bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2434: Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis
Authors: Chatterjee Aniruddha , McCall John , Guilford Parry , Pattison Sharon , Rodger Euan , Ajithkumar Priyadarshana 
Published: September 2020

Abstract
Colorectal cancer (CRC) is one of the biggest cause of death world-wide and the second-highest cause of death by cancer in New Zealand with a mortality rate of approximately four deaths/day and 90% of CRC-associated mortality is caused by metastasis. Development of metastasis and the modifications that occur during various stages in the CRC metastatic cascade remains obscure. Emerging evidence indicates that epigenetic alterations have a greater influence on the development of metastasis than genetic mutations, but the current understanding of epigenetic drivers that cause CRC metastasis is limited. In the current project, as a first step in identifying epigenetic drivers for metastasis, whole-genome methylation and transcriptome profiles from paired primary and metastatic samples from the same NZ patients will be compared. Subsequently, the epigenetic drivers will be validated, and it can potentially be used as CRC metastatic markers. Circulating tumour cells (CTCs) represent the conduit between primary and metastatic tumours. They could be used to identify driver epigenetic changes associated with epithelial-to-mesenchymal transition (EMT), migration and invasion which occur during the metastatic cascade. CTCs are obtained from the blood and would provide a clinically less invasive test rather than requiring tissue samples. The recent development of single-cell sequencing technologies has now made it feasible to identify epigenetic changes in individual CTCs. In the second part of this project, protocols for single-cell DNA methylation and expression analysis will be developed using human colon cancer cell lines as a model. These optimised protocols will then be applied to CTCs from patient blood samples to identify epigenetic changes that predispose patients to metastasis. Identification of epigenetic drivers will help to detect individuals at high risk for metastasis prior to metastasis being clinically apparent, allowing for more personalised treatment and contribute to improving survival.
Citation Format: Aniruddha Chatterjee, John McCall, Parry Guilford, Sharon Pattison, Euan Rodger, Priyadarshana Ajithkumar. Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2434.
Abstract 2445: Epigenetic modulation of tumor cell tolerance to chemotherapy
Authors: Santos Jephesson Alex Alex dos , Santo Carolina Nunes , Pereira Luiza Cherobini 
Published: September 2020

Abstract
Cancer represents a set of diseases that are dynamic and heterogeneous. Their cells are phenotypically and genetically different from each other and, exhibit stochastic fluctuations in their phenotype. Investigations of isogenic populations of tumor cells reveal that tolerance also may emerge through non-genetic mechanisms, often through stochastic fluctuations in key factors in response to drugs. The objective of this work is to measure the dynamics of chemotherapy tolerance phenotype and modulate these dynamics in tumor cells to potentiate the therapy. Glioma cell lines and primary gliomas were plated at low density and after the formation of colonies, cells were treated with Temozolomide (TMZ) for 3 days. Growth rate of individual colonies was measured and the variance of growth rate of colonies with the same size was used to determine the dynamics of the phenotype. We developed a method to measure the dynamics of phenotype called DynaFit and defined a proportion of phenotypic chance in fitness until generation 6 called rCoDyFF (relative Colony DynaFit Factor 6), which is a value between 0% (stable phenotype) and 100% (dynamic phenotype). The untreated glioma cell lines (U251 and A172) and primary gliomas (LS12 and LS17) had rCoDyFF6 of 40%, 33%, 41%, 33% and treatment with 50uM TMZ produced an rCoDyFF6 67% in U251 cells. However, when cells were treated with a combination of epigenetic modulators and chemotherapy agent, 0.5µM Saha plus 3µM Azacytidine, and 50µM TMZ, the rCoDyFF6 result in - 23%, showing a drastic reduction, indicating stabilization of the phenotype. This treatment also increases sensitivity to TMZ. The result demonstrates that epigenetic modulators were able to induce a phenotypically stable state in U251 cells, leading to the copy of the tolerance phenotype of the colony founder cell to all the cells in the colony. Here we showed tumor cells fluctuate their fitness levels in the absence or presence of chemotherapy and this fluctuations can be stabilized by the inhibition of chromatin and DNA modifying enzymes.
Citation Format: Jephesson Alex Alex dos Santos, Carolina Nunes Santo, Luiza Cherobini Pereira. Epigenetic modulation of tumor cell tolerance to chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2445.
Abstract 2447: Analyses of epigenetic marks and mechanisms in disease: your guide to a successful CUT and RUN assay
Authors: Chen Fang , Comeau Christopher R. , Mason Jillian , Brown Carrie A. , Fry Christopher J. 
Published: September 2020

