bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 427: Upregulation of KLF4 and inhibition of HDAC activity by methylseleninic acid in human esophageal squamous cells
Authors: Hu Chenfei , Zhang Wei , Xu Qing , Chen Lechuang , Ma Kai , Liu Mei , Zhu Hongxia , Xu Ningzhi
Published: May 2015

In the present study, we investigated the potential mechanisms of the inhibition of cell growths induced by methylseleninic acid, MSA. Our result showed that MSA could induce cell growth inhibition and apoptosis in esophageal squamous cell carcinoma (ESCC) cells and inhibit tumor growth measured by xenograft volume in nude mice. Upon treated with MSA, HDACs activity was decreased and GCN5, one of histone acetyltransferases, upregulated as well in human esophageal squamous cell lines. Meanwhile a significant increase of H3K9 acetylation (H3K9ac) was detected. Notably,upregulation of KLF4, which has been wildly reported usually downregulated expression in cell lines and tumor tissues of ESCC, was also observed after MSA treatment. Additionally the acetylated histone H3 could locate more at KLF4 promoter region than non-acetylated one did, showed by chromatin immunoprecipitation assay. Moreover knockdown of GCN5 reduced the protein levels of both H3K9ac and KLF4, along with less cell growth inhibition. Taken all, our result indicates that MSA can exert its anti-tumor effects, at least in part, by up-regulation KLF4, probably via inhibiting the HDAC activity and up-regulation GCN5. By our best of knowledge, this is the first report of selenium-induced selective, post-translational acetylation of histone H3 at Lys9, which may depend on the activities of and the balance between HDACs and HATs.

Note: This abstract was not presented at the meeting.

Citation Format: Chenfei Hu, Wei Zhang, Qing Xu, Lechuang Chen, Kai Ma, Mei Liu, Hongxia Zhu, Ningzhi Xu. Upregulation of KLF4 and inhibition of HDAC activity by methylseleninic acid in human esophageal squamous cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 427. doi:10.1158/1538-7445.AM2014-427
Abstract 4273: An enhanced microRNA backbone for potent single-copy RNAi
Authors: Fellmann Christof , Hoffmann Thomas , Sridhar Vaishali , Hopfgartner Barbara , Lai Dan Y. , Zuber Johannes
Published: May 2015

Short-hairpin RNA (shRNA) technology enables stable and regulated gene repression through RNA interference (RNAi). Synthetic shRNAs can be embedded into the context of an endogenous microRNA (miRNA) to create natural substrates of RNAi pathways and minimize cellular toxicities. While such “shRNAmir” reagents provide an experimentally versatile RNAi system, their design remains challenging due to our limited understanding of miRNA processing factors. Consequently, shRNAmir reagents often fail to trigger potent target knockdown, especially when expressed from a single genomic integration (“single-copy”), a common scenario in pool-based screens and other key applications. To tackle this issue, we recently developed a high-throughput Sensor assay that enables functional identification of effective shRNAs and has been used to establish shRNA-specific prediction rules. Following these advances in design of the synthetic shRNA stem, here we take a systematic approach to improve the experimental miR-30 backbone. Among several sequence features enhancing knockdown efficiency, we identify a conserved element 3′ of the basal stem that is critically required for optimal shRNAmir processing. Implementation of this feature in an optimized backbone termed “miR-E” results in a 10-30 fold increase in mature shRNA levels, and generally boosts knockdown efficacy. Importantly, combination of the enhanced miR-E backbone with Sensor-based design rules establishes a validated and easily accessible platform for generating single-copy effective shRNAs for pool-based RNAi screening and transgenic animals, and will facilitate the functional annotation of the genome.

Citation Format: Christof Fellmann, Thomas Hoffmann, Vaishali Sridhar, Barbara Hopfgartner, Dan Yu Lai, Johannes Zuber. An enhanced microRNA backbone for potent single-copy RNAi. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4273. doi:10.1158/1538-7445.AM2014-4273
Abstract 4277: Alternative splicing in cancer transcriptomes
Authors: Shen Shihao , Wu Ying Nian , Xing Yi
Published: May 2015

Alternative splicing of precursor mRNA is a key mechanism of eukaryotic gene regulation. Cancer-associated changes in alternative splicing can modulate gene function or activity, and consequently affect downstream molecular pathways critical for tumorigenesis or metastasis. RNA-Seq has become a powerful technology for genome-wide analysis of alternative splicing. We have developed sensitive and robust computational methods for quantitative analysis of alternative splicing using RNA-Seq data. Using these methods, we have analyzed the alternative splicing profiles of >4000 cancer samples across multiple types of cancer. Our analysis has identified important alternative splicing events that may contribute to the development and progression of cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Shihao Shen, Ying Nian Wu, Yi Xing. Alternative splicing in cancer transcriptomes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2014-4277
Abstract 4289: Noninvasive monitoring of blood-brain barrier permeability for antibodies by apoptosis imaging: optimization of treatment strategies for maximal brain delivery
Authors: Weber Thomas G. , Mathejczyk Julia , Galbán Stefanie , Rehemtulla Alnawaz , Scheuer Werner
Published: May 2015

Purposes: Large molecules like antibodies cannot penetrate the intact blood-brain barrier (BBB) in adequate amounts to deploy efficacy. Brain shuttle antibodies for enhanced BBB crossing are considered to overcome this obstacle. Thus, we established a non-invasive technology that accelerates quantification of brain tumor delivery and pharmacodynamics of antibodies.

