bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4290: c-Myc and Ras differentially regulate mitochondrial apoptotic priming and chemosensitivity.
Authors: Sarosiek Kristopher A. , Ni Chonghaile Triona , Karst Alison , Patel Luv , Drapkin Ronny , Letai Anthony
Published: August 2014

Successful treatment of most human cancers is dependent on tumor cell sensitivity to chemotherapy yet the basis for chemosensitivity is poorly understood. We have previously shown that a cancer's proximity to the apoptotic threshold, a property we call "apoptotic priming," governs its chemosensitivity in patients with multiple myeloma, ALL, AML and ovarian cancer. The genetic and molecular events that underlie the differential priming in tumors, however, are currently unknown. Utilizing in vitro ovarian, kidney and lung tumorigenesis models we find that oncogenes are powerful modulators of apoptotic priming and are consequently responsible for differential chemosensitivity. For example, forced expression of the c-Myc oncogene increases priming and chemosensitivity of immortalized normal epithelium. In contrast, Ras activation in isogenic cells dramatically decreases both priming and chemosensitivity. Our in vitro findings are validated by clinical observations. Apoptotic priming, an important determinant of patient outcomes, is modulated in contrasting manners by oncogenes.

Citation Format: Kristopher A. Sarosiek, Triona Ni Chonghaile, Alison Karst, Luv Patel, Ronny Drapkin, Anthony Letai. c-Myc and Ras differentially regulate mitochondrial apoptotic priming and chemosensitivity. [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 4290. doi:10.1158/1538-7445.AM2013-4290
Abstract 43: Detection of bladder cancer-associated gene methylation using next-gen bisulfite sequencing.
Authors: Dunn Andrew R. , Li Shuqiang , Fernandez Cecilia A. , Shuber Anthony P.
Published: August 2014

DNA methylation profiling of bladder cancer specific gene promoters has been shown to be a sensitive and specific diagnostic tool for early- and late-stage bladder cancer. We have recently shown that a urine-based assay that combines DNA and protein biomarkers can accurately triage patients undergoing evaluation for bladder cancer. As part of this assay, hypermethylation of Twist1 and Nid2 is performed by conventional methylation-specific PCR (cMSP). To increase throughput and lower cost, we have now developed an approach to determine the methylation at single-base resolution of CpG sites using next generation bisulfite sequencing. In addition to Twist1 and Nid2, methylation of the Vimentin promoter, which has been shown to be associated with epithelial to mesenchymal transition (EMT) in bladder cancer, was also evaluated using this technique. To determine the sensitivity of this approach, amplification was performed on control bisulfite converted genomic DNA that was either enzymatically methylated or chemically unmethylated at CpG sites using non-methylation specific PCR. Twist1, Nid2 and Vimentin PCR products were subsequently used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Methylation status was determined by aligning sequencing reads to unmethylated reference sequences. In this assay, 0.4% methylated DNA could be reliably detected in 10 ng of bisulfite converted DNA, more than doubling the sensitivity of the current assay as well as potentially generating a single-base resolution mapping of additional CpG sites not yet quantified for Twist1, Nid2 and Vimentin. The clinical performance of the Next-Gen bisulfite sequencing assays for Twist1, Nid2 and Vimentin is currently being tested using urine samples from patients with bladder cancer as well as from age matched individuals who present with hematuria but who are found to be cancer-free. The technique we have developed could be used to detect other hypermethylated genes in a variety of bodily fluids where percent methylation may be much lower than in tumor tissue samples.

Citation Format: Andrew R. Dunn, Shuqiang Li, Cecilia A. Fernandez, Anthony P. Shuber. Detection of bladder cancer-associated gene methylation using next-gen bisulfite sequencing. [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 43. doi:10.1158/1538-7445.AM2013-43
Abstract 4306: NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates MAPK activation and cellular protection against chemical stress.
Authors: Patrick Brad A. , Jaiswal Anil K.
Published: August 2014

NAD(P)H: quinone oxidoreductase (NQO1) is a cytosolic redox-regulating enzyme modulated by Nrf2-mediated stress signaling pathways, with an established role in regulation of stress-related transcription factors. In these studies we investigate the role of NQO1 in regulation of mitogen-activated protein kinase (MAPK) signaling pathways. Quantitative PCR and western blot studies showed that constitutive levels of several MAPK members are upregulated in NQO1-null mouse skin as well as in skin-derived keratinocytes, relative to WT. However, though total protein levels are increased, inducibility of these kinases by known MAPK inducers such as EGF and benzo[a]pyrene (BP) is significantly decreased in NQO1-null cells. NQO1 regulation of MAPK signaling was confirmed by studies showing that NQO1 knockdown by siRNA led to decreased MAPK activation, and overexpression of NQO1 protein led to increased MAPK activation by EGF. Similarly, downregulation of activity via dicoumarol caused loss of ERK phosphorylation, while NQO1 activation via t-BHQ caused increased ERK phosphorylation, which was blocked by MAPK pathway-specific inhibitors. To investigate the functional role of MAPK deactivation in skin-derived keratinocytes, studies with BP and menadione showed that lack of NQO1 expression led to increased susceptibility to ROS generation, and that this ROS generation causes increased toxicity. Taken together, these results show a significant role for NQO1 in regulation of MAPK signaling pathways, which ultimately lead to protection against ROS-mediated apoptosis.

