bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5660: Cell-free generation and characterization of ErbB2-nanoparticle conjugates.
Authors: He Wei , Saldana Matthew , Scharadin Tiffany , Gellner Candice , Li Yuanpei , Lam Kit , Carraway Kermit , Henderson Paul , Coleman Matthew
Published: August 2014

We have developed a rapid process for cell-free production of full-length ErbB2, a receptor tyrosine kinase that contributes to the onset and progression of breast and other tumor types. The receptor is solubilized and stabilized via incorporation into nanolipoprotein particles (NLPs). NLPs are discoidal structures, approximately 20 nm in diameter, that arise from the self-assembly of apolipoproteins around a phospholipid bilayer that acts as a mimic of the cell membrane. The ErbB2-NLP complex was soluble and could be recognized by antibodies, such as Trastuzumab, that indicate the presence of correctly folded ErbB2 extracellular domains. Kinase function was confirmed using Lapatinib, which inhibited tyrosine autophosphorylation during cell-free synthesis. The cell-free synthesis and biochemical characterization data support the potential use of ErbB2-NLPs and related variants for novel biochemistry and drug screening studies.

Citation Format: Wei He, Matthew Saldana, Tiffany Scharadin, Candice Gellner, Yuanpei Li, Kit Lam, Kermit Carraway, Paul Henderson, Matthew Coleman. Cell-free generation and characterization of ErbB2-nanoparticle conjugates. [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 5660. doi:10.1158/1538-7445.AM2013-5660
Abstract 5676: Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-Ribose)polymerase inhibitors.
Authors: Gangloff Anthony R. , Brown Jason , De Jong Ron , Erickson Philip , Grimshaw Charles , Hixon Mark , Jennings Andy , Kamran Ruhi , Kiryanov Andre , O'Connell Shawn , Taylor Ewan , Vu Phong
Published: August 2014

Poly(ADP-ribose) polymerase (PARP) is a critical DNA repair enzyme involved in DNA single-strand break repair via the base excision repair pathway. PARP inhibitors sensitize tumors to DNA-damaging agents and also inhibit the growth of cancers with BRCA1/2 loss. The synthesis, enzymatic, cellular & pharmacodynamic activities of a series of PARP-1 inhibitors with a novel 1,4-benzoxazin-3-one scaffold are described. A small screening fragment was elaborated through structure-based drug design, ultimately producing several vectors for optimization including interaction with a hydrophobic shelf and extension into the back pocket. The synthesis of 40 compounds is outlined. Optimization of the screening fragment improved LLE's to > 5.0. Several of the resulting inhibitors displayed greater than 1000-fold enhancement of temozolimide (TMZ)-mediated DNA damage in cellular assays. Pharmacodynamic effects in SW620 tumor bearing nude mice demonstrated inhibition of poly-ADP-ribosylation through 24 hours following a single dose.

PARP-1 Binding Potencies and Efficiency Metrics for Benzoxazinones 2-3CmpdaR1R2R3PARP-1 Kd (uM)PARP-1 LLE2aHHH>500-2dMeHH322.32gMeMeH>500-2bHH6-Formyl1702.02fHH6-Ethyl1002.03aHH6-PhenylNHCH2262.23bHH6-BenzylNHCH2113.53cMeH6-BenzylNHCH25.23.23dHH6- (4-(Me2N)piperidine)CH2153.03eMeH6- (4-(Me2N)piperidine)CH20.0964.63fHH6- (4-(Phenyl)piperidine)CH20.2963.43gMeH6- (4-(Phenyl)piperidine)CH20.0463.73hMeH7- (4-(Phenyl)piperidine)CH2101.43iHH6- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH20.0154.53jMeH6- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH20.0034.93kMeH7- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH21000.23lEtH6- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH21000.1  Enzymatic and Cellular Activity for Benzoxazinones 10CmpdR1R2R3PARP-1 IC50 (nM)Cell EC50 w/TMZ (uM)Cell EC50 w/o TMZ (uM)Cell PF5010aHHMe2915141.010bHHEt2215422.810cHClEt1841002010dMeHMe1711756.810eMeHEt699410.410fMeHcPr3351002010gOMeHMe29272519.310hOmeHEt23161519.410iOMeHcPr30161621010jMeClMe4914453.210kMeClEt8914473.410lMeClcPr760.0143430010mMeMeMe389788.710nMeMeEt569556.110oMeMecPr52181005.610pOmeMeMe360.06100166710qOMeMeEt320.0144440010rOMeMecPr1470.0277385010sOEtMeMe350.0226130010tOEtMeEt550.02100500010uOEtMecPr410.021005000ABT-888---16316056AZD-2281---60.0616660MK-4827---350.2031160

Citation Format: Anthony R. Gangloff, Jason Brown, Ron De Jong, Philip Erickson, Charles Grimshaw, Mark Hixon, Andy Jennings, Ruhi Kamran, Andre Kiryanov, Shawn O'Connell, Ewan Taylor, Phong Vu. Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-Ribose)polymerase inhibitors. [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 5676. doi:10.1158/1538-7445.AM2013-5676
Abstract 582: hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects.
Authors: Liu Tiantian , Björkholm Magnus , Xu Dawei
Published: August 2014

