bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4723: Mesenchymal status promotes metastatic colonization via a cancer cell-stroma crosstalk which uncouples EMT and stemness
Authors: Del Pozo Martin Yaiza , Park Danielle , Sahai Erik , Malanchi Ilaria
Published: August 2015

During metastatic colonisation tumour cells must establish a favourable microenvironment, or niche, that will sustain their growth. However, both the temporal and molecular details of this process are poorly understood.

Here, using breast cancer models, we find that metastatic initiating cells (MICs) in a mesenchymal phenotype exhibit high niche activation ability as a result of Thrombospondin 2 (THBS2) secretion, which prompts lung fibroblasts activation. Importantly, inhibiting the mesenchymal phenotype of MICs, by blocking the epithelial to mesenchymal transition (EMT)-associated kinase AXL during the early stages of cancer cell infiltration, reduces THBS2 secretion, niche activation ability, and consequently metastatic establishment. Subsequently, cancer cells start expanding at the target site and revert to a more epithelial phenotype. Interestingly, the activated fibroblasts of the newly formed metastatic niche trigger the reversion of cancer cell mesenchymal phenotype without compromising self-renewal. Preventing this phenotypic reversion by enhancing AXL expression during colonization block metastasis.

In summary, we demonstrate that during metastatic colonization the two events of niche activation and EMT are functionally linked at the target site. However, in this context, EMT becomes uncoupled from the ‘stemness’ required for metastatic growth. Further, once fibroblasts within the niche are activated they promote the mesenchymal to epithelial transition (MET) of tumour cells, leading to the outgrowth of metastases with similar epithelial characteristics and stromal cell networks to their original primary tumours.

Citation Format: Yaiza Del Pozo Martin, Danielle Park, Erik Sahai, Ilaria Malanchi. Mesenchymal status promotes metastatic colonization via a cancer cell-stroma crosstalk which uncouples EMT and stemness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4723. doi:10.1158/1538-7445.AM2015-4723
Abstract 4766: DNA methylation changes in Lynch syndrome associated colorectal adenomas and carcinomas
Authors: Valo Satu , Kaur Sippy , Ristimäki Ari , Renkonen-Sinisalo Laura , Järvinen Heikki , Mecklin Jukka-Pekka , Nyström Minna , Peltomäki Päivi
Published: August 2015

Colorectal cancer (CRC) develops via multiple steps which involve genetic changes, such as mutations in growth-regulatory genes, and epigenetic alterations, such as CpG island hypermethylation. Lynch syndrome (LS) is one of the most common inherited cancer susceptibility syndromes and is associated with inherited defects of the DNA mismatch repair (MMR) genes, which together with other genetic and epigenetic changes are known to accelerate tumorigenesis. In this investigation we explored the timing of CpG island methylation changes in the course of transformation of normal colonic mucosa to pre-cancerous and cancerous lesions in genetically predisposed individuals.

Colorectal neoplastic lesions including low-grade adenomas, high-grade adenomas and carcinomas together with matching normal tissue were gathered from 43 LS mutation carriers enrolled in colonoscopy surveillance. Frequency of CpG island methylator phenotype (CIMP) in different tumor types was analyzed with the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) which detects simultaneously the methylation status of eight CIMP marker genes. Additionally, we analyzed the methylation status of seven selected tumor suppressor genes previously associated with early colon oncogenesis. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions.

We show that the expression of the MMR protein corresponding to the germline mutation decreases along with increasing dysplasia in tumors. Similarly, methylation increases in LS adenomas and carcinomas together with dysplasia grade in comparison to normal colonic mucosa. A proportion of low-grade adenomas could already be classified CIMP positive, and the frequency of CIMP further increased in high-grade adenomas and carcinomas. Our results offer new insights into the molecular mechanisms through which DNA methylation can affect colorectal carcinogenesis in LS mutation carriers.

Citation Format: Satu Valo, Sippy Kaur, Ari Ristimäki, Laura Renkonen-Sinisalo, Heikki Järvinen, Jukka-Pekka Mecklin, Minna Nyström, Päivi Peltomäki. DNA methylation changes in Lynch syndrome associated colorectal adenomas and carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4766. doi:10.1158/1538-7445.AM2015-4766
Abstract 4778: HP1-mediated spatiotemporal control of estrogen-responsive transcription in breast cancer cells
Authors: Hsu Pei-Yin , Hsu Hang-Kai , Jin Victor X. , Sharp Zelton D. , Huang Tim H.-M.
Published: August 2015

Recruitment of transcriptional machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13 were frequently amplified (50∼60 copies) and translocated to other chromosomes in ERα-positive breast cancer cells. In this study, we used three-dimensional interphase fluorescence in situ hybridization to decipher spatiotemporal gathering of multiple DEREs in the nucleus. Upon estrogen stimulation, the majority of 20q13 DEREs were mobilized to form regulatory depots for synchronized regulation of target loci. A chromosome conformation capture assay coupled with chromatin immunoprecipitation further uncovered that ERα-bound regulatory depots are tethered to heterochromatin protein 1 (HP1) for coordinated chromatin movement and subsequent facilitation of histone modifications of target loci, resulting in transcription repression. Neutralizing HP1 function dissembled these regulatory depots and reversed this transcription inactivation of candidate tumor-suppressor genes. Deletion of amplified DEREs using the CRISPR/Cas9 genomic-editing system profoundly altered transcriptional profiles of JAK/STAT signaling networks, leading to proliferation inhibition of cancer cells with beneficial outcome of breast cancers. These findings reveal a formerly uncharacterized feature of oncogenic amplicons that multiple copies of the amplicon congregate as transcriptional unions in the nucleus for synchronous regulation of function-related loci in tumorigenesis. Disruption of their assembly can be a new strategy for treating breast cancers and other malignancies.

