bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5377: Intragenic DNA methylation and increased gene expression: New directions from pan-cancer analysis
Authors: McGuire Michael H. , Herbrich Shelley , Wu Sherry , Wang Ying , Rupaimoole Rajesha , Yao Hui , Gharpure Kshipra , Lopez-Berestein Gabriel , Baggerly Keith , Sood Anil
Published: July 2017

Purpose: While the impact of DNA methylation at the promoter region as a repressive influence is well-known, the vast majority of DNA methylation actually occurs outside of the promoter region. The function of intragenic DNA methylation remains incompletely understood. Here, we investigate how intragenic DNA methylation impacts gene expression, and how these modifications differ between normal and corresponding cancer tissues.

Methods: A pan-cancer analysis of gene expression array data was integrated with Illumina 450k methylation array data across ten tumor types to produce correlation values between DNA methylation and gene expression at distinct loci on a genome-wide basis. The probes exhibiting a significant correlation between gene expression and DNA methylation were selected for further analysis.

Results: We uncovered robust positive correlations between gene expression and 3’ methylation in 3,200 genes, with 590 genes in bladder cancer, and 256 genes in squamous cell lung carcinoma displaying a correlation coefficient of >0.5. Furthermore, we observed 44 genes with a correlation coefficient of >0.7 in at least two tissue types. The majority of genes with the strongest correlations are transcription factors known to play roles in differentiation and development, with an enrichment of zinc finger and homeobox-containing genes. Further analysis of these genes exhibit divergent 3’

methylation when comparing normal and tumor tissues. Importantly, the extent of 3’ methylation of these genes is associated with patient overall survival in 5 of the 10 tumor types analyzed, strongly suggesting that this process plays a role in cancer pathogenesis.

Conclusion: DNA methylation of the 3’ region is a functionally and clinically relevant epigenetic modification, and may serve as a novel target for inhibiting tumorigenesis and tumor progression.

Citation Format: Michael H. McGuire, Shelley Herbrich, Sherry Wu, Ying Wang, Rajesha Rupaimoole, Hui Yao, Kshipra Gharpure, Gabriel Lopez-Berestein, Keith Baggerly, Anil Sood. Intragenic DNA methylation and increased gene expression: New directions from pan-cancer analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5377. doi:10.1158/1538-7445.AM2017-5377
Abstract 5380: Inactivation of endogenous genes in cancer cells using targeted promoter DNA methylation via CRISPR-DNMT3a fusion protein
Authors: Spivack Simon D.
Published: July 2017

Background: Developing technologies for precise manipulation of individual DNA methylation loci is an attractive challenge in cancer and lung biology, because aberrant expression via hyper- or hypo-methylation is so common. Projects such as ENCODE and the Roadmap Epigenetics Project, have identified thousands of epigenetic marks from human genome. However, the functional evaluation of these marks has been largely limited to determining their associations with gene expression. Technologies for targeting manipulation of epigenetic marks would allow direct experimental testing of the impact of DNA methylation at specific residues, mechanisms of gene regulation, and potential use as interventions, for example to silence constitutively active oncogenes.

Methods: Here, we developed a CRISPR Cas9-based tool for specific DNA methylation in which the catalytic domain of DNMT3a (DNMT3a-CD) is fused to the carboxy-terminus of Cas9 D10A-H840A mutant (dCas9). Both construct promoter strength and transfection strategies were optimized.

Results: We demonstrated targeted and consistent CpG methylation in 30~100bp regions downstream of the PAM (binding) site of the gRNA guided fusion protein in HEK293 cells. The multiple guide RNAs could target the dCas9-DNMT3A to multiple sites consistently. DNA methylation activity was specific for the targeted region and was heritable across cell divisions. We also found directed promoter DNA methylation of DAL1, GATA5 and C-MYC could decrease the corresponding mRNA expression by up to 80%; different DNA methylation positions had consequently different effects on gene expression. We have now performed C-MYC methylation-mediated knockdown in A549 lung cancer cells and are evaluating resulting cell cycle and apoptosis phenotypes.

Conclusion: We believe this new targeted epigenetic technology can be directed to cancer cells for the purpose of reprogramming lung cancer, and unregulated stromal and microenvironment cells, for preventive and therapeutic uses.

Note: This abstract was not presented at the meeting.

Citation Format: Simon D. Spivack. Inactivation of endogenous genes in cancer cells using targeted promoter DNA methylation via CRISPR-DNMT3a fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2017-5380
Abstract 5385: Colorectal cancer methylome and laterality
Authors: Hirabayashi Sho , Hayashi Masamichi , Nakayama Goro , Kurimoto Keisuke , Tanabe Hiroshi , Kanda Mitsuro , Takami Hideki , Niwa Yukiko , Iwata Naoki , Kobayashi Daisuke , Tanaka Chie , Yamada Suguru , Sugimoto Hiroyuki , Koike Masahiko , Fujii Tsutomu , Fujiwara Michitaka , Kodera Yasuhiro
Published: July 2017

Background: Colorectal cancers are anatomically derived from Midgut area (from cecum to mid transverse colon) and Hindgut area (from mid transverse to anal canal). The laterality of colorectal cancers was reported as one of the prognosis markers of these cancers. Midgut originated cancers had statistically poorer overall survivals than Hind gut (Yahagi et al, J Gastrointest Surg, 2016). In addition to clinical reasons, for example its symptomless nature of the bowel obstruction on the right side, the difference of genetic and/or epigenetic profiles between both side colorectal cancers may exists.