Telomerase and telomerase reverse transcriptase (hTERT) confer cancer cells sustained proliferation and survival potentials through telomere lengthening dependent and independent activities. Targeting telomerase or hTERT is thus a novel anti-cancer strategy. However, telomerase/hTERT inhibition alone has minimal clinical efficacy. We explored the relationship between hTERT and oncogenic cyclooxygenase 2 (COX2) and evaluated the synergistic anti-cancer effects of targeting both hTERT and COX2. hTERT depletion in gastric and cervical cancer cells led to robust increases in mRNA and protein levels of COX2. The COX2 promoter activity increased substantially in hTERT-depleted cancer cells. Mechanistically, hTERT depletion led to the activation of p38 MAPK that was responsible for the stimulation of COX2 gene transcription. hTERT depletion or the COX2 inhibitor celecoxib alone did not affect gastric cancer cell survival, while their combination synergistically killed them both in vitro and in vivo. Taken together, hTERT induces COX2 expression in cancer cells and simultaneously targeting hTERT and COX2 synergistically kills cancer cells, which may help path a new avenue in telomerase-based cancer therapeutics.

Citation Format: Tiantian Liu, Magnus Björkholm, Dawei Xu. hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects. [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 582. doi:10.1158/1538-7445.AM2013-582

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 584: Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy.
Authors: Osaki Shuhei , Sasaki Tsuyoshi , Tazawa Hiroshi , Hasei Joe , Yamakawa Yasuaki , Hashimoto Yuuri , Kunisada Toshiyuki , Urata Yasuo , Ozaki Toshifumi , Fujiwara Toshiyoshi
Published: August 2014

Osteosarcomas are the most common malignant bone tumors. Although the multi-agent chemotherapy has improved the long-term survival rate of osteosarcoma patients, some patients show a poor response to chemotherapy, leading to recurrence and poor prognosis. Therefore, the enhancement of chemosensitivity is required to cure patients with osteosarcomas. We recently developed a telomerase-speciﬁc replication-competent oncolytic adenovirus, Telomelysin (OBP-301), and confirmed the antitumor effect of OBP-301 in human osteosarcoma cells. A phase I clinical trial in US has also shown the safety of OBP-301 in cancer patients. In this study, we investigated whether OBP-301 enhances the antitumor effect of chemotherapeutic agents, doxorubicin and cisplatin, that are used for the treatment of osteosarcomas, and the molecular mechanism of chemotherapy- and OBP-301-mediated cell death pathway, autophagy and apoptosis, in human osteosarcoma cell lines. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS2. OBP-301 is an attenuated adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter element drives expression of E1A and E1B genes, and causes tumor-selective lysis in a variety of human malignant tumor cells with high telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents and the calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. To analyze the molecular mechanism in the synergistic effect induced by combination therapy, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Combination of OBP-301 increased both apoptosis (the cleavage of PARP) and autophagy (conversion of LC3-I to LC3-II and p62 downregulation) in the chemotherapeutic agents-treated human osteosarcoma cells. In contrast, the replication of OBP-301 was not suppressed by chemotherapeutic agents. Moreover, combination therapy of chemotherapeutic agents with OBP-301 signiﬁcantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. These results suggest that combination therapy of chemotherapeutic agents with OBP-301 provides a novel therapeutic strategy for human osteosarcomas.

Citation Format: Shuhei Osaki, Tsuyoshi Sasaki, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy. [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 584. doi:10.1158/1538-7445.AM2013-584
Abstract 593: Embelin induces reactive oxygen species-mediated apoptosis in PTC cells.
Authors: Uddin Shahab , Ahmed Maqbool , Hussain Azhar R. , Al-Kuraya Khawla S.
Published: August 2014

The X-linked inhibitor of apoptosis (XIAP) is a promising new molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells to. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Embelin, identified primarily from the Embelia ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity. In this study we found tthat embelin generated a dose dependent Reactive oxygen species (ROS) in PTC cell lines. Pretreatment of PTC cell with N-acetyl-Lcysteine, an scavenger of ROS prevented embelin mediated inhibition of cell proliferation and apoptosis. Further more, NAC pretreatment also abrogated embeling mediated mitochondrial apoptosis. In addition treatment of PTC cell lines with embelin causes activation of p38, a stress related map kinases leading to up-regulation of death receptor 5 (DR5). Interestingly NAC pretreatment prevented its activation as well as up-regulation of DR5. Altogether, these data suggest a novel function for embelin causes inhibition of cell proliferation and induction of apoptosis via generation of ROS a suppressor of XIAP pathway via generation of ROS in PTC cells, and raise the possibility that this agent may have a future therapeutic role in PTC and possibly other malignancies with up-regulated XIAP pathway.