Experimental Procedures: The technology implies intracranial implantation of a split-luciferase apoptosis reporter cell line (Caspase-3/7 GloSensor) in SHO mice. An agonistic Death Receptor 5 antibody (anti-DR5) was used as model substance. To quantify BBB crossing of systemically applied anti-DR5, we compared apoptosis inductions after i.c. and i.v. application at different dosages.

Results: Assuming a 100 % drug delivery after i.c. application, the amount of antibody delivery after i.v. application was calculated relative to its apoptosis induction. We found that less than 1 % of the unmodified anti-DR5 passed the BBB dose-dependently. These data were confirmed by 3-dimensional fluorescence microscopy of explanted brain tissue. Significant anti-tumor efficacy was detected when anti-DR5 is given in adequately high dosages. We further demonstrate in different brain tumor models that unspecific IgG antibody penetration into brain tumors is presumably enhanced by a slightly impaired BBB.

Conclusions: Our technology facilitates the quantification of BBB penetration by measuring apoptosis non-invasively without using radiolabeling approaches. This orthotopic model accelerates the assessment of i.v. dosage optimization and brain shuttle systems for maximal anti-tumor efficacy in brain tumors.

Citation Format: Thomas G. Weber, Julia Mathejczyk, Stefanie Galbán, Alnawaz Rehemtulla, Werner Scheuer. Noninvasive monitoring of blood-brain barrier permeability for antibodies by apoptosis imaging: optimization of treatment strategies for maximal brain delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4289. doi:10.1158/1538-7445.AM2014-4289
Abstract 429: HERP2 as a novel molecular marker and therapeutic target in glioblastoma
Authors: Velpula Kiran Kumar , Asuthkar Swapna , Lee William , Tsung Andrew J.
Published: May 2015

Glioblastoma (GBM) remains one of the most lethal and difficult-to-treat cancers of the central nervous system despite a multi-modality approach. The poor prognosis in GBM patients is due to therapeutic resistance which calls for identifying novel molecular therapeutic targets. Recently, HES-related repressor protein 2 (HEPR2), a mediator of notch signaling has been identified as a molecular target implicated in chemoresistance. This current study aims at both the epigenetic and transcription factor regulation as the potential mechanisms resulting in the expression of HES-related repressor protein 2 (HERP2) in GBM patient specimens and cell lines. Using immunohistochemistry and immunoblot analysis, we observed that the HERP2 protein is highly expressed in human GBM tissues and glioma cells (4910, U251 and 5310) and is significantly correlated with O6-alkylguanine DNA alkyltransferase (MGMT) levels. We also observed that the expression of HERP2 was dependent on the methylation status of a HERP2 promoter-associated CpG island region as evidenced by prominent PCR products using methylation-specific PCR (MSP) analysis. Interestingly, treatment of GBM cells with demethylating agent, 5-aza-2’-deoxycytidine resulted in hypomethylation, whereas sodium butyrate-treated cells remained specific for methylated CpG islands. Additionally, we also demonstrated a positive regulatory loop between HERP2 and NOTCH1 using rescue experiments. Further, we show the anti-tumorigenic role of siHERP2 to be mediated by the negative regulation of NOTCH1 protein. Our studies indicate that targeting HERP2 in cancer patients undergoing chemotherapy will have favorable therapeutic implications.

Citation Format: Kiran Kumar Velpula, Swapna Asuthkar, William Lee, Andrew J. Tsung. HERP2 as a novel molecular marker and therapeutic target in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 429. doi:10.1158/1538-7445.AM2014-429
Abstract 4295: A 3D culture model for screening of cancer therapeutics
Authors: Patel Kalpana , O'Clair Belinda , O'Callaghan Tim , Appledorn Daniel M. , Trezise Derek
Published: May 2015

3D cell culture models are currently of great interest to the bioscience community. This is most notable in the cancer biology area, where a growing body of evidence suggests that more relevant and translational observations can be made by testing compounds in a physiological relevant 3D model compared to a 2D model. In this study, we take a kinetic, live-cell imaging approach to measure the growth or shrinkage of non-adherent tumor spheroids using Ultra Low Attachment (ULA) 96-well and 384-well microtiter plates (Corning®). These round-bottomed, hydrogel coated plates facilitate the formation of spheroids by promoting self-adherence as opposed to adherence to the plate surface. In a single cell type, monoculture model, A549 lung epithelial carcinoma cells developed into single spheroid structures in each well of 96 and 384-well plates within 48-96 hours in culture. In both plate types, the resulting spheroid size, ranging from 330 µm - 600 µm, was proportional to the number of cells (1K - 5K cells per well). Post formation, spheroids remained untreated or were subjected to three test agents, staurosporine, oxaliplatin or cycloheximide. The increase or decrease in spheroid size was monitored over 3 - 10 days using the IncuCyte™ ZOOM Live Content Imaging system. Using the red fluorescent channel, spheroid growth was analyzed using fluorescent area and intensity metrics. Whereas staurosporine and oxaliplatin significantly decreased spheroid size at high concentrations, cycloheximide treatment resulted in a cytostatic response, effectively inhibiting spheroid growth in a concentration dependent manner, while not reducing total spheroid area at any tested concentration. Interestingly, in side-by-side comparisons of kinetic 2D and 3D assays, we observed a significant, 10-fold (one log) shift in the pharmacology of cycloheximide. In a 3D co-culture breast cancer model, this study also illustrates the effect of Lapatinib on SK-BR-3 cells grown in the presence of mammary derived stromal cells (fibroblasts). Together, this study describes a kinetic, live-cell approach to measuring spheroid size over time, illustrates a differential response of cycloheximide in a 3D system compared to identically treated cells grown in a 2D monolayer, and details a complex co-culture method for elucidating the effect of Lapatinib in a physiologically relevant 3D model of breast cancer.