Citation Format: Pei-Yin Hsu, Hang-Kai Hsu, Victor X. Jin, Zelton D. Sharp, Tim H.-M. Huang. HP1-mediated spatiotemporal control of estrogen-responsive transcription in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4778. doi:10.1158/1538-7445.AM2015-4778
Abstract 4811: Pro-oncogenic role of NOTCH1 in early tongue squamous cell carcinoma
Authors: Upadhyay Pawan , Aich Jyotirmoi , Sethunath Vidyalakshmi , Dani Prachi , Kannan Sadhana , Chandrani Pratik , Sonawane Kavitha , Agarwal Beamon , Kane Shubhada , Nair Sudhir , Dutt Amit
Published: August 2015

Notch pathway play a context-dependent tumor-suppressive or pro-oncogenic role in solid tumors and hematological malignancies. In head and neck cancer, inactivating mutations in NOTCH1 are associated with poor prognosis. Here we perform meta-analysis of DNA copy number and gene expression profiling of NOTCH signaling pathway genes in 35 paired primary T1 and T2 stage tongue tumors, comprising of Notch ligands, receptors and target genes. We find 32% of primary tongue squamous cell carcinoma (TSCC) tumors amplify NOTCH signaling pathway components; and over express activated NOTCH1based on immuno-histochemical staining in an additional series of 50 surgically resected paired TSCC tumors. Interestingly, these alterations: amplification at Notch ligand DLL3 (χ2 = 7.575, P = 0.023); over expression of Notch pathway target gene HEY2 transcript (χ2 = 9.885, P = 0.007); and expression of activated NOTCH1 (χ2 = 7.575, P = 0.023) positively correlate with N+ve and non- smoking status of patients. In a parallel effort, we characterized four HNSCC cell lines– NT8E, OT9, AW13516, and AW8507– established from Indian patients using whole exome, whole transcriptome sequencing and SNP array assays. Significant amplification and over expression of NOTCH signaling pathway components were observed in AW13516 and NT8E cells. Inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 could inhibit transformation, survival, migration and invasion of NT8E cells. Taken together, we present that activation of Notch signaling pathway in a subset of TSCC patients is associated with nodal positivity and non-smoking status among early tongue cancer patients.

Citation Format: Pawan Upadhyay, Jyotirmoi Aich, Vidyalakshmi Sethunath, Prachi Dani, Sadhana Kannan, Pratik Chandrani, Kavitha Sonawane, Beamon Agarwal, Shubhada Kane, Sudhir Nair, Amit Dutt. Pro-oncogenic role of NOTCH1 in early tongue squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4811. doi:10.1158/1538-7445.AM2015-4811
Abstract 4813: High-depth sequencing of 800 cancer-associated genes in 48 matched tumor-normal tissues to identify somatic alterations including low-frequency variants in colorectal cancer
Authors: Chua Clarinda , Ho Dan Liang , Suzuki Yuka , Malik Simeen , McPherson John , Gan Anna , Koh Dennis , Tang Choon Leong , Ng Sarah Boon Hsi , Tan Patrick , Rozen Steve , Tan Iain Beehuat
Published: August 2015

Purpose: Colorectal cancer is the 3rd most common cancer globally accounting for 10% of the global cancer burden. Catalogues of somatic mutations in cancer (e.g. The Cancer Genome Atlas (TCGA)) are based upon exome sequencing, typically performed at depths of ∼ 80x, which will miss sub-clonal low-frequency somatic variants. We performed high depth sequencing of 800 cancer-associated genes across 48 tumor/normal pairs of CRC to catalogue somatic variants including those present at low variant allele frequencies.

Method: We used the Agilent SureSelect Target Enrichment system to design a customized enrichment panel comprising all coding exons of ∼800 cancer-associated genes. From genomic DNA extracted from frozen tissue specimens (matched primary tumor and adjacent normal), we performed library construction, target enrichment and illumina next generation sequencing. We used 3 variant-calling algorithms - a Genome Analyzer Toolkit (GATK) based pipeline, LoFreq and MuTect to identify somatic variants including sub-clonal variants present at low variant allele frequencies.

Results: Sequencing was performed to a median of 302-fold and 310-fold coverage across target regions in the tumor tissues and normal tissues respectively. The use of 3 bioinformatics variant-calling algorithm allowed sensitive and comprehensive identification of variants down to a variant allele frequency of 2.5%; selected variants were validated by an orthogonal sequencing platform (Ion Torrent). The validation rate was > 97%. Each patient had a variant in a median of 19 genes (range 8-100) with a different complement of clonal and sub-clonal variants. Recurrent alterations (e.g. APC, KRAS, TP53) tended to be clonal. Broad regions of allelic imbalance (based on heterozygous SNPs identified in each patient's normal tissue) could also be mapped in each patient.