Citation Format: Shahab Uddin, Maqbool Ahmed, Azhar R. Hussain, Khawla S. Al-Kuraya. Embelin induces reactive oxygen species-mediated apoptosis in PTC 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 593. doi:10.1158/1538-7445.AM2013-593
Abstract 606: Inhibition of epithelial to mesenchymal transition and advancement of therapeutic effect in oral cancer by the flavonoid compound, Quercetin: pivotal roles of Snail repression and RKIP induction.
Authors: Chang Yun-Ching , Chiou Shih-Hwa , Nieh Shin
Published: August 2014

Oral squamous cell carcinoma (OSCC) is prevalent worldwide, and survival in OSCC has not improved significantly in the last few decades. Cancer prevention using natural products has become an integral part of cancer control. Quercetin is a plant-derived flavonoid known to possess anti-inflammatory property, but its use as a chemopreventive substance has been reviewed quite recently. Several reports have assessed the pro-apoptotic action of Quercetin in cancer cells similar to many other flavonoids, although the mechanisms involved are not clear. We here reported that Quercetion-mediated sensitization may result from modulation of the EMT inducer Snail and the EMT suppressor/ immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Quercetin regulated EMT, invasion, cell survival and apoptosis via inhibition of NF-κB inactivation as well as leaded to the induction of RKIP through downregulation of its transcriptional repressor Snail. Quercetin-induced RKIP upregulation potentiates further modulated mitochondria-mediated apoptosis, thus leading to enhance therapeutic effect. These findings demonstrate the role of Quercetin in the regulation of EMT via inhibition of NF-κB and Snail and induction of RKIP and also identified Quercetin as targets for resistance via NF-κB/Snail/RKIP circuitry.

Citation Format: Yun-Ching Chang, Shih-Hwa Chiou, Shin Nieh. Inhibition of epithelial to mesenchymal transition and advancement of therapeutic effect in oral cancer by the flavonoid compound, Quercetin: pivotal roles of Snail repression and RKIP induction. [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 606. doi:10.1158/1538-7445.AM2013-606
Abstract 610: Short peptides for targeted apoptosis in cancer cells: development & evaluation of therapeutic applications.
Authors: Boyko Oleksandr , Riabowol Karl T.
Published: August 2014

The ING proteins are type II tumor suppressors that are involved in the regulation of gene transcription, DNA repair, cellular senescence, tumorigenesis and apoptosis. Inactivation of apoptosis pathways is frequently observed in cancer cells, and it leads to a drastic decrease in the efficiency of available cancer treatments. Inactivation of INGs during the suppression of apoptosis tumors and greater sensitivity of cancer versus normal cells to the elevation of ING1 levels suggest that the INGs could serve as viable targets for cancer therapy. In this study we expand our previous observations that all ING proteins share a conserved sequence domain, the Lamin Interacting Domain (LID), which is unique to INGs in the human proteome. The overexpression of a recombinant peptide containing multiple fragments of the LID domain was previously shown to induce apoptosis in a rapid and efficient way in several cancer cell line models. Together, rather limited knowledge of the molecular mechanisms that mediated these apoptosis-inducing effects and the large size of the recombinant LID peptide precluded use of the LID for therapeutic purposes. Here we define the minimal portion of the LID domain that is capable of efficiently inducing apoptosis, and devise approaches to efficiently introduce it into cancer cells where we predict it will show a good therapeutic index by virtue of inducing apoptosis in cancer cells much more efficiently than in normal surrounding cells. Our preliminary in silico analysis of the LID protein structure indicated that LID peptides could interfere with several “master regulators” of cell cycle control and apoptosis, including Retinoblastoma and SMAC proteins. Overexpression of LID peptides could also interfere with the formation of functional ING1-HDAC chromatin modifying protein complexes by disrupting ING1-Lamin A interactions, thus leading to the loss of HDAC targeting and apoptosis. Our ongoing studies should help to identify the molecular mechanism(s) of LID-mediated apoptosis induction and identify the minimal required sequence of LID peptide that still displays potent apoptosis-inducing activity against tumor cells. Our long term goal is to further expand current understanding of apoptosis pathways and develop LID-based therapeutic for the selective induction of apoptosis in cancer cells.

Citation Format: Oleksandr Boyko, Karl T. Riabowol. Short peptides for targeted apoptosis in cancer cells: development & evaluation of therapeutic applications. [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 610. doi:10.1158/1538-7445.AM2013-610
Abstract 611: AID/APOBEC cytidine deaminases can mimic the phenomenon of localized hypermutation in cancer or kataegis.
Authors: Nik-Zainal Serena , Taylor Ben , Wu Yee Ling , Wedge David , Rada Cristina , Stratton Mike , Neuberger Michael
Published: August 2014

Recently, using whole-genome sequencing data of breast cancer genomes we revealed localized regions of dense somatic hypermutation, called kataegis, in which substitutions at C:G base pairs occurring within a distinctive sequence context were found associated with clusters of genomic rearrangements. The mechanism of kataegis is unknown. On the basis of similarities in mutation class and sequence context in experimental systems, members of the AID/APOBEC family of cytidine deaminases were implicated.