Methods: Surgically resected 30 colorectal cancers (Midgut n=9, Hidgut n=21) were included. Any clinicopathological factors except for tumor site was not statistically different between 2 groups. We decided to focus on colon cancer methylome and laterality in this study. Two novel methylation markers of colon cancers (PAX5 and VGF) which were extracted from microarray analysis were used, and quantitative methylation-specific PCR (QMSP) assay was performed for each marker.Results: Both PAX5 and VGF methylations were extremely tumor-specific markers (P<0.001 and P=0.011). Interestingly, Both markers tended to have high methylation frequency in Midgut derived cancers. High frequency of PAX5 promoter methylation was found in 9/9 cases (100%) of Midgut and 15/21 cases (71%) of Hindgut (P=0.141), while that of VGF was found in 6/9 cases (67%) of Midgut and 4/21 cases (19%) of Hindgut (P=0.030). In addition, three multiple colon cancers were found in the cohort. All of them were derived from Midgut area (T1/T1, T3/Tis and T3/Tis), and had high frequency of both PAX5 (2/3, 67%) and VGF (3/3, 100%) methylation.

Conclusion: Cancer methylome between Midgut and Hindgut seemed to be different, and it may affect tumor malignancy, plurality and chemotherapy sensitivity. Now we are in progress of increased colorectal cancer cases (over 100) and methylation markers.

Citation Format: Sho Hirabayashi, Masamichi Hayashi, Goro Nakayama, Keisuke Kurimoto, Hiroshi Tanabe, Mitsuro Kanda, Hideki Takami, Yukiko Niwa, Naoki Iwata, Daisuke Kobayashi, Chie Tanaka, Suguru Yamada, Hiroyuki Sugimoto, Masahiko Koike, Tsutomu Fujii, Michitaka Fujiwara, Yasuhiro Kodera. Colorectal cancer methylome and laterality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5385. doi:10.1158/1538-7445.AM2017-5385
Abstract 539: MAEL promotes colorectal cancer cell growth and migration by activating EGFR pathway
Authors: Liu Lulu , Tong Zhou , Jiang Weiqin , Zheng Yi , Zhao Peng , Fang Weijia
Published: July 2017

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system. It is generally accepted that CRC pathogenesis is a long-term process involving accumulation of multiple genetic alterations. Herein, we aim to explore the molecular biological mechanisms of CRC initiation and progression. We have been committed to investigating MAEL function in digestive neoplasms and have found MAEL is a new candidate oncogene. Previously, overexpression of MAEL was frequently detected in CRC tissues and significantly associated with poor 5-year outcome. By MTT assay and transwell assay, functional study demonstrated that overexpression of MAEL promoted cell growth and cell migration in DLD1 and THC8307 cell lines. Furthermore overexpression of MAEL in DLD1 cell line could promote tumor formation in nude mice. Further study found that MAEL overexpression in DLD1 and THC8307 elevated the protein expression of p-EGFR, p-Raf-1, p-ERK 1/2, p-PI3K and p-AKT. These data indicated that MAEL enhanced the activity of EGFR/Ras/Raf/MEK/ERK1/2 and EGFR/PI3K/Akt pathways. However, more effort is needed to investigate MAEL function and how MAEL activate EGFR pathways.

Citation Format: Lulu Liu, Zhou Tong, Weiqin Jiang, Yi Zheng, Peng Zhao, Weijia Fang. MAEL promotes colorectal cancer cell growth and migration by activating EGFR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 539. doi:10.1158/1538-7445.AM2017-539
Abstract 5390: Broad Genomics and Cancer Program development of comprehensive blood biopsy sequencing capabilities to support direct-to-patient cancer research initiatives
Authors: Cibulskis Carrie , Hollinger Andrew
Published: July 2017

Genomic profiling of cell free tumor DNA (Blood Biopsy) offers the potential to revolutionize cancer precision medicine. As a proxy for tumor tissue profiling, successful blood biopsy analysis can help select appropriate patients for clinical trials, provide useful data for treatment monitoring, and discover genomic mechanisms of disease resistance. Recognizing these potential benefits, we aim to enable large-scale genomic data generation and analysis of cfDNA samples to support the patient driven Metastatic Breast Cancer Project (mbcproject.org) and many other such efforts. The widespread adoption of cfDNA sequencing technology in clinical research will require low cost, efficient, and in some cases clinically-validated processes. Commercial offerings in this field are focused on analysis of small targeted hotspot panels due to the challenges of more complete sequencing approaches for cfDNA (e.g. variability in the fraction of tumor present and high false positive rates). Working in collaboration with the Broad Institute Cancer Program, we have developed a process to overcome these challenges and provide high quality comprehensive exome analysis of cfDNA. Our process includes screening samples using ultra low pass, UMI-enabled whole genome sequencing to quantify the % of tumor DNA available for sequencing. Qualified samples are then moved into automated processes for exome sequencing and somatic mutation detection. In our initial validation study of 23 breast and prostate cancer patients with matched cfDNA and tumor tissue sequencing, we are able to identify 90% of clonal and 45% of subclonal mutations found in the matched tumor biopsy. In our effort to scale this process, and fully characterize the benefits and limitations of this technology we will present additional validation analysis. We will evaluate the relative importance of using paired sample analysis (tumors and matched normals) with analysis of tumor sequencing alone and assess sensitivity and specificity of somatic alterations as a function of tumor fraction and sequencing depth.

