- Email: [email protected]
Sa1903 Differential Expression and Function of BMP4 in the Progression to Esophageal Adenocarcinoma
AGA Abstracts
nucleosomal DNA (3rd) fractions were separated. Indexed DNA fragment library sequencing was performed on SOLiD IV system. The sequencing yielded 50 nt long reads a total of 86.6 Gbases. Sequence alignments were performed by SHRiMP and Bowtie aligners. To validate our results 900 independent cases downloaded from SRA database were aligned to bacteria genomes. RESULTS: On average above 70% of the reads were mapped to the human reference genome. SEPT9 gene derived region (chr17:75369567-75369656) has shown over 1000 times average coverage in CRC samples compared to normal, adenoma also showed elevated level. This position is exactly matched with region detected by EpiProColon Kit (Epigenomics), a method based on the enrichment of methylated fragments. The unmapped reads were aligned to genomes of various other organisms. Significant portion of the sequence tags (0.5-50 ppm) can be mapped back with high confidence to the genome of Enterobacteriaceae (Escherichia coli, Shigella sonnei) and commonly consumed food substances such as potato. Characteristic differences were found among the disease groups in the foreign DNA content. CONCLUSION: Using high throughput next-generation sequencing data we have identified traces of foreign DNA molecule in the human cfDNA samples. The foreign DNA fragments are large enough to carry complete genes and they align to various bacteria of the human microbiome and to edible plant genomes. Since many of identified bacteria are normally present in the human gut, the results suggest that the isolation between the gut and the circulatory system is not perfect. These findings can fundamentally change our view of DNA digestion and transport processes and can be a potential blood base biomarker and monitoring tool. Sa1902 SEPT9 and SFRP1 DNA Hypermethylation in Colorectal Cancer: Restriction to a Single CpG Island and Expansion to the Gatekeeper Myofibroblasts Barbara K. Barták, Reinhold Wasserkort, Alexandra Kalmár, Gabor Valcz, Kinga Tóth, Zsolt Tulassay, Theo deVos, Béla Molnár
Fecal Recovery of Ingested Slmll Marker as Function of Amplicon Size
Background Several genes are regulated by DNA methylation and the aberrant alteration of this mechanism can lead to cancer formation. Septin 9 (SEPT9) and secreted frizzledrelated protein 1 (SFRP1) are known to play a role in colorectal cancer, however, the DNA methylation pattern of these genes in the epithelial and stromal cells in colorectal cancer remains unknown. Aims Our aims were to analyze DNA methylation patterns in DNA methylation-regulated genes in healthy and diseased colonic epithelial and stromal cells. Methods Colonic epithelial and stromal cells were collected separately using laser capture microdissection (LCM). Microdissected samples were subjected to bisulfite treatment and direct bisulfite sequencing was used to analyze the methylation status of ten regions within SEPT9, and in one region within SFRP1 in tissue samples obtained from normal (n=3), adenomatous (n=3) and colorectal cancer (n=3) samples. In addition SEPT9 and SFRP1 protein expression was assessed using immunohistochemistry on independent healthy (n= 10), adenomatous (n=14) and CRC (n=13) samples. Stromal myofibroblast cells were detected by alpha-SMA immunohistochemistry, the immunopositive cells were LCM separated and SFRP1 DNA methylation was assessed by high resolution melting (HRM) analysis. Results The regions analysed in SEPT9 were unmethylated in normal tissues except for a methylation boundary detected downstream of the largest CpG island within SEPT9. In adenoma and tumor samples, the epithelial cells displayed markedly increased methylation levels (>80%, p<10-4), but only within one of the CpG islands investigated. In stromal cells increased methylation (up to 50%, p<10-4) was only seen in tumor patients and in histologically normal tissue close to the tumor, but not in adenoma. The analyzed region of SFRP1 also showed a remarkable increase in both adenoma and tumor epithelial cells. Protein levels of SEPT9 and SFRP1 showed a significant (p<0,05) decrease in adenoma and tumor tissue samples compared to healthy controls. Alpha-SMA immunopositive myofibroblast cells were identified as the main source of stromal SFRP1 protein, that was found to be downregulated by DNA hypermethylation only in carcinoma, but not in adenoma. Conclusions Hypermethylation of SEPT9, SFRP1 could be detected in the analyzed adenoma and cancer samples compared to the healthy control samples. According to the results of the laser microdissected samples, the DNA methylation alterations originated in epithelial cells, while stromal cells appear to acquire hypermethylation only subsequently via field effects. The DNA hypermethylation of the analyzed genes result in decreased protein level, that can contribute to colorectal cancer formation and invasion.
