bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 4430: Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells
Authors: Senicourt Blanche 
Published: July 2016

Abstract
Background: The Hedgehog (HH) pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC), an organelle not expressed in the normal colonic epithelium which recruits the functional components and regulates the pathway.
Aims: Since the intestinal epithelium is known to be a non-ciliated tissue, the HH pathway as related to the PC has not been explored. We investigated the presence of PC in colon cancer tumors and cell lines. Our hypothesis is that PC formation controls the HH activation that has been reported in numerous colon cancer studies.
Materials and methods: We used cellular models to look at HH activation, focusing on the final effector Gli1. The link between PC and the HH pathway was shown by the recruitment of the Smo receptor in the PC. We looked for PC in a subset of 63 tumors from a Tissue Micro Array and in 4 colorectal cell lines by immunofluorescence using two well-known markers, acetylated α-tubulin and polyglutamylated tubulin. 3D deconvoluted pictures were obtained to characterize the shape and size of PC. Using cellular models we investigated HH pathway expression by qPCR. We assessed the functional link between HH pathway and PC through localization of the Smo receptor in the PC using immunofluorescence.
Results: We observed the presence of the PC in the epithelium of primary colorectal tumors at all stages but not in their normal counterparts. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the PC membrane.
Conclusion: We conclude that the PC directly participates in the HH pathway in colorectal cancer cells which is the first observation of PC expression in tumors arising from a non-ciliated tissue. Functional studies are required to determine the mode of action of this organelle in carcinogenesis.
Citation Format: Blanche Senicourt. Implication of the primary cilium in the Hedgehog pathway 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 4430.
Abstract 4432: Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation
Authors: Fukushige Shinichi , Mizuguchi Yasuhiko , Chakma Kanchan , Saiki Yuriko , Horii Akira 
Published: July 2016

Abstract
DNA demethylating agents are useful therapeutic drugs for human cancer; 5-aza-2’-deoxycytidine and 5-azacytidine for myelodysplastic syndrome are good examples. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects. However, precise mechanisms for anticancer activity of these demethylating reagents have yet to be established, mainly due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulates more genes than ones expressing TET1-CDwt without MBD (TET1-CDwt) or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site (TSS). Interestingly, 65% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with 5-azacytidine, DNA demethylating agent. These results suggested a fact that gene reactivation by both methods was primarily based on DNA demethylation. In order to examine growth inhibitory effects of DNA demethylation to cancer cells, we utilized a tetracycline inducible system to regulate the expression of MBD-TET1-CDwt using a prostate cancer cell line. Induction of MBD-TET1-CDwt demethylated and upregulated the glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells. Flow cytometry analysis showed decreased cells in S phase and increased cells in G1 phase by MBD-TET1-CDwt expression. Our present results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation, and that our methods allow us to analyze the effects of global DNA demethylation in a wide variety of cancer cells.
Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Kanchan Chakma, Yuriko Saiki, Akira Horii. Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation. [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 4432.
Abstract 444: Hyaluronan-mediated motility receptor (RHAMM) in ovarian cancer
Authors: Buttermore Stephanie , Kruk Patricia 
Published: July 2016