Citation Format: Carrie Cibulskis, Andrew Hollinger. Broad Genomics and Cancer Program development of comprehensive blood biopsy sequencing capabilities to support direct-to-patient cancer research initiatives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5390. doi:10.1158/1538-7445.AM2017-5390
Abstract 5396: An NGS workflow to detect down to 0.1% allelic frequency in cfDNA for breast and colon cancers
Authors: Dhingra Dalia , Chien Richard , Gu Jian , Brinza Dumitru , Chaudhary Ruchi , Banjara Kunal , Li Yanchun , Ballesteros-Villagrana Efren , Bramlett Kelli
Published: July 2017

Noninvasive detection of rare mutations in blood could allow tumor monitoring for research purposes. Research studies have suggested that cfDNA contains DNA from tumor cells with somatic mutations that could inform on tumor progression and therapeutic resistance. Here, we demonstrate a complete workflow from a single tube of blood through data analysis for research samples down to a 0.1% allelic frequency.

The low abundance tumor mutations found in cfDNA requires sensitive and accurate mutation detection. We have developed two panels that utilize an amplification-based assay that generates tagged DNA copies, which allows detection of low abundance tumor mutations found in cfDNA. The two panels allow multiplex interrogation of primary driver and resistance mutations specific to ctDNA from breast and colon cancer. The Oncomine Colon cfDNA panel targets 236 hotspots within 14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in 10 genes. This workflow was validated from matched single blood tubes, Streck and K2EDTA. Additionally, the utility for cancer research was demonstrated with concordance studies using matched FFPE and plasma from oncology samples.

To further characterize these panels we have developed an oncology control for cfDNA with nucleosome fragment sizing and minimal sonication damage. This engineered control contains SNPs and indels at 0.1% allelic frequencies, orthogonally confirmed with TaqMan based Rare Mutation assays. With this control, the Oncomine Breast cfDNA panel had over 81% sensitivity and 99.9% specificity. The Oncomine Colon cfDNA panel had over 85% sensitivity and 100% specificity. The Oncomine Breast cfDNA panel and Oncomine Colon cfDNA panel integrated into a complete workflow starting from a single tube of blood can advance oncology research with the ability to detect blood based cancer biomarkers present at 0.1%.

Citation Format: Dalia Dhingra, Richard Chien, Jian Gu, Dumitru Brinza, Ruchi Chaudhary, Kunal Banjara, Yanchun Li, Efren Ballesteros-Villagrana, Kelli Bramlett. An NGS workflow to detect down to 0.1% allelic frequency in cfDNA for breast and colon cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5396. doi:10.1158/1538-7445.AM2017-5396
Abstract 5398: High-throughput clonal analysis of AML tumors with droplet microfluidics
Authors: Eastburn Dennis , Pellegrino Maurizio , Treusch Sebastian , Sciambi Adam , Hyun Bill , Yates Jamie
Published: July 2017

Single cell analysis tools are crucial to better understand the role that rare or heterogeneous cancer cells play in the evolution of tumor progression. Although, it is now feasible to perform single-cell RNA-Seq on thousands of, several challenges remain for high-throughput single-cell DNA sequencing. To address these challenges and enable the characterization of genetic diversity in cancer cell populations, we developed a novel approach that barcodes amplified genomic DNA of individual cells confined to microfluidic droplets. The barcodes are used to reassemble the genetic profiles of individual cells from next generation sequencing data. A key feature of our approach is the “two-step” microfluidic workflow that releases genomic DNA from cellular proteins prior to amplification. The microfluidic workflow first encapsulates individual cells in droplets, lyses the cells and prepares the lysate for genomic DNA amplification using proteases. Following this lysate preparation step, the proteases are inactivated and droplets containing the genomes of individual cells are then paired with molecular barcodes and PCR reagents. We demonstrate that the two-step microfluidic approach is vastly superior to workflows without the two-step process for efficient DNA amplification on tens of thousands of individual cells per run with high coverage uniformity and low allelic dropout of targeted genomic regions. To apply our single-cell sequencing technology to the study of acute myeloid leukemia (AML), we developed a targeted panel to sequence genes frequently mutated in AML including TP53, DNMT3A, FLT3, NPM1, NRAS, KRAS, JAK2, IDH1 and IDH2. Using this panel, we were able to identify clonal populations from AML research samples; moreover, the single-cell nature of our approach enabled the correlation of multiple mutations within subclones and determination of whether the mutations existed as as a homozygote or heterozygote. Collectively, our results show a greater degree of heterogeneity in AML tumor samples than is commonly appreciated with bulk sequencing methods.