Sa1900 CD44v9 Results in Resistance to Trastuzumab via Induction of ROS Resistance Hideki Mori, Hidekazu Suzuki, Juntaro Matsuzaki, Hitoshi Tsugawa, Sawako Okada, Seiichiro Fukuhara, Tatsuhiro Masaoka, Takanori Kanai, Hideyuki Saya Background: Twenty percent of advanced gastric cancers are HER2-positive. Trastuzumab(Tmab), a monoclonal antibody that interferes with the HER2 receptor, combined with cisplatin and a fluoropyrimidine has been shown to increase survival in HER2-positive gastric cancer. Tmab induces reactive oxygen species (ROS) in lung adenocarcinoma in vitro, and may have similar effects in other cancer types as well. CD44 variant 9 (CD44v9) expression is associated with a poor prognosis in gastric adenocarcinomas. The CD44v9 isoform interacts with and stabilizes xCT, a glutamate-cystine transporter, resulting in increased intracellular levels of reduced glutathione (GSH), and increased defense against ROS. The relationship between CD44v9 expression and HER2 status in the response of gastric cancer to various treatments, including Tmab has not been reported. The present study was conducted to investigate the role of CD44v9 in the effectiveness of Tmab treatment on gastric adenocarcinomas. We hypothesize that CD44v9 causes resistance to Tmab via induction of ROS resistance, and targeting CD44v9 may increase effectiveness of Tmab therapy. Methods: NCI-N87(high HER2), a gastric cancer cell line, was used for this study. Anti-HER2 siRNA was used to knockdown HER2 expression in NCI-N87 cells. The pRc/CMV expressing plasmid encoding human CD44v9 was transfected into NCI-N87 cells (CD44v9-NCI-N87). N-acetyl cysteine (NAC) (15mM) was used as a ROS scavenger for 72h in vitro. Cells were incubated for 72h with different concentrations of Tmab (a range of doses between 0, 10, 50 μg/ml), and cell viability was determined by MTS assay. Intracellular GSH levels were measured by luminescent based assay. Intracellular ROS were measured by flow cytometry with CM-H2DCFDA. Cells were cultured for 24h with different concentrations of Tmab (a range of doses between 0, 40, 80, 120, 200 μg/ml) in this assay. Results: Tmab caused a dose-dependent decrease in cell viability in NCI-N87 cells(10μg/ml 88.2%(p=0.015), 50μg/ml 73.3%(p<0.001)), but did not in anti-HER2 siRNA treated NCI-N87(10μg/ml 99.0%(p=0.879), 50μg/ml 93.0%(p= 0.495) and CD44v9-NCI-N87(10μg/ml 104.4%(p=0.442), 50μg/ml 97.3%(p=0.623)). Tmab increased intracellular ROS level in NCI-N87 cells (p=0.007), but did not in anti-HER2 siRNA treated NCI-N87 and CD44v9-NCI-N87. Intracellular GSH levels were significantly higher in CD44v9-NCI N87 than NCI-N87 (p=0.004). NAC decreased Tmab-induced cell death in NCI-N87. These data indicated that CD44v9 is associated with increased intracellular GSH level, and may protect against Tmab induced ROS damage and cell death. Conclusion: Knock down of CD44v9 expression in HER2 positive gastric cells restores Tmab sensitivity through the restoration of ROS mediated cell death.