Citation Format: Brad A. Patrick, Anil K. Jaiswal. NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates MAPK activation and cellular protection against chemical stress. [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 4306. doi:10.1158/1538-7445.AM2013-4306
Abstract 4315: Extracellular signal regulated kinase 5 inpathogenesis of malignant mesothelioma.
Authors: Shukla Arti , Miller Jill , Cason Christopher , Sayan Mutlay , MacPherson Maximilian , Beuschel Stacie , Mossman Brooke
Published: August 2014

Malignant mesothelioma (MM) is a neoplastic disease of the pleural, peritoneal or pericardial cavity. Currently, there is no therapy for MM as it is resistant to most of the common chemotherapies and therefore, there is a need for new treatment strategies. In the present study we demonstrated that ERK5 is important in MM tumor growth and can potentially be targeted in combination with the chemotherapeutic drugs for more effective treatment. Human MM cell lines exhibit constitutive activation of ERK5.

Addition of doxorubicin to these MM cell lines resulted in further activation of ERK5. ERK5 silencing by small hairpin RNA (shERK5) increased DOX-induced cell death and DOX retention in human MM cells. In addition, shERK5 MM lines exhibited both attenuated colony formation on soft agar and invasion of MM cells in vitro. Most importantly, injection of shERK5 MM cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the IP model of tumor growth, proving our hypothesis that ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in MM patients. This work is supported by Mesothelioma

Applied Research Foundation (AS), NIEHS-RO1ES021110 (AS) and T32 ES07122 (BM) grants.

Citation Format: Arti Shukla, Jill Miller, Christopher Cason, Mutlay Sayan, Maximilian MacPherson, Stacie Beuschel, Brooke Mossman. Extracellular signal regulated kinase 5 inpathogenesis of malignant mesothelioma. [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 4315. doi:10.1158/1538-7445.AM2013-4315
Abstract 4328: Phenotypic selection for identification of functional antibodies and development of high throughput screening assays.
Authors: Hay Carl , Rust Steven , Sult Erin , Clarke Lori , Rosenthal Kim , Guillard Sandrine , Lowne David , Flynn Matt , Jermutus Lutz , Minter Ralph , Hollingsworth Robert , Sachsenmeier Kris
Published: August 2014

Discovery of therapeutic antibodies requires the continued identification of tractable targets, and we have employed a phenotypic screening strategy to this end. In this study, antibodies from phage display libraries were screened for their abilities to inhibit MDA-MB-231 (‘triple negative’) breast cancer cells. The screening cascade included tests for specific cell binding, antibody internalization, and cytotoxicity as an antibody-drug conjugate. Targets of these antibodies then were identified using immunoprecipitation and mass spectrometry, and confirmed by showing diminished antibody binding after siRNA-mediated knockdown of the putative target gene. One target was identified as NT5E (also known as CD73), a 5’-ectonucleotidase. NT5E catalyzes the conversion of adenosine monophosphate to adenosine, and its function has been linked with immunity, angiogenesis, and cancer. To screen for additional inhibitors of this target, a novel, high-throughput ectonucleotidase assay was developed. NT5E activity is typically assayed either by high performance liquid chromatography or by quantification of free phosphate using malachite green, and neither of these methods is suitable for high-throughput screening. One inhibitory antibody was found to significantly slow growth of MDA-MB-231 xenograft tumors in mice. In summary, our phenotypic screening approach provides a mechanism for rapidly discovering and evaluating new antibody targets and is being used to accelerate the discovery of new cancer drugs.

Citation Format: Carl Hay, Steven Rust, Erin Sult, Lori Clarke, Kim Rosenthal, Sandrine Guillard, David Lowne, Matt Flynn, Lutz Jermutus, Ralph Minter, Robert Hollingsworth, Kris Sachsenmeier. Phenotypic selection for identification of functional antibodies and development of high throughput screening assays. [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 4328. doi:10.1158/1538-7445.AM2013-4328
Abstract 4329: Novel HER2 targeting antibody-drug conjugates based on DNA-interacting duocarmycin and an unique linker technology with great potential in breast cancer and NSCLC.
Authors: Dokter Wim , Beusker Patrick , Verheijden Gijs , Ubink Ruud , van der Lee Miranda , van den Dobbelsteen Diels , Goedings Peter , Lemmens Jacques , de Groot Vincent , Timmers Marco
Published: August 2014