Here we expressed members of this gene family in yeast and demonstrate that mutation clusters mimicking many of the features of kataegis in cancer, can be generated. Because the genomic location of each stretch of kataegis is different between individual human cancer genomes, it was reasoned that kataegis is triggered by a local stochastic event, such as a DNA double strand break. Here, we have introduced double strand breaks into the yeast genome and shown that kataegis is elicited in its vicinity. Furthermore, kataegis is substantially diminished in the absence of uracil-DNA glycosylase, indicating that base excision DNA repair is implicated in its genesis. Additionally, the mutation sequence context preferences revealed by these yeast experiments suggest that APOBEC3B and APOBEC3A are the deaminases most likely responsible for the kataegis observed in human cancer. The results therefore provide insights into the mechanism of kataegis and indicate a contribution of APOBEC3B/A-catalyzed DNA deamination to genome mutation in cancer. Finally, we show using an algorithm developed to allow efficient detection of kataegis in other publicly available whole-genome sequenced datasests that this phenomenon is not restricted to breast cancer.

Citation Format: Serena Nik-Zainal, Ben Taylor, Yee Ling Wu, David Wedge, Cristina Rada, Mike Stratton, Michael Neuberger. AID/APOBEC cytidine deaminases can mimic the phenomenon of localized hypermutation in cancer or kataegis. [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 611. doi:10.1158/1538-7445.AM2013-611
Abstract 626: DNA damage responses in different cell types of nonproliferating human prostate tissue.
Authors: af Hällström Taija M. , Jäämaa Sari , Sankila Anna , Mirtti Tuomas , Rantanen Ville , Koistinen Hannu , Stenman Ulf-Håkan , Zhang Zhewei , Yang Zhiming , De Marzo Angelo M. , Taari Kimmo , Ruutu Mirja , Andersson Leif C. , Laiho Marikki
Published: August 2014

DNA damage response (DDR) pathways have been extensively studied in cancer cell lines and mouse models, but little is known about how DNA damage is recognized by different cell types in nonmalignant, slowly replicating human tissues. Here, we assess, using ex vivo cultures of human prostate tissue, DDR caused by cytotoxic drugs (camptothecin, doxorubicin, etoposide, and cisplatin) and ionizing radiation (IR) in the context of normal tissue architecture. Using specific markers for basal and luminal epithelial cells, we determine and quantify cell compartment-specific damage recognition. IR, doxorubicin, and etoposide induced the phosphorylation of H2A.X on Ser(139) (gH2AX) and DNA damage foci formation. Surprisingly, luminal epithelial cells lack the prominent gH2AX response after IR when compared with basal cells, although ATM phosphorylation on Ser(1981) and 53BP1 foci were clearly detectable in both cell types. The attenuated gH2AX response seems to result from low levels of total H2A.X in the luminal cells. Marked increase in p53, a downstream target of the activated ATM pathway, was detected only in response to camptothecin and doxorubicin. These findings emphasize the diversity of pathways activated by DNA damage in slowly replicating tissues and reveal an unexpected deviation in the prostate luminal compartment that may be relevant in prostate tumorigenesis. Detailed mapping of tissue and cell type differences in DDR will provide an outlook of relevant responses to therapeutic strategies. TSC model presented can also be used for functional genomic studies, cellular and tumor biology as well as for drug response testing.

Citation Format: Taija M. af Hällström, Sari Jäämaa, Anna Sankila, Tuomas Mirtti, Ville Rantanen, Hannu Koistinen, Ulf-Håkan Stenman, Zhewei Zhang, Zhiming Yang, Angelo M. De Marzo, Kimmo Taari, Mirja Ruutu, Leif C. Andersson, Marikki Laiho. DNA damage responses in different cell types of nonproliferating human prostate tissue. [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 626. doi:10.1158/1538-7445.AM2013-626
Abstract 640: Human MutS homologue 4 (hMSH4) interacts with eIF3f and inhibits NHEJ-mediated DNA repair.
Authors: Wu Xiling , Chu Yen-Lin , Her Chengtao
Published: August 2014

DNA mismatch repair (MMR) proteins participate in diverse cellular functions including DNA damage response and repair. As a member of the MMR family, hMSH4 plays an important role in meiotic recombination, likely through the formation of hMSH4-hMSH5 heterocomplex that interacts with homologous recombination DNA intermediate structures. It is known that hMSH4 is expressed in an array of somatic cells; however, its involvement in mitotic processes is poorly defined. We have identified a new hMSH4 interacting partner eIF3f, a protein that functions not only in translation but also in the regulation of apoptosis and tumorigenesis. Our studies have demonstrated that hMSH4-eIF3f interaction is mediated through the N-terminal regions of both proteins. The interaction with eIF3f stabilizes hMSH4 protein, which in turn sustains DNA double strand break (DSB)-induced γ-H2AX foci and compromises cell survival in response to ionizing radiation (IR). These effects could be attributed to the inhibition of NHEJ activity by hMSH4. Furthermore, hMSH4 reduces IR-induced AKT activation, thereby facilitating eIF3f-mediated bypass of S phase arrest, and ultimately promoting G2/M arrest in response to IR treatment. In short, this study revealed a potential role of hMSH4 in the maintenance of genomic stability through suppressing error-prone DSB repair.