Sa1903 Differential Expression and Function of BMP4 in the Progression to Esophageal Adenocarcinoma Silvia Calpe, Chiu Ting Lau, Kausilia K. Krishnadath Background: The BMPs are a family of growth factors that constitute a group of pivotal morphogenetic signals, that orchestrate tissue architecture throughout the body, including intestinal homeostasis. It has become clear that besides its homeostatic function in normal adult tissues, BMP4 has a key role in carcinogenesis and metastasis. Our group previously demonstrated that the BMP4/pSMAD pathway plays a key role in the development of Barrett's esophagus (BE), yet its role in the progression towards esophageal adenocarcinoma (EAC) is unclear. BMP4 signalling is complex. After binding its receptors, it transduces its signals through canonical (SMAD) and non-canonical pathways (such as PI3K/Akt and MAPK). Although non-canonical signalling have been involved in relevant aspects of the BMP physiology, nothing is known about their involvement in the progression from BE to EAC. The aim of this study was to explore the clinical significance and the role of BMP-4 in the progression of malignant transformation of the esophageal epithelium. Methods: First, expression of BMP4 by Immunohistochemistry (IHC), was assessed on 21 tissue specimens of both differentiated (n=13) and undifferentiated (n=8) EAC. This data was further validated by ELISA and Western Blot in four esophageal adenocarcinoma cell lines with varying degrees of malignant differentiation (OE33, OE19, Flo-1, SKGT-4). Functions such as proliferation, invasion and metastasis were studied in these cell lines after BMP4 stimulation. Finally, BMP4-mediated signalling pathways and their significance in BMP4 function were elucidated. Results: IHC of tumor biopsies of EAC patients, revealed a heterogeneous pattern of BMP4 expression in EAC, in which higher expression was associated to differentiation status. A similar expression pattern was observed in the EAC cell lines. Our experiments
Sa1901 Bacterial DNA and Methylated Host DNA Sequences in Plasma Samples of Healthy and Colorectal Cancer Patients: A Whole-Metagenome Analysis Barbara K. Barták, Sandor Spisak, Péter Ittzés, András Bodor, Dániel Kondor, Gábor Vattay, Zsofia B. Nagy, Alexandra Kalmár, Zsolt Tulassay, István Csabai, Béla Molnár INTRODUCTION: The exact nucleotide composition of the circulating cell-free DNA (cfDNA) is not fully understood. Methylated and non-methylated cfDNA can be derived from both normal and tumor cells, through apoptosis, necrosis, or direct secretion. Furthermore, we are constantly exposed to foreign DNA from various sources like benign or malicious microbes in and on our body, pollens in the inhaled air and as the largest amount with the daily food supply. Here we report a new possible mechanism, which can contribute the quantity and quality of the cfDNA. AIMS: Our aims were to determine the DNA content of cfDNA from healthy, IBD, colorectal adenoma (AD) and -cancer (CRC) patients by NGS sequencing technology, and identify differences among these clinical groups. Moreover, we aimed to analyze the unmapped reads. METHODS: CfDNA was extracted from plasma samples which were collected (TUKEB 2009/037) from 50-50 normal, IBD, AD and CRC samples. Fractions were size separated via electrophoresis and DNA fragments were recovered from the gel slices. Intact DNA above 10kb (1st), the fraction is between 200bp to 10kb (2nd) and
AGA Abstracts
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showed differential preference for certain signaling pathways that was dependant on the malignant status of these cell lines. Furthermore, our data uncovered opposing functions for BMP4 in the progression from BE to EAC. Whereas BMP4 inhibited proliferation of all the cell lines, regardless of their differentiation status, it was able to promote metastasis, invasion and esophageal to mesenchymal transition, only in the more differentiated EAC cell lines. Further analyses, revealed that the SMAD pathway was responsible for the BMP4mediated proliferation, and the non-canonical pathways were responsible for the other functions. Conclusion: We have shown that the opposing functions of BMP4 might be a result of differential use of signaling pathways. Because its expression is highly correlated to a differentiated status, and its metastasis-driven functions are mainly predominant in the differentiated cells, it is tempting to speculate that BMP4 might be involved in the progression from early to late stage EAC.