Citation Format: Dennis Eastburn, Maurizio Pellegrino, Sebastian Treusch, Adam Sciambi, Bill Hyun, Jamie Yates. High-throughput clonal analysis of AML tumors with droplet microfluidics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5398. doi:10.1158/1538-7445.AM2017-5398
Abstract 54: The many faces of antibody in G protein-coupled receptor biology
Authors: Chen Lei , Cheung Wing-Tai
Published: July 2017

G protein-coupled receptors (GPCRs), the largest family of cell-surface receptor proteins mediating signal transmission, play a pivotal role in many physiological functions and are involved in multiple diseases. Although receptor activities of GPCRs are successfully modulated by many small molecules which represent 30% of all marketed drugs today, in fact only a few GPCR members are targeted and many more GPCRs of interest are intractable targets of small molecules, like orphan GPCRs and GPCRs with large binding sites. Besides, small molecule drugs rarely trigger GPCR-mediated apoptosis or induce direct cell killing, which is crucial for cancer therapies. Therefore, for these GPCRs, antibody-based drug would be a good alternative. Despite being eagerly sought for, the production of monoclonal antibody (mAb) targeting GPCR is hindered by the low expression of recombinant GPCR on cell surface, and relatively small exposed regions with glycosylation and conformational heterogeneity. Therefore those conventional methods that were well established for generation of mAbs targeting soluble proteins usually showed frustrating results in the case of GPCRs. To bypass the production and purification of recombinant GPCR proteins, we developed a novel approach of transplanting immunogenic conformational epitopes of GPCR into an antibody scaffold to make a water soluble surrogate antigen of GPCR, named as GPCR-antigenized antibody which can be easily and abundantly produced in bacteria for animal immunization. In coupled with phage display technology and cell panning strategy, which allow the retrieval of specific binders from a huge number of candidates, we have successfully isolated scFv antibodies for two human GPCRs, the MAS1 receptor and the chemokine receptor CXCR4. These antibodies showed good specificity in immunofluorescent staining, flow cytometry analysis, western protein immunoblot and immunoprecipitation assay. The results demonstrated this novel approach may offer a generic and effective method to generate specific monoclonal antibody targeting GPCR for diagnostic and therapeutic applications. [The project is partly funded by a CUHK direct grant (4054300)]

Citation Format: Lei Chen, Wing-Tai Cheung. The many faces of antibody in G protein-coupled receptor biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2017-54
Abstract 5407: Integrated analysis of genome-wide microRNA and mRNA profiling reveals unregulated miR-125b associated with poor prognosis by reducing cell apoptosis in gastric cancer
Authors: Liu Ben , Yang Da , Song Fengju , Zhang Xining , Guo Yan , Zhao Yanrui , Zheng Hong , Zhang Wei , Chen Kexin
Published: July 2017

Background & Aims: In our prior study, an integrated analysis of microRNA and mRNA profiling of gastric cancer (GC) revealed a mRNA-regulatory network. It defined a GC microRNA subtype that was associated with poor survival in 90 GC cases. In this study, we further demonstrate that miR-125b, a key node in this microRNA regulatory network, is upregulated in GC and associated with poor overall survival by interfering in the apoptosis pathway.

Methods: To further investigate the role of miR-125b in GC, we conducted both an in vitro experiment and clinical study (cohort of 373 GC samples). miR-125b and targets in the network were validated through RT-PCR and tissue microarray GC cases. The miR-125b targeted gene and related pathway were identified by a gene expression profile analysis in miR-125b transfected GC cell lines. The Annexin V-FITC and TUNEL assay were used to assess apoptosis. MTT and flow cytometry were performed to detect the effect of miR-125b on proliferation and cell cycle. The wound healing test and transwell assay were used to analyze migration and invasion.

Results: The markly upregulated expression of miR-125b was validated in 373 GC samples.. Univariate and multivariate analyses find that the miR-125b was associated with poor prognosis (p=0. 011). Pathway analysis shows that the predicted targets of miR-125b are highly involved in apoptosis/program death pathway. The robust apoptosis genes, BIK and CASP6 are validated as the directed targets of miR-125b. miR-125b can suppress apoptosis and increas cell growth, migration and invasion in GC cell lines. Furthermore, low expression of BIK (p=0. 034) and CASP6 (p=0.048) in GC were associated with poor disease-free survival.

Conclusions: The apoptosis pathway was found to be dominant in the pathway construction based on target genes of miR-125b. Our findings suggest that miR-125b-regulated apoptosis pathway is important to GC progression and that the miR-125 may be an important clinical biomarker for GC survival and possible therapeutic target for GC treatment.