Conclusion: High Depth sequencing of 800 cancer-associated genes in 48 matched tumor normal tissues permits identification of somatic alterations including low-frequency variants and sub-clonal alterations in patients with colorectal cancer.

Citation Format: Clarinda Chua, Dan Liang Ho, Yuka Suzuki, Simeen Malik, John McPherson, Anna Gan, Dennis Koh, Choon Leong Tang, Sarah Boon Hsi Ng, Patrick Tan, Steve Rozen, Iain Beehuat Tan. High-depth sequencing of 800 cancer-associated genes in 48 matched tumor-normal tissues to identify somatic alterations including low-frequency variants in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4813. doi:10.1158/1538-7445.AM2015-4813
Abstract 4815: Tumor heterogeneity of advanced primary lung cancer evaluated by multiregion sequencing
Authors: Um Sang-Won , Joung Je-Gun , Lee Su Yeon , Bae Joon Seol , Park Jinha , Park Woong-Yang
Published: August 2015

Background: Cancers are composed of cells with distinct genetic and phenotypic characteristics. Tumor heterogeneity in each patient may influence on personalized medicine strategies that depend on results from single tumor-biopsy samples. However, tumor heterogeneity in advanced primary lung cancer has not been well studied. The purpose of this study was to investigate the tumor heterogeneity in advanced primary lung cancer.

Methods: We sequenced fourteen regions from four patients who were suspicious for primary lung cancer with multiple lymph node metastasis. Tumor samples were obtained from primary tumor and metastatic lymph node(s) or distant metastasis using bronchoscopic forcep biopsy, endobronchial ultrasound-guided transbronchial needle aspiration, and percutaneous needle biopsy. The profiles of somatic mutations, copy number alterations and gene expression were compared among primary tumor and metastatic sites in each patient. Clonal evolution was evaluated using the concept of cancer cell fraction by SciClone analysis.

Results: We performed multiregion whole exome sequencing and RNA sequencing on three non-small cell lung cancers (2 adenocarcinomas [TH1 and TH4] and 1 squamous cell carcinoma [TH2]) and one small cell lung cancer (TH3). EML4-ALK fusion was noticed in TH1 and TH4. There was profound tumor heterogeneity in terms of somatic mutation, copy number alteration, or gene expression in each patient. The frequency of private somatic mutation from spatially distinct regions were 59% in TH1, 44% in TH2, 13% in TH3, and 30% in TH4. Known driver mutation was also heterogeneous in TH1 and TH2. Analysis of the clonal architecture confirmed the presence of clonal heterogeneity in each patient. Phylogenetic reconstruction revealed the evidence of branched clonal evolution.

Conclusions: Tumor heterogeneity was noticed from spatially distinct regions from advanced primary lung cancers. The single biopsy specimens may not represent the whole landscape of genetic alteration in advanced primary lung cancers.

Citation Format: Sang-Won Um, Je-Gun Joung, Su Yeon Lee, Joon Seol Bae, Jinha Park, Woong-Yang Park. Tumor heterogeneity of advanced primary lung cancer evaluated by multiregion sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4815. doi:10.1158/1538-7445.AM2015-4815
Abstract 4819: Differential evolution of tumor heterogeneity in leiomyosarcomas and liposarcomas suggested by genomic profiling
Authors: Amin-Mansour Ali , Sioletic Stefano , Carter Scott L. , Garraway Levi A. , Demetri George D. , George Suzanne , Van Allen Eliezer M. , Wagner Andrew J.
Published: August 2015

Sarcomas represent a heterogeneous set of malignant tumors originating from mesenchymal cells. Liposarcomas (LPS) and Leiomyosarcomas (LMS) are two of the most common histotypes, accounting for approximately 25% and 30% of all soft tissue sarcomas, respectively. The most frequent form of LPS is represented by a spectrum of disease including well-differentiated LPS (WDLPS), a non-metastasizing but locally recurrent disease with adipocytic differentiation, and de-differentiated LPS (DDLPS), an aggressive, frequently metastasizing high grade sarcoma which can arise in conjunction with elements of WDLPS. Besides the known oncogenic KIT or PDGFRA driver mutations in GIST, somatic alterations in RB1, deletions in PTEN, MDM2 amplifications in LPS, and TP53 mutations in LMS are the most commonly reported genomic aberrancies identified in sarcomas. Both WDLPS and DDLPS share amplification of 12q13-15, a genomic region containing the MDM2 and CDK4 loci, yet little is understood about the other genetic processes that yield such different phenotypes. In LMS, recurrent mutations in TP53 have been identified, but a detailed analysis of primary and metastatic tumors has not previously been described.

To evaluate genetic events in these transitions, we performed whole exome sequencing (WES) on trios comprising normal tissue, WDLPS, and DDLPS obtained from individual patients (n = 19), as well as on trios of normal tissue, primary and metastatic LMS tumors obtained from separate individual patients (n = 8). Consistent with prior reports of the molecular characterization of LPS, we observed arm-level amplifications in Chr 12 in all LPS samples, and Chr 17 amplification in many LMS cases. There were no shared somatic alterations across paired WDLPS and DLPS pairs from individual patients aside from the common Chr 12 amplification (MDM2 & CDK4). A higher mutation rate was observed in LMS, and there were many clonal somatic alterations shared between the primary and metastatic lesions of individuals.