We have generated cleavable linker-duocarmycin payloads that previously have been shown to be highly stable in human plasma and to cause excellent in vitro and in vivo efficacy when conjugated to antibodies directed against multiple targets in different models. In our HER2 program, we have used these linker-duocarmycin payloads to generate HER2-targeting antibody-drug conjugates (ADCS) and prepare these for FIM studies. HER2 is a validated target in breast (BC) and gastric cancer (GC) that is currently used to target trastuzumab towards HER2-positive BC and GC tumors. We have generated a set of cysteine-coupled ADCs using newly developed duocarmycins conjugated to a therapeutic HER2/neu antibody. In vitro studies with the anti-Her2 ADCs showed that compared to the naked Ab, the ADCs tested have gained a more than 100-fold potency and more than two-fold efficacy in cell killing capacity. Studies in HER2 positive and negative cell lines indicated that cytotoxicity was HER2- mediated. HER2 binding and ADCC activity was not affected by conjugation. Single dose and multiple dose xenograft studies in mice using a human HER2-positive breast cancer cell line showed dose-dependent anti-tumor activity of the ADCs in vivo. Patient-derived xenografts (PDx) using primary tumors from HER2-positive breast cancer and NSCLC patients demonstrated the ability of the ADC to induce complete remission of tumors that remained unresponsive to the naked Ab trastuzumab. Also taken into account toxicology studies in rodents and cynomolgous monkeys, we conclude that we have generated a new class of HER2-based ADCs that have great potential to become effective treatment for HER2-positive tumors, including breast cancer and NSCLC. From a series of compounds, a preclinical candidate was selected that is currently being prepared for Phase I clinical trials that will start in 2014.

Citation Format: Wim Dokter, Patrick Beusker, Gijs Verheijden, Ruud Ubink, Miranda van der Lee, Diels van den Dobbelsteen, Peter Goedings, Jacques Lemmens, Vincent de Groot, Marco Timmers. Novel HER2 targeting antibody-drug conjugates based on DNA-interacting duocarmycin and an unique linker technology with great potential in breast cancer and NSCLC. [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 4329. doi:10.1158/1538-7445.AM2013-4329
Abstract 433: mTOR overexpression is associated with radio-resistance and worse prognosis of cervical cancer.
Authors: Nishimura Masato , Yoshida Kanako , Furumoto Hiroyuki , Irahara Minoru
Published: August 2014

mTOR is an important factor for cell proliferation. It is reported that overexpression of mTOR is associated with poor prognosis in many cancers. We examined mTOR expression in cervical cancer and association with clinico-pathological findings. 34 patients were examined about mTOR expression by immuno-histochemistry. The result was classified into 3 categories, i.e negative, weak positive and strong positive. 25 patients were treated with concurrent chemoradiotherapy, 8 ptients were treated with hysterectomy and one patient was treated with chemotherapy as main treatment. Overexpression of mTOR was recognized in 24(71%) patients. The frequency of parametrial invasion and lymphnode metastasis in mTOR positive and negative were 17/24 (71%) versus 3/10 (30%) (p=0.027) and 14/24 (58%) versus 2/10 (20%) (p=0.041). In 25 cases treated with radiotherapy, mTOR overexpression was seen in 19 cases (76%). Averages of tumor diameter in mTOR positive and negative cases were 4.8 cm and 5.0cm respectively (n.s). In 5 local recurrent cases treated with chemo-radiotherpy, tumor diameters of pre-treatment were 0, 3.5, 5.5, 7 and 7cm. Small tumor was included in local recurrence in mTOR positive cases. It is suggested that mTOR overexpression promotes lymphnode and parametrial metastasis and is associated with radio-resistance. In case of mTOR overexpression, we need to consider treatment by hysterectomy if the tumor is resectable or usage of mTOR inhibitor at chemo-radiation, and control of mTOR expression is important in treatment of cervical cancer.

Citation Format: Masato Nishimura, Kanako Yoshida, Hiroyuki Furumoto, Minoru Irahara. mTOR overexpression is associated with radio-resistance and worse prognosis of cervical 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 433. doi:10.1158/1538-7445.AM2013-433
Abstract 4337: Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing.
Authors: Whelan Rebecca J. , Kapur Arvinder , Felder Mildred , Nie Jeff , Patankar Manish S.
Published: August 2014

The mucin, MUC16, facilitates metastasis and immuneprotection of ovarian tumors. Therefore, agents that effectively target MUC16 expressing ovarian cancer cells will be useful for diagnostic and therapeutic applications. Here, we have utilized the Cell-based Systematic Evolution of Ligands by Exponential Enrichment (cell-SELEX) approach to generate high affinity single-stranded DNA (ssDNA) aptamers as agents for detection and treatment of MUC16-positive ovarian tumors. An ssDNA aptamer library composed of ∼100 trillion sequences was subjected to ten iterative rounds of negative and positive selection using the MUC16-knockdown and MUC16-positive ovarian cancer cell line, OVCAR-3. Aptamers from each round were amplified by asymmetric PCR and subjected to high throughput NextGen sequencing. Eight ssDNA aptamers enriched through the selection process were identified by bioinformatics analysis and their selectivity and affinity for MUC16-positive cells was determined by flow cytometry. Two of these aptamers (Apt-1 and Apt-8) showed significant binding to the MUC16-positive OVCAR-3 with a Kd of 24 and 28 nM, respectively. In contrast, Apt-1 and Apt-8 only weakly recognized the MUC16-knockdown OVCAR-3 cells. M-Fold analysis indicated that Apt-1 and Apt-8 had defined secondary structures resulting from ordered base pairing of the ssDNA. The inclusion of NextGen sequencing techniques has therefore allowed rapid identification of theranostic aptamers from an exhaustive library of ssDNA sequences. This study provides a streamlined method for development of theranostic aptamers that can be used to recognize and target MUC16 expressing ovarian cancer cells.