Sa1906
Background: Colorectal cancer (CRC) and adenomatous polyps (AP) are frequently diagnosed at colonoscopy. Recently, some reports have shown the detection of circulating microRNA (miRNA) in plasma of CRC and AP patients. However, the optimal miRNA panels are not clear yet. Since colonoscopy is invasive and the adherence rate is low, detection of circulating miRNA in plasma may significantly improve current approaches of CRC screening and aid in early diagnosis. The focus of this study is to have a better picture of circulating plasma miRNA in detection of CRC and AP. Methods: Patients (matched with age, sex and race) were grouped into three categories. Group A=CRC patients, group B= patients with adenomatous polyps >1cm, and group C=healthy controls with no adenomatous polyps. Plasma from group A (drawn prior to any primary treatment), B and C were used. For the discovery phase, plasma of 5 patients from each group was pooled separately. miRNA sequencing was done to discover genome-wide circulating miRNA profile which might differentiate patients with CRC and AP from patients without polyps. After this phase, quantitative real-time PCR was conducted to validate the expressions of selected plasma miRNAs in group A (n=22), B (n=23) and C (n=52). Results: At the discovery stage, 166 miRNAs were detected that were differentially expressed between groups A, B and C. 170 miRNAs were differentially expressed between groups B and C. Of these 170 miRNAs, 107 miRNAs were differentially expressed in group B and A but not in group C. Of the 107 overlap markers, 60 miRNAs were up-regulated in both group A and in group B, and conversely 47 miRNAs were downregulated in both group A and B. In the validation, we selected 5 miRNAs and confirmed the expression level in the 15 discovery samples by using qPCR. We further observed that miR-152 was down regulated in CRC patients with 3.54-fold change when compared with healthy controls (p value =0.0007), but not in AP patients (1.21-fold change, p value = 0.4). Conclusions: 66 miRNAs were isolated from the plasma which were different between patients with AP and those without AP. Both plasma up-regulated miRNA (N=60) and downregulated miRNA (N=47) can be used to distinguish patients with advanced adenomatous polyps and patients with CRC from matched controls. There is high rate of overlap of miRNA differentially regulated in CRC from healthy controls likewise preferentially regulated in AP versus controls 107/170 (62.9%). mir-152 is preferentially down-regulated in CRC alone but not in healthy controls or those with advanced adenomatous polyps. While prior noninvasive methodologies have been limited in their ability to detect advanced AP, it appears that numerous plasma miRNAs hold promise to differentiate patients with AP as well as CRC from healthy.
Sa1904 HER-2 Amplifications Correlate With Genetic Instability and Appear Late During Malignant Progression of Barrett's Esophagus Silvia Calpe, Akueni Davelaar, Chiu Ting Lau, Sybren L. Meijer, Paul Fockens, Kausilia K. Krishnadath Objective: In Barrett's associated esophageal adenocarcinoma (EAC), amplification of the Her-2 proto-oncogene correlates with poor prognosis. The exact mechanisms leading to Her-2 gene abnormalities in these cancers are unknown. Aneuploidy and p53 abnormalities are major and frequent events in Barrett's esophagus (BE), and are indicative for genetic instability. In several cancers genetic instability is associated with Her-2 amplification, yet this has not been studied in the Barrett's associated esophageal adenocarcinomas. The aim of this study was to gain more insight in genetic events related to genetic instability that frequently occur during BE progression by evaluating the correlation of p53 abnormalities, aneuploidy and Her-2 amplifications. Methods: Her-2 locus (17q11.2) amplification, TP53 loss and aneuploidy (CEP7/CEP17 gains) were evaluated by DNA fluorescent in situ hybridization (FISH) in a population of 174 BE patients with different degrees of dysplasia and 28 EAC patients. Results were validated by analysing the p53 mutational and FISH profiles of a panel of nine well characterized esophageal cell lines representing the Barrett's carcinogenesis sequence, including: the normal esophageal (EPC-htert), the Barrett-derived non-dysplastic (CPA) and dysplastic (CPB, CPC, CPD) and EAC cell lines with different degrees of differentiation (Flo-1, SKGT4, OE33, OE19). Results: We found that p53 losses and gain of chromosome 17 are earlier events, already detectable at early stages of metaplasia, significantly increasing with dysplasia stage, and reaching a high frequency in EAC. Her-2 locus amplification, however, was absent in BE and only detectable in High Grade Dysplasia (HGD) and EAC cases. Concordantly, a relatively small number of abnormalities (p53 mutations and CEP17/ 7 gains) were observed in the normal squamous (EPC-htert) or early BE cell line (CPA). The frequency of these abnormalities progressively increased throughout the BE-EAC sequence reaching almost 100% in the undifferentiated malignant EAC cell lines. In contrast, and in keeping with the human data, Her-2 amplifications were only observed in cell lines characterized by a malignant phenotype (OE33, OE19). Conclusion: Our studies have unravelled a sequence of genetic abnormalities that associates with malignancy. They support that gains of chromosome 17 may to Her-2 amplifications but only in case of genetic instability manifested by p53 abnormalities or/and aneuploidy. The concordance of the genetic abnormality sequence between our panel of cell lines and the human samples renders this panel of cell lines as a unique in vitro model to study molecular events in the progression and development of Barrett's cancers. These abnormalities could be used as prognostic clinical tools for evaluation of the biological behaviour of BE and EAC and for patient outcome.