Citation Format: Xiling Wu, Yen-Lin Chu, Chengtao Her. Human MutS homologue 4 (hMSH4) interacts with eIF3f and inhibits NHEJ-mediated DNA 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 640. doi:10.1158/1538-7445.AM2013-640
Abstract 644: Identification of a novel hypermethylated tumor suppressor gene in human bladder cancer and its implication in non-invasive cancer detection using voided urine.
Authors: Yeh Chia-Ming , Chen Pi-Che , Chen Yu-Hsin , Hung Lin-Chien , Wang Yi-Ting , Tsai Ming-Hsuan , Huang Martin MS , Shen Cheng-Huang , Hsu Cheng-Da , Chan Michael WY
Published: August 2014

Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Although bladder cancer patients have a low mortality rate, long term follow-up with repeated cystoscopy is required due to the high recurrence nature of the tumor. Therefore, a non-invasive detection assay is urgently required for bladder cancer patients. Aberrant promoter hypermethylation which is considered as a hallmark of cancer for over a decade, plays an important role in controlling cancer progression. Nevertheless, detection of promoter hypermethylation in bodily fluid has been implicated as a non-invasive and sensitive tool for cancer diagnosis. In this regard, by using illumina 27K CpG island methylation array, we identified a novel gene that are hypermethylated in 7 bladder cancer patient samples but not in primary normal human urothelim. In vitro promoter methylation assay and epigenetic treatment confirmed that the expression of this gene is epigenetically controlled in bladder cancer cell lines. Bisulphite pyrosequencing demonstrated a significant correlation between higher methylation level of this gene and cancer grade (P<0.01) and recurrence free survival (P<0.05) in bladder cancer patient samples (n=100). To investigate the diagnostic potential of this novel epigenetic marker in non-invasive cancer detection, we performed qMSP assay in voided urine samples from bladder cancer patients (n=27) as well as normal healthy control (n=19). The sensitivity and specificity of this gene in cancer detection is 42.3% and 100% respectively. The sensitivity can increase to 93.3% when using combination of this gene and our previously identified epigenetic markers (IRF8, p14, sFRP1). Re-expression of this gene in bladder cancer cell line inhibited cancer growth in soft agar colony formation assay and invasion assay. In conclusion, this gene may be a potential tumor suppressor in bladder cancer and its potential in non-invasive diagnosis of bladder cancer using voided urine warrants further investigation.

Citation Format: Chia-Ming Yeh, Pi-Che Chen, Yu-Hsin Chen, Lin-Chien Hung, Yi-Ting Wang, Ming-Hsuan Tsai, Martin MS Huang, Cheng-Huang Shen, Cheng-Da Hsu, Michael WY Chan. Identification of a novel hypermethylated tumor suppressor gene in human bladder cancer and its implication in non-invasive cancer detection using voided urine. [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 644. doi:10.1158/1538-7445.AM2013-644
Abstract 645: DNA methylation marker discovery for the prediction of nodal metastases in head and neck cancer.
Authors: Clausen Martijn , Melchers Lieuwe J. , Bruine de Bruin Leonie , Mastik Mirjam F. , Slagter-Menkema Lorian , Groen Harry J. , van der Vegt Bert , Van der Laan Bernard F. , De Meyer Tim , Van Criekinge Wim , Wisman G Bea , Roodenburg Jan L. , Schuuring Ed
Published: August 2014

Annually 650,000 new patients are diagnosed with Head and Neck cancer, making it the sixth most common cancer worldwide. The 5-year survival is only 60% due to inaccurate detection of nodal metastases in the cervical or submandibular region (N-status). Our goal is to find new DNA methylation markers in the primary tumor that predict nodal metastases to improve diagnostics and treatment. For biomarker discovery we used a new approach combining global methylation assessment, next generation sequencing, quantitative methylation and microarray expression profiling. Here we report the success of this new approach and the further validation of novel biomarkers. We performed MethylCap-Seq on DNA from 6 oral squamous cell carcinomas (OSCC) with nodal metastases (N+) and 6 OSCC without nodal metastases (N0) to measure global methylation levels. After comparison of the sequenced reads a list was composed of the 5000 most differentially methylated genes between the two groups. The most significantly differentially methylated genes were validated using Quantitative Methylation Specific PCR (QMSP) and Pyrosequencing on a independent series of 20 N+ and 20 N0 OSCC. In addition, the MethylCap-Seq methylation data was combined with microarray expression data of 696 genes for a N-status validation cohort containing 222 OSCC (Hoof et al.; J Clin Oncol. 2012 Oct 8). Based upon the assumption that hypermethylation is associated with downregulation of gene expression, we selected hypermethylated and downregulated genes distinguishing between the two groups. The effect of DNA methylation on gene expression will be measured by (Q)RT-PCR and immunohistochemistry on a N-status validation panel. The most promising methylation markers will be further validated by Pyrosequencing and QMSP on DNA of a N-status validation cohort containing 463 cases for which complete clinicopathological and follow-up data are available. These markers might contribute to better diagnosis, improved quality of life and may be associated with increased survival.

This research was supported by the Center for Translational Molecular Medicine (project AIRFORCE).