Citation Format: Kalpana Patel, Belinda O'Clair, Tim O'Callaghan, Daniel M. Appledorn, Derek Trezise. A 3D culture model for screening of cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2014-4295
Abstract 4297: Quantifying three-dimensional cellular morphology and its heterogeneity in epithelial cancers by single-cell optical tomography
Authors: Nandakumar Vivek , Helland Stephanie , Han Jessica H. , Davis Geriann , Ashcroft Brian A. , Tran Thai H. , Li Ting , Anderson Karen S. , Pannala Rahul , Mikhael Joseph R. , Fleischer David E. , Johnson Roger H. , Bussey Kimberly J. , Meldrum Deirdre R.
Published: May 2015

The three-dimensional (3D) structure of the cell and its nucleus is intricately associated with cellular function. Aberrations and heterogeneity in cellular and nuclear morphology serve as diagnostically important cues to identify malignant transformation. However, current cytopathology practice is predominantly based on a qualitative, 2D assessment of cellular morphology that may bias the diagnosis. We utilized the Cell-CT® platform to perform single-cell, optical tomography imaging and generate 3D cell images with isotropic, sub-micron spatial resolution. We developed custom image analysis tools to accurately quantify 3D cell and nuclear morphology. We used these measures to characterize the morphological heterogeneity within and among epithelial cell populations, and to identify morphological descriptors that are reliably predictive of cell state. We assessed the efficacy of our approach relative to conventional 2D assessment (at 40x magnification) to detect malignant transformation in immortalized and primary normal-cancer cell line pairs from human esophagus and breast epithelia. We observed the 3D method of assessment to provide a consistently superior performance (sensitivity and specificity) in predicting malignant transformation. Further, some widely used diagnostic parameters, such as nuclear size or nucleus-to-cytoplasm ratio, did not always exhibit the highest predictive power in 3D space. The availability of 3D morphological information also provided novel insights on intra-population morphological heterogeneity in both normal and cancer cell populations. Our results emphasize the need to incorporate 3D morphological information into standard pathological practice.

Citation Format: Vivek Nandakumar, Stephanie Helland, Jessica H. Han, Geriann Davis, Brian A. Ashcroft, Thai H. Tran, Ting Li, Karen S. Anderson, Rahul Pannala, Joseph R. Mikhael, David E. Fleischer, Roger H. Johnson, Kimberly J. Bussey, Deirdre R. Meldrum. Quantifying three-dimensional cellular morphology and its heterogeneity in epithelial cancers by single-cell optical tomography. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4297. doi:10.1158/1538-7445.AM2014-4297
Abstract 4302: The development study of hypoxia responsive chemiluminescent probe for tumor hypoxia imaging
Authors: Okuda Kensuke , Ban Toru , Hirayama Tasuku , Nagasawa Hideko
Published: May 2015

Introduction: The tumor microenvironment is recognized as a major factor that influences not only the response to conventional anticancer therapies but also the potential for malignant progression and metastasis. Therefore, it is considered that tumor hypoxia imaging is critical for cancer diagnosis and therapy. As optical modalities offer the advantages of non-invasive, easy-to-handle, and cost-effective imaging, much effort has been done to develop synthetic fluorescent imaging probes for tumor hypoxia. In particular, we focus our attention on chemiluminescence imaging which can avoid interference from tissue autofluorescence and scattering light. We therefore show the development of hypoxia responsive chemiluminescent probe, which has the advantage of lower detection limits and improved signal-to-noise ratio compared to existing fluorescent probes.

Results and discussion: In our molecular design, luminescence is derived from 1,2-dioxetane cleavage triggered by hypoxia-selective enzymatic reduction of electron-deficient aromatics. At the start, nitrobenzyl moiety was chosen as a bioreductively cleavable protective group linked to 1,2-dioxetane moiety through phenyl ether linker to construct synthetic probe NB-Adm. The compound was synthesized from 2-adamantanone in 19% (overall yield, 6 steps). Then NB-Adm was attempted to be reduced by nitroreductase, however, no luminescence was observed. HPLC analysis of the reaction mixture indicated that our expected reduction did not occur, probably because bulky adamantane moiety prevented the enzymatic reaction owing to steric hindrance. Therefore, a self-immolative linker was employed to keep away 1,2-dioxetane moiety from enzyme recognition moiety. In this time, quinone moiety was chosen as a protective group which was expected to be better substrate than nitrophenyl one in terms of their redox potential. This newly designed compound, TMQ-Adm was synthesized from 2-adamantanone in 16% (overall yield, 6 steps). By the use of TMQ-Adm, we successfully observed luminescent emission from the reaction with nitroreductase. This implied that our bioredctively activatable probe can be applicable for chemiluminescence imaging for tumor hypoxia. Now we are investigating to develop chemiluminescent probe to realize in vivo imaging of tumor hypoxia by improving luminescent quantum yield under physiological condition.