Sa1907 Cohesin Overexpression Is a Frequent Event in Hepatocellular Carcinogenesis: Implications for Chemoprevention Richard Kalman, Mart DeLaCruz, Ramesh K. Wali, Lisa I. Jepeal, David Nunes, Hemant K. Roy Background: Hepatocellular Carcinoma (HCC) is the third most common cause of cancer related mortality worldwide and a feared complication of chronic liver disease. One challenge in HCC diagnosis and prevention has been its genetic complexity, with an average of 39 mutations per cancer (Vogelstein et al., Science 2013). A hallmark of HCC is dysregulation of proliferation, metastasis and angiogenesis. These changes are driven predominantly by genetic and more frequently epigenetic events altering gene expression. These include microRNA methylation which can be potentially targeted by chemopreventive agents. In other malignancies, a recently discovered modality of gene regulation has been through chromatin looping controlled by the cohesin family of proteins (e.g. Solomon et al., Nature Gen 2013). The cohesin multimer includes structural maintenance proteins (SMC1A and SMC3) which interact with the zinc-finger DNA binding protein CTCF. The role of cohesins in HCC has been unexplored. Our goal was to elucidate the role of cohesins in HCC development and chemoprevention For the latter, we used metformin given it's established risk reduction in HCC (~50%, Singh et al., Am J Gas 2013). Methods: We performed immunohistochemical (IHC) analysis on HCC and normal liver (n=96) with slides scored on a 4 point scale. We measured the expression of cohesin SMC3, and SA-1 and SA-2 (two homologs of Scc3, a cohesin protein), as well as CTCF. Cell culture studies were performed on the human HCC cell line HepG2. These cells were grown in 6-well plates and treated with 20mM of metformin or vehicle for 48hrs and subjected to Western blot analysis. Results: IHC revealed expression of SA1, SMC-3 and CTCF. As demonstrated in table 1, there was a marked upregulation in all three proteins in HCC when compared with nonneoplastic hepatocytes. There was no difference between staining intensity among tumor grades, suggesting that cohesin overexpression may be an early event. In HepG2 cells, Western blot analysis revealed a robust expression of SA1, SMC-3 and CTCF, mirroring the IHC data. As noted in figure 2, metformin treatment led to a statistically significant decrease in SA-1, SMC-3 and CTCF when compared to vehicle-treated controls (64%, p<4.5x10-4 and 44%, p<-4.84x10-5 and 51%, p<1.78x10-5 respectively). Conclusion: We demonstrated that SA-1 and SMC-3, along with CTCF were dramatically upregulated in HCC. Thus, all facets of the cohesin complex are comparably altered, supporting their role in high order chromatin alterations and hence transcriptional activation in HCC. Importantly, this appears to be a therapeutic target as indicated by the profound decrease with the chemopreventive agent metformin. This supports the significant role of cohesin as both a biomarker for HCC and a putative "druggable" target. Furthermore, this may give novel insights into HCC biology. Immunohistochemistry Staining Intensity
Sa1905 Psoriasin Expression in Human Colorectal Cancer Lucy Satherley, Lin Ye, Rachel Hargest, Wen G. Jiang Introduction Psoriasin (S100A7) is a calcium binding protein initially identified in psoriatic keratinocytes. Elevated psoriasin expression has been found in a variety of human cancers including bladder, breast, prostate and lung cancer. Overexpression of psoriasin has been associated with increased cancer cell growth and invasion but studies of cell adhesion have shown conflicting results. The aim of the present study was to investigate the expression of psoriasin in colorectal cancer tissue and to determine whether there is any association between psoriasin expression and stage of disease or prognosis. Methods Primary colorectal cancer tissue (n=90) and normal colorectal tissue (n=67) were collected at operation and examined by a consultant pathologist. Frozen sections of each tissue sample were prepared from which RNA was extracted. Reverse transcription was then used to generate cDNA which was analysed using quantitative transcript analysis (qPCR) to determine psoriasin expression. Patients were routinely followed up clinically and radiologically after surgery and the median follow up period was 65 months. The expression profile was then analysed against the clinical, pathological and outcome data. Results Psoriasin expression was significantly higher in colorectal cancer tissue compared with normal colorectal tissue, both for the whole cohort (p<0.005) and for paired samples (p<0.005). Median transcript number increased significantly with grade of differentiation and there was a trend towards increasing psoriasin expression with nodal status, although this did not reach significance. Psoriasin expression was significantly higher in tumour tissue across all TNM stages, Dukes' stages and T stages compared with normal colorectal tissue. Although psoriasin expression appeared to be higher in patients with distant metastases and those who subsequently developed local recurrence or who died of their disease, this was not significant. Conclusions To our knowledge, this is the first study examining psoriasin expression in colorectal cancer. Psoriasin expression is significantly higher in colorectal cancer tissue compared with normal colorectal tissue in keeping with previous studies which show increased levels of the protein in a range of malignancies. Our results suggest that psoriasin expression may be associated with distant disease and poorer prognosis but further work is required to confirm this. Future studies are required to determine the functional role of psoriasin in colorectal cancer cells.
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AGA Abstracts
AGA Abstracts
Circulating Plasma MicroRNA As Markers in the Early Detection of Colorectal Cancer Hankui Chen, Sandeep S. Nayak, Helu Liu, Phillip Engen, Junmei Ai, Yan Li, Joshua E. Melson, Youping Deng
nucleosomal DNA (3rd) fractions were separated. Indexed DNA fragment library sequencing was performed on SOLiD IV system. The sequencing yielded 50 nt long reads a total of 86.6 Gbases. Sequence alignments were performed by SHRiMP and Bowtie aligners. To validate our results 900 independent cases downloaded from SRA database were aligned to bacteria genomes. RESULTS: On average above 70% of the reads were mapped to the human reference genome. SEPT9 gene derived region (chr17:75369567-75369656) has shown over 1000 times average coverage in CRC samples compared to normal, adenoma also showed elevated level. This position is exactly matched with region detected by EpiProColon Kit (Epigenomics), a method based on the enrichment of methylated fragments. The unmapped reads were aligned to genomes of various other organisms. Significant portion of the sequence tags (0.5-50 ppm) can be mapped back with high confidence to the genome of Enterobacteriaceae (Escherichia coli, Shigella sonnei) and commonly consumed food substances such as potato. Characteristic differences were found among the disease groups in the foreign DNA content. CONCLUSION: Using high throughput next-generation sequencing data we have identified traces of foreign DNA molecule in the human cfDNA samples. The foreign DNA fragments are large enough to carry complete genes and they align to various bacteria of the human microbiome and to edible plant genomes. Since many of identified bacteria are normally present in the human gut, the results suggest that the isolation between the gut and the circulatory system is not perfect. These findings can fundamentally change our view of DNA digestion and transport processes and can be a potential blood base biomarker and monitoring tool. Sa1902 SEPT9 and SFRP1 DNA Hypermethylation in Colorectal Cancer: Restriction to a Single CpG Island and Expansion to the Gatekeeper Myofibroblasts Barbara K. Barták, Reinhold Wasserkort, Alexandra Kalmár, Gabor Valcz, Kinga Tóth, Zsolt Tulassay, Theo deVos, Béla Molnár
Fecal Recovery of Ingested Slmll Marker as Function of Amplicon Size
Background Several genes are regulated by DNA methylation and the aberrant alteration of this mechanism can lead to cancer formation. Septin 9 (SEPT9) and secreted frizzledrelated protein 1 (SFRP1) are known to play a role in colorectal cancer, however, the DNA methylation pattern of these genes in the epithelial and stromal cells in colorectal cancer remains unknown. Aims Our aims were to analyze DNA methylation patterns in DNA methylation-regulated genes in healthy and diseased colonic epithelial and stromal cells. Methods Colonic epithelial and stromal cells were collected separately using laser capture microdissection (LCM). Microdissected samples were subjected to bisulfite treatment and direct bisulfite sequencing was used to analyze the methylation status of ten regions within SEPT9, and in one region within SFRP1 in tissue samples obtained from normal (n=3), adenomatous (n=3) and colorectal cancer (n=3) samples. In addition SEPT9 and SFRP1 protein expression was assessed using immunohistochemistry on independent healthy (n= 10), adenomatous (n=14) and CRC (n=13) samples. Stromal myofibroblast cells were