Citation Format: Ben Liu, Da Yang, Fengju Song, Xining Zhang, Yan Guo, Yanrui Zhao, Hong Zheng, Wei Zhang, Kexin Chen. Integrated analysis of genome-wide microRNA and mRNA profiling reveals unregulated miR-125b associated with poor prognosis by reducing cell apoptosis in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5407. doi:10.1158/1538-7445.AM2017-5407
Abstract 5411: Demonstrated improvements in tumor profiling with enzymatic ribosomal RNA depletion and streamlined RNA-sequencing library preparation
Authors: Nabilsi Nancy H. , Cheney Drew , Cloete Leendert , Jaarsveld Ida van , Kasinskas Rachel , Pavlica Jennifer
Published: July 2017

High resolution RNA analysis using next-generation sequencing (RNA-seq) is a rapidly growing application in cancer research. Formalin-fixed paraffin-embedded (FFPE) tissue is a ubiquitous resource for clinical studies. However, the ability to generate high quality libraries is confounded by the low quality and yield of RNA extracted from FFPE specimen. To enable optimal interrogation of such a clinically-relevant tissue resource, it is critical to identify RNA-seq library construction workflows that improve performance using challenging and degraded sample types. In this study, the effects of RNA type, quality, and quantity on RNA-seq library construction are assessed, and expectations regarding sequencing data quality are addressed. Included are recommendations for quality control measures and input-specific modifications that may improve performance Three workflows employing either bead-based or enzymatic strategies for the removal of ribosomal RNA content were used to prepare RNA-seq libraries from degraded paired breast normal and tumor samples. Enzymatic depletion followed by library preparation using the KAPA RNA HyperPrep1 workflow identified the highest number of transcripts for each sample, translating to a 4.9-fold increase in number of differentially expressed transcripts detected between normal and tumor samples. Of the transcripts identified as differentially expressed exclusively by Kapa, 174 are protein-coding RNAs, many of which are frequently dysregulated in breast cancer. In contrast, only two protein-coding RNAs were identified exclusively by the alternative workflows. RealTime Ready Custom panels were used for independent quantitative rt-PCR assessment. Of a subset of 71 transcripts identified exclusively by Kapa, up to 69 (97%) were validated as differentially expressed. Additional performance improvements of the KAPA RNA HyperPrep workflow include coverage balance across transcripts, lower duplication rate, technical replicate reproducibility, agreement across input quantities, and gene expression concordance between paired fresh frozen and FFPE samples. 1For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Nancy H. Nabilsi, Drew Cheney, Leendert Cloete, Ida van Jaarsveld, Rachel Kasinskas, Jennifer Pavlica. Demonstrated improvements in tumor profiling with enzymatic ribosomal RNA depletion and streamlined RNA-sequencing library preparation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5411. doi:10.1158/1538-7445.AM2017-5411
Abstract 5412: Expression and inhibition of monocarboxylate transporters in canine osteosarcoma
Authors: Gardner Heather L. , Fenger Joelle M. , Sandanayaka Vincent P. , London Cheryl A.
Published: July 2017

Background: A distinguishing feature of cancer cells is their ability to undergo aerobic glycolysis, even in the presence of oxygen. This process generates lactic acid, which must be removed from the cell in order to maintain proliferation and survival. Monocarboxylate transporters (MCTs) move lactic acid across the plasma membrane, providing a mechanism for tumor cells to meet their bioenergetic needs during glycolysis in a variety of limiting microenvironments. The objective of this work is to determine the biologic effects of modulating cellular metabolism through inhibition of MCT1 and MCT4 in canine osteosarcoma (OS), a well-established spontaneous large animal model of the human disease.

Methods: MCT1 and MCT4 expression was assessed in canine OS cell lines using qRT-PCR and western blotting. Immunohistochemistry and qRT-PCR were performed to determine relative MCT1 and MCT4 expression in primary canine OS tissues. The effects of small molecule inhibitors (NGY066 and NGY008) and shRNA approaches targeting MCT1 and MCT4 on proliferation and survival of canine OS were examined using the CyQUANT Cell Proliferation and Caspase-3/7 assays, respectively. The impact of MCT1/4 inhibition on cellular oxygen consumption was evaluated using the Seahorse Cell Mito Stress Test. Lastly, potential synergistic effects of combining metformin and doxorubicin with MCT1/4 inhibition in canine OS cell lines were investigated using the CyQUANT Assay.

Results: We found that while both MCT1 and MCT4 are expressed in canine OS lines and tissues, MCT1 is expressed at higher levels relative to MCT4 in the cell lines. Small molecule inhibitors directed against MCT1 and MCT4 did not significantly inhibit cell proliferation or induce apoptosis even in the presence of limiting cell culture conditions. Maximal respiratory capacity was not increased in canine OS cell lines following inhibition of MCT1 and MCT4 function. However, synergistic anti-proliferative activity was observed when OS cells were treated with doxorubicin or metformin in the presence of MCT1 and MCT4 inhibitors.

Conclusions: MCT1 and MCT4 are expressed in canine OS, and therefore represent a good model to evaluate the therapeutic potential of manipulating lactic acid transport. Importantly, drug combinations with synergistic activity may be necessary to realize the full potential of MCT1/4 inhibition.