Citation Format: Rebecca J. Whelan, Arvinder Kapur, Mildred Felder, Jeff Nie, Manish S. Patankar. Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing. [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 4337. doi:10.1158/1538-7445.AM2013-4337
Abstract 4350: Low frequencies of SF3B1 mutations indicate SF3B1 inhibitor as a novel moleculartargeted drug for colorectal cancer.
Authors: Yamano Tomoki , Nakajima Hidenori , Tsuda Satoshi , Kubo Shuji , Hamanaka Mie , Kobayashi Masayoshi , Yamagishi Daisuke , Kuno Takashi , Tsukamoto Kiyoshi , Tamamoto Astuko , Noda Masafumi , Matsubara Nagahide , Hoon Dave SB , Tomita Naohiro
Published: August 2014

Purpose: FR901464 (FR) is an antitumor agent that specifically binds to and inhibits the function of splicing factor 3B subunit 1 (SF3B1). Higher frequencies of SF3B1 gene mutations and their relevance to cancer progression have been shown in studies of hematological malignancies.

We assessed the frequencies of SF3B1 gene mutations in colorectal cancer (CRC) and the correlation of SF3B1 mutations and FR sensitivity in CRC.

Experimental Design: We measured FR sensitivity in CRC cell lines to establish FR-resistant clones and performed DNA sequencing of all

25 SF3B1 exons in nine CRC cell lines, FR-resistant clones, and 16 CRC patients.

On the basis our results, we performed further DNA sequencing in 80 CRC patients.

Results: The FR IC50 values in all CRC cell lines were < 1ng/ml. Six FR-resistant clones were established: three from DLD1, two from HCT116, and one from LoVo cells. The FR IC50 of the FR-resistant clones was > 500-fold greater than that of their parental cells, although the IC50 of oxaliplatin or 5-fuluorouracil were the same. We found nonsense mutations in exon 10 of DLD1 cells and no significant mutations in the other samples. All the FR-resistant clones had the missense mutations in codon 1073 of exon 22. We found no mutation in exons 10, 22, and 12-15, which were previously reported in hematological malignancies in 80 CRC patients.

Conclusion: Our data indicated the usefulness of the SF3B1 inhibitor as a novel molecular targeted drug for CRC and the other malignancies without codon 1073 mutations.

Citation Format: Tomoki Yamano, Hidenori Nakajima, Satoshi Tsuda, Shuji Kubo, Mie Hamanaka, Masayoshi Kobayashi, Daisuke Yamagishi, Takashi Kuno, Kiyoshi Tsukamoto, Astuko Tamamoto, Masafumi Noda, Nagahide Matsubara, Dave SB Hoon, Naohiro Tomita. Low frequencies of SF3B1 mutations indicate SF3B1 inhibitor as a novel moleculartargeted drug for 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 4350. doi:10.1158/1538-7445.AM2013-4350
Abstract 4356: EphB4 as a novel therapeutic target for head-neck SCC.
Authors: Liu Ren , Yen Hai-yun , Masood Rizwan , Sinha Uttam , El-Khoueiry Anthony , Liu Stephen , Gitlitz Barbara , Gill Parkash S.
Published: August 2014

Introduction: Receptor tyrosine kinase EphB4 and its cognate trans-membrane ligand EphrinB2 regulate venous-artery determination and are critically required for fusion of vein and arterial compartments, as well as vessel maturation in development and in pathological angiogenesis. Aberrant induction of EphB4 in certain tumors and pro-tumor cell survival function independent of its role in tumor angiogenesis have been reported. We have studied the expression and function of EphB4 in head neck squamous cell cancer.

Methods: EphB4 expression in 78 freshly frozen head/neck squamous cell carcinoma tumor samples were studied by immunostaining, western blot analysis, gene copy number, and correlated with stage. Head/neck tumor cell lines also were studied for expression and significance of EphB4 expression on survival by knock down studies using EphB4 lentiviral shRNA. Inhibition of EphB4 activation using decoy soluble EphB4 receptor fused in frame at the C-terminus with human albumin (sEphB4-HSA) was evaluated for in vivo studies of human tumor xenografts.

Results: EphB4 was expressed in all human tumor tissues, and the levels correlated directly with stage. High EphB4 also predicted shortened survival. EphB4 protein levels varied in tumor cell lines, and loss of cell viability with EphB4 shRNA knock down was highest in cell lines with highest EphB4 levels (range 25-78%). Tumor xenografts in sEphB4-HSA treated mice were reduced over 80% compared to the control PBS treatment. Tissue analysis revealed reduction in tumor vessel density, reduced recruitment of pericytes, and decreased perfusion of the tumor vessels. sEphB4-HSA treatment also decreased tumor tissue PI3K activity manifested by reduced phosphorylated AKT and S6 level. Tumor cell death by apoptosis was widespread consistent with combined anti-angiogenic activity and direct tumor cell toxicity.