detected by alpha-SMA immunohistochemistry, the immunopositive cells were LCM separated and SFRP1 DNA methylation was assessed by high resolution melting (HRM) analysis. Results The regions analysed in SEPT9 were unmethylated in normal tissues except for a methylation boundary detected downstream of the largest CpG island within SEPT9. In adenoma and tumor samples, the epithelial cells displayed markedly increased methylation levels (>80%, p<10-4), but only within one of the CpG islands investigated. In stromal cells increased methylation (up to 50%, p<10-4) was only seen in tumor patients and in histologically normal tissue close to the tumor, but not in adenoma. The analyzed region of SFRP1 also showed a remarkable increase in both adenoma and tumor epithelial cells. Protein levels of SEPT9 and SFRP1 showed a significant (p<0,05) decrease in adenoma and tumor tissue samples compared to healthy controls. Alpha-SMA immunopositive myofibroblast cells were identified as the main source of stromal SFRP1 protein, that was found to be downregulated by DNA hypermethylation only in carcinoma, but not in adenoma. Conclusions Hypermethylation of SEPT9, SFRP1 could be detected in the analyzed adenoma and cancer samples compared to the healthy control samples. According to the results of the laser microdissected samples, the DNA methylation alterations originated in epithelial cells, while stromal cells appear to acquire hypermethylation only subsequently via field effects. The DNA hypermethylation of the analyzed genes result in decreased protein level, that can contribute to colorectal cancer formation and invasion.
Sa1900 CD44v9 Results in Resistance to Trastuzumab via Induction of ROS Resistance Hideki Mori, Hidekazu Suzuki, Juntaro Matsuzaki, Hitoshi Tsugawa, Sawako Okada, Seiichiro Fukuhara, Tatsuhiro Masaoka, Takanori Kanai, Hideyuki Saya Background: Twenty percent of advanced gastric cancers are HER2-positive. Trastuzumab(Tmab), a monoclonal antibody that interferes with the HER2 receptor, combined with cisplatin and a fluoropyrimidine has been shown to increase survival in HER2-positive gastric cancer. Tmab induces reactive oxygen species (ROS) in lung adenocarcinoma in vitro, and may have similar effects in other cancer types as well. CD44 variant 9 (CD44v9) expression is associated with a poor prognosis in gastric adenocarcinomas. The CD44v9 isoform interacts with and stabilizes xCT, a glutamate-cystine transporter, resulting in increased intracellular levels of reduced glutathione (GSH), and increased defense against ROS. The relationship between CD44v9 expression and HER2 status in the response of gastric cancer to various treatments, including Tmab has not been reported. The present study was conducted to investigate the role of CD44v9 in the effectiveness of Tmab treatment on gastric adenocarcinomas. We hypothesize that CD44v9 causes resistance to Tmab via induction of ROS resistance, and targeting CD44v9 may increase effectiveness of Tmab therapy. Methods: NCI-N87(high HER2), a gastric cancer cell line, was used for this study. Anti-HER2 siRNA was used to knockdown HER2 expression in NCI-N87 cells. The pRc/CMV expressing plasmid encoding human CD44v9 was transfected into NCI-N87 cells (CD44v9-NCI-N87). N-acetyl cysteine (NAC) (15mM) was used as a ROS scavenger for 72h in vitro. Cells were incubated for 72h with different concentrations of Tmab (a range of doses between 0, 10, 50 μg/ml), and cell viability was determined by MTS assay. Intracellular GSH levels were measured by luminescent based assay. Intracellular ROS were measured by flow cytometry with CM-H2DCFDA. Cells were cultured for 24h with different concentrations of Tmab (a range of doses between 0, 40, 80, 120, 200 μg/ml) in this assay. Results: Tmab caused a dose-dependent decrease in cell viability in NCI-N87 cells(10μg/ml 88.2%(p=0.015), 50μg/ml 73.3%(p<0.001)), but did not in anti-HER2 siRNA treated NCI-N87(10μg/ml 99.0%(p=0.879), 50μg/ml 93.0%(p= 0.495) and CD44v9-NCI-N87(10μg/ml 104.4%(p=0.442), 50μg/ml 97.3%(p=0.623)). Tmab increased intracellular ROS level in NCI-N87 cells (p=0.007), but did not in anti-HER2 siRNA treated NCI-N87 and CD44v9-NCI-N87. Intracellular GSH levels were significantly higher in CD44v9-NCI N87 than NCI-N87 (p=0.004). NAC decreased Tmab-induced cell death in NCI-N87. These data indicated that CD44v9 is associated with increased intracellular GSH level, and may protect against Tmab induced ROS damage and cell death. Conclusion: Knock down of CD44v9 expression in HER2 positive gastric cells restores Tmab sensitivity through the restoration of ROS mediated cell death.