Citation Format: Heather L. Gardner, Joelle M. Fenger, Vincent P. Sandanayaka, Cheryl A. London. Expression and inhibition of monocarboxylate transporters in canine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5412. doi:10.1158/1538-7445.AM2017-5412
Abstract 5416: Downregulation of PRMT6 promotes the Warburg effect in hepatocellular carcinoma via PKM2
Authors: Wong Tin Lok , Chan Lok Hei , Ma Stephanie
Published: July 2017

Hepatocellular carcinoma (HCC) is one of the top ranked causes of cancer death in the world, accounting for more than 740,000 deaths in 2012. Treatment of HCC is hindered by recurrence and resistance to chemotherapy. Altered metabolism in cancer has been linked to increased tumorigenicity and resistance to chemotherapeutics. One of the main features of altered metabolism is the Warburg effect, which is widely observed in solid tumor including HCC. Recently, our group have identified protein arginine methyltransferase 6 (PRMT6) as a tumor suppressor protein in HCC. Protein arginine methylation is a post-translational modification implicated in a variety of cellular functions including signaling and regulation of gene expression.Downregulation of PRMT6 expression was observed in HCC patients and knockdown of PRMT6 in HCC cell lines increased the tumorigenicity and sensitivity to both chemo- and molecular-targeted therapies. Other members of the Type 1 PRMT protein has previously been reported to be involved in glucose metabolism. Microarray profiling of HCC cells stably knockdown and overexpressed of PRMT6 showed enrichment of genes in glycolysis. We therefore hypothesize that reduced expression of PRMT6 in HCC drives the Warburg effect to support cancer progression. Metabolome profiling of HCC cells stably knockdown and overexpressed of PRMT6 showed enrichment of metabolites in glycolysis. Using Seahorse Extracellular Flux Analyzer, we show that stable knockdown of PRMT6 in HCC cell line drives glycolysis. This enhanced glycolysis rate is coupled with increased uptake of glucose, lactate production and pyruvate kinase activity. mRNA expression level of several glycolysis-related genes was screened by real-time quantitative PCR and we identified PKM2, a well-known regulator of the Warburg effect, as a potential link between PRMT6 and enhanced glycolysis. Knockdown of PRMT6 leads to an increase in mRNA and protein expression level of PKM2, but not PKM1 and PKLR, suggesting that PKM2 is the sole contributor for enhanced pyruvate kinase activity. In addition, by immunofluorescence staining, PKM2 is shown to accumulate in the nucleus following PRMT6 knockdown, where it has been shown to drive the expression of other glycolysis-related genes. In conclusion, PRMT6 regulates the expression level PKM2 and glycolysis of HCC cell. Ongoing studies are in progress to delineate the underlying mechanisms by which PRMT6 regulates PKM2 to drive Warburg effect.

Citation Format: Tin Lok Wong, Lok Hei Chan, Stephanie Ma. Downregulation of PRMT6 promotes the Warburg effect in hepatocellular carcinoma via PKM2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2017-5416
Abstract 5421: Differential expression of lysosomal acid lipase (LAL) between benign and malignant tissues among human organs
Authors: Gassim Mohamed EI , Mao Jinghe , Ma Tangen , Zhou Xinchun
Published: July 2017

Background: Lysosomal acid lipase (LAL), a lysosomal enzyme is able to degrade cholesteryl esters to free fatty acids and cholesterol. The information on the association of LAL with malignancies is limited. This study is to determine the difference in expression level of LAL between benign and malignant tissues among human organs.

Method: Immunohistochemistry (IHC) for human LAL was performed on A tissue microarray (TMA) containing 360 benign and malignant tissues from 36 human organs. The expression level of LAL was represented by an IHC score calculated by multiplying area score (0-3) with intensity score (0-3), ranged from 0 to 9. Student’s t-test was used to compare the mean IHC score between benign and malignant tissues in same organs.

Results: Eighteen out of 36 human organs have IHC stains on both benign and malignant tissues. As shown in Table 1, LAL expression level was higher in malignancy than in benign tissues in 9 organs. The difference in LAL expression level was significantly (or nearly lower in malignant tissues of adrenal gland, stomach, liver, testis and kidney.

Conclusion: The expression level of LAL varies among human organs. As compared with elevation of LAL expression level, decreased expression level of LAL in malignancies could be of more significance.

Table 1Difference in expression level of LALOrganmalignantBenignpM/BnMeanSEnMeanSEvalueRatioAdrenal Gland31.670.73560.950.02-1.36Stomach131.170.4583.130.820.035-2.7Liver131.350.3483.560.490.001-2.6Larynx80.250.0940.630.380.22-2.5Testis41.630.4743.750.750.054-2.3Uterus131.620.4272.570.970.302-1.6Kidney122.920.3134.50.870.051-1.5Prostate152.970.51441.22.389-1.3Urinary Bladder110.680.1750.70.30.9561Cervix41.630.4751.250.560.6361.3Colon162.340.3991.50.280.1451.6Esophagus41.630.992100.6951.6Ovary43.631.0741.940.930.2781.9Skin83.591.3251.850.720.3491.9Breast142.860.5861.380.550.1392.1Pancreas101.80.5590.830.140.1262.2Brain32.170.4450.80.560.144`2.7Lung81.970.7450.600.290.193.3

Citation Format: Mohamed EI Gassim, Jinghe Mao, Tangen Ma, Xinchun Zhou. Differential expression of lysosomal acid lipase (LAL) between benign and malignant tissues among human organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5421. doi:10.1158/1538-7445.AM2017-5421
Abstract 5426: LPA stimulates glycolytic shift in ovarian cancer
Authors: Ha Ji Hee , Radhakrishnan Rangasudhagar , Ward Jeremy D. , Jayaraman Muralidharan , Dhanasekaran Danny N.
Published: July 2017