Citation Format: Kensuke Okuda, Toru Ban, Tasuku Hirayama, Hideko Nagasawa. The development study of hypoxia responsive chemiluminescent probe for tumor hypoxia imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2014-4302
Abstract 4309: Whole slide image analysis quantification in a mouse lung metastasis model
Authors: Sandusky George E. , Surface Ronne , Tonsing-Carter Eva , Silver jayne , Sinn Tony , Pollok Karen
Published: May 2015

Digital whole slide imaging is the technique of digitizing a microscope slide at the highest resolution to produce a “digital virtual microscope slide”. This digital image can be viewed in three or four fields, from low to high power fields, which can be commonly used to evaluate the tissue. Many of these systems have a whole slide software image analysis system.

The goal of this study was to determine if the Aperio positive pixel algorithm (image analysis) could effectively quantitate metastatic mouse lung tumors in a lung section using a H&E stain.

Lung sections from a mouse lung metastasis model with 8 mice in each group were evaluated: control, 50mg/kg, and 75mg/kg carboplatin. H&E and Ki67 immunostain slides were scanned using the Aperio whole slide scanning system (Scanscope CS). A single field of view from each slide representing a whole lung lobe with multiple lung metastases was selected for image analysis. The standard positive pixel algorithm was altered to read the H&E slides. Various histology slides were used to validate the altered algorithm. The immunostain (Ki67) was generated using the standard positive pixel algorithm analysis.

The Aperio automated positive pixel count for a Ki67 immunostain was consistent with the H&E image analysis. The values went down with a dose dependent treatment (control vs. 50mg/kg, and 75mg/kg Carboplatin) and were (H&E) 37%, 28%, 22%, and (Ki67) 9%, 5%, 3%, respectively.

The analysis had decreasing values for both the H&E and Ki67 analysis on a dose dependent drug treatment. The metastases decreased in both treatment groups compared to controls with both the H&E and Ki67 analyses. The Aperio Image Analysis positive pixel algorithm allows large areas of the lung tissue section to be examined and NOT just a single 25x or 40x field like many common image analyses.

Citation Format: George E. Sandusky, Ronne Surface, Eva Tonsing-Carter, jayne Silver, Tony Sinn, Karen Pollok. Whole slide image analysis quantification in a mouse lung metastasis model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2014-4309
Abstract 431: CYP26C1 gene is highly methylated and correlated with chemoradiation therapy in esophageal squamous cell carcinoma
Authors: Watanabe Yoshiyuki , Oikawa Ritsuko , Yoshida Yoshihito , Yamamoto Hiroyuki , Itoh Fumio
Published: May 2015

Background: Systemic chemo- and chemoradiation- therapy is widely accepted in esophageal squamous cell carcinoma (ESCC) patients. However resistance to these therapies remaining pretty high and multicentric occurrence of ESCC often can see after treatment. Multiple studies have shown that promoter methylation of tumor suppressor genes underlie esophageal carcinogenesis. But, we still do not know that epigenetic changes after chemoradiation therapy (CRT) in ESCC. Our aim is to identify how change DNA methylation in tumor site (T) but also adjacent normal site (ADJ) before/after CRT. Methods: Tumor and adjacent normal biopsy specimens were obtained before/after treatment from 34 patients (124 samples) enrolled in a uniform CRT protocol. Genomewide DNA methylation analysis was performed using 12 non-treated test samples (T and matched ADJ: 6 each) by methylated CpG island amplification and microarray (MCAM) method (4x44K, Agilent custom array), and selected candidate genes that were highly methylated in T than ADJ. We next analyzed one of the candidate genes CYP26C1 (cytochrome P450, family 26, subfamily C, polypeptide 1) DNA methylation using 112 validation samples by quantitative bisulfate pyrosequencing. Results: CYP26C1 was selected by unsupervised hierarchical clustering after MCAM in test samples. CYP26C1 DNA methylation was significantly high in T than ADJ in both test and validation samples (p<0.04, T: 60.7±7.6, ADJ: 44.5±11.5). However some ADJ also showed high methylation in validation samples. Moreover, CYP26C1 methylation in both T and ADJ were significantly depressed after CRT (T: p<0.01 (45.1±18.3 to 31.6±14.9), ADJ: p<0.0001 (43.8±10.3 to 30.5±13.8)). Conclusions: CYP26C1 has a CpG island in promoter area and was highly methylated in T of ESCC patients. And, some ADJ showed high CYP26C1 methylation, suggesting possibility of multicentric occurs of ESCC. Moreover, CYP26C1 methylation depress after CRT both T and ADJ, suggesting potential clinical application of CRT sensitive biomarker.