Abstract
Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The CUT&RUN assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation, making it a much faster and more efficient method for enriching protein-DNA interactions and identifying target genes. CUT&RUN can be performed in as little as one to two days, from live cells to purified DNA, and has been shown to work with as few as 500-1000 cells per assay (1,2). Instead of fragmenting all of the cellular chromatin as done in ChIP, CUT&RUN utilizes an antibody-targeted digestion of chromatin, resulting in much lower background signal than seen in the ChIP assay. As a result, CUT&RUN requires only 1/10th the sequencing depth that is required for ChIP-seq assays. Finally, the inclusion of simple spike-in control DNA allows for accurate quantification and normalization of target-protein binding between samples. This provides for effective normalization of signal between samples and between experiments. I will discuss the basics of the CUT&RUN assay and important factors to consider when setting up your experiment. In addition, I will introduce Cell Signaling Technology's new CUT&RUN Assay Kit and CUT&RUN pAG-MNase enzyme, and present data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple cell types.
Citation Format: Fang Chen, Christopher R. Comeau, Jillian Mason, Carrie A. Brown, Christopher J. Fry. Analyses of epigenetic marks and mechanisms in disease: your guide to a successful CUT and RUN assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2447.
Abstract 2466: A novel role for the transcription factor SNAIL in alternative splicing
Authors: Ray Atrayee , Rudraraju Smruthi , Krueger Shawn , kumar Janani , Perley Danielle , Scheidegger Adam , Dhasarathy Archana 
Published: September 2020

Abstract
The transcription factor SNAIL is a master regulator of the Epithelial to Mesenchymal Transition (EMT), a hallmark event during cancer metastasis. SNAIL repressed transcription of several epithelial cell-specific genes such as E-cadherin, leading to a mesenchymal phenotype that is less attached and more migratory. As a DNA-binding transcription factor, SNAIL binds consensus E-box motifs in gene promoters, and recruits epigenetic modifiers to repress or activate gene expression. Previous studies from our lab and others have indicated that SNAIL and its family member SLUG have several gene targets in common but also bind to unique gene targets. Whether SNAIL or SLUG share protein-interacting partners is unknown. To identify interactome partners of SNAIL and SLUG, we performed co-immunoprecipitation followed by iTRAQ mass spectrometry. The protein interactome of SNAIL, but not SLUG, was highly enriched for splicing proteins, of which the top hit was the Serine-Arginine Rich-Splicing Factor, SRSF1. SRSF1 participates in regulation of alternative splicing, a mechanism through which a cell can generate multiple proteins from the same gene. Alternative splicing events play a central role in EMT progression, and SRSF1 is overexpressed in many cancers. We explored the role of SNAIL and SRSF1 in driving alternative splicing events during EMT. First, we confirmed the association of SNAIL and SRSF1 by co-immunofluorescence, PLA assay and co-immunoprecipitation experiments during TGF-beta induction of EMT. RNA-IP (RIP) experiments demonstrated association of SNAIL with mesenchymal specific but not epithelial-specific splice isoforms, further suggesting an RNA binding role for SNAIL. Lastly, RNA-EMSA experiments suggest specificity for some but not all four Zinc fingers of SNAIL in binding to RNA. Based on these observations, we hypothesize that SNAIL regulates alternative splicing events during EMT at least in part through association with SRSF1 on RNA. As transcription and splicing are closely coupled, we predict that the regulation of splicing by transcription factors through RNA binding is universally utilized by other biological systems to alter phenotype in response to external stimuli. We are currently working on identifying the genome-wide changes in alternative splicing regulated by SNAIL, and the mechanism by which SNAIL interacts with the spliceosome to regulate alternative splicing.
Citation Format: Atrayee Ray, Smruthi Rudraraju, Shawn Krueger, Janani kumar, Danielle Perley, Adam Scheidegger, Archana Dhasarathy. A novel role for the transcription factor SNAIL in alternative splicing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2466.
Abstract 2476: Role of retinoblastoma gene in maintenance of osteoblast function and communication
Authors: Pendleton Elisha , Ketner Anthony , Bodenstine Thomas , Chandar Nalini 
Published: September 2020