Citation Format: Martijn Clausen, Lieuwe J. Melchers, Leonie Bruine de Bruin, Mirjam F. Mastik, Lorian Slagter-Menkema, Harry J. Groen, Bert van der Vegt, Bernard F. Van der Laan, Tim De Meyer, Wim Van Criekinge, G Bea Wisman, Jan L. Roodenburg, Ed Schuuring. DNA methylation marker discovery for the prediction of nodal metastases in head and neck 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 645. doi:10.1158/1538-7445.AM2013-645
Abstract 662: Role of FAM3B as a novel tumor suppressor gene in gastric cancer.
Authors: Kim Hyojung , Park Sujin , Kang Jin Muk , Kim Staci Jakyong , Kim Seong-Jin
Published: August 2014

PANcreatic-DERived factor (PANDER, FAM3B) is a member of the FAM3 family of cytokine molecules. FAM3B expression is originally identified in the endocrine pancreas and also expressed in in colon and stomach. Initial characterization of FAM3B revealed a potential role in pancreatic islet apoptosis. However, the biological function of FAM3B has still remained elusive. Here we show that FAM3B level is decreased in 11 out of 14 cancer tissues samples (78.5%) as well as in gastric cancer cell lines. Ectopic expression of FAM3B leads to inhibition of cell growth and reduction of single cell colony forming activity and tumorigenicity of gastric cancer cells. FAM3B not only induces reduction of AKT phosphorylation, which is involved in cell survival, but also upregulation of p21, an inhibitor of cell cycle progression. We also demonstrated that FAM3B gene is silenced by epigenetic modification in gastric cancer cell lines. MS-275, a synthetic inhibitor of histone deacetylase, induced FAM3B gene expression. In conclusion, our data suggest the role of FAM3B as a putative tumor suppressor through downregulation of oncogenic AKT signaling pathway in gastric cancer.

Citation Format: Hyojung Kim, Sujin Park, Jin Muk Kang, Staci Jakyong Kim, Seong-Jin Kim. Role of FAM3B as a novel tumor suppressor gene in gastric 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 662. doi:10.1158/1538-7445.AM2013-662
Abstract 668: DNA methylation of apoptosis-related genes as clinical biomarker in rectal cancer.
Authors: Benard Anne , Zeestraten Eliane C.M. , Goossens-Beumer Inès J. , Putter Hein , Kuo Christine , van de Velde Cornelis J.H. , Kuppen Peter J.K. , Hoon Dave S.B.
Published: August 2014

Background: To prevent over- and undertreatment of rectal cancer patients, there is a need for the development of new biomarkers that can complement the current staging system. We have previously shown that apoptosis plays an important role in local tumor recurrence after TME surgery. We studied the epigenetic regulation of several key apoptosis genes in rectal cancer tissues, using methylation-sensitive restriction enzymes.

Materials and methods: DNA was obtained from frozen specimens of 138 patients representative for the non-irradiated arm of the Dutch Total Mesorectal Excision (TME) trial. Isoschizomers HpaII (CpG methylation sensitive) and MspI (not sensitive) were used for restriction enzyme digestion of tumor DNA, followed by real-time PCR for apoptosis genes Apaf1, Bcl2, TrailR2, Fas and p53. A pilot series consisted of 50 patients with tumor stages I-III and 10 normal rectal tissues. For the validation study stage III patients (n=88) and 18 normal rectal tissues were selected.

Results: Single marker analyses showed a significant correlation between methylation status and apoptotic status (measured by immunohistochemical staining of M30) of Fas (p=0.005) and Apaf1 (p=0.052). Combining intrinsic pathway markers Apaf1, Bcl2 and p53, significant differences were observed between the methylation groups in survival and recurrence analyses (log rank test), with overall survival (p=0.054), cancer specific survival (p=0.014) and distant recurrence free survival (p=0.006).

Conclusion: Methylation of apoptosis genes is representative for the apoptotic status of a tumor and is correlated to survival and recurrence in rectal cancer patients. The apoptotic status of a tumor may be used to guide treatment strategies for individual patients.

[A.B. and E.C.M.Z contributed equally to this work.]

Citation Format: Anne Benard, Eliane C.M. Zeestraten, Inès J. Goossens-Beumer, Hein Putter, Christine Kuo, Cornelis J.H. van de Velde, Peter J.K. Kuppen, Dave S.B. Hoon. DNA methylation of apoptosis-related genes as clinical biomarker in rectal 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 668. doi:10.1158/1538-7445.AM2013-668
Abstract 671: Identification of potent BET bromodomain inhibitors for treatment of cancer.
Authors: Sasmal Sanjita , Shrimali Rajeev Kumar , Abbineni Chandrasekhar , Arumalla Kamala Kumari , Lakshminarasimhan Anirudha , Narasingapuram Arumugam Karthikeyan , Tiwari Nirbhay Kumar , Rao Narasimha K. , AB Aravind , Hosahalli Subramanya
Published: August 2014

Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression. Histone lysine acetylation is one of the most abundant epigenetic modifications central to control of gene transcription. Bromodomains are the only known readers of this specific lysine acetylation code, playing an important role in transcriptional regulation of diverse cellular processes such as inflammatory gene expression, mitosis and viral/host interactions. Recently, the human BET family bromodomains which consists of BRD2, BRD3, BRD4 and BRDT has emerged as new druggable target class for the development of specific protein interaction inhibitors, enabling a novel strategy for the development of new therapies for various diseases. Here we report the identification of potent BET bromodomain inhibitors using structure based drug design principle. Multiple distinct series of compounds have been identified with low nM potency in biochemical binding assay. Crystal structures of BRD4 in complex with hit compounds have been solved to assist in optimization. The lead compounds showed very good cell based activity and favorable ADME properties. The compounds demonstrated dose dependent inhibition of c-Myc expression confirming the mechanism of action. Further optimization of these compounds and profiling in relevant pre-clinical disease models is in progress.