Citation Format: Yoshiyuki Watanabe, Ritsuko Oikawa, Yoshihito Yoshida, Hiroyuki Yamamoto, Fumio Itoh. CYP26C1 gene is highly methylated and correlated with chemoradiation therapy in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 431. doi:10.1158/1538-7445.AM2014-431
Abstract 4311: Fluorescence-guided surgery of pancreatic cancer using activatable cell penetrating peptides (ACPPs) in orthotopic mouse models
Authors: Metildi Cristina A. , Kaushal Sharmeela , Felsen Csilla N. , Nguyen Quyen T. , Hoffman Robert M. , Tsien Roger Y. , Bouvet Michael
Published: May 2015

Surgical resection with complete removal of tumor remains one of the most effective modalities in the treatment of cancer. The aim of this study was to evaluate the efficacy of using activatable cell penetrating peptides (ACPPs) conjugated to Cy5 fluorophores to accurately label primary pancreatic tumor for fluorescence-guided surgery (FGS) in orthotopic mouse models. Orthotopic mouse models of human pancreatic cancer using surgical implantation of tumor fragments of the MiaPaca-2-GFP pancreatic cancer cell line were established. Two weeks after tumor implantation, the tumors were resected with bright light surgery (BLS) or FGS. FGS was performed with a florescence-dissecting microscope (MVX10, Olympus) 2 hours after periorbital injection of ACPPs. Completeness of resection was assessed from postoperative images obtained with the Maestro CRI Small Imaging System. Postoperatively, we performed whole body imaging of the mice to assess for recurrence. At eight weeks, mice were sacrificed to evaluate pancreatic tumor burden, as well as local and distant recurrence. Cy-5-conjugated ACPPs, cleavable by MMP-2 and MMP-9 permitted clear visualization of tumor margins for fluorescence-guided resection. With FGS, no residual tumor was detected in the postoperative surgical site in all 16 mice. One mouse out of 16 undergoing BLS had evidence of residual tumor postoperatively. At 8 weeks, mice in the BLS group demonstrated larger primary tumors compared to mice in the FGS group (1.72 g ± SE 0.58 vs 0.25 g ± SE 0.14, p=0.026). Mean disease-free survival (DFS) was significantly lengthened from 5.33 weeks in the BLS group to 7.38 weeks in the FGS group (p=0.02). Furthermore, recurrence rates were lower in the FGS group (38% vs 73%, p=0.049). This translated into a lower local and distant recurrence rate for FGS (31% vs 67% and 25% vs 60%, respectively). ACPPs can provide an alternative method to accurately and effectively label cancer cells for improved resection of tumors that do not express specific tumor antigens and result in better outcomes post-resection.

Citation Format: Cristina A. Metildi, Sharmeela Kaushal, Csilla N. Felsen, Quyen T. Nguyen, Robert M. Hoffman, Roger Y. Tsien, Michael Bouvet. Fluorescence-guided surgery of pancreatic cancer using activatable cell penetrating peptides (ACPPs) in orthotopic mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4311. doi:10.1158/1538-7445.AM2014-4311
Abstract 4321: Effect of pretreatment, dose and route of administration of BPM31510 (Coenyzme Q10 containing proprietary formulation) alone or in combination with gemcitabine improves survival in pancreatic cancer
Authors: Narain Niven , Mauro Lucia , Lens Assuan , Akmaev Viatcheslav , Sarangarajan Rangaprasad , Jimenez Joaquin
Published: May 2015

In-vitro studies have demonstrated the ability of BPM31510, a Coenzyme Q10 (CoQ10) containing proprietary formulation to effectuate a metabolic switch from glycolysis to mitochondrial OXPHOS resulting in the activation of apoptotic pathways specifically in cancer cells. The effectiveness of BPM31510 in combination with standard of care chemotherapy agents in in-vitro and in-vivo models has been previously established. The current study was to determine the effect of dose, route of administration and combination with gemcitabine on survival in an animal model of pancreatic cancer. BPM31510 administered in three divided doses (50mg/kg vs 75mg/kg body weight daily, intravenous) was associated with dose dependent increase in survival and was significantly better when combined with gemcitabine. Continuous infusion of BPM31510 significantly improved survival rates compared to three divided doses (50mg/kg or 75mg/kg body weight) of BPM31510, with best outcomes at 200mg/kg body weight dose. Sixty day pretreatment with BPM31510 alone followed by combination with gemcitabine was also associated with improved survival outcomes with either gemcitabine or BPM31510 alone. The data suggests that dose and route of administration of BPM31510 alone or in combination with standard of care chemotherapy agents influences and improves survival in animal model of pancreatic cancer.

Citation Format: Niven Narain, Lucia Mauro, Assuan Lens, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Joaquin Jimenez. Effect of pretreatment, dose and route of administration of BPM31510 (Coenyzme Q10 containing proprietary formulation) alone or in combination with gemcitabine improves survival in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4321. doi:10.1158/1538-7445.AM2014-4321
Abstract 4323: Mitochondrial 22 tRNAs alterations in Iranian breast cancer patients
Authors: Eslamizadeh Sara , Ghaffarpour Massoud , Arabzadeh Mohammad , Houshmand Massoud
Published: May 2015

Introduction: Mitochondrial dysfunction is related to the genesis of many types of cancers, including breast cancer. Mitochondrial tRNA genes perform several functions including processing and translation in which are essential components of mitochondrial protein synthesis. Until now only few somatic mitochondrial tRNA mutations have been reported in cancer cells. The aim of this study was to find out the relationship between mitochondrial DNA mutations and breast cancer.