Abstract
Retinoblastoma gene (Rb1) loss of function mutations is commonly seen in osteosarcomas resulting in poorly differentiated tumors. In addition to cell cycle regulation, Rb1 function has been shown to be important in cell fate determination. It is known that loss of Rb1 function in mesenchymal stem cells inhibits osteoblast differentiation and predisposes them towards adipocytic lineage. Recent studies have suggested a role for Rb1 in cell adhesion but mechanistic details are missing. As cadherin 11 (Cad11) and gap junctional protein connexin43 (Cx43), play important roles in adhesion and communication in osteoblasts, we studied these proteins in MC3T3-E1 mouse calvaria osteoblast cell line. We compared osteoblasts with reduced Rb1 expression to vector-transfected osteoblasts to assess the relationship between Rb1, Cad11, and Cx43. Decreased RNA and protein expression of Cad11 were noted during differentiation of Rb1 deficient osteoblasts when compared to controls. Gap junctional intercellular communication was also studied and found to be reduced with Rb1 loss. Rb1 deficient cells had a higher steady-state level of adipocyte transcription factors and adipocyte markers when compared to control. Interestingly no changes were observed in Cx43 mRNA or protein expression when comparing the two lines. Immunofluorescence analysis demonstrated that a decrease in Cad11 may have affected its co-localization with Cx43. This, in turn, may have affected the proper formation of intercellular channels and thereby communication. These observations suggest that loss of Rb1 affects adhesion and communication in osteoblasts resulting in the expression of an adipocyte phenotype.
Citation Format: Elisha Pendleton, Anthony Ketner, Thomas Bodenstine, Nalini Chandar. Role of retinoblastoma gene in maintenance of osteoblast function and communication [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2476.
Abstract 2481: Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer
Authors: Huang Chi-Cheng , Tu Chao-Chiang , Tsai Mong-Hsun , Lai Liang-chuan , Huang Ching-Shui 
Published: September 2020

Abstract
Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer
Introduction
Breast cancer is molecularly heterogeneous and at least five molecular subtypes have been defined according to intrinsic genes transcription. Currently microarrays, RT-PCR, and next generation RNA sequencing have been adopted for subtyping in addition to the original nanoString nCounter assay. This study aimed to evaluate the consistency in subtyping Taiwanese breast cancers.
Methods
A total of 109 Taiwanese breast cancers (24 with adjacent normal breast) were assayed with Affymetrix Human Genome U133 plus 2.0 arrays and 96 were assayed with the customized 145-gene nanoString Codeset BCeCSig, of which the academic PAM50 was investigated. Of note, 64 patients were assayed for both platforms. Subtyping with the nearest centroid (single sample prediction) was called for each patient; Spearman's rank correlation coefficients were used, and samples were unclassified if correlation coefficients to all 5 centroids were less than 0.1.
Results
Table 1 showed the distributions of molecular subtypes with clinical IHC results. For 24 matched normal breast tissues, 21 were classified as normal breast-like. For 64 breast cancers (one unclassified) tested for both platforms, 41 (65%) were classified with identical subtype, resulting in a fair Kappa statistic of 0.55. Taking nCounter as gold standard, classification accuracy was 50% (3/6), 72% (13/18), 53% (10/19), 87% (13/15), and 40% (2/5) for basal-like, HER2-enriched, luminal-A, luminal-B, and normal breast-like subtype using Affymetrix microarray. ER-/HER2- phenotype and basal-like subtype experienced worse relapse free survival at 8-year follow-up (55.6% and 63.6% censored).
Table 1.Molecular subtypes and IHC phenotypes (two unclassified and one missing IHC result).Subtype IHCBasal-likeHER2-enrichedLuminal-ALuminal-BNormal breast-likeHR+/HER2+276174HR+/HER2-6442429HR-/HER2+625209HR-/HER2-144003
Conclusions:
Using public domain microarrays as a handy shortcut for breast cancer molecular subtyping is luring in scientific communities. Our experiments showed that fundamental discrepancy existed between distinct approaches, and more cation should be taken without too much extrapolation. On the other hand, adjacent normal breast tissue did display normal breast-like character, and this subtype should be viewed as a continuum of surrounding stroma.
Citation Format: Chi-Cheng Huang, Chao-Chiang Tu, Mong-Hsun Tsai, Liang-chuan Lai, Ching-Shui Huang. Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2481.
Abstract 249: Wild-type IDH2 is a potential therapeutic target for acute myeloid leukemia
Authors: Zeng Peiting , Huang Peng 
Published: September 2020