Citation Format: Sanjita Sasmal, Rajeev Kumar Shrimali, Chandrasekhar Abbineni, Kamala Kumari Arumalla, Anirudha Lakshminarasimhan, Karthikeyan Narasingapuram Arumugam, Nirbhay Kumar Tiwari, Narasimha K. Rao, Aravind AB, Subramanya Hosahalli. Identification of potent BET bromodomain inhibitors for treatment of 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 671. doi:10.1158/1538-7445.AM2013-671
Abstract 685: Epigenetic modulators and calcium-sensing receptor expression in colorectal cancer cells.
Authors: Fetahu Irfete Sh. , Höbaus Julia , Aggarwal Abhishek , Tennakoon Samawansha , Mesteri Ildiko , Baumgartner-Parzer Sabina , Kallay Enikoe
Published: August 2014

Introduction: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer. The antiproliferative action of calcium in colon might be mediated by the calcium-sensing receptor (CaSR). The CaSR expression is lost in human colorectal cancer. We hypothesized that DNA methylation and histone modifications may be the cause of CaSR silencing in tumors.

Material and methods: We analyzed CaSR mRNA and protein expression in colorectal tumors and cell lines by real time qRT-PCR and immunofluorescence. We determined the methylation pattern in the second promoter of the CaSR that contains a large CpG island by bisulfite sequencing. To induce the expression of the CaSR we treated colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC) and RG108, two DNA methyltransferase inhibitors, three different histone deacetylase inhibitors (HDACIs) Trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate in the presence or absence of a lysine specific demethylase 1 (LSD1) inhibitor to prevent demethylation of mono- and dimethylated histone H3 lysine 4.

Results: In our patient cohort we observed significantly less CaSR mRNA expression (n=57, P<0.001) in colorectal tumors compared with adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of CaSR protein in tumors. The CaSR promoter was stronger methylated in tumors compared with adjacent mucosa and we found an inverse correlation between extent of methylation and mRNA expression (n=20, Spearman's rho = - 0.3, P<0.05). Treatments with 5-aza-dC and the HDACIs restored the expression of CaSR in a compound and cell line dependent manner. However, treatments with the LSD1 inhibitor caused only modest increase of CaSR expression.

Conclusion: In summary, DNA methylation and histone deacetylation seem to be important factors in silencing the expression of CaSR in colorectal cancer, although additional mechanism or factors might be also involved.

Citation Format: Irfete Sh. Fetahu, Julia Höbaus, Abhishek Aggarwal, Samawansha Tennakoon, Ildiko Mesteri, Sabina Baumgartner-Parzer, Enikoe Kallay. Epigenetic modulators and calcium-sensing receptor expression in colorectal 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 685. doi:10.1158/1538-7445.AM2013-685
Abstract 687: Variations in promoter methylation levels of DNA repair genes and breast cancer risk in the New York site of the Breast Cancer Family Registry.
Authors: Kappil Maya A. , Terry Mary Beth , Liao Yuyan , Santella Regina
Published: August 2014

Reduced DNA repair capacity contributes to genomic instability, a hallmark of carcinogenesis. The link between DNA repair capacity and cancer is especially relevant in breast cancer, where women with suboptimal repair capacity have been reported to have up to a five-fold increase in breast cancer risk compared to women with better repair capacity. We investigated promoter methylation levels of DNA repair-related genes and their association with breast cancer risk among participants of the New York site of the Breast Cancer Family Registry, comparing sisters affected with breast cancer to their unaffected sisters. We analyzed DNA extracted from viable mononuclear cells from 218 sisters (N=538 women) using pyrosequencing assays designed to target CpG islands in the promoter regions of BRCA1, MSH2, and MLH1. Breast cancer risk was not significantly associated with the promoter methylation level of BRCA1 (OR=1.09 [0.98-1.20]), MLH1 (OR=1.18 [0.90-1.54]) or MSH2 (OR=0.87 [0.47-1.59]). We assessed the Spearman correlation between methylation and previously generated data on expression level of BRCA1 and MSH2, where we had observed a significant increase in breast cancer risk among women in the lowest quartile of expression for MSH2. Among the affected women, no significant correlation was observed between methylation and expression level for MSH2 (-0.04, p=0.39) or BRCA1 (r=-0.05, p=0.39). These findings suggest that while MSH2 may have implications for breast cancer risk, as suggested by the previously observed increase in risk with decrease in expression level of MSH2, the lack of an association between gene promoter methylation levels of MSH2 and breast cancer risk and the lack of a correlation with expression level in the current study, indicate that other mechanisms impacting the reduction in expression level of this gene may be more relevant to explain the observed increase in breast cancer risk.