Conclusion: EphB4 receptor tyrosine kinase is highly expressed in head/neck squamous cell cancer. EphB4 provides survival signal to the tumor cells directly. Decoy EphB4 inhibitor sEphB4-HSA is highly potent in in vivo tumor xenograft models. sEphB4-HSA is thus a candidate for investigation in human trials and the clinical trials are underway.

Citation Format: Ren Liu, Hai-yun Yen, Rizwan Masood, Uttam Sinha, Anthony El-Khoueiry, Stephen Liu, Barbara Gitlitz, Parkash S. Gill. EphB4 as a novel therapeutic target for head-neck SCC. [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 4356. doi:10.1158/1538-7445.AM2013-4356
Abstract 4374: Identification of novel, biologically relevant, G-quadruplexes formed within the critical promoter of kRAS as promising molecular targets.
Authors: Hockings Chi-Fan , Greer Kristen M. , Brooks Tracy A.
Published: August 2014

To date, kRAS is one of, if not the, most established oncogenic anomalies identified. It is either upregulated or mutationally activated in a variety of cancers, most notably pancreatic adenocarcinoma, non-small cell lung and colon cancers. Pancreatic cancer carries the highest mortality rate of all cancers, with only a 3-6 month median survival, and a 5-year survival of <5%. Lung and colon cancers harboring a mutant kRAS are associated with a worsened prognosis and shorter disease-free survival, and such mutations are independent factors for poor prognosis. To date no attempts to modulate kRAS activity, localization, or signaling have produced clinically active moieties; there is a desperate need for new, kRAS-focused therapeutics. Various silencing technologies have demonstrated that decreasing kRAS expression is sufficient to produce cancer cell kill, and the presented works describe a novel target to downregulate kRAS expression - secondary structures within the proximal promoter, which contains a unique string of G-rich DNA. Negative superhelicity induced by transcription results in this region opening up to form unique secondary structures called G-quadruplexes (G4s). These G4s most often act as silencer elements, forming globular structures that mask binding sites for transcriptional factors, allowing for specific molecular targeting by small molecule drugs, modulating transcription, and protein expression. The G-rich region of the kRAS promoter is extensive, containing three separate putative G4-forming regions. Initial works described the near G4 as a unique ‘kinked’ structure, as well as described two compounds that stabilize different isoforms of this structure, but do not appear to contribute much to the transcriptional control of kRAS. Further elucidation of promoter activity has highlighted new, and more biologically important, G4s existing more distally from the transcriptional state site. Stabilization of this region in a plasmid system significantly decreases luciferase expression, and the mid-G4-forming region forms several stable, complex, structures. These formations have been examined by EMSA, polymerase stop, and footprinting methods. From these data, the elucidated predominant structures are currently the target of further studies with the ultimate goal of describing a new molecular structure, and its dynamic regulation, for future drug development.

Citation Format: Chi-Fan Hockings, Kristen M. Greer, Tracy A. Brooks. Identification of novel, biologically relevant, G-quadruplexes formed within the critical promoter of kRAS as promising molecular targets. [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 4374. doi:10.1158/1538-7445.AM2013-4374
Abstract 4382: Genome-Wide Gene Expression analysis and molecular portraits regulated by NASEp, an inhibitor of CREB and CBP interaction, in non-small cell lung cancer.
Authors: Park Sin-Aye , Platt James , Park Hee Sun , Lee Jong Woo , Stern David F. , Herbst Roy S. , Koo Jaseok Peter
Published: August 2014

Cyclic-AMP response element-binding protein (CREB)-cAMP response element (CRE) transcriptional pathway plays a critical role in the growth and survival of non-small cell lung cancer (NSCLC) cells. Recruitment of CREB binding protein (CBP) is essential for the transcriptional activity of CREB. However, there is no specific drug in use blocking CREB-CRE transcriptional pathway which may become a new therapeutic target in anti-cancer strategy. Naphthol AS-E phosphate (NASEp) was identified as an inhibitor of CREB transcriptional activity by binding to the KIX domain of CBP and blocking its access to the KID domain in CREB. Our objective was to identify which genes and molecular pathways could be affected by NASEp that may alter target molecules through the regulation of a subset of CRE-containing genes. The effects of the CREB inhibitor NASEp on A549 lung carcinoma cells were examined on an Affymetrix HuGene 1.0 expression microarray and a list of significantly differentially regulated genes was generated. Gene Set Enrichment Analysis (GSEA) shows down-regulation of multiple cancer-related pathways including proliferation, RAS signaling, drug resistance and metastasis. Most significantly, a strong negative correlation with Rosty Cervical Cancer Proliferation Cluster, shows that NASEp inhibits the same set of genes which are up regulated by the E6 and E7 viral oncogenes. These viral elements are known to interact with CBP, which is the direct target of NASEp. Since CBP is known to be a cofactor to multiple transcription factors, we performed an additional round of GSEA using the motif-based Transcription Factor Targets gene set collection and represented the results using a Cytoscape network diagram to illustrate the multifactorial effects of NASEp. Particularly, we focused that several transcription factors regulating cell cycle-related genes. This study implicates potential combinational blockage of CREB and cell cycle-regulated proteins as a cancer therapy.