Patients and Methods: In current this study, 22 mitochondrial tRNAs genes in 30 tumoral tissues and adjacent non-tumoral tissues of patients with breast cancer and 100 blood samples of healthy controls were investigate using PCR-Sequencing methods.

Results and conclusions: From 30 breast cancer cases, twelve genetic variations were found in 22 tRNA MtDNA. Six of twelve (50%) were novel and remaining was polymorphism. Among new variants, which incloding C1631A (tRNA Val), T1633A (tRNA Val), A5565G (tRNA Trp), G5849A (tRNA Tyr) were at hetroplasmic state and remaining which including C12187 (tRNA His) and A15948G (tRNA Thr) were at homoplasmic. Among variants, A12308G, a polymorphic mutation in V-loop (tRNALeu(CUN)), was found in 23% breast tumoral tissues (7 of 30) and 3%in healthy blood controls (3 of 100), indicating a statistically significant association of the A12308G alteration between tumoral tissues and controls(p < 0.05).Our finding indicate that , the A12308G alteration may increase the risk of breast cancer through its impact on the tRNA structure result in mitochondrial dysfunction. Therefore, it may consider as a pathogenic predisposition factor for the breast cancer risk.

Note: This abstract was not presented at the meeting.

Citation Format: Sara Eslamizadeh, Massoud Ghaffarpour, Mohammad Arabzadeh, Massoud Houshmand. Mitochondrial 22 tRNAs alterations in Iranian breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4323. doi:10.1158/1538-7445.AM2014-4323
Abstract 4324: BNip3 connects energy sensing to hepatic lipid metabolism and mitophagy
Authors: Boland Michelle L. , Huang He , Shah Ramilla , Ali Almas , Zhao Yingming , Rhodes Christopher J. , Macleod Kay F.
Published: May 2015

BNip3 is a hypoxia-inducible member of the Bcl-2 family that integrates into the outer mitochondrial membrane as stable homodimers where it acts as a receptor for mitochondrial engulfment through a conserved LC3 interacting region. Hepatic BNip3 levels are constitutively elevated relative to other tissues, and dramatically increase upon nutrient deprivation or glucagon stimulation, suggesting a key role for BNip3 in the fasting response. We have determined that the fasting-induced increase in hepatic BNip3 protein levels is not solely attributable to transcriptional induction and have identified post-translational modification of BNip3 that affects protein stability and function. In a mouse model of BNip3 loss, we observe metabolic defects including increased hepatic lipid synthesis and reduced fatty acid oxidation. Consistent with BNip3's role in mitophagy, defects in liver metabolism were linked to increased mitochondrial mass, but decreased mitochondrial function, including reduced oxygen consumption and loss of mitochondrial membrane potential. Delivery of a mitophagy deficient mutant BNip3 to the BNip3 null liver indicates that defects in lipid metabolism are not simply due to the accumulation of defective mitochondria, but rather BNip3 has another role to regulate lipid metabolism based on nutrient status. Of relevance, BNip3 is epigenetically silenced in the more aggressive and common form of HCC (sub-type A) and thus this work identifies potential mechanisms by which this protein may act as a tumor suppressor.

Citation Format: Michelle L. Boland, He Huang, Ramilla Shah, Almas Ali, Yingming Zhao, Christopher J. Rhodes, Kay F. Macleod. BNip3 connects energy sensing to hepatic lipid metabolism and mitophagy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4324. doi:10.1158/1538-7445.AM2014-4324
Abstract 4332: Diagnostic applications of fatty acid synthase monoclonal antibodies
Authors: Carney Walter P. , Zhang Wendy , Jarosz David , Muraca Patrick , TAM Sunny
Published: May 2015

Fatty acid synthase (FASN), is an enzyme capable of de novo fatty acid synthesis and highly expressed and activated in most human carcinomas. Fatty acid synthase is a multi-enzyme protein that catalyzes fatty acid synthesis and is not a single enzyme but a whole enzymatic system composed of two identical 272 KDa multifunctional polypeptides. Fatty-acid synthesis is now associated with clinically aggressive tumor behavior, tumor-cell growth and is associated with poor prognosis in prostate and breast cancer. Its inhibition is selectively cytotoxic to human cancer cells. Thus, FASN and fatty acid metabolism have become an important focus for the diagnostic and treatment of cancer. We have developed a panel of anti-human FASN Mab for application to several diagnostic platforms. Recombinant full length intact FASN protein (rFASN) was used to immunized C57BL/6 mice, sera were collected from pre- and post-immunized mice and tested by ELISA and Western blot on rFASN protein or rFASN immobilized on Western blot. Spleens from mice with the highest anti-FASN antibody titer was fused with a mouse myeloma cell line Sp2/0-Ag14 for hybridoma production. Among the panel of positive anti-FASN clones selected, two clones with isotype of IgG1kappa have shown strong reactivity to the rFASN and native FASN protein on Western analysis. Upon further Western analyses with cell lysates, we have demonstrated specific detection of FASN protein in human embryonic kidney cells (HEK), HEK cells overexpressing the rFASN protein, HOP-62 cells which express low levels of FASN and MALME cells, which express high levels of FASN. Additional biochemical characterization of these antibodies is ongoing, and includes epitope mapping and reactivity to various FASN protein fragments. These anti-FASN antibodies will be tested by IHC, ELISA to compare tissues expression levels of FAS by IHC or circulating levels of the FASN in both normal and cancer patients. With the increasing development of FAS inhibitors these antibodies will be tested as Companion Diagnostics and tested to see if the rise and fall of the FASN circulating levels correspond with cancer progression or therapy response.

