.Thus, cellular and xenograft studies of CG200745 using pancreatic cancer cell lines have been carried out in our laboratories. In these studies, treatments of CG200745 combining with gemcitabine and/or erotinib show clear synergistic effects. These results indicate that the combination of CG200745 with existing therapy can be a promising intervention for pancreatic cancer.

Citation Format: Seonggu Ro, Jang-Won Hong, Sool-Ki Kwon, Hyunju Cha, Dong Sub Jung, Ho-Jin Chang, Joong Myung Cho. Synergistic effects of CG200745 combining with Gemcitabine and/or Erotinib in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 682. doi:10.1158/1538-7445.AM2013-682
Abstract 689: Methylation of miRNA genes is a useful biomarker for detection of bladder cancer.
Authors: Suzuki Hiromu , Shimizu Takashi , Nojima Masanori , Yamamoto Eiichiro , Maruyama Reo
Published: August 2014

Background and aim: Dysregulation of microRNAs (miRNAs) has been implicated in bladder cancer (BCa), though the mechanism is not fully understood. We aimed to explore the involvement of epigenetic alteration of miRNA expression in bladder cancer. Materials and methods: Two BCa cell lines (T24 and UM-UC-3) were treated with 5-aza-2’deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which their miRNA expression profiles were analyzed using a TaqMan Array. Bisulfite pyrosequencing was used to assess miRNA gene methylation in 5 cancer cell lines, 83 primary tumors, and 120 preoperative and 47 postoperative urine samples. Receiver operating characteristic curve analysis was used to assess the diagnostic performance of the miRNA gene panel. Results: Of 664 miRNAs examined, 146 were upregulated by 5-aza-dC plus PBA. CpG islands were identified in the proximal upstream of 23 miRNA genes, and 12 of those were hypermethylated in cell lines. Among them, miR-137, -124-2, -124-3 and -9-3 were frequently and tumor-specifically methylated in primary cancers (miR-137, 68.7%; miR-124-2, 50.6%; miR-124-3, 65.1%; miR-9-3, 45.8%). Methylation of the same four miRNAs in urine specimens enabled BCa detection with 81% sensitivity and 89% specificity; the area under the ROC curve was 0.916. Ectopic expression of silenced miRNAs in BCa cells suppressed growth and cell invasion. Conclusions: Our results indicate that epigenetic silencing of miRNA genes may be involved in the development of BCa and that methylation of miRNA genes could be a useful biomarker for cancer detection.

Citation Format: Hiromu Suzuki, Takashi Shimizu, Masanori Nojima, Eiichiro Yamamoto, Reo Maruyama. Methylation of miRNA genes is a useful biomarker for detection of bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 689. doi:10.1158/1538-7445.AM2013-689
Abstract 695: The generation of a mono-selective CDK7 inhibitor.
Authors: Eickhoff Jan E. , Zischinsky Gunther , Choidas Axel , Habenberger Peter , Wolf Alexander , Degenhardt Carsten , Baumann Matthias , Unger Anke , Ghallab Ahmad , Werchau Doreen , Hengstler Jan , Nussbaumer Peter , Klebl Bert M.
Published: August 2014

Cyclin-depending kinase (CDK) family members that trigger passage through the cell cycle have been considered as attractive cancer targets for at least two decdes Yet, the first generation of pan-specific CDK inhibitors failed to show clinical efficacy due to their small therapeutic window. CDK family members that control processes such as transcription have caught less attention, although experimental evidence for an involvement in different pathological processes is emerging. CDK7 (or CAK) has essential roles in both the cell-division cycle and transcription, and serves as a direct link between the regulation of basal transcription and the cell cycle. In addition to its role as a transcription activator, CDK7 has been reported to act on specific transcription events by phosphorylating a series of specific transcription factors, such as the estrogen receptor and p53. These findings indicate that CDK7 is a potential target for cancer therapy, and perhaps inflammation. The detailed molecular characterization of the biological role of CDK7, as well as its therapeutic validation, is dependent on the development of highly specific and potent CDK7 inhibitors. We have developed a series of CDK7 inhibitors with picomolar potency on CDK7, more than 1000-fold selectivity over any other CDK, and single digit nM inhibition of sensitive cancer cell lines. Interestingly, a broad screen of >200 different tumor cell lines lead to the identification of responder and non-responder cell lines. Non-responder tumor lines, similar to primary cells are at least by a factor of 1000 less sensitive to the inhibition with the mono-selective CDK7 inhibitor. Our findings further indicate that CDK7 is neither essential for general transcription, nor for the progression through the cell cycle. It rather coordinates cell line specific biological processes. The effects of selective CDK7 inhibitors on tumor cell lines are clearly distinct from existing, nonselective pan-CDK inhibitors. The responder status for a subset of cancer cell lines can be predicted with a distinct biomarker. Chemical optimization of the selective inhibitor lead to a compound which shows dose-dependent pharmacological efficacy in mouse xenograft models after oral administration, with no signs of toxicity at the efficacious doses.