Citation Format: Sin-Aye Park, James Platt, Hee Sun Park, Jong Woo Lee, David F. Stern, Roy S. Herbst, Jaseok Peter Koo. Genome-Wide Gene Expression analysis and molecular portraits regulated by NASEp, an inhibitor of CREB and CBP interaction, in non-small cell lung 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 4382. doi:10.1158/1538-7445.AM2013-4382
Abstract 4383: Transglutaminase-2 mediates the pro-malignant effects of oncostatin-M receptor overexpression in cervical squamous cell carcinoma.
Authors: Caffarel Maria M. , Chattopadhyay Anasuya , Scarpini Cinzia G. , Coleman Nicholas
Published: August 2014

An important feature of cervical squamous carcinogenesis is genomic instability caused by deregulated expression of HPV oncogenes in proliferating epithelial cells. A genomic imbalance that is commonly selected in advanced cervical squamous cell carcinoma (SCC) is copy number gain of chromosome 5p. One gene that is likely to drive selection of this abnormality is the oncostatin-M receptor (OSMR; located at 5p13.1), which is commonly up-regulated in cervical SCC and produces a significantly worse clinical outcome when over-expressed. Cervical SCC cells that over-expressed OSMR showed enhanced responsiveness to the major ligand OSM, which induced multiple pro-malignant effects, including increased cell migration and invasiveness. We now show that transglutaminase-2 (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in cervical carcinoma cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of SCC of the cervix and head and neck. TGM2 depletion in cervical SCC cells abrogated the increased migration and invasiveness induced by OSM. TGM2 was shown to interact with integrin-α5β1 and fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin-α5β1 and fibronectin were over-expressed in cervical SCC samples, where levels correlated with those of OSMR. We conclude that an OSMR-TGM2-integrin-α5β1 pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.

Citation Format: Maria M. Caffarel, Anasuya Chattopadhyay, Cinzia G. Scarpini, Nicholas Coleman. Transglutaminase-2 mediates the pro-malignant effects of oncostatin-M receptor overexpression in cervical 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 4383. doi:10.1158/1538-7445.AM2013-4383
Abstract 4385: The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5.
Authors: Das Amlan , Banerjee Snigdha , De Archana , Haque Inamul , Maity Gargi , McEwen Matt , Banerjee Sushanta K.
Published: August 2014

Background: Epithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasion. Recent studies had shown the importance of CCN5 (also known as WISP-2,Wnt-1-induced signaling protein-2) in the regulation of various carcinomas including the breast cancer. Recent studies had showed that ectopic expression of CCN5 can reverse Epithelial-Mesenchymal transition (EMT) and inhibit cancer metastasis. Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis. However, the mechanism of action of EGCG in breast carcinoma is uncertain.

Objective: The objective of the present study is to determine whether CCN5 plays any significant role in EGCG-mediated cytotoxicity in triple negative breast cancer cells.

Results: Exposure of triple negative human breast cancer (TNBC) cells, MDA-MB-231 and HCC70 to EGCG resulted in a dose-dependent inhibition of proliferation after 72h and the IC50 was observed around 75μM for MDA-MB-231 cells and 50μM for HCC70 respectively. We found EGCG-treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose-dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG-treated MDA-MB-231-tumor xenograft in nude mouse model.

Conclusion: EGCG imparts its anti-cancer activity in both TNBC cells as well as MB-231-tumor xenografts via induction of CCN5.

Citation Format: Amlan Das, Snigdha Banerjee, Archana De, Inamul Haque, Gargi Maity, Matt McEwen, Sushanta K. Banerjee. The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5. [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 4385. doi:10.1158/1538-7445.AM2013-4385
Abstract 4390: Aurora kinases: potential therapeutic targets in K-Ras-induced lung cancer.
Authors: dos Santos Edmilson O. , Aoki Mateus N. , Bassères Daniela S.
Published: August 2014

K-Ras-induced lung cancer is a very common disease, for which there are currently no effective therapies. Intense efforts are underway to identify K-Ras targets that play a crucial role in oncogenesis. We have previously shown that K-Ras-induced lung tumorigenesis is potentiated by the transcription factor NF-κB; and others have shown that Aurora kinases, not only play a significant role in tumorigenesis, but also can activate NF-κB. Therefore, we hypothesized that Aurora A and/or Aurora B are important K-Ras targets in lung cancer. In order to test this hypothesis, we first determined whether oncogenic K-Ras induces Aurora kinase expression. For that purpose, we used three different cell-based models: (1) an immortalized primary lung epithelial cell line and its isogenic K-Ras-transformed counterpart, (2) H1703 lung cancer cell line engineered to express oncogenic K-Ras inducibly, and (3) K-Ras positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting K-Ras. In all cases, K-Ras expression positively correlated with Aurora A and Aurora B expression. In order to validate Aurora A an B as therapeutically relevant K-Ras targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate Aurora A or B. Inducible shRNA-mediated knockdown of Aurora A or B, as well as treatment with a dual Aurora A and B inhibitor (AI II), decreased growth, viability and migration of K-Ras positive H358 and A549 cell lines. Interestingly, in our primary isogenic model and in our H1703 K-Ras-inducible cell line, AI II reduced viability and induced apoptosis in an oncogenic K-Ras-dependent manner. This suggests that Aurora kinase inhibition therapy can specifically target K-Ras transformed cells. In conclusion, our results support our hypothesis that Aurora kinases are important K-Ras targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for K-Ras-induced lung cancer therapy.