This study demonstrates the heterogeneity of evolution between different subtypes of sarcomas using whole exome sequencing of clinical specimens. This finding has implications for better understanding of the molecular drivers of these tumors and opportunities to identify subtype-specific therapeutic targets.

Citation Format: Ali Amin-Mansour, Stefano Sioletic, Scott L. Carter, Levi A. Garraway, George D. Demetri, Suzanne George, Eliezer M. Van Allen, Andrew J. Wagner. Differential evolution of tumor heterogeneity in leiomyosarcomas and liposarcomas suggested by genomic profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4819. doi:10.1158/1538-7445.AM2015-4819
Abstract 4828: Differential gene expression profile of tissue factor pathway inhibitor-2 (TFPI-2) in malignant pleural mesothelioma
Authors: Wali Anil , Wang Ying , Tarca Adi , Lonardo Fulvio , Liu Zhandong , Rishi Arun K. , Pass Harvey I.
Published: August 2015

Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy characterized by rapid progressive local growth, late metastases, and poor prognosis despite aggressive strategies involving surgery, chemotherapy, and radiation. The tissue factor pathway inhibitor-2 (TFPI-2) has been shown to play an important role in the regulation of extracellular matrix digestion and remodeling, and its reduced synthesis is implicated in various pathophysiological processes such as inflammation and tumor growth/metastasis. In this study we have elucidated the gene expression profile of TFPI-2 among 22 MPM tumor samples using Affymetrix U133AB chip platform. We noticed 13/22 tumors had more than 3-fold reduction in their TFPI-2 gene expression patterns compared to three normal peritoneums. Decreased TFPI-2 expression was observed in majority of epithelial histotypes that exhibited short-term recurrence (STR <12 months) compared to longer time to relapse (LTR after surgery), group of MPM tumor samples. TFPI-2 expression was profoundly decreased (>4-fold) among 5 out of 7 autologous normal pleura-MPM tumor samples. We also found > 3-fold decreased TFPI-2 gene expression pattern in three out of six MPM patient derived cell lines. A robust 2-fold induction in TFPI-2 gene expression was found as a consequence of 5′-aza-2′-deoxycytidine (5-AzaC) and trichostatin-A combination treatment of two MPM patient derived cell lines. Studies are underway to assess whether TFPI-2 promoter hypermethylation is involved in the down-regulation of its expression pattern seen among fifty percent of MPM tumor samples. Further studies are needed to understand the regulation and interaction of TFPI-2 in mesothelioma carcinogenesis that could be utilized for future early detection and therapeutic strategies to combat this insidious disease.

Note: This abstract was not presented at the meeting.

Citation Format: Anil Wali, Ying Wang, Adi Tarca, Fulvio Lonardo, Zhandong Liu, Arun K. Rishi, Harvey I. Pass. Differential gene expression profile of tissue factor pathway inhibitor-2 (TFPI-2) in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4828. doi:10.1158/1538-7445.AM2015-4828
Abstract 4854: A computational framework for prioritizing noncoding regulatory variants in cancer
Authors: Fu Yao , Liu Zhu , Lou Shaoke , Colonna Vincenza , Bedford Jason , Mu Xinmeng , Yip Kevin Y. , Kang Hyun Min , Lappalainen Tuuli , Sboner Andrea , Yu Haiyuan ,   1000 Genomes Project Consortium, Rubin Mark , Tyler-Smith Chris , Khurana Ekta , Gerstein Mark
Published: August 2015

Mutations in key regulatory sequences have been suggested to cause oncogenesis. However, identification of noncoding cancer “drivers” from thousands of somatic alterations is a difficult and unsolved problem. We report a computational framework, FunSeq, to annotate and prioritize these mutations. The framework combines an adjustable data context integrating large-scale genomics (e.g. ENCODE) and cancer resources with a streamlined variant-prioritization pipeline. The pipeline has a weighted scoring system combining: inter- and intra-species (we used patterns of natural polymorphisms to identify human-specific conserved elements) conservation; loss- and gain-of function events for transcription-factor binding; enhancer-gene linkages and network centrality; and per-element recurrence across samples. We further highlight putative drivers with information specific to a particular sample, such as differential gene expression. When applied to an individual tumor genome, our method is able to prioritize the TERT promoter mutation. We then evaluated our framework on a larger-scale first by doing various comparisons with other existing noncoding variant-prioritization tools. Next, we used the recurrence of somatic mutations to validate some of our prioritized mutations. Finally, we developed the recurrence analysis into a database combining all whole-genome sequenced cancer samples and used this to provide higher confidence in mutation prioritization. FunSeq is available from funseq.gersteinlab.org.

Note: This abstract was not presented at the meeting.