Abstract
This study focuses on Hyaluronan Mediated Motility Receptor (RHAMM) protein expression in ovarian carcinoma (OC). RHAMM protein increases migration, invasion and is associated with poor prognosis when overexpressed. It is a dualistic oncogenic protein located within and on the cell surface acting as a receptor for Hyaluronan (HA). RHAMM is not generally overexpressed in normal tissues, but is upregulated in a number of cancers and is associated with poor clinical outcome. However, the role of RHAMM in OC progression is unknown.
Hypothesis: Elevated RHAMM levels promote OC progression and represent a novel urinary biomarker for OC.
Procedures: Measuring RHAMM expression in OC cell protein lysate and conditioned media (CM) was done by western blot (WB) and densitometry. With institution approval, patient cohort urine from male and female healthy controls and patients with cancer of the ovary, lung, breast, prostate, cervix, colorectum, skin, endometrium, head and neck, and brain were acquired from the H. Lee Moffit Cancer Center at the University of South Florida. Enzyme-linked immunosorbent assay (ELISA) designed for RHAMM detection, was used to measure patient urine expression levels.
Results: Cultured OC cells overexpress and secrete RHAMM. Using OC, SV-40 large T-antigen transfected human ovarian surface epithelial (IOSE) cells (HIO118, IOSE-121) and human dermal fibroblasts (HDF), we found &gt;2x the expression of RHAMM protein in BC (MCF7) and OC (OVCAR5, OV2008, C13) cells by WB. WB of CM indicated &gt;3x RHAMM levels secreted by OC compared to IOSE cells.
Urinary levels of RHAMM are elevated in OC patients. Since RHAMM is not an integral membrane protein, but rather loosely tethered to the plasma membrane, we determined whether RHAMM could be detected in bodily fluids of OC patients. ELISA showed detection of urinary RHAMM protein levels averaged 0.140 ng/ml (n = 26) in healthy patient controls whereas OC patients averaged 0.411 ng/ml (n = 132).
Conclusions: Prior to this study, overexpression and secretion of RHAMM by OC cells had not been reported. This has expanded our knowledge of OC etiology by delineating a functional role for RHAMM-driven OC progression. Validating elevated urinary levels of RHAMM suggests it is a potential diagnostic marker alone or in combination with other markers for OC detection. Further, the ability to monitor OC through the course of disease using urinary RHAMM levels could indicate recurrent disease or therapeutic efficacy. Understanding RHAMM expression in OC may lead to a possible diagnostic/prognostic indicator or therapeutic target for OC.
Citation Format: Stephanie Buttermore, Patricia Kruk. Hyaluronan-mediated motility receptor (RHAMM) in ovarian cancer. [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 444.
Abstract 4441: Non-pathogenic bacteria change host DNA methylation in vivo
Authors: Sun Ang , Jelinek Jaroslav , Maegawa Shinji , Jobin Christian , Issa Jean-Pierre 
Published: July 2016

Abstract
Introduction: More than a thousand species of bacteria live in a normal person's gastrointestinal tract. Epigenetic effects on the colon directly caused by the commensal bacteria remain elusive. In the current study, we explore the bacterial effects on DNA methylation of the host colon. Methods: Eight non-pathogenic bacterial species were introduced to the gut of germ-free (GF) mice to make specific-pathogen free (SPF) mice. Our study consisted of the following groups (n = 6 for each): wildtype (WT) GF, WT SPF, IL-10 knockout (KO) GF, IL-10 KO SPF, and IL-10 KO GF or SPF treated with azoxymethane (AOM). We analyzed DNA methylation in genomic DNA extracted from the proximal colon using Digital Restriction Enzyme Analysis of Methylation (DREAM) and compared methylation levels at CpG sites among different groups. Results: 1) Principal component analysis (PCA) performed on methylomes of all the samples separated the mouse samples based on presence or absence of bacteria, IL-10 KO status or AOM treatment into 6 distinct groups. 2) We analyzed CpG sites differentially methylated between GF and SPF mice at p&lt;0.05 significance level. In WT mice, there were 1871 CpG sites (11% out of 17475 analyzed sites), in IL10 KO mice, there were 5986 CpG sites (33% out of 18254 analyzed sites) and in IL10 KO AOM-treated mice there were 6755 CpG sites (31% out of 21697 analyzed sites) differentially methylated. 3) There were 756 common CpG sites differentially methylated in all SPF vs GF comparisons. Interestingly, these sites were enriched 2.6-fold for aging-related CpG sites. 4) WT SPF mice showed increased methylation at CpG sites hypomethylated in WT GF mice and, conversely, decreased methylation at CpG sites hypermethylated in WT GF mice. This pattern of DNA methylation change was also observed by comparing GF with SPF in IL-10 KO mice with or without AOM treatment. 5) Both in GF and SPF mice the DNA methylation changes were more pronounced in IL10 KO while AOM had little additional effect. 6) IL10 KO resulted in genome-wide increase of methylation. However, the introduction of bacteria in SPF mice resulted in the methylation change mainly at CpG islands. Conclusions: Non-pathogenic bacteria introduced in the colon cause methylation gains at CpG sites with low DNA methylation and methylation losses at sites with high DNA methylation in a pattern similar to the changes observed in aging and cancer.
Citation Format: Ang Sun, Jaroslav Jelinek, Shinji Maegawa, Christian Jobin, Jean-Pierre Issa. Non-pathogenic bacteria change host DNA methylation in vivo. [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 4441.
Abstract 4447: EZH2 contributes to breast cancer metastasis
Authors: Yomtoubian Shira , Ryu Seongho , Lee Sharrell , Havel Lauren , Gao Dingcheng , Mittal Vivek 
Published: July 2016

Abstract
While the 5-year relative survival rate of localized stage breast cancer is 99%, once the cancer spreads to distant lymph nodes and organs the survival rate falls to 25%. In order to develop strategies to combat metastasis, we have been studying epigenetic factors that contribute to the aggressive nature of breast cancer. We have focused on enhancer of zeste homolog 2 (EZH2), a histone methyl transferase, because breast cancer patients expressing increased EZH2 exhibit highest metastatic recurrence. We have found through in vivo studies that blocking EZH2 activity not only prevents metastasis formation, but also targets established metastases. Genome wide ChIP-seq analysis revealed that EZH2 may impact breast cancer metastasis through regulating the expression of EMT marker, E-cadherin, and stem cell markers, GATA3 and DKK2. Mammosphere assays from tumors treated with pharmacological blockade of EZH2 and limiting dilution analysis validates that EZH2 inhibition impacts the tumor-initiating cell population. Our data suggests that inhibition of EZH2 activity may have anti-metastatic therapeutic potential and may complement current standards of breast cancer therapy.
Citation Format: Shira Yomtoubian, Seongho Ryu, Sharrell Lee, Lauren Havel, Dingcheng Gao, Vivek Mittal. EZH2 contributes to breast cancer metastasis. [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 4447.
Abstract 4448: Development of DNA methylation biomarkers for pancreatic cancer diagnosis
Authors: Shinjo Keiko , Katsushima Keisuke , Nagae Genta , Aburatani Hiroyuki , Yamao Kenji , Kondo Yutaka 
Published: July 2016

Abstract
Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop minimally invasive biomarkers for detection. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. The presence of cell-free DNA (cfDNA) in blood stream has been known and cfDNA possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system might be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We validated these methylation markers in different FNA sample set (n = 19) by bisulfite pyrosequencing or quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC = 0.95). Finally, we performed methylation analysis in cfDNA from pancreatic cancer patients. From our preliminary data, we could detect aberrant DNA methylation in cfDNA from pancreatic cancer patients.
We identified potential DNA methylation biomarkers for the diagnosis of pancreatic cancers and these markers might be applicable for detecting cancer in cfDNA.
Citation Format: Keiko Shinjo, Keisuke Katsushima, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of DNA methylation biomarkers for pancreatic cancer 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 4448.
Abstract 445: Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues
Authors: Krimmel Jeffrey D. , Schmitt Michael W. , Kennedy Scott R. , Harrell Maribel I. , Agnew Kathy J. , Loeb Larry A. , Swisher Elizabeth M. , Risques Rosa Ana 
Published: July 2016

Abstract
The detection of subclonal tumor-specific somatic mutations in clinical samples could revolutionize cancer diagnostics, but is limited by insufficiently sensitive sequencing methods. Duplex Sequencing is a novel next-generation sequencing (NGS) technology that implements single-molecule barcoding of both strands of DNA to allow internal error correction. This modification reduces the error rate of NGS from 1 in 1000 to less than 1 in 10 million nucleotides, an unprecedented sensitivity that enables accurate ultra-deep sequencing for clinical applications. In this study we sought to determine whether Duplex Sequencing could detect extremely rare TP53 mutated cancer cells disseminated into the peritoneal cavity of women with high-grade serous ovarian carcinoma (HGSOC). HGSOC is the most common and most aggressive type of ovarian cancer, shows early transperitoneal dissemination, and is characterized by near-universal prevalence of driver TP53 mutations. We analyzed 17 peritoneal fluid samples from women with HGSOC and known TP53 tumor mutation (cases) and 20 peritoneal fluid samples from women without cancer (controls). The tumor-specific TP53 mutation was detected in matched peritoneal fluid from 94% of cases (16/17), including 2 patients with occult tubal intraepithelial neoplasia, 7 patients with early stage HGSOC, and 8 with negative peritoneal fluid cytopathology. Tumor-specific alleles were detected as low as &lt;1/25,000. Surprisingly, we also detected additional extremely low-frequency somatic TP53 mutations in peritoneal fluid from nearly all (35/37) cases and controls. These mutations were more abundant in cases than in controls, and in the latter correlated with age. The total burden of somatic TP53 mutations (tumor specific and non-specific) in a peritoneal fluid sample could distinguish cases from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Duplex Sequencing also revealed low-frequency, age-associated somatic TP53 mutations in 100% (15/15) of matched peripheral blood samples. Our results demonstrate the ability of Duplex Sequencing to detect very rare cancer cells, but also provide evidence of widespread, low frequency somatic TP53 mutation in non-cancerous tissue. This compromises the specificity of TP53 mutation tests for cancer detection in liquid biopsies.
Citation Format: Jeffrey D. Krimmel, Michael W. Schmitt, Scott R. Kennedy, Maribel I. Harrell, Kathy J. Agnew, Larry A. Loeb, Elizabeth M. Swisher, Rosa Ana Risques. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues. [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 445.
Abstract 4452: Oxdative stress suppresses the expression of 15-hydroxyprostaglandin dehydrogenase via upregulation of DNMT3a/Snail in human colon epithelial cells
Authors: Lee Ja-Young , Lee Jeong-Eun , Na Hye Kyung 
Published: July 2016