Sa1903 Differential Expression and Function of BMP4 in the Progression to Esophageal Adenocarcinoma Silvia Calpe, Chiu Ting Lau, Kausilia K. Krishnadath Background: The BMPs are a family of growth factors that constitute a group of pivotal morphogenetic signals, that orchestrate tissue architecture throughout the body, including intestinal homeostasis. It has become clear that besides its homeostatic function in normal adult tissues, BMP4 has a key role in carcinogenesis and metastasis. Our group previously demonstrated that the BMP4/pSMAD pathway plays a key role in the development of Barrett's esophagus (BE), yet its role in the progression towards esophageal adenocarcinoma (EAC) is unclear. BMP4 signalling is complex. After binding its receptors, it transduces its signals through canonical (SMAD) and non-canonical pathways (such as PI3K/Akt and MAPK). Although non-canonical signalling have been involved in relevant aspects of the BMP physiology, nothing is known about their involvement in the progression from BE to EAC. The aim of this study was to explore the clinical significance and the role of BMP-4 in the progression of malignant transformation of the esophageal epithelium. Methods: First, expression of BMP4 by Immunohistochemistry (IHC), was assessed on 21 tissue specimens of both differentiated (n=13) and undifferentiated (n=8) EAC. This data was further validated by ELISA and Western Blot in four esophageal adenocarcinoma cell lines with varying degrees of malignant differentiation (OE33, OE19, Flo-1, SKGT-4). Functions such as proliferation, invasion and metastasis were studied in these cell lines after BMP4 stimulation. Finally, BMP4-mediated signalling pathways and their significance in BMP4 function were elucidated. Results: IHC of tumor biopsies of EAC patients, revealed a heterogeneous pattern of BMP4 expression in EAC, in which higher expression was associated to differentiation status. A similar expression pattern was observed in the EAC cell lines. Our experiments
Sa1901 Bacterial DNA and Methylated Host DNA Sequences in Plasma Samples of Healthy and Colorectal Cancer Patients: A Whole-Metagenome Analysis Barbara K. Barták, Sandor Spisak, Péter Ittzés, András Bodor, Dániel Kondor, Gábor Vattay, Zsofia B. Nagy, Alexandra Kalmár, Zsolt Tulassay, István Csabai, Béla Molnár INTRODUCTION: The exact nucleotide composition of the circulating cell-free DNA (cfDNA) is not fully understood. Methylated and non-methylated cfDNA can be derived from both normal and tumor cells, through apoptosis, necrosis, or direct secretion. Furthermore, we are constantly exposed to foreign DNA from various sources like benign or malicious microbes in and on our body, pollens in the inhaled air and as the largest amount with the daily food supply. Here we report a new possible mechanism, which can contribute the quantity and quality of the cfDNA. AIMS: Our aims were to determine the DNA content of cfDNA from healthy, IBD, colorectal adenoma (AD) and -cancer (CRC) patients by NGS sequencing technology, and identify differences among these clinical groups. Moreover, