bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4458: Discovery of novel epigenetic biomarkers in premalignant/malignant oral cavity lesions by methylation arrays
Authors: Demokan Semra , Comert Sevde , Ozkoklesen Cansu , Ulusan Murat , Ak Gulsum , Alatli Canan , Dalay Nejat
Published: July 2016

Purpose: The incidence of the oral squamous cell carcinoma (OSCC) is around 50% of all head and neck cancer (HNC), the sixth most common type of cancer in the world. Aberrant hyper/hypo-methylation in the promoter region of some known or putative tumor suppressor/protoonco-genes occurs frequently during the development of various cancers including OSCC. Both of genetic and environmental factors play roles in the carcinogenesis of malignant lesions. Etiological factors predisposing to OSCC are smoking, alcohol and tobacco consumption, snuffuse, viral factors, chronic irritation, iron and various vitamin deficiencies, poor oral hygiene and diseases such as syphilis. Despite the advances in cancer diagnosis and treatment, the 5-year survival rate of OSCC patients is poor and because of advanced stage of disease, it has high mortality and morbidity. Another the most important ethiological factor of OSCC is oral premalignant lesions (OPML), since OPML is related with malignant transformation. In our study, we included three different OPML associated with different risks of leading to malignancy: Oral leukoplakia has a high risk of conversion into malignancy, whereas oral lichen planus and oral lichenoid dysplasia are associated with lower risk. We aimed to investigate the epigenetic characterization of OSCC and OPML relative to the epigenetic changes in these groups.

Experimental Design: Tumor and corresponding healthy tissues from 12 and 10 Turkish patients with OPML and OSCC, respectively were collected. The samples were analyzed histopatologically and after DNA extraction and bisulfite modification, methylation patterns of the samples were investigated by using the Illumina Infinium HumanMethylation450 (450K) BeadChip array.

Results: According to our preliminary results obtained from the methylation array experiments, CpG regions in the UOX, ABCC12, FCRLB, ABL2, LGR6 and SLC22A18 genes were found unmethylated while CpGs located in YPEL-3 were highly methylated (p<0.005).

Conclusion: We concluded the UOX, ABCC12, FCRLB, ABL2, LGR6, SLC22A18 and YPEL-3 may be potential biomarker candidate genes playing role in the transformation mechanism of OPML/OSCC. In the further studies, we will compare the methylation patterns between OPML subgroups to discriminate the potential candidates.

*This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK-SBAG-114S497).

Citation Format: Semra Demokan, Sevde Comert, Cansu Ozkoklesen, Murat Ulusan, Gulsum Ak, Canan Alatli, Nejat Dalay. Discovery of novel epigenetic biomarkers in premalignant/malignant oral cavity lesions by methylation arrays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4458.
Abstract 4465: Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma
Authors: Phelps Michael , Bailey Jenna , Vleeshouwer-Neumann Terra , Chen Eleanor
Published: July 2016

Embryonal rhabdomyosarcoma (ERMS), one of the most common and lethal pediatric sarcomas, is characterized by an arrest in myogenic differentiation. We have previously shown that treatment of ERMS cells with pan histone deacetylase (HDAC) inhibitors promotes myogenic differentiation. However, the underlying mechanism specific to each HDAC in ERMS remains unknown. We have completed a high-efficiency CRISPR/Cas9 gene knockout screen of class I and II HDAC genes, revealing HDAC3 as a major driver of myogenic suppression in ERMS. HDAC3-deficient ERMS cells showed robust terminal myogenic differentiation with the formation of multinucleated myotubes. Expanding on our in vitro studies, we developed a novel tamoxifen-inducible CRISPR gene-targeting system in ERMS xenografts established in immune-compromised mice. Disruption of HDAC3 in vivo significantly suppressed tumor growth by inducing myogenic differentiation of ERMS cells. By combining inducible CRISPR gene targeting with HDAC3 structure-function analysis, we showed that HDAC3 functions in concert with the nuclear receptor co-repressor (NCOR1 and NCOR2) complex to regulate gene transcription. HDAC3/NCOR binding and HDAC3 deacetylase activity are both required for suppressing myogenic differentiation in ERMS. RNA sequencing analysis of differentially expressed genes in HDAC3-deficient ERMS cells highlighted key pathways dysregulated in ERMS. Our results suggest that epigenetic silencing of the myogenic program is a major mechanism required for ERMS tumor growth. Identification of the essential interaction between HDAC3 and the NCOR transcriptional repressor complex as well as downstream genes and pathways provides important insights into the biology of ERMS tumorigenesis. Our findings highlight potential new drug targets specific to the activity of HDAC3 in order to improve treatment of ERMS patients.