Abstract
Overproduction of prostaglandin E2 (PGE2) is implicated in pathogenesis of inflammation-associated carcinogenesis. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme that catalyzes the conversion of PGE2 to biologically inactive 15-keto-PGE2 metabolite. 15-PGDH has been considered to be a tumor suppressor as its expression is frequently repressed in human malignancies via hypermethylation of its promoter. In our previous study, oxidative stress caused by Helicobacter pylori infection suppressed the expression of 15-PGDH. Reactive oxygen species (ROS) can induce methylation as well as mutation of genes including those that encode tumor suppressor proteins, thereby contributing to carcinogenesis. This prompted us to investigate whether 15-PGDH expression could be down-regulated by oxidative stress in normal colon epithelial CCD841 cells. When CCD841 cells were treated with H2O2, the expression level of 15-PGDH was significantly reduced in both concentration and time dependent manners. Hydrogen peroxide (300 μM) induced the expression of DNA methyltransferase (DNMT) 3a, which was blocked by an inhibitor of DNMT, 5-Aza-2′-deoxycytidine (5-Aza). The 5-Aza abrogated the down-regulation of 15-PGDH expression induced by H2O2. The antioxidant, N-acetylcysteine (NAC) abrogated the down-regulation of 15-PGDH and upregulation of DNMT 3a expression induced by H2O2. The transcriptional repressor Snail plays a central role in the suppression of 15-PGDH expression. H2O2 treatment also elevated expression of Snail, which was abrogated by NAC and 5-Aza. Taken together, these finding suggest that ROS suppresses the expression of 15-PGDH through hypermethylation of CpG island in the 15-PGDH promoter.
Citation Format: Ja-Young Lee, Jeong-Eun Lee, Hye Kyung Na. Oxdative stress suppresses the expression of 15-hydroxyprostaglandin dehydrogenase via upregulation of DNMT3a/Snail in human colon epithelial 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 4452.
Abstract 4475: SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate
Authors: Eggert Erik , Stellfeld Timo , Korr Daniel , Stresemann Carlo 
Published: July 2016

Abstract
The SET and MYND domain-containing protein 2 (SMYD2) has been reported to mono-methylate several lysine residues on histone and non-histone proteins. Overexpression of SMYD2 has been reported in cancer cell lines as well as in esophageal squamous cell carcinoma (ESCC), bladder carcinoma, gastric cancer and pediatric acute lymphoblastic leukemia patients and correlates with lower survival rates. Although several studies uncovered important roles of SMYD2 in promoting tumorgenesis by protein methylation of key proteins (e.g. p53, PTEN, Rb, PARP), the biology of SMYD2 and its possible role in cancer biology is still far from being fully understood. Therefore, we employed the SMYD2 inhibitor BAY-598 (AACR-2015 abstract 2829, publication in preparation), and used proteomics to find novel substrates of SMYD2 in cancer cells.
We confirmed with both BAY-598 and by knockdown experiments that the giant scaffold protein AHNAK is a novel major substrate of SMYD2. Different studies have tried to decipher the function of AHNAK in normal and diseased tissues. AHNAK forms multi-protein complexes acting as a structural scaffold involved in different processes, including calcium channel regulation, cell contact signaling, migration, and tumor metastasis. Interestingly, we found AHNAK to be mono-methylated by SMYD2 on multiple sites in its central repeated domain as well as on its C-terminal domain. BAY-598 potently inhibited methylation at these sites on AHNAK. To further determine the prevalence of AHNAK methylation, we analyzed different cancer cell lines and tissues from mice. AHNAK methylation correlated with SMYD2 expression, but not with AHNAK expression, suggesting tissue-specific regulation of AHNAK methylation by SMYD2.
In summary, the novel finding of AHNAK regulation by methylation further complements the understanding of the roles of SMYD2 in cancer biology and underlines the utility of using probe inhibitors like BAY-598 to explore potential therapeutic targets in cancer.
Citation Format: Erik Eggert, Timo Stellfeld, Daniel Korr, Carlo Stresemann. SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate. [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 4475.