Citation Format: Edmilson O. dos Santos, Mateus N. Aoki, Daniela S. Bassères. Aurora kinases: potential therapeutic targets in K-Ras-induced lung 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 4390. doi:10.1158/1538-7445.AM2013-4390
Abstract 4408: A functional genomics approach for identification of sirolimus sensitizer genes regulated by HDAC inhibitors.
Authors: Gamache Benjamin J. , Simmons John , Patel Jyoti , Ou Lihui , Michalowski Aleksandra , Sullivan Patrick , Wei Bih-Rong , Simpson R. Mark , Zhang Shuling , Zhang Ke , Kuehl W. Michael , Landgren Ola , Caplen Natasha , Mock Beverly
Published: August 2014

The molecular pathogenesis of many cancer types, including multiple myeloma (MM), involves alterations in the PI3K/Akt/mTOR and cyclin/CDK/CDKI/Rb (Rb) pathways. We have shown the combination of a Class I-specific HDAC inhibitor (entinostat, MS-275) with the mTOR inhibitor sirolimus to be synergistic in a large panel of B cell tumor cell lines including multiple myeloma, mantle cell and Burkitt's lymphoma. While tumor outgrowth occurred in both single agent arms, the combination effectively controlled in vivo tumor growth in long-term preclinical studies. The combination was also highly effective in a tumor-cell specific bioluminescence assay where myeloma cells are co-cultured with bone marrow stromal cells to mimic the protective effects of the tumor microenvironment. We found the combination antagonized the oncogenic activation of the AKT pathway associated with single-agent rapamycin treatment, inhibited the ERK/MAPK pathway, decreased pro-survival signaling, and increased growth inhibitory signals to a much greater extent than either single agent alone. To further understand the molecular mechanisms contributing to the synergy of this combination, a functional genomics approach utilizing high-throughput RNAi screening and gene expression profiling was taken. Several cell cycle-specific kinases were identified that upon inhibition enhance the activity of sirolimus and are significantly down-regulated by entinostat. Thus far, we have confirmed by western blot that PLK1 and AURKB are decreased in a dose-dependent manner by entinostat. Specific inhibition of these targets synergizes with sirolimus. Additionally, we found very low (<EC20) entinostat/sirolimus combination doses to hyper-sensitize tumor cells to a number of cell cycle inhibitors. These findings are further supported by increased cell cycle arrest seen in the combination treatment compared to either drug alone, as well as marked reduction in the cell cycle progression marker phospho-Histone H3. Taken together, our findings indicate the anti-tumor synergy of combined HDAC/mTOR inhibition is the result of multiple cooperative effects.

Citation Format: Benjamin J. Gamache, John Simmons, Jyoti Patel, Lihui Ou, Aleksandra Michalowski, Patrick Sullivan, Bih-Rong Wei, R. Mark Simpson, Shuling Zhang, Ke Zhang, W. Michael Kuehl, Ola Landgren, Natasha Caplen, Beverly Mock. A functional genomics approach for identification of sirolimus sensitizer genes regulated by HDAC 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 4408. doi:10.1158/1538-7445.AM2013-4408
Abstract 4414: Differential effects of functionalized and non-functionalized short multi-wall carbon nanotubes on survival and cell signaling in pancreatic cancer cells.
Authors: Zhang Yao , Lai James C.K. , Bhushan Alok ,   Biomedical and Pharmaceutical Sciences Department, College of Pharmacy, Division of Health Sciences.
Published: August 2014

Pancreatic cancer is an aggressive type of cancer with poor prognosis and low five-year survival rate. Because chemotherapeutic and radiation treatments are not effective, new treatments for this cancer are needed to improve its prognosis. Carbon nanotubes (CNTs) constitute one novel and important nanomaterial extensively examined for potential biomedical applications (e.g., drug delivery vehicles and diagnostic agents). Modification of CNTs with functional groups improves solubility and may serve as attachment site of other molecules and/or drugs. This study elucidated the cytotoxicity induced by CNTs and the underlying mechanisms. We employed MTT assay to determine survival of PANC1 cells treated with different concentrations of short-multi-wall CNTs (SMWCNTs) or SMWCNTs-COOH for 24, 48, and 72 hrs. Our results suggested that functionalization with carboxyl group to SMWCNTs lowers their cytotoxicity. Therefore we hypothesized that the induced cytotoxicity by both SMWCNTs and SMWCNTs-COOH is a result of either up-regulation of apoptotic pathways or down-regulation of cellular survival/proliferation/differentiation pathways or a combination of both. We carried out western-blot analysis to examine the effects of CNTs on ERK, phospho-ERK, AKT, phospho-AKT signal transduction pathways and on the NF-κB pathway. PANC1 cells were treated with 2, 20, and 200 μg/ml SMWCNTs-COOH or SMWCNTs for 48hrs. The expressions of phospho-ERK were decreased in cells treated with 2 μg/ml SMWCNTs-COOH. However, as the concentration of SMWCNTs-COOH increased from 2 to 200 μg/ml, the phospho-ERK expression was increased: however, the total ERK expression was unaffected. The NF-κB expression in the cells was increased compared to control group as the concentration of SMWCNTs-COOH increased from 2 to 200 μg/ml. Our study demonstrates that both SMWCNTs and SMWCNTs-COOH induced cytotoxicity in pancreatic cancer cells and functionalization with carboxyl group lowered their cytotoxicity. One mechanism underlying their cytotoxicity may be the altered regulation of cellular survival/proliferation pathways such as ERK. Thus, our results may have implications in cytotoxicity of SMWCNTs in pancreatic cells and in designing therapies for pancreatic cancer.