Citation Format: Michael Phelps, Jenna Bailey, Terra Vleeshouwer-Neumann, Eleanor Chen. Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4465.
Abstract 4470: ST3GAL1-associated transcriptomic program portends poor prognosis in glioblastoma
Authors: Chong Yuk Kien , Sandanaraj Edwin , Koh Lynnette , Thangaveloo Moogaambikai , Tan Melanie SY , Koh Geraldene , Toh Tan Boon , Lim Grace GY , Holbrook Joanna , Kon Oi Lian , Nadarajah Mahendran , Ng Ivan , Ng Wai Hoe , Tan Nguan Soon , Lim Kah Leong , Tang Carol S. , Ang Beng Ti
Published: July 2016

Cell surface sialylation has been associated with tumor cell invasiveness in several cancers. Patients with grade IV glioblastoma multiforme (GBM) often show a median survival period of fifteen months, even with the current standard-of-care treatment. Among the reasons for this poor prognosis lies in the cellular and molecular heterogeneity of tumor cells. The Cancer Genome Atlas efforts have shown that histologically identical GBM tumors can be divided into four molecular subtypes based on gene expression, with each subtype corresponding to unique genomic aberrations and clinical outcome. These findings highlight the limitation of relying solely on morphological approaches to diagnose and subsequently treat patients. We conducted a lectin screen with the goal of identifying candidates that stained normal and tumor cells differentially. We identified a sialyltransferase, ST3Gal1 as a mediator of the binding pattern of one such lectin, Peanut Agglutinin. We demonstrate that ST3Gal1 promotes tumor cell invasiveness and enriches for self-renewal potential in the mesenchymal tumor subclass of patients, typified by highly aggressive and recurrent profiles. Depletion of ST3Gal1 extends tumor latency and prolongs survival in mice. Importantly, an enrichment of the ST3GAL1 transcriptomic program portends poor prognosis in glioma patients. Moving forward, since there are no mutations or changes in copy number of ST3GAL1, we explored epigenetic regulation as a possible mechanism. By tapping into data from the NIH Roadmap Epigenome, and transcriptomic analysis of our patient tumor-derived cells, we identified a histone clustering signature that correlates with survival outcome and disease progression. We are currently investigating one such histone modifier, lysine (K)-specific demethylase 1A (LSD1) and its role at modifying ST3GAL1 transcription and activity.

Citation Format: Yuk Kien Chong, Edwin Sandanaraj, Lynnette Koh, Moogaambikai Thangaveloo, Melanie SY Tan, Geraldene Koh, Tan Boon Toh, Grace GY Lim, Joanna Holbrook, Oi Lian Kon, Mahendran Nadarajah, Ivan Ng, Wai Hoe Ng, Nguan Soon Tan, Kah Leong Lim, Carol S. Tang, Beng Ti Ang. ST3GAL1-associated transcriptomic program portends poor prognosis in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4470.
Abstract 4484: Differentially methylation between ER negative and ER positive breast cancer identifies master regulators to expose potential epigenetic drivers of aggressive disease
Authors: Worsham Maria J. , Chen Kang Mei , Datta Indrani , Stephen Josena K. , Chitale Dhananjay , Divine George
Published: July 2016

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has yet to configure in epigenetics. There has been relatively little advancement in changing the management of women with ER negative BC, mainly due to a dearth of actionable therapeutic targets. Our drill-down approach to identified an ER negative-specific 16 gene methylation signature (AHNAK, ALPL, ANXA2R, CCND1, CIRBP, CPQ, DST, EGFR, ESR1, GPRC5B, HERC5, IL22RA2, MITF, OBSL1, POU3F3, RB1CC1) starting from a discovery approach (Illumina Infinium HumanMethylation450 BeadChip, 40 ER negative vs. 40 ER positive BC) followed by expression verification, significant rankings in biological pathways (Ingenuity Pathway Analysis), and confirmation by targeted sequencing using Illumina MiSeq.

Causal Networks are small hierarchical networks of regulators that control the expression/methylation of dataset targets. They can enhance understanding of the effect of master regulators on disease or function.

The objective of this study was to identify regulatory networks utilizing IPA's Causal Network Analysis (CNA) in order to illuminate possible causes and mechanisms underlying the biological activities of the 16 candidate gene signature differentiating ER negative from ER positive BC. To reflect expected gene expression direction implied by methylation changes for the 16 candidate genes, the inverse of the methylation ratio from ER negative vs. ER positive tissue was used for CNA.

CNA software identified 4 hierarchical networks and their corresponding master regulatory molecules, diethylstilbestrol, MSH2, 15-ketoprotaglandin E2, and transcription regulator SP1. Diethylstilbestrol and SP1 had direct regulatory influence (depth level 1) to the candidate molecules ALPL, CCND1, EGFR, ESR1 and CCND1, CIRBP, EGFR, ESR1, respectively. CNA raised the profile of ALPL, CCND1, CIRBP, EGFR, ESR1 (5/16 candidate genes) for further consideration as potential epigenetic drivers of ER negative BC.

Support: Komen Foundation: KG110218

Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, George Divine. Differentially methylation between ER negative and ER positive breast cancer identifies master regulators to expose potential epigenetic drivers of aggressive disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4484.
Abstract 4500: Normal tissue and data resources for cancer research from the GTEx program
Authors: Guan Ping , Rao Abhi , Volpi Simona , Koester Susan , Moore Helen M. ,   GTEx consortium
Published: July 2016

The NIH Common Fund's Genotype-Tissue Expression (GTEx) project aims to study gene expression and regulation across multiple human tissues from approximately 1000 healthy normal postmortem donors. GTEx will provide valuable insights into gene regulation and its tissue specificity, to identify correlations between genetic variations and variations in gene expression levels as expression quantitative trait loci (eQTLs), and to help understand inherited susceptibility to disease.

Initiated in 2010, the GTEx program has generated a large volume of data associated with each donor, including clinical and histopathological, as well as genotyping and gene expression data from whole genome sequencing, whole exome sequencing, expression array, and RNAseq data. The program has published research results in multiple scientific journals over the past year.

The following public resources are available from the GTEx program:

1. GTEx Portal: an open access database of GTEx expression data and analysis results: http://www.gtexportal.org

2. Database of Genotypes and Phenotypes (dbGaP): controlled access of comprehensive GTEx clinical data and raw sequencing data: http://www.ncbi.nlm.nih.gov/gap

3. Access to residual GTEx biospecimens for research: http://www.gtexportal.org/home/samplesPage

4. SOPs and best practices from GTEx biospecimen collections: http://biospecimens.cancer.gov/resources/sops/library.asp

5. GTEx histological image viewer: http://biospecimens.cancer.gov/resources/tissue_image_library.asp

6. GTEx donors’ families website: a lay description of the GTEx project tailored to the GTEx donors’ families: http://www.genome.gov/gtex

GTEx data can be used in combination with other data sets such as The Cancer Genome Atlas (TCGA) and genome-wide association studies (GWAS) to further explore the genetic causes and biology of cancer. The eQTL data from multiple tissues will help prioritize candidate genes within GWAS-associated loci; allow evaluation of tissue specificity of associated loci, and pinpoint target tissues for disease studies. Researchers are using GTEx resources to: study cancer heterogeneity by subtracting normal tissue expression; identify mutations in protein-truncating variants which may cause cancer; and explore gene activity from multiple donors across multiple tissue types to better understand the age/gender bias in cancer and other diseases.

Citation Format: Ping Guan, Abhi Rao, Simona Volpi, Susan Koester, Helen M. Moore, GTEx consortium. Normal tissue and data resources for cancer research from the GTEx program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4500.
Abstract 4503: Actionable mutations and mutational burden in renal cell carcinoma
Authors: Mockus Susan M. , Patterson Sara E. , Pettus Jason R. , Tsongalis Gregory J.
Published: July 2016

Clear cell renal cell carcinoma (CCRCC), the most common subtype of renal cell carcinoma, is a heterogeneous cancer with variable outcomes and molecular aberrations. Further elucidation of mutational profiles and actionable mutations may improve patient outcomes in CCRCC. Somatic mutational profiling via next-generation sequencing was conducted on 30 CCRCC whole section FFPE patient samples to determine the mutational burden as compared to other solid tumor types. The sequencing failure rate for these samples was 20% (6/30). A 358-gene panel was also run on an additional 57 non-CCRCC solid tumor FFPE patient samples (81 samples in total). FASTQ files generated from Illumina's CASAVA software were submitted to the JAX Clinical Genome Analytics (CGA) data analysis pipeline to perform automated read quality assessment, alignment, and variant calling. Identified variants were then submitted for clinical curation using the JAX Clinical Knowledgebase (CKB) for actionable mutational analysis and overall mutational burden. Of the CCRCC sample set, 33% had one or more actionable mutations. This was low compared to actionable mutations in colon (16/16, 100% actionable), TNBC (18/20, 90% actionable), squamous lung (8/9, 89% actionable), and pancreatic (8/12, 67% actionable). Furthermore, the CCRCC set had a low overall mutational burden with 22 ± 6.7 nonsynonymous SNV's as compared to colon (29 ± 11), squamous lung (43 ± 17), and TNBC (31.5 ± 12). However, the pancreatic set had even lower SNV's at 12.7 ± 3.4. The number of indels was comparable across the five sets, except for colon, which averaged 6.9 ± 8.3. Significant copy number variations (CNV’s; amplification validated at greater than or equal to 6 copies) also had a wide range with squamous lung at the highest (11 ± 11), followed by TNBC (5.6 ± 5.0), and then colon (2.3 ± 3.0). Both the CCRCC and pancreatic sets had few CNV's at 0.8 ± 1.5 and 0.17 ± 0.40, respectively. Common mutations identified in the CCRCC set included EPHB6 duplications near S166, which were identified in 25% of samples. Gene specific mutations in CCRCC were also identified in VHL (54%) and SETD2 (21%). These three genes, EPHB6, VHL, and SETD2 have been previously implicated in CCRCC pathogenesis. Six variants identified in VHL were C77*, P86R, Q132fs, A149D, R177fs, and L184fs. Due to the low mutational burden and/or actionable mutations in CCRCC, even large NGS panels may not be adequate to profile somatic mutations and whole exome or whole genome may be more conducive for molecular subtyping of renal cell carcinoma.