Citation Format: Walter P. Carney, Wendy Zhang, David Jarosz, Patrick Muraca, Sunny TAM. Diagnostic applications of fatty acid synthase monoclonal antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4332. doi:10.1158/1538-7445.AM2014-4332
Abstract 4335: A p53/HIF-1alpha-mediated metabolic response to stress
Authors: Ganapathy Suthakar , Yuan Zhi-Min
Published: May 2015

p53 is a key player in the cellular stress response. Apart from its canonical role in cell checkpoint functions, p53 also regulates cellular metabolism by blocking glycolysis and promoting oxidative phosphorylation. The hypoxia-inducible factor 1(HIF-1), on the other hand, is a master transcription factor promoting glycolysis. We describe a previously unrecognized metabolic response to low levels of stress, including low doses of ionizing radiation or arsenic. By using an integrated approach, we demonstrate that low-level stress induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis leading to increased stress tolerance in both cell and animal models. Low-level stress-induced metabolic changes are mediated by a functional cooperation between p53 and HIF-1α. Our work provides a novel metabolic mechanism of the stress response.

Citation Format: Suthakar Ganapathy, Zhi-Min Yuan. A p53/HIF-1alpha-mediated metabolic response to stress. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2014-4335
Abstract 4336: Design of a chamber for studying glucose metabolism by anoxic cancer cells
Authors: Lankelma Jan , Ahmed Mohamed , Nadal Adrià López , Dorsman Josephine C. , Joenje Hans , Westerhoff Hans V.
Published: May 2015

Many tumors contain anoxic areas. In vitro studies of anoxia can easily be hampered by contamination with traces of oxygen. In order to find out how temporal depletion of extracellular glucose effects anoxic cell death in vitro we have developed a chamber in which 50 000-500 000 cells can be exposed to approximately 30 μl of medium, resulting in rapid depletion of oxygen and nutrients. Before the experiments, cancer cells were seeded on a glass plate, which was the lower glass plate after clamping with an upper plate that contained an 80-100 μm thick silicone spacer. The medium of the cavity between the two flat glass plates could be flushed through an inlet and outlet hole in the upper plate, located in the corners of the cavity. Samples were collected from the outlet hole for analysis.

Most cancer cell lines use both anaerobic and aerobic metabolism to generate ATP, resulting in a more rapid depletion of oxygen than of glucose. We found that after 3 hours the glucose was completely consumed by the MCF-7 human breast cancer cells and that overnight exposure resulted in cell death, except in the immediate proximity of the holes, where oxygen could diffuse into the medium. An interesting observation was that the lactate produced was more than double the glucose used from the medium, suggesting that also intracellular glycogen stores had been consumed by the cancer cells.

We are especially interested in using this system to simulate the effect of dietary restriction on individual dormant breast tumors that have not (yet) started angiogenesis. These are hypothesized to contain an outer ring of anoxic cells, that depend on and rapidly consume the lowest concentrations of glucose “at the end of the glucose gradient” from the nearest capillary blood vessel. A temporary decrease of blood glucose may kill these anoxic cells and perhaps affect non-Warburg cells less. The time dependence of cell killing on this decrease will be investigated using this system.

Citation Format: Jan Lankelma, Mohamed Ahmed, Adrià López Nadal, Josephine C. Dorsman, Hans Joenje, Hans V. Westerhoff. Design of a chamber for studying glucose metabolism by anoxic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2014-4336
Abstract 4341: Metabolomic analysis of HER2-positive breast cancer cells
Authors: Sahni Malika K. , Ravindran Resmi K. , Khan Imran H. , Bush Jason A. , Krishnan V. V.
Published: May 2015

Purpose: HER2-positive breast cancer accounts for more than 20% of the diagnosed cases and is characterized by aggressive growth, increased disease recurrence, and poor prognosis. While much of the signaling mechanism(s) have been elucidated, the impact of HER2 positivity on cellular metabolism is not well understood. We examined (1) gene expression profiles of mammalian breast cancer cell lines with increasing HER2 expression levels (MCF10A, MDA-MB-468, MDA-MB-453, HCC1954, HCC2218) and correlated changes to metabolic expression profiles and (2) the effect of serum starvation and pervanadate treatment on the metabolic profiles of these HER2-expressing cell lines.

Methods: Our study used a combination of 1H-NMR and 2D-NMR (TOCSY), bioinformatics analyses, and protein expression validation in human breast cancer cell lines (untreated, serum-starved, serum-starved with pervanadate treatment).