Cancer cells resort to aerobic glycolysis to meet the increased energy consumption and growth requirement during active proliferation. While it has been observed that the cancer cells preferentially shift to aerobic glycolysis, the extraneous growth factors involved in this process is largely unknown. Since our previous studies have shown that lysophosphatidic acid (LPA) plays a critical role in the pathophysiology of ovarian cancers, we investigated the role of LPA in the metabolic reprogramming of ovarian cancer cells. Our results using XFe96 analyzer indicate that LPA stimulates aerobic glycolysis in multiple ovarian cancer cell lines as well as in patient-derived ovarian cancer cells. Physiological role for LPA in this process is demonstrated by the observation that the inhibitors of LPA-receptors attenuate the glycolytic shift stimulated by the ascitic fluid derived cancer patients. We further demonstrate that the silencing Gαi2, a downstream signaling mediator for LPA, inhibits LPA-stimulated glycolytic shift in ovarian cancer cells. Further analyses indicated that LPA-stimulated metabolic reprogramming involves the generation of reactive oxygen species (ROS). Treatment of cells with N-Acetyl Cysteine (NAC), a ROS-scavenger, inhibited the glycolytic shift stimulated by LPA. In addition, our studies point to a role for LPA-stimulated HIF1α activation in this process. These results indicate that LPA-stimulated metabolic reprogramming in ovarian cancer cells involve ROS-generation via LPA-Gαi2-HIF1α-dependent signaling pathway. Together with the findings that the glycolytic shift is crucially involved in tumor cell survival our studies point to LPA-Gαi2-HIF1α signaling axis as a potential target for therapy in ovarian cancer.

Citation Format: Ji Hee Ha, Rangasudhagar Radhakrishnan, Jeremy D. Ward, Muralidharan Jayaraman, Danny N. Dhanasekaran. LPA stimulates glycolytic shift in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5426. doi:10.1158/1538-7445.AM2017-5426
Abstract 5431: Androgen receptor regulates the mitochondrial pyruvate carrier to fuel oncometabolism in prostate cancer
Authors: Bader David A. , Putluri Nagireddy , Hartig Sean M. , McGuire Sean E.
Published: July 2017

The metabolic underpinnings of androgen receptor (AR)-driven growth in prostate cancer (PCa) are underexplored, hindering the development of strategies to leverage the metabolic properties differentiating PCa from normal tissue. To this end, we discovered a subunit of the mitochondrial pyruvate carrier (MPC), MPC2, is a direct AR target gene that is increased in PCa specimens and associated with clinical outcomes. Our observation suggests a targetable link between AR signaling and carbon trafficking in PCa. Indeed, MPC inhibition delays proliferation and alters metabolic properties in hormone sensitive and castrate resistant AR-driven PCa models. Next, using 13C metabolic flux analysis coupled with a reverse phase protein array, we determined MPC inhibition results in activation of the eIF2α/ATF4 integrated stress response pathway to partially compensate for MPC inhibition by coordinating increased glutamine incorporation into the citric acid cycle. Therefore, to amplify the effect of MPC inhibition, we restricted glutamine availability during MPC inhibition and found this combination resulted in proliferative arrest. Last, we extended our efforts in-vivo and demonstrated combinatorial MPC/glutaminase inhibition resulted in a significant reduction in castrate-resistant prostate tumor xenograft growth in mice with no overt host toxicity. We anticipate our findings will accelerate the development of clinical strategies to therapeutically manipulate carbon flux to starve clinically lethal castrate-resistant PCa.

Citation Format: David A. Bader, Nagireddy Putluri, Sean M. Hartig, Sean E. McGuire. Androgen receptor regulates the mitochondrial pyruvate carrier to fuel oncometabolism in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5431. doi:10.1158/1538-7445.AM2017-5431
Abstract 5432: Characterization of metabolic fuel dependency in multidrug resistant breast cancer cells
Authors: Kam Yoonseok , Romero Natalia , Swain Pamela , Dranka Brian P.
Published: July 2017

Multidrug resistance (MDR) is a common resistant mechanism of cancer cells to cytotoxic drugs in systemic therapy. MDR is characterized by increased expression of ATP-dependent drug exporting pumps which remove cytotoxic compounds from the cytosol. However, the mechanism whereby cancer cells rapidly respond to this increased ATP demand is not fully understood although the glycolytic nature of MDR phenotype has been evidenced repeatedly by previous reports. We thus hypothesized that flexibility and dependency on mitochondrial fuels would be altered in concert with the switch to glycolysis. In order to more accurate analyze glycolytic pathway, we newly developed an assay to improve glycolytic rate measurements by accounting for TCA-dependent CO2 contribution to extracellular acidification and to correlate Seahorse extracellular flux data with lactate production. In a comparison of glycolytic rates between MCF7 breast cancer cell line and its MDR variant MCF7/Dox using this new assay, we confirmed that in MCF7/Dox cells glycolysis rate under basal conditions is significantly increased compared to wild type cells. Furthermore, MCF7Dox presents a significant increase in compensatory glycolysis when mitochondrial ATP production is blocked. This metabolic profile switch is accompanied by a decreased dependency on glutamine to fuel mitochondrial respiration an increased tolerance of the MCF7/Dox cells to glutamine deprivation as compared to the MCF7 wild type. In contrast, dependency on glucose and fatty acids mitochondrial-oxidation was largely unchanged in MDR cells. Additionally, an increase in long chain fatty acid oxidation is observed when glucose and glutamine oxidation is blocked indicating that MDR cells has higher mitochondrial flexibility to compensate for inhibition of alternative fuels utilization although it preferentially uses glucose. Together these data demonstrate that acquisition of multidrug resistance in MCF7 cells fundamentally changes their metabolism from a glutamine-driven, oxidative phosphorylation dependent phenotype to a highly glycolytic and glucose-dependent phenotype. These findings have potential therapeutic relevance in the context of inhibition of specific mitochondrial fuel pathways to prevent therapy resistance.