Citation Format: Susan M. Mockus, Sara E. Patterson, Jason R. Pettus, Gregory J. Tsongalis. Actionable mutations and mutational burden in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4503.
Abstract 4520: The effect of EPZ011989, an enhancer of Zeste homolog 2 inhibitor, in acute myeloid leukemia
Authors: Fobare Sydney , Miller Cecelia , Orwick Shelley J. , Keilhack Heike , Hertlein Erin , Byrd John C.
Published: July 2016

Enhancer of Zeste Homolog 2 (EZH2) is a catalytic core subunit of the Polycomb repressive complex 2, PRC2. EZH2 catalyzes the tri-methylation of lysine 27 on histone H3 (H3K27me3) which often results in the silencing of associated gene promoters. The over-expression of EZH2 is associated with cancer progression and poor prognosis in solid tumors and hematological malignances. EZH2 mutations in lymphoma generally are activating whereas those in myelodysplasia and acute myeloid leukemia are typically loss of function. Additionally, EZH2 mutations are not isolated to a hotspot and therefore discerning phenotype is challenging. The purpose of this study was to determine if the inhibition of EZH2 induces cell differentiation or apoptosis in AML, and to determine the significance of EZH2 mutations in AML patients. As H3K27me3 deposition is catalyzed by EZH2, we hypothesized that analysis of H3K27me3 levels could differentiate an inactivating EZH2 mutation. We analyzed H3K27me3 levels in AML patient samples with wild type (N = 5) and mutant (N = 3) EZH2, and found that overall H3K27me3 is reduced in the mutant patient samples. Furthermore, the sample with the highest variant allele frequency (VAF) of the EZH2 mutation had noticeably lower levels of H3K27me3 compared to those that had a lower VAF. This finding demonstrates that diminished H3K27me3 levels can predict the presence of an inactivating EZH2 mutation in AML cells. We next assessed if the EZH2 tool molecule EPZ011989 produced a similar phenotype in wild-type EZH2 AML cell lines (Kasumi-1, MOLM-13, and MV4-11). EPZ011989 inhibited H3K27me3 in all of the cell lines at a concentration of 0.625 μM after only four days of treatment. At these concentrations and this time point we demonstrated no change in viability among these three cell lines and only minimal proliferation inhibition in MV4-11 and MOLM-13 cells. Longer time points are currently being investigated as EZH2 inhibitors often induce anti-proliferative effects only after longer incubation periods.

Therefore, our results support that inactivation of EZH2 with EZP011989 in AML cell lines induces the same cell-biochemical consequence (i.e. H3K27me3 loss) associated with EZH2 mutations in AML patients. This potentially will allow diverse pharmacologic studies in cell lines relative to gene targets and also synergistic lethality studies utilizing genetic and pharmacologic screening techniques to potentially develop therapeutic agents to treat this subtype of AML.

Citation Format: Sydney Fobare, Cecelia Miller, Shelley J. Orwick, Heike Keilhack, Erin Hertlein, John C. Byrd. The effect of EPZ011989, an enhancer of Zeste homolog 2 inhibitor, in acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4520.
Abstract 4527: Cellular stress response 1 down-regulates the expression of epidermal growth factor receptor and platelet-derived growth factor receptor through inactivation of splicing factor 3A3
Authors: Luo Jian-Hua , Zuo Ze-Hua , Yu Yan P.
Published: July 2016

Cellular stress response 1 (CSR1) is a tumor suppressor gene that plays an important role in regulating cell death. In this report, we showed that the N-terminus of CSR1 interacts with splicing factor 3A, subunit 3 (SF3A3). The SF3A3 binding motif was identified in the region of amino acids 62-91 of CSR1 through cell-free binding analyses. The interaction between CSR1 and SF3A3 led to migration of SF3A3 from nucleus to cytoplasm. The cytoplasmic redistribution of SF3A3 significantly reduced the splicing efficiency of epidermal growth factor receptor and platelet-derived growth factor receptor. Induction of CSR1 or down-regulation of SF3A3 also significantly reduced the splicing activity of oxytocin reporter gene both in vivo and in vitro. Mutant CSR1 that lacks the SF3A3 binding motif contained no RNA splicing regulatory activity, while the peptide corresponding to the SF3A3 binding motif in CSR1 interfered with the wild type CSR1 mediated inhibition of RNA splicing. Interaction of CSR1 and SF3A3 is essential for CSR1 mediated cell death. To our knowledge, this is the first report demonstrating that RNA splicing is negatively regulated by redistribution of a splicing factor.

Citation Format: Jian-Hua Luo, Ze-Hua Zuo, Yan P. Yu. Cellular stress response 1 down-regulates the expression of epidermal growth factor receptor and platelet-derived growth factor receptor through inactivation of splicing factor 3A3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4527.
Abstract 4538: Heterochromatin protein 1 alpha modulates the invasive potential of malignant cells
Authors: Cui Xiaoyan , Stimpson Lance , Hale Tracy , Contreras Alejandro
Published: July 2016

Altered chromatin patterning is observed in neoplastic cells, with loss of heterochromatin associated with aggressive malignancies. Heterochromatin is the tightly packed form of chromatin characterized by limited transcription and is identified by the localization of Heterochromatin Protein 1 alpha (HP1 alpha) and Heterochromatin Protein 1 beta (HP1 beta). HP1 alpha and HP1 beta help to maintain chromatin organization during the cell cycle and regulate gene expression. We have previously shown that HP1 alpha and HP1 beta are both downregulated in papillary thyroid carcinomas and that both proteins are lost to a greater degree in 3 out of 4 metastatic lesions of papillary thyroid carcinomas. In addition, others have shown a loss of HP1 alpha is associated with increased invasive potential and metastatic lesions in a range of human tumors.