Results: We found that both gene and metabolic expression profiles of HER2-positive cell lines correlated with leucine and isoleucine biosynthesis (p-value < 0.05). Comparative analyses of profiles from untreated, serum-starved, and serum-starved with pervanadate-treated cell samples revealed that lactate concentration (p-value <0.05) dropped significantly with serum starvation and pervanadate treatment consistent with reduced glycolytic metabolism and potentially mediated through suppression of tyrosine phosphatase activity.

Conclusion: This study highlights important new metabolites that are potential biomarkers of HER2-positive breast cancer and suggests alternate/novel pathways to consider for future understanding and treatment.

Citation Format: Malika K. Sahni, Resmi K. Ravindran, Imran H. Khan, Jason A. Bush, V. V. Krishnan. Metabolomic analysis of HER2-positive breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4341. doi:10.1158/1538-7445.AM2014-4341
Abstract 4344: Tumor suppressive miR-137 targets Aurora-A and leads to apoptosis in colorectal cancer cells
Authors: Huang Yu-Chuan , Hung Liang-Yi
Published: May 2015

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and the third leading cause of death in the world. Early diagnosis, early therapy, finding of new biomarkers and specific therapeutic targets are urgent necessities for CRC patients. Previous reports indicated that loss of miR-137 expression, which is a potential tumor suppressor microRNA and always loses its expression in cancer cells, may contribute to the progression of CRC. However, the detailed underlying mechanism is still unclear. Here, we demonstrated that Aurora-A, a centrosomal serine/threonine kinase that plays essential roles in regulating various important events during cell cycle progression, is one of the targets of miR-137. By luciferase reporter assay, Q-PCR, and Western blot analysis, we demonstrated that miR-137 can target Aurora-A and inhibit its expression. Overexpression of miR-137 results in cell apoptosis in colorectal cancer cells. Expression of miR-137 and Aurora-A in colorectal cancer tissues and colon polyps, the pre-lesion colon tissue, is negative correlated. In addition, the down-regulated miR-137 expression in colon polyps is positive correlated with its size. The treatment of cells with methyltransferase inhibitor 5-Azacytidine induces the expression of miR-137. Our results suggest that losses of miR-137 expression may contribute to the early stage neoplasia in CRC progression. The investigation of the regulatory mechanism of miR-137 mediated Aurora-A inhibition may provide a novel therapeutic strategy for CRC.

Citation Format: Yu-Chuan Huang, Liang-Yi Hung. Tumor suppressive miR-137 targets Aurora-A and leads to apoptosis in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2014-4344
Abstract 4347: Interphase reduction in ZEB1 and tubulin isotypes in breast cancer cells associated with antimitotic drug treatment
Authors: Lobert Sharon , Graichen Mary E.
Published: May 2015

We recently demonstrated that paclitaxel treatment (10X IC50) reduces mRNA for the epithelial to mesenchymal protein ZEB1 and β-tubulin isotype classes I, III and IVB in MDA-MB-231 breast cancer cells, commonly used as a model for triple-negative breast cancers [Lobert et al., Biochemistry Aug 13;52(32):5482-9]. This work showed that miR-200c, which regulates ZEB1, contributes to the changes in β-tubulin isotype mRNA. The current question is whether these parallel changes in ZEB1 and β-tubulin isotype mRNA were unique to antimitotic drug treatment. We treated MDA-MB-231 cells with equipotent (3X IC50) concentrations of paclitaxel, vincristine, eribulin or doxorubicin for 24 hours. Flow cytometry demonstrated that 27-30% of cells were in G2/M (paclitaxel, vincristine and eribulin) compared to 25% of control cells. Thus the cells had similar cell cycle profiles and were not blocked in mitosis. At 24 hours, there were no measurable changes in total tubulin protein by Western blotting. As shown previously, paclitaxel treatment significantly reduced ZEB1 and β-tubulin isotype classes I, III and IVB mRNA. Eribulin treatment significantly reduced ZEB1 and β-tubulin isotype classes I and IVB mRNA, but not β-tubulin class III. Vincristine treatment only reduced β-tubulin class I mRNA. These data together indicate that although antimitotic drugs all target tubulin, they alter signaling pathways that do not overlap. Doxorubicin did not reduce ZEB1 or β-tubulin isotypes; nor did it increase levels of miR-200c, suggesting that these changes may require tubulin or microtubules as a mediator of the effects. These data show that signaling during interphase is altered by antimitotic drug treatment, leading to changes in β-tubulin isotype mRNA levels. A reduction in β-tubulin isotype class III mRNA in interphase cells is associated specifically with paclitaxel treatment. Next generation sequencing for micro-RNAs comparing control cells to those treated with paclitaxel under the conditions described above suggests that nineteen micro-RNAs are either up or downregulated, indicating that the efficacy of paclitaxel may in part be due to micro-RNA regulation of mRNA. (supported by funding from UMMC School of Nursing)

Note: This abstract was not presented at the meeting.

Citation Format: Sharon Lobert, Mary E. Graichen. Interphase reduction in ZEB1 and tubulin isotypes in breast cancer cells associated with antimitotic drug treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4347. doi:10.1158/1538-7445.AM2014-4347