Citation Format: Yao Zhang, James C.K. Lai, Alok Bhushan, Biomedical and Pharmaceutical Sciences Department,College of Pharmacy, Division of Health Sciences. Differential effects of functionalized and non-functionalized short multi-wall carbon nanotubes on survival and cell signaling in pancreatic 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 4414. doi:10.1158/1538-7445.AM2013-4414
Abstract 4415: Differential effects of biochanin A on cell proliferation and ROS-dependent pathways in estrogen receptor positive and HER-2 positive breast cancer cells.
Authors: Dickerson Lindsy , Sehdev Vikas , Crowell Pamela L. , Lai James C.K. , Bhushan Alok
Published: August 2014

The naturally occurring isoflavone biochanin A has been reported to have chemo-preventative properties towards cancer. Biochanin A exhibits antioxidant properties. Because of its antioxidant properties, biochanin A may be used to target breast cancer cells with high intracellular levels of reactive oxygen species (ROS). In this study, we investigated the effects of biochanin A on two cell lines — an estrogen receptor (ER) positive and a HER-2 positive breast cancer cell line. Biochanin A is implicated to affect both hormonal receptors and receptor tyrosine kinases. Overstimulation of ER is a basis for tumor progression due to the disruption of the cell cycle and apoptosis in estrogen positive breast cancer cells. Activation of HER-2 initiates cell growth, survival and invasion promoting cell signaling events in HER-2 positive breast cancer cells. MCF-7 (estrogen positive) and SK-BR-3 (HER-2 positive) cells were treated with biochanin A (2-100 μM) for 72 hours. Then MTT (cell survival) assay was carried out to determine the effects of biochanin A on cell survival. Our results suggest that biochanin A induces a dose-related inhibition of MCF-7 and SK-BR-3 cell viability at 50 μM. MCF-7 and SK-BR-3 cells were also treated with biochanin A (50 μM) for 12 hours and subsequently treated with hydrogen peroxide (H2O2)(2 μM) for 30 minutes: then western blotting was carried out to determine their expression of ERK and transcription factor NFκB. We found that biochanin A inhibits phospho-ERK pathway in SK-BR-3 but not in MCF-7 cells. However, expression of the transcription factor NFκB was decreases in both SK-BR-3 and MCF-7 cells. Thus, biochanin A may be an effective chemotherapeutic agent for breast cancer because of its ability to affect both hormonal receptors and receptor tyrosine kinases and to decrease intracellular ROS levels. (Supported by NIH INBRE P20RR016454, URC and MSTMRI)

Citation Format: Lindsy Dickerson, Vikas Sehdev, Pamela L. Crowell, James C.K. Lai, Alok Bhushan. Differential effects of biochanin A on cell proliferation and ROS-dependent pathways in estrogen receptor positive and HER-2 positive 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 4415. doi:10.1158/1538-7445.AM2013-4415
Abstract 4422: Methylcytosine topology: potential of a DNA landmark in pre-clinical epigenetic drug toxicology.
Authors: Tajbakhsh Jian
Published: August 2014

Cellular reprogramming through drug-based epigenetic intervention has become a novel approach in the treatment of complex diseases and in regenerative medicine towards reconstitution of normal cell, tissue and organ function. Within this context, non-specific and off-target global DNA demethylation — either directly induced by DNA demethylating drugs or indirectly caused by histone modifiers or any other agent with epigenetic consequences — could impact the cells’ DNA methylation pattern and lead to unwanted cryptic DNA methylation toxicity and carcinogenesis: by influencing chromatin conformation, genome organization, and cellular gene expression programs. The Translational Cytomics Group at the Cedars-Sinai Medical Center (Los Angeles) has developed 3D quantitative DNA Methylation Imaging (3D-qDMI), a non-disruptive image-cytometrical method that measures the differential load and spatial distribution of methylated cytosine (MeC) within individual nuclei of thousands of cells in a cell-by-cell fashion. Thus the technology has the additional capability of capturing cellular heterogeneity regarding global DNA methylation properties in response to drug exposure. Utilizing 3D-qDMI we are currently evaluating these topological parameters, which translate to differential DNA methylation phenotypes (MeC phenotypes), as potential chromatin signatures (textures) to be utilized in the characterization of cells during reprogramming, and for genotoxicity assessment of drugs.

Citation Format: Jian Tajbakhsh. Methylcytosine topology: potential of a DNA landmark in pre-clinical epigenetic drug toxicology. [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 4422. doi:10.1158/1538-7445.AM2013-4422