By modulating the level of HP1 alpha in breast cancer cell lines we demonstrate a causal role for HP1 alpha in regulating invasive potential. To identify the molecular pathways through which HP1 alpha regulates this phenotype, RNA-seq was used to measure changes in both miRNA and mRNA expression after knock-down of HP1 alpha in the poorly invasive breast cancer cell line, MCF7. Reduction of HP1 alpha levels increased their invasive potential by 44% and this corresponded with differential expression of genes that promote an epithelial to mesenchymal transition (EMT). In addition, a tissue microarray containing 56 invasive breast carcinomas was immunohistochemically stained for HP1 alpha and HP1 beta. A majority of the breast carcinomas showed decreased expression of HP1 alpha when compared to HP1 beta (32/56 tumors). Taken together, these data suggest that loss of HP1 alpha results in a phenotype that is an intermediate of the EMT, and is supported by their altered cytoskeleton, cell-cell and cell-matrix interactions. HP1 alpha loss may confer a more aggressive, invasive potential in breast carcinomas.

By exploring these HP1 alpha −δϵπϵνδϵντ pathways, we seek to further elucidate the regulatory signals that allow a malignant epithelial cell to undergo EMT and begin to invade surrounding tissue.

Citation Format: Xiaoyan Cui, Lance Stimpson, Tracy Hale, Alejandro Contreras. Heterochromatin protein 1 alpha modulates the invasive potential of malignant cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4538.
Abstract 4583: Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells
Authors: Balan Murugabaskar , Pal Soumitro
Published: July 2016

Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins that act as co-receptors for class III semaphorins and several members of the vascular endothelial growth factor (VEGF) family. In conjunction with VEGF receptors and plexins, NRPs and semaphorins modulate various biological responses. Recent studies suggest that NRPs are up-regulated in many types of cancer cells, and may play a major role in tumor growth. In addition to semaphorins and VEGF, NRP1 and NRP2 can also bind to hepatocyte growth factor (HGF), which is the ligand for the tyrosine kinase receptor c-MET. c-MET is highly over-expressed in renal cancer cells; however, the impact of NRPs on c-MET-mediated tumorigenic pathways have not been studied. Recently, we have reported that HGF-/c-MET-induced signaling increases the expression of immunosuppressive and negative-co-stimulatory molecule programmed death-1 ligand (PD-L1) on renal cell carcinoma (RCC) cells, and it protects tumor cells from immune-mediated killing. In this study, we investigated whether NRPs and its ligands can modulate the expression of HGF-/c-MET-induced PD-L1. We have observed that NRP2, and to a lesser extent NRP1, is over-expressed in human RCC cell lines (786-O, ACHN and Caki-1) compared with normal renal epithelial cells at both mRNA and protein level. siRNA-mediated silencing of NRP1 and NRP2 promoted apoptosis of renal cancer cells, suggesting possible role(s) of NRPs in tumor growth. The knock-down of NRP2 down-regulated the cell surface expression of PD-L1 possibly through the reduced interaction of growth-promoting ligands (like, HGF) with NRP2. Interestingly, SEMA3F, a class III semaphorin ligand for both NRP1 and NRP2, has been reported to have anti-tumorigenic effects. We observed that SEMA3F was significantly down-regulated in RCC cells compared with normal renal epithelial cells; and when we added SEMA3F to the cultures of renal cancer cells, it down-regulated both basal and HGF-/c-MET-induced PD-L1 expression. Together, our findings suggest that NRPs (primarily NRP2) can play a critical role in regulating HGF-/c-MET-induced PD-L1 expression in renal cancer cells to mediate immune escape of tumor cells. Therefore targeting NRP2 and/or its activation through SEMA3F/synthetic ligands may restrict c-MET-PD-L1-induced tumor promoting pathways in renal cancer cells.

Citation Format: Murugabaskar Balan, Soumitro Pal. Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4583.
Abstract 4587: Dynactin dysregulation-induced increase in EGFR on basal-like breast cancer cells
Authors: Chaudhuri Gautam , Misra Smita
Published: July 2016