Abstract
Isocitrate dehydrogenase 2 (IDH2) is an important mitochondrial enzyme, which catalyzes the metabolic conversion between isocitrate and alpha-ketoglutarate (α-KG) in the TCA cycle. Somatic point mutations in the gene encoding for IDH2 have been identified in a variety of human cancers including certain glioma and acute myeloid leukemia (AML), and are correlated with a favorable prognosis compared with those with wild-type IDH2. However, the role of wild-type IDH2 in cancer development largely remains unknown. Here, we show that wild-type IDH2 is up-regulated in AML, and may play an important role in promoting AML cells survival and proliferation. Database analysis reveals that high expression of IDH2 seems to be correlated with poor survival of AML patients. We show that pharmacological inhibition of wild-type IDH2 in AML cell lines harboring wild-type IDH2 or genetically silencing of IDH2 expression by shRNA could impair cell viability, suppress the proliferation and colony formation capacity of the leukemia cells, and promote apoptosis, leading to significant inhibition of leukemia growth. Metabolic study suggests that disturbance of α-KG metabolism might play a major role in affecting AML cell viability and proliferation. Specifically, suppression of IDH2 caused a significant increase in α-KG and a decrease in isocitrate and citrate, suggesting that the reductive segment of the TCA cycle might be abnormally active in AML cells. Thus inhibition of IDH2 would lead to a severe disruption of the leukemia cellular metabolism due to suppression of α-KG to isocitrate conversion. Furthermore, genetic silencing of the wild-type IDH2 expression in AML cells suppressed in vivo leukemia growth in two xenograft mouse models. Overall, our study has identified wild-type IDH2 as an important molecule that is critical for AML cell survival and proliferation, and suggests that wild-type IDH2 could potentially serve as a new therapeutic target for the potential treatment of acute myeloid leukemia.
Citation Format: Peiting Zeng, Peng Huang. Wild-type IDH2 is a potential therapeutic target for acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 249.
Abstract 2507: Understanding endometrial cancer heterogeneity at single-cell resolution
Authors: Villarreal Enrique I. Velazquez , Carpten John D. , Craig David W. 
Published: September 2020

Abstract
Endometrial cancer is the fourth most common gynecologic malignancy in the US. Tumor- associated genetic signatures are known to be associated with poorer prognosis of patients with endometrial cancer. In general, frequent gene alterations are enrichments of p53 and KRAS, PTEN, PIK3CA and PI3K/AKT mutations with variable CNV. Considerable evidence for intratumor heterogeneity indicate that bulk genomic sequencing approaches may be insufficient since endometrial cancer is characterized by extensive tumor heterogeneity. Here, to get new insight about endometrial cancer heterogeneity, we applied single cell sequencing since it has the potential to improve our understanding of this genetic feature by providing clonotype and variant information in high resolution. We examined cancer heterogeneity using single cell DNA sequencing Copy Number Variation (sc-DNAseq-CNV) analysis and clonotype detection in three tumors from patients with endometrial cancer. The technology used for processing single cell was 10x ChromiumTM Technology that provides detection of 100 Kb CNV events, calling clonotypes down to 10 of 1000 cell inputs. By processing these samples resulted in the sequencing of around 800 cells. Genome-wide ploidy analysis of the tumor cells showed variable median ploidies per sample. The regional copy number was estimated by processing our data in 20 Kb cases of reads. Multiple sub-clones were identified per sample number one where four clusters of sub-clones characterized cancer heterogeneity. Copy number changes on entire chromosome arms and mutations at variant detection level were detected since the resolution provided by single cell technology applied. Among the variant detection we identified previously reported mutations in POLE, POLE2, TP53, FGFR2, ARID1A, CTNNBI, PIK3CA, PIK3RI, KRAS and SWI/SNF in specific sub clones. Our study addresses the predominant challenge to determine tumor heterogeneity by performing better approaches to detect genomic differences and identifying molecular signatures among women with endometrial cancer. Better understanding of tumor heterogeneity may benefit patients diagnosed with endometrial cancer, or patients with specific molecular characteristics linked to endometrial cancer.
Citation Format: Enrique I. Velazquez Villarreal, John D. Carpten, David W. Craig. Understanding endometrial cancer heterogeneity at single-cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2507.