Citation Format: Jan E. Eickhoff, Gunther Zischinsky, Axel Choidas, Peter Habenberger, Alexander Wolf, Carsten Degenhardt, Matthias Baumann, Anke Unger, Ahmad Ghallab, Doreen Werchau, Jan Hengstler, Peter Nussbaumer, Bert M. Klebl. The generation of a mono-selective CDK7 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 695. doi:10.1158/1538-7445.AM2013-695
Abstract 698: MCL1 dependent cells are sensitive to the CDK inhibitor Dinaciclib.
Authors: Hatch Harold , Booher Robert , Perera Samanthi , Nguyen Thi , Dolinski Brian , Al-Assaad Samer , Harmonay Lauren , Schuller Alwin , Angagaw Minilik , Long Brian , Qu Xianlu , Miselis Nathan , Ayers Mark , Nebozhyn Michael , Hirsch Heather , Greenawalt Danielle , Loboda Andrey , Graef Thorseten , Im Ellie , Blanchard Rebecca , Zawel Leigh , Strack Peter
Published: August 2014

Dinaciclib is a potent inhibitor of cyclin dependent kinases (CDKs) 1, 2, 5, and 9 and is currently in Phase 3 for the treatment of refractory chronic lymphocytic leukemia (CLL). To further understand the mechanism of action, identify predictive biomarkers, and find additional cancer types which may benefit from dinaciclib, we evaluated cell viability following 24 hours treatment across a panel of ∼500 cells lines. Hematopoietic cell lines were on average 3-times more sensitive than solid tumor lines. In agreement with previous findings, mRNA expression of the anti-apoptotic family member BCL-xL or the ratio of MCL1-to-BCL-xL continue to be the best predictor of dinaciclib sensitivity in both hematopoietic and solid tumor cell lines. MCL1 appears to be an important target of dinaciclib particularly in MCL1 amplified cell lines. Dependence on MCL1 was established in a panel of 19 breast, NSCLC and SCLC cell lines by depletion of the protein by either dinaciclib treatment or MCL1 RNAi. The NSCLC line H23 was highly dependent on MCL1, as RNAi knockdown decreased viability to <20% and could be rescued by introduction of a non-targeted MCL1 expression construct. In the H23 xenograft, dinaciclib diminished MCL1 levels and induced tumor apoptosis resulting in >80% tumor regression. Cell lines which lacked pro-apoptotic proteins BAX / BAK or harbored a BAX mutation were insensitive to the inhibitor. Using apoptosis defective lines we demonstrate that 24 hours of dinaciclib treatment still impacted cell count by blocking cell cycle progression as measured by FACS. These data demonstrate that both cell cycle block and induction of apoptosis contribute to dinaciclib's mechanism of action. However, the observation that MCL1 and BCL-xL were top genes associated with sensitivity suggests that induction of apoptosis is the predominant mechanism of dinaciclib's anti-tumor effect and warrants further investigation of MCL1 amplification as a predictive biomarker in future clinical studies.

Citation Format: Harold Hatch, Robert Booher, Samanthi Perera, Thi Nguyen, Brian Dolinski, Samer Al-Assaad, Lauren Harmonay, Alwin Schuller, Minilik Angagaw, Brian Long, Xianlu Qu, Nathan Miselis, Mark Ayers, Michael Nebozhyn, Heather Hirsch, Danielle Greenawalt, Andrey Loboda, Thorseten Graef, Ellie Im, Rebecca Blanchard, Leigh Zawel, Peter Strack. MCL1 dependent cells are sensitive to the CDK inhibitor Dinaciclib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 698. doi:10.1158/1538-7445.AM2013-698
Abstract 701: Characterization of two novel quinoline derivatives that induce apoptosis in a cancer-specific manner.
Authors: Lindamulage Indeewari K. , Vu Hai-Yen , Lee Yi-Fang , Lee Hoyun , Trivedi Piyush
Published: August 2014

Many quinoline-based compounds possess pharmacological properties that are beneficial for the control of various human diseases, including microbial infections, inflammatory-related disorders, cardiovascular conditions, and cancer. The anticancer activities of quinoline compounds are often rendered by their property of inhibiting protein kinases, proteasome activity, tubulin polymerization/depolymerization and DNA repair. To further improve the efficacy and specificity, we created and characterized several quinoline derivatives, which led us to identify CTRI-17 and CTRI-20 as promising leads. We examined their anti-proliferative properties using three human breast cancer cell lines (MDA-MB-468, MDA-MB-231 and MCF-7) and two matching non-cancer breast cell lines (184B5 and MCF10A). In addition, we also examined their efficacy using one leukemic cell line (K-562) and one cervical cell line (HeLa). Data from this study showed that the IC50 values of CTRI-17 and CTRI-20 were 0.1-0.3 μM and 1.2-2.4 μM ranges on cancer and non-cancer cell lines, respectively. Thus, both CTRI-17 and CTRI-20 killed cancer cells 10-20 times more effectively than non-cancer cells. This differential cell killing effects on cancer and non-cancer cells is a highly desirable property for potential anticancer therapeutics. We further found that cancer cells treated with the two compounds resulted in increases of pro-apoptotic proteins such as p53 and Bax, and decreases of anti-apoptotic proteins such as Bcl-2 and survivin. Our data obtained from flow cytometry and confocal microscopy showed that CTRI-17 and CTRI-20 induced prolonged cell cycle arrest at G2/M phase prior to causing apoptosis in cancer cells. However, the two compounds neither induced substantial cell cycle arrest nor caused high levels of apoptosis in non-cancer cells at the same drug concentration. We are currently in the process of determining the detailed functional mechanisms of CTRI-17 and CTRI-20.

Citation Format: Indeewari K. Lindamulage, Hai-Yen Vu, Yi-Fang Lee, Hoyun Lee, Piyush Trivedi. Characterization of two novel quinoline derivatives that induce apoptosis in a cancer-specific manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2013-701