Aggressive triple-negative basal-like breast cancer (BLBC) cells are often characterized with overabundance of the epidermal growth factor receptor (EGFR). Major objective of our research is to explore the possibility that dysregulation of the function of the dynactin complex as a mechanism of over-abundance of EGFR on BLBC cell surface. Our notion is that molecular defects in the dynactin-mediated retrograde EGFR recycling pathway in the BLBC cells causes the accumulation of this receptor on their cell surface making them hyper-responsive to EGF signaling. Our comparative transcriptome analysis between BLBC cells with higher levels of EGFR and the non-BLBC cells with lower levels of EGFR revealed that the expression of dynactin complex subunits 1, 2, 3, 4 and 6 (DCTN1, 2, 3, 4, 6), are significantly reduced in the EGFR-high aggressive BLBC cells. In addition, EGFR-high BLBC cells also have lower levels of two other dynactin-associated proteins, CLIP1 and LRRK1. Interestingly, elevation of surface EGFR levels in BLBC cells are associated with higher levels of the long non-coding RNA PVT1. Six miRNAs are generated from the precursor of PVT1 RNA during cis-splicing. They are miR-1204, miR-1205, miR-1206, miR-1207-3p, miR-1207-5p and miR-1208. We found that relative levels of these miRNAs are significantly higher in EGFR-high BLBC cells and tissues in comparison to the non-aggressive EGFR-low BLBC cells. Bioinformatics analysis followed by experimental validation revealed that miR-1207-5p inhibits DCTN1, DCTN3 and ACTR1A; DCTN2 is inhibited by miR-1205 and miR-1208. MiR-1208 also inhibits DCTN6 and CLIP1 whereas LRRK1 and CAPZA1 are inhibited by miR-1207-3p and miR-1206, respectively. Moreover beta-spectrin (SPTB), a protein associated with dynactin complex, is also inhibited by miR-1205 and miR-1207-3p. Treatment of the cells with the antagomirs of the miRNAs increased the levels of their respective target proteins with concomitant decrease in the surface level of EGFR. Our data suggest that BLBC cells maintain a robust level of EGFR on the cell surface by repressing the retrograde transport of EGFR through the repression of the proteins in the dynactin complex and thus sustain their higher growth rates. This research is supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC.

Citation Format: Gautam Chaudhuri, Smita Misra. Dynactin dysregulation-induced increase in EGFR on basal-like breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4587.
Abstract 4589: Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells
Authors: Bashir Asma’U I.J. , Jones Sarah , Perry Christopher J. , Safrany Stephen T. , Nicholl Iain D.
Published: July 2016

BACKGROUND

Colorectal cancer (CRC) is a global phenomenon with over 135 million new cases and over 600,000 deaths worldwide annually. The EGFR regulates normal growth and differentiation of cells, however its dysregulation is the cause of a wide range of cancers, including CRC. Activation of the EGFR requires phosphorylation at specific sites, which then leads to the recruitment of proteins and ultimately its internalization.

The use of low-dose aspirin as a preventative agent is a controversial topic, particularly as the mechanism of action is hotly disputed. We have previously determined that novel aspirin analogues dramatically reduce EGFR phosphorylation in SW480 CRC cells; these cells express neither COX-1 nor COX-2, the perceived target for aspirin-like compounds. We have seen these aspirin analogues to also perturb the internalization of this receptor.

METHODS

SW 480 CRC cells were cultured in Leibovitz's L-15 medium on glass slides and starved for 48 hours. The cells were then treated with 0.5 mM aspirin analogues (ortho-aspirin, meta-aspirin, para-aspirin, ortho-thioaspirin, meta-thioaspirin and para-thioaspirin) for 30 minutes and then treated with EGF-tagged with AlexaFluor 555 for an hour. The reaction was the quenched with cold PBS, fixed and mounted on microscope slides with VectaShield + DAPI. Confocal microscopy was used to study the effect of these aspirin analogues on EGFR internalization.

RESULTS

EGF increased phosphorylation at all sites tested. We have peviously shown that these effects were prevented by preincubation with thioaspirins more potently than with aspirin and its positional isomers. These novel thioaspirins are also more potent in inhibiting proliferation of SW480 CRC cells. We now show these analogues prevent the internalization of EGFR. Instead they permit the formation of clusters. Aspirin and its analogues can perturb EGFR internalization and signalling in SW 480 cells.

CONCLUSION

The EGFR signalling pathway is a target for aspirin and aspirin analogues. The inhibition of receptor phosphorylation is accompanied by the loss of receptor internalization.

Citation Format: Asma’U I.J. Bashir, Sarah Jones, Christopher J. Perry, Stephen T. Safrany, Iain D. Nicholl. Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4589.
Abstract 459: The use of circulating SAA and CXCL4 to predict outcome of osteosarcoma at diagnosis
Authors: Flores Ricardo J.
Published: July 2016

Background: Osteosarcoma is the most common malignant bone tumor in children. The survival rate of osteosarcoma is 60-70%, which has remained stagnant for the past four decades. Identifying biomarkers that can better prognosticate osteosarcoma patients and guide therapies would improve survival of this disease.

Material and Methods: In this study, we used ELISA to evaluate the expression of two previously identified circulating biomarkers, namely Serum Amyloid A (SAA) and CXC Chemokine Ligand 4 (CXCL4), in a large cohort of serum samples (n = 233) obtained at from the Children's Oncology Group at initial diagnosis. The biomarkers’ expression was correlated with survival and evaluated for prognostic significance in the OS patients.