Citation Format: Maya A. Kappil, Mary Beth Terry, Yuyan Liao, Regina Santella. Variations in promoter methylation levels of DNA repair genes and breast cancer risk in the New York site of the Breast Cancer Family Registry. [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 687. doi:10.1158/1538-7445.AM2013-687
Abstract 707: MK1775-mediated gemcitabine sensitization correlates with prolonged DNA damage signaling in S-phase cells.
Authors: Parsels Leslie A. , Parsels Joshua D. , Morgan Meredith A. , Lawrence Theodore S. , Maybaum Jonathan
Published: August 2014

Pancreatic cancer remains among the most lethal cancers with an overall 5-year survival of <5%. Wee1 inhibition has emerged as a potential approach to sensitize pancreatic cancer to gemcitabine (Gem), the current standard therapy for both local control and metastatic disease. In this study, we first characterized the ability of MK1775, a small molecule Wee1 inhibitor now entering Phase II clinical trials, to sensitize pancreatic cancer cells to Gem. Using a clonogenic survival assay, we tested the effects of MK1775 in a panel of pancreatic cancer cells and found MK1775-mediated Gem sensitization ranged from 80-fold in MiaPaCa2 cells to 12-fold in Panc1 cells. This sensitization was associated with aberrant mitotic entry in MiaPaCa2 and Panc1 cells, but not in the other cells lines tested. In addition, MK1775 potentiated and prolonged Gem-induced DNA damage signaling (γ-H2AX) as determined by 2-parameter flow cytometry. For example, 48 h post-Gem, 35±12% of BxPC3 cells remain γ-H2AX-positive compared to 76±3.7% of cells treated with Gem + MK1775. Using immunofluorescent staining, we further determined that Wee1 inhibition with MK1775 qualitatively changed Gem-induced γ-H2AX nuclear foci to high intensity pan-nuclear γ-H2AX pan-staining. Finally, we tested the ability of the CDK inhibitor roscovitine (Ros) to attenuate MK1775-mediated chemosensitization. Ros not only prevented aberrant mitotic entry in MiaPaCa2 and Panc1 cells, but in each cell line sensitized to Gem by MK1775, Ros inhibited both high-intensity γ-H2AX staining after treatment with Gem + MK1775 and chemosensitization. These results suggest the effects of MK1775 on DNA repair in S-phase cells may be more important for Gem chemosensitization than the effects of MK1775 on the G2-M checkpoint.

Citation Format: Leslie A. Parsels, Joshua D. Parsels, Meredith A. Morgan, Theodore S. Lawrence, Jonathan Maybaum. MK1775-mediated gemcitabine sensitization correlates with prolonged DNA damage signaling in S-phase 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 707. doi:10.1158/1538-7445.AM2013-707
Abstract 71: Meningioma: response to retinoids and radiation therapy.
Authors: Ren Xuan , Lo Hannah , Vanderwaal Robert , Jaboin Jerry
Published: August 2014

Meningioma represents one third of all primary central nervous system neoplasms. Most meningiomas are effectively addressed by surgical resection. However unresectable, partially resected, recurrent and high grade meningiomas are also typically treated with adjuvant or definitive radiation therapy.

Retinoids are a class of compounds structurally related to Vitamin A. Retinoic acid (RA), the active derivative of vitamin A and the first diffusible morphogen in vertebrates, has recently been evaluated for its chemotherapeutic and chemopreventive attributes in solid tumors. Additionally, a novel orally bioavailable synthetic retinoid, fenretinide, has been tested with different pharmacokinetics, side effect profile, and mechanism of action than the classic retinoids, all-trans retinoic acid (ATRA) and 13-cis-retinoic acid (13CRA). In this project, we study the effects of retinoic acid agents alone and in combination with ionizing radiation in meningioma cell lines (IOMM-Lee and CH157MN).

We demonstrate that ATRA, 13CRA and fenretinide have similar (increasing) toxicity profiles in both cell lines at doses above 1μM; but below this concentration there are minimal changes on cellular proliferation. Pretreatment of meningioma cell lines with ≤1μM doses of ATRA and 13CRA protected cells from radiation-induced cell death, while pre-treatment with fenretinide had no effect. Using flow cytometric analysis, we demonstrated that RA inhibited radiation-induced G2/M phase accumulation. Western blot result showed that ionizing radiation increased Phospho-Chk1 and -Chk2 levels, and decreased the levels of cdc25c, Phospho-cdc25c (Ser126) and Phospho cdc25c(THr68), Pre-treatment with RTRA reversed each of these effects in a dose- and time-dependent manner.

These data suggest a role for the classic RAs in radiation protection through modulation of DNA repair through cell cycle checkpoint pathways. Fenretinide, however, works through a different mechanism, and does not result in radiation protection. These divergent roles suggest that fenretinide may escape the concerns of radiation protection of earlier retinoids making it a promising agent for tumor treatment in the periradiotherapy period.

Citation Format: Xuan Ren, Hannah Lo, Robert Vanderwaal, Jerry Jaboin. Meningioma: response to retinoids and radiation therapy. [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 71. doi:10.1158/1538-7445.AM2013-71