Citation Format: Yoonseok Kam, Natalia Romero, Pamela Swain, Brian P. Dranka. Characterization of metabolic fuel dependency in multidrug resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5432. doi:10.1158/1538-7445.AM2017-5432
Abstract 5433: β-Tubulin inhibitors reduce GLUT1 membrane trafficking to attenuate tumorigenesis in glioblastoma subtypes
Authors: Labak Collin M. , Guda Maheedhara R. , Asuthkar Swapna , Jain Neha , Lu Yining , Purvis Ian , Tuszynski Jack , Nakano Ichiro , Tsung Andrew J. , Velpula Kiran K.
Published: July 2017

Glioblastoma is an aggressive, high-grade tumor with poor prognosis due to lack of sound therapeutic options. Mesenchymal subtype GBMs are particularly difficult to treat because they tend to proliferate in the sub ventricular zone, an area that breeds stem-like neural cells and proves nearly impossible to access for surgical resection. They also contribute to creating a hypoxic microenvironment and switch to glycolytic metabolism in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting the glucose transporter, GLUT1, through cytoskeletal components that traffic it to the cell membrane may reverse the Warburg Effect, attenuating further tumorigenesis and decreasing the stemness of such cells. Datamining studies and immunoblot analysis conducted on human glioblastoma patient specimens (hGBM) demonstrated that GLUT1 is highly upregulated. Limiting dilution assay conducted using GLUT1 inhibitors- fasentin and 2-Deoxy-D-glucose reduced the proliferation in mesenchymal cancer stem cell (CSC) subtype in in vitro culture. Simultaneously, mass spectrometric analysis revealed significant association between GLUT1 and β-tubulin 4 (TUBB4) in mesenchymal subtyped cells. This association was further confirmed by large-sample datamining and in immunoprecipitation studies from hGBM specimens, suggesting that TUBB4 may be a viable target in deterring the trafficking of upregulated GLUT1 to the membrane. Collectively, these studies confirm that GLUT1 is associated with TUBB4, and that targeting TUBB4 via siRNA or colchicine derivatives could prove effective in reversing the Warburg Effect in GBM cells and ultimately improve patient outcomes.

Citation Format: Collin M. Labak, Maheedhara R. Guda, Swapna Asuthkar, Neha Jain, Yining Lu, Ian Purvis, Jack Tuszynski, Ichiro Nakano, Andrew J. Tsung, Kiran K. Velpula. β-Tubulin inhibitors reduce GLUT1 membrane trafficking to attenuate tumorigenesis in glioblastoma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2017-5433
Abstract 5434: Tumor necrosis factor receptor-associated protein 1 (TRAP1) as a potential target for glutamine addicted cancer cells
Authors: Vo Vu T. , Phan Ai N. , Hua Tuyen N. , Jeong Yangsik , Kang Byoung Heon , Kim Hyun-Won , Choi Jong-Whan
Published: July 2017

Glutamine, a non-essential amino acid, is an important nutrient which is involved in many biochemical pathways such as energy production, macromolecular synthesis, and oxidative stress scavenging. Glutamine metabolism is dysregulated in many cancers which mostly display glutamine addiction for cell proliferation. Thus, glutamine metabolism has become a potential target for treating cancer. Here we report that glutamine-dependent cancer cells are more susceptible for inhibiting cell proliferation with inhibitor treatment of Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1), a downstream factor of oncogenic c-Myc involved in glutamine metabolism. Using cell proliferation and cell viability assays, we examined growth inhibitory effects of TRAP1 inhibitor, gamitrinib-triphenylphosphonium (G-TPP), on two groups of cell lines, glutamine-deprivation sensitive versus resistant cell lines. Included are cell lines for each group: HCC827 acquired gefitinib resistance and A549 for the sensitive; HuH7 for the resistant group. Glutamine-deprivation sensitive cell lines showed significant growth inhibition with TRAP1 inhibitor treatment while the corresponding resistant group of cell lines showed no growth inhibitory effects in the same treatment condition. This suggests a potential mechanistic connection of TRAP1 to c-Myc regulation in glutamine metabolism. Taken together, this finding provides a better understanding of oncogene-driven glutamine metabolism as well as evidence that TRAP1 is a promising therapeutic target for glutamine addicted cancer cells.

Citation Format: Vu T. Vo, Ai N. Phan, Tuyen N. Hua, Yangsik Jeong, Byoung Heon Kang, Hyun-Won Kim, Jong-Whan Choi. Tumor necrosis factor receptor-associated protein 1 (TRAP1) as a potential target for glutamine addicted cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5434. doi:10.1158/1538-7445.AM2017-5434