Results: Analysis showed that patients with high SAA and low CXCL4 levels had significantly poorer outcome (p = 0.014, HR = 1.68), which was independent of initial metastasis status. The 5-year overall survival rates for the “high-risk” and “low-risk” groups were 47% (95% CI; 36% - 62%) and 64% (95% CI; 57% - 72%), respectively. In addition, low tumor expression of CXCL4 correlated with poor survival (p = 0.017) in an osteosarcoma tissue microarray. In conclusion, this study reports a novel prognostic model consisting of high SAA and low CXCL4 levels in sera that prognosticates osteosarcoma patients at initial diagnosis. This model could serve as the basis for future biomarker-guided clinical trials by incorporating targeted therapy for the poor prognostic population.

Citation Format: Ricardo J. Flores. The use of circulating SAA and CXCL4 to predict outcome of osteosarcoma at diagnosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 459.
Abstract 4600: Regulation of Wnt/β-catenin signaling by Wnt co-receptor LRP6 in colorectal cancer cells
Authors: Li Yonghe , Lu Wenyan , King Taj D.
Published: July 2016

Mutations of APC and CTNNB1 are two major factors for Wnt/β-catenin signaling over-activation in colorectal cancer, however recent studies indicate that the levels of Wnt/β-catenin signaling in colorectal cancer cells could be modulated on the cell surface. The low-density lipoprotein-related protein 6 (LRP6) is an essential Wnt co-receptor for the Wnt/β-catenin signaling pathway. Herein, we reported that LRP6 expression is up-regulated in colorectal cancer. Moreover, LRP6 silencing significantly reduced Wnt/β-catenin signaling in colorectal cancer HCT116 cells (harboring a CTNNB1 mutation) and DLD-1 cells (harboring an APC mutation) and inhibited cancer cell viability. In addition, treatment of HCT116 cells with Wnt3A resulted in LRP6 phosphorylation and activation of Wnt/β-catenin signaling, which was blocked by LRP6 antagonist Mesd. Finally, we found that salinomycin, a potent small molecule inhibitor of LRP6, suppressed LRP6 expression and phosphorylation, inhibited Wnt/β-catenin signaling in colorectal cancer HCT116 and DLD-1 cells, and repressed cancer cell viability and colony formation within the same concentration range. Together, these results indicate that LRP6 plays an important role in over-activation of Wnt/β-catenin signaling in colorectal cancer cells, and that LRP6 is a potential therapeutic target for colorectal cancer.

Citation Format: Yonghe Li, Wenyan Lu, Taj D. King. Regulation of Wnt/β-catenin signaling by Wnt co-receptor LRP6 in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4600.
Abstract 4626: In-vitro growth inhibitory effects of diosmetin on human prostate cancer cells
Authors: Pakradooni Rebecca , Patel Riddhi , Srivastava Janmejai K. , Shukla Sanjeev
Published: July 2016

Characterization of effective chemo-preventive agents for treatment of prostate cancer is an utmost priority. Diosmetin (5, 7-Trihydroxy-4′-methoxyflavone), a natural flavonoid present in citrus plant, has anti-mutagenic and anti-allergic properties. However, the anticancer properties of diosmetin have not been explored in prostate cancer. Present study demonstrates, cell growth inhibition and induction of apoptosis in human prostate carcinoma LNCaP (androgen-responsive) and PC-3 (androgen-refractory) cells by diosmetin, however, no significant growth inhibition observed in normal prostate epithelial cells (RWPE1). Cell growth is a fundamental biological process, where mTOR (mammalian target of rapamycin) pathway has its role, regulates cell growth by coordinating energy and nutrient signals with growth factor. mTOR protein kinase have two different complexes; complex-I contains major component raptor; a target of rapamycin and complex II; insensitive to rapamycin, has major component rictor. Rictor is important as it phosphorylates Ser-473 of Akt/PKB, which is essential for full Akt/PKB activation. We observed diosmetin treated prostate cancer cells was able to inhibit growth factor (IGF-1) and cytokine (IL-6) induced rictor expression. Furthermore, diosmetin (20μM) treatment to prostate cancer cells prevented rictor nuclear localization and subsequently inhibited phospho-Akt (Ser-473), which subsequently resulted in increased FOXO3a nuclear presence. Downstream pathway p70S6 kinase, which has a critical role in cell cycle, growth and survival, was also inhibited after diosmetin treatment. These effects were associated with a marked decrease in the protein expression of cyclin D1 and their activating partner, cyclin-dependent kinase (cdk) 2 and 4 with concomitant upregulation of KIP1/p27 and INK4a/p16. Additionally diosmetin treatment to these cells modulated cell survival machinery by down regulating key molecules viz., c-Myc, Survivin and XIAP (X-Linked Inhibitor of Apoptosis). Diosmetin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with an increase in cleaved caspase-3. Taken together, our studies demonstrate that diosmetin-mediated growth inhibition and induction of apoptosis in both LNCaP and PC-3 cells were due to deregulated levels of rictor and Akt signaling cascade and modulation of cell-cycle machinery. We are documenting these evidences for the first time that diosmetin acts against potential molecular targets to alter cellular events to elicit anticancer effects in prostate cancer cells.