Citation Format: Yao Fu, Zhu Liu, Shaoke Lou, Vincenza Colonna, Jason Bedford, Xinmeng Mu, Kevin Y. Yip, Hyun Min Kang, Tuuli Lappalainen, Andrea Sboner, Haiyuan Yu, 1000 Genomes Project Consortium, Mark Rubin, Chris Tyler-Smith, Ekta Khurana, Mark Gerstein. A computational framework for prioritizing noncoding regulatory variants in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4854. doi:10.1158/1538-7445.AM2015-4854
Abstract 486: BET protein inhibition by JQ1 blocks EWS-FLI1 activity in Ewing sarcoma
Authors: Bledsoe Krista L. , Stonestrom Aaron , Kadauke Stephan , Quick Laura , Young Robert , Blobel Gerd A. , Chou Margaret M.
Published: August 2015

Ewing sarcoma is the second most common bone cancer in children, with an estimated 5-year survival rate of 20% for metastatic cases. These tumors harbor the characteristic chromosomal translocation t(11;22)(q24;q12), which leads to expression of the oncogenic chimeric transcription factor, EWS-FLI1. EWS-FLI1 drives Ewing sarcoma tumorigenesis, and is an attractive therapeutic target because it is expressed exclusively in tumor cells. However, directly targeting EWS-FLI1 has proved difficult. Thus, despite advances in our understanding of EWS-FLI1 function, treatment options remain limited, and to date, no targeted therapeutics have been developed.

In this study, we investigated the effects of the epigenetic modifier drug, JQ1, on Ewing sarcoma pathogenesis. JQ1 is a small molecule inhibitor of the BET family of bromodomain proteins, which bind to acetylated histones and transcription factors to regulate gene expression. JQ1 selectively inhibits these acetylation-dependent BET protein interactions. Recent studies reveal that JQ1 has potent anti-neoplastic activity against multiple cancers, with c-MYC downregulation being a key mechanism by which it induces cell death in several tumor types. Because Ewing sarcoma cells express high levels of c-MYC, we predicted that they would be sensitive to the cytotoxic effects of JQ1.

Our results demonstrate that JQ1 indeed inhibits growth of Ewing sarcoma cells, but unexpectedly, it does not function through inhibition of c-MYC. Using multiple patient-derived Ewing sarcoma cell lines, we find that JQ1 induces apoptosis as measured by PARP cleavage. Preliminary data further indicate that JQ1 suppresses growth of Ewing sarcoma xenografts in vivo. Mechanistic studies and microarray analysis reveal that JQ1 inhibits EWS-FLI1 activity, with multiple targets of the oncogene being rapidly downregulated upon JQ1 treatment. Studies are currently underway to identify the specific BET family members required for EWS-FLI1 function, and to determine the mechanism by which they regulate EWS-FLI1 activity. Given the emergence of numerous JQ1-derivative compounds targeting specific BET proteins, it is hopeful that this class of compounds may ultimately be used as targeted therapies for the treatment of Ewing sarcoma.

Citation Format: Krista L. Bledsoe, Aaron Stonestrom, Stephan Kadauke, Laura Quick, Robert Young, Gerd A. Blobel, Margaret M. Chou. BET protein inhibition by JQ1 blocks EWS-FLI1 activity in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 486. doi:10.1158/1538-7445.AM2015-486
Abstract 488: High DKC1 expression supports neuroblastoma tumor cell proliferation and is strongly associated with poor patient outcomes
Authors: O'Brien Rosemary , Maritz Michelle F. , Kong Cheng Fei , Purgato Stefania , Liu Bing , Yang Chen , Russell Amanda , Murray Jayne , Flemming Claudia , Haber Michelle , Perini Giovanni , Norris Murray D. , Fletcher Jamie I. , Mackenzie Karen L.
Published: August 2015

Dyskerin is a nucleolar RNA-binding protein that functions as a core component of the telomerase holoenzyme, as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. This study investigates the clinical and functional relevance of dyskerin expression in the paediatric cancer neuroblastoma. Here it is shown, in two independent neuroblastoma patient cohorts, that high expression of the gene encoding dyskerin (DKC1) very strongly correlates with poor event-free and overall survival. The prognostic power of DKC1 expression was independent of the established indicators used in the management of this disease, namely diagnosis age > 1.5 yr, stage of disease and amplification of the MYCN oncogene (P<0.0001). Dyskerin mRNA and protein expression were also found to be elevated, relative to normal cells, across a panel of neuroblastoma cell lines. Chromatin immunoprecipitation showed that the DKC1 promoter region was directly targeted by both MycN and c-Myc in neuroblastoma cells. Depletion of dyskerin using RNAi severely impaired proliferation, inhibited anchorage independent growth and dramatically slowed tumour growth in a mouse model of neuroblastoma (P<0.01, Two-way Anova). In p53 competent cells, the proliferative arrest induced by dyskerin depletion was accompanied by an accumulation of p53 and p21cip1, accumulation in G1 phase of the cell cycle and cell death. However in the absence of p53 function, an alternate mechanism operated to impair proliferation when dyskerin was downregulated. The mechanism responsible for the proliferative arrest also appeared to be telomerase-independent, as it was not rescued by ectopic overexpression of telomerase. Taken together, these data identify DKC1 is a crucial MYC target gene in neuroblastoma, and show that neuroblastoma cells become addicted to high DKC1 expression. The results support further investigation of DKC1 as a therapeutic target in the treatment of refractory neuroblastoma.