Citation Format: Rebecca Pakradooni, Riddhi Patel, Janmejai K. Srivastava, Sanjeev Shukla. In-vitro growth inhibitory effects of diosmetin on human prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4626.
Abstract 463: Immunohistochemical study of TROP-2 protein expression in thyroid lesions
Authors: Liao Zhiming
Published: July 2016

Introduction

Tumor-associated calcium signal transducer 2 (TROP-2) is a transmembrane glycoprotein overexpressed in various types of epithelial carcinomas from lung, breast, stomach, ovary, cervix, colon and pancreas. A recent immunohistochemical (IHC) study indicated that TROP-2 is also overexpressed in thyroid papillary carcinoma, but the sensitivity is only 82% in formalin-fixed and paraffin-embedded thyroid (FFPE) tissues when a polyclonal anti-TROP-2 antibody was used. A high quality of TROP-2 antibody for IHC applications is needed to improve the sensitivity of the detection of thyroid papillary carcinoma in FFPE tissues.

Methodology

A rabbit monoclonal antibody (clone SP295) against human TROP-2 protein was developed. Western blot was performed using SK-BR-3 cell lysates to verify the specificity of the TROP-2 antibody. An IHC study of TROP-2 was performed on normal tissue and tumor arrays from the thyroid and other organs. TROP-2 staining intensity was semi-quantitatively scored using a 0-3 scale. The sensitivity and specificity of the TROP-2 IHC assay for detecting papillary carcinoma were determined using 65 cases of thyroid papillary carcinoma, 29 cases of follicular carcinoma, and 31 cases of other thyroid conditions (normal, adenoma, Grave's disease, thyroiditis, Goiter, medullary carcinoma, clear cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma).

Results

A single band at 33 kDa was detected by SP295 on a western blot. In normal tissues, TROP-2 is detectable by IHC mostly in squamous epithelia of many normal organs (skin, pharynx, esophagus, and cervix) and cuboidal and columnar epithelium (breast, kidney, liver, uterus, pancreas, prostate and transitional epithelium of the bladder). In thyroid lesions, the TROP-2 positive rate is 100% (65/65) in papillary adenocarcinoma and it is negative in all other thyroid conditions (normal, adenoma, Grave's disease, thyroiditis, Goiter, medullary carcinoma, clear cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma). The sensitivity and specificity of TROP-2 IHC for detecting papillary carcinoma were both 100% in this study.

Conclusions

Rabbit monoclonal TROP-2 antibody (clone SP295) is highly sensitive and specific for detecting TROP-2 in FFPE tissues and it can be used to identify thyroid papillary carcinoma.

Citation Format: Zhiming Liao. Immunohistochemical study of TROP-2 protein expression in thyroid lesions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 463.
Abstract 4637: Chk1 inhibition: a promising targeted treatment for head and neck squamous cell carcinoma
Authors: van Harten Anne M. , Stigter-van Walsum Marijke , Buijze Marijke , Braakhuis Boudewijn Jm , Brakenhoff Ruud H.
Published: July 2016

Over 90% of all malignant tumors in the head and neck region are squamous cell carcinomas (HNSCC), ranking within the top 10 of most common cancers worldwide. HNSCC arise in the mucosal linings of the upper aerodigestive tract, and known risk factors are tobacco use, excessive alcohol consumption and infection with high risk types of the human papillomavirus (HPV). Despite the application of intensive treatment protocols, the 5-years survival rate of HNSCC patients has hardly increased last decades and remained at approximately 50%. Therefore, new therapeutic possibilities are urgently awaited.

Previously, we identified CHEK1 as a candidate therapeutic target for HNSCC in a whole genome siRNA screen (Martens-de Kemp et al, Clin Cancer Res 2013). Additionally, a functional screen using a microRNA expressing library indicated that knockdown of ATM expression may cause a lethal effect in HNSCC cell lines as well (Lindenbergh-van der Plas et al, Clin Cancer Res 2013). The aim of the present study was to investigate whether interference with the ATM/ATR/CHEK1/CHEK2 DNA damage response pathway is indeed a promising therapeutic approach in HNSCC.

RNA interference using siRNAs targeting ATM, ATR, CHEK1 and CHEK2 confirmed that particularly CHEK1 is essential for the survival of a subset of HNSCC cell lines, whereas the cell viability of primary oral keratinocytes and fibroblasts was not compromised. This was confirmed in vitro by treatment with a small molecule inhibitor targeting Chk1, and a therapeutic window between cancer and normal oral mucosal cells was observed. Functional assays indicated an S-phase delay in Chk1 inhibited HNSCC cells, followed by DNA damage. The latter was indicated by elevated phosphorylation of ATM Ser1981 and γH2Ax Ser139, as well as the appearance of chromosomal breaks. The DNA damage response initiated caspase-mediated apoptosis, resulting in programmed cell death.

In summary, our data show that HNSCC cells appear to depend strongly on a functional Chk1 protein during DNA replication. Therefore, Chk1 inhibition seems an interesting therapeutic strategy for HNSCC, at least in a subset of tumors.

Citation Format: Anne M. van Harten, Marijke Stigter-van Walsum, Marijke Buijze, Boudewijn Jm Braakhuis, Ruud H. Brakenhoff. Chk1 inhibition: a promising targeted treatment for head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4637.