Citation Format: Rosemary O'Brien, Michelle F. Maritz, Cheng Fei Kong, Stefania Purgato, Bing Liu, Chen Yang, Amanda Russell, Jayne Murray, Claudia Flemming, Michelle Haber, Giovanni Perini, Murray D. Norris, Jamie I. Fletcher, Karen L. Mackenzie. High DKC1 expression supports neuroblastoma tumor cell proliferation and is strongly associated with poor patient outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2015-488
Abstract 4904: Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology
Authors: Wang Li-chong , Pan Liuliu , Chang Kuang-Jung , Kim Daniel , Wu Xingyong , Wang Hongwei , Kernag Casey , Zhang Bingqing , He Mingxiao , Su Nan , Ma Xiao-Jun , Luo Yuling
Published: August 2015

Heat induced target retrieval (HITR) is a widely adopted approach for Immunohistochemistry (IHC) and in situ hybridization (ISH) applications on formalin fixed and paraffin embedded (FFPE) tissues to facilitate target access and increase detection sensitivity. A variety of factors, such as fixative type, fixation time, tissue type, epitope abundance, antibody affinity, and probe length greatly affect the efficiency of HITR. Given such variability, over the decades, multiple HITR buffer formulations and protocols were invented to fit the needs in different experimental scenarios.

We have developed a novel HITR solution that allows highly efficient target retrieval for RNA ISH application based on the RNAscope technology. When tested on 15 different tumor types, the new HITR solution resulted in significantly better sensitivity and/or tissue morphology comparing to using traditional HITR buffers, including Citrate, EDTA, and Tris based buffers with various pH. Furthermore, this new HITR solution is highly tolerant to sample variability. When used on multiple human tumor tissue microarrays (TMAs), including breast, colon, lung, and gastric cancers, the new HITR solution improved overall signal-to-noise ratio and reduced core disqualification rate, compared to traditional citrate based HITR buffer for ISH. Moreover, when tested on multiple mouse tissues fixed with different types of fixatives for varying periods of time (6 - 72 hours), a single experimental condition with the new HITR solution showed comparable results in both detection sensitivity and morphological integrity in all the samples. It also allowed detection of a target (Tbp) with extremely low abundance (<5 copies/cell).

Given that all the RNAscope probes are synthetic oligonucleotide probes with length shorter than 60 bases, the new HITR solution, in combination with the RNAscope technology, will allow universal detection of any RNA targets with single molecule detection sensitivity in FFPE tissues with minimal requirement for analytical condition optimization.

Citation Format: Li-chong Wang, Liuliu Pan, Kuang-Jung Chang, Daniel Kim, Xingyong Wu, Hongwei Wang, Casey Kernag, Bingqing Zhang, Mingxiao He, Nan Su, Xiao-Jun Ma, Yuling Luo. Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2015-4904
Abstract 4915: Tracking genomic alterations in young patients with head and neck carcinomas using whole-exome sequencing
Authors: Miranda Priscila Mayrink , Marchi Fabio A. , Pinheiro Maísa , Kowalski Luiz Paulo , Rogatto Silvia Regina
Published: August 2015

Head and neck squamous cell carcinomas (HNSCC) often affect patients over 60 years, although epidemiological studies have shown an increased incidence in young adults (18-45 years), especially those having oropharynx and oral cavity tumors. There is no consensus regarding the etiology, clinical course and prognosis of HNSCC in young patients that often do not have the risk factors frequently described as associated with the disease (alcohol consumption, smoking and HPV infection). Despite its importance, studies with this group of patients are scarce, mainly focusing on the molecular aspects involved in the development and progression of the disease. In this study, we evaluated 36-paired samples (6 oral cavity, 12 oropharyngeal carcinomas and 18 matched blood samples) by whole exome sequencing (WES) aiming to identify molecular markers useful to clinical practice. Libraries were prepared (Illumina Nextera exome Enrichment kit) for WES using the high quality DNA from tumor tissue and peripheral blood of each patient (HiSeq2500, Illumina). The coverage reached an average of 55x per sample. We used an in-house pipeline for cleaning, mapping and SNP calling revealing an average of 36,494 SNPs per sample. A paired comparison of normal and tumor samples was performed considering only the exclusive variants in tumors, after exclusion of the common variants, based on 1000 Genomes (http://www.1000genomes.org/), project and 6500 exomes (evs.gs.washington.edu/). It was identified 248 variants distributed in 169 genes, with 184 (74%) classified as new (variant not described) and 64 (26%) as rare (population frequency ≤0.1%). Three genes, ZNF717, HYDIN and KCNJ12, presented higher frequency of SNPs and were enriched to head and neck carcinoma (p = 1,9×10-9) and calcium (p = 1.82×10-3) pathways. Considering only tongue samples (4 cases) from non-smokers and HPV negative patients, the ZNF717 and HYDIN genes remained among those with the highest frequency of SNPs [non-smokers: HYDIN (23 SNPs) and ZNF77 (24 SNPs); HPV-negative: HYDIN (6 SNPs) and ZNF77 (5 SNPs)]. These genes are putative drivers in young patients with HNSCC.

Financial Support: FAPESP (2013/06897-2) and INCiTO (FAPESP: 2008/57887-9 and CNPq 573589/08-9)

Citation Format: Priscila Mayrink Miranda, Fabio A. Marchi, Maísa Pinheiro, Luiz Paulo Kowalski, Silvia Regina Rogatto. Tracking genomic alterations in young patients with head and neck carcinomas using whole-exome sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4915. doi:10.1158/1538-7445.AM2015-4915
Abstract 4933: Sanger sequencing of low amount of genomic DNA and FFPE DNA with PCR primers derived from the Ion AmpliSeq cancer hotspot panel
Authors: Schreiber Edgar H. , Broomer Adam , Andersen Mark , Varma Kamini
Published: August 2015

The introduction of defined Ion AmpliSeq™ panels for detection and characterization of actionable mutations occurring in tumor tissue has the potential to revolutionize translational oncology research.

The Ion Ampliseq™ cancer hotspot panel version 2 (CHP v2) by Ion Torrent includes 207 actionable mutation targets present in 50 genes and the more comprehensive Ion Oncomine™ cancer panel (OCP) contains over 2000 mutations. A hallmark of these Ion Torrent Ampliseq cancer panels is the low amount of input DNA needed which is critical when the clinical specimen material is limited such as with fine needle biopsy or FFPE samples. Typically, 10 ng of DNA obtained from these sources is sufficient to produce informative sequencing data. Often, cancer-causing or promoting mutations are detected at relatively low allele frequencies like 10-20% compared to the major normal allele. Many researchers wish to verify these findings of low frequency mutations by an orthologous method such as traditional dye-fluorescent Sanger sequencing on a capillary electrophoresis (CE) instrument such as the Applied Biosystems 3500 genetic analyzer. To that end, we have developed a workflow that enables the amplification and traditional Sanger sequencing of individual Ion AmpliSeq targets directly from the AmpliSeq library starting material.

The method requires a retainer of 1 μl (∼ 5%) of the original AmpliSeq preamplification material. A dilution of this aliquot is used as template source for individualized PCR/sequencing reactions. We show that a random selection of 48 targets from the CHPv2 panel could be successfully amplified and Sanger-sequenced from an Ion Torrent Ampliseq library originally prepared from 10 ng of FFPE DNA. Furthermore, we show the successful Sanger-re-sequencing of all individual 24 targets covering the TP53 exons from the same sample processed and pre-amplified with the OncoMine AmpliSeq panel.

Taken together, this method will enable researchers to reflex-test potential mutations of interest from very material-limited specimen using Sanger CE sequencing.

Citation Format: Edgar H. Schreiber, Adam Broomer, Mark Andersen, Kamini Varma. Sanger sequencing of low amount of genomic DNA and FFPE DNA with PCR primers derived from the Ion AmpliSeq cancer hotspot panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4933. doi:10.1158/1538-7445.AM2015-4933
Abstract 494: MYC, dominant negative p53 and AKT transform human neural stem cells into primitive neuro-ectodermal tumors sensitive to glutaminase inhibitors
Authors: Taylor Isabella , Ahsan Sama , Price Antoinette , Eberhart Charles , Raabe Eric H.
Published: August 2015

Primitive neuroectodermal tumors (PNET) are highly aggressive tumors that can arise in different regions of the brain. A subset of PNET have genomic amplification of MYC, and the MYC-regulator LIN28A is known to define a group of PNET. Both MYC and LIN28 regulate metabolism in normal and malignant cells. We hypothesized that a combination of dominant negative R248Wp53, MYC, AKT and hTERT would transform human neural stem cells into PNET-like tumors. We used lentiviral vectors to infect human neural stem cells derived from hindbrain and cortex with the same packages of oncogenic elements. Hindbrain neural stem cells transduced with MYC/R248Wp53/AKT/hTERT proliferated at an increased rate compared to control cells as measured by BrdU incorporation (Hindbrain pBABE vs. Hindbrain MYC/R248Wp53/AKT/hTERT: 4% vs. 50% BrdU positive, t-test p = 0.001). Similar results were observed in cortex derived human neural stem cells transduced with MYC/R248Wp53/AKT/hTERT (Cortex pBABE vs Cortex MYC/R248Wp53/AKT/hTERT: 5% vs. 24% BrdU positive, t-test p = 0.027). Both hindbrain and cortex derived human neural stem cells transformed with MYC /R248Wp53/hTERT, and MYC/R248Wp53/hTERT/AKT formed aggressive orthotopic xenograft tumors with histologic characteristics of PNET. The latency of tumor formation decreased with increasing addition of oncogenes. We detected increased glutaminase (GLS) in our MYC-driven human neural stem cell models compared to SV40 immortalized neural stem cells, and hypothesized that GLS inhibitors would inhibit the growth of MYC-driven and LIN28 driven brain tumors. The glutaminase inhibitors DON (6-Diazo-5-oxo-L-norleucine) and acivicin suppressed the growth of our MYC-driven neural stem cell models of PNET as well as the PNET cell line PFSK (p = 0.003) as measured by BrdU and MTT growth assay. Cell cycle analysis of MYC-transduced human neural stem cell models showed increased cell death as measured by sub G1 population after treatment with DON and acivicin. This data shows that glutaminase inhibition may be a useful therapeutic modality in PNET.

Citation Format: Isabella Taylor, Sama Ahsan, Antoinette Price, Charles Eberhart, Eric H. Raabe. MYC, dominant negative p53 and AKT transform human neural stem cells into primitive neuro-ectodermal tumors sensitive to glutaminase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 494. doi:10.1158/1538-7445.AM2015-494
Abstract 4958: Melanoma transition is frequently accompanied by a loss of cytoglobin, a putative tumor suppressor, in melanocytes
Authors: Fujita Yoshihiko , Koinuma Satoshi , De Velasco Marco , Jan Bolz , Togashi Yosuke , Terashima Masato , Hayashi Hidetoshi , Matsuo Takuya , Nishio Kazuto
Published: August 2015

Cytoglobin (CYGB), a new member of the globin family, remains to be characterized. To assess its expression in human malignancies, we screened a number of cell lines originated from many tissues, and found that several, but not all, melanoma cell lines expressed CYGB mRNA and protein at much higher levels than cells of other origins. Melanocytes, the primary origin of melanoma, also expressed CYGB at a high level. Bisulfite-modified genomic sequencing revealed that several melanoma cell lines that abrogated CYGB expression were found to be epigenetically regulated by hypermethylation in the promoter region of CYGB gene. The RNAi-mediated knockdown of the CYGB transcript in CYGB expression-positive melanoma cell lines resulted in increased proliferation in vitro and in vivo. Flow cytometric analysis using an indicator of reactive oxygen species (ROS) revealed that the CYGB knockown enhanced the cellular ROS level, which may be involved in the antiproliferative effect of CYGB. Thus, CYGB appears to play a tumor suppressive role as a ROS regulator, and its epigenetic silencing, as observed in CYGB expression-negative melanoma cell lines, might function as an alternative pathway in the melanocyte-to-melanoma transition.

Citation Format: Yoshihiko Fujita, Satoshi Koinuma, Marco De Velasco, Bolz Jan, Yosuke Togashi, Masato Terashima, Hidetoshi Hayashi, Takuya Matsuo, Kazuto Nishio. Melanoma transition is frequently accompanied by a loss of cytoglobin, a putative tumor suppressor, in melanocytes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4958. doi:10.1158/1538-7445.AM2015-4958
Abstract 4969: Metformin causes AR degradation via Skp2-mediated ubiquitination
Authors: Yang Joy C. , Gao Allen C. , Evans Christopher P.
Published: August 2015

One of the mechanisms for prostate cancer cells to escape the new androgen receptor (AR) signaling inhibitor Enzalutamide (Enza) is through autophagy. Combination of anti-diabetic drug metformin with Enza demonstrated improved tumor suppression in the LNCaP C4-2B orthotopic model. Through delineating the mechanism in the metformin benefit, we discovered degradation of AR in cells treated with metformin alone and more in the combination with Enza and Abiraterone (Abi). The mRNA for AR in metformin-treated cells did not change significantly by qPCR assays. This degradation was inhibited when proteasome inhibitor MG132 was added prior to the treatments. Use of the FDA approved proteasome inhibitor Bortezomib also protected AR from degradation in the presence of metformin. Immunoprecipitation of AR with its antibodies showed ubiquitination; the reverse immunoprecipitation with anti-ubiquitin followed by anti-AR probing also showed similar results; suggesting ubiqutination responsible for AR degradation. With a paper by Li et al. proposing Skp2 being a novel E3 ubiquitin ligase targeting AR for ubiquitination, we attempted to knock-down Skp2 with transient siRNA transfection. Degradation of AR was lessened with Skp2 knockdown. This study supports that use of metformin in combination with Enza or other ARSI drugs may not only block autophagy survival but also cause AR degradation that leads to PC cell death.

Citation Format: Joy C. Yang, Allen C. Gao, Christopher P. Evans. Metformin causes AR degradation via Skp2-mediated ubiquitination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4969. doi:10.1158/1538-7445.AM2015-4969
Abstract 4988: A7 nicotinic acetylcholine receptor mediated the chemosensitivity of gastric cancer cells
Authors: Huang Chien-Yu , Chang Yu-Jia
Published: August 2015

Background: Gastric cancer is important healthy issue in the worldwide. A7-nAChR has become a potential therapeutic target for cancer. Highly expression of A7-nAChR caused the benefit of cell survival in gastric cancers. However, it is unclear for the role of A7-nAChR in chemotherapy therapy on gastric cancer. Methods: In this study, we generated A7-nAChR knockdown (A7-nAChR-KD) AGS cells by a short interfering RNA (siRNA) technique in gastric cancer cells. The anti-proliferation effects of chemotherapy drugs were determined by biosensor system, MTT assay, TUNEL assay, and cell cycle determination.

Results: We determined the IC50 of 5-FU, taxane, epirubicin, cisplastin, gemcitabine, labtinibine, sunitinib in both A7-nAChR-Kd cells and scrambled control cells by MTT assay. We found that there is no difference between A7-nAChR-KD cells and scrambled control cells for epirubicin, cisplastin, labtinibine, sunitinib treatments. Interestingly, A7-nAChR-KD cells were less resistant to taxane treatment compared with the scrambled control cells. The apoptosis cell population was increased less in scrambled control cells treated with taxane than A7-nAChR-KD cells in cell cycle distribution and TUNEL assays. Conclusions: We conclude that A7-nAChR may be a key biomarker in mediating the chemotherapy efficacy on gastric cells.

Citation Format: Chien-Yu Huang, Yu-Jia Chang. A7 nicotinic acetylcholine receptor mediated the chemosensitivity of gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4988. doi:10.1158/1538-7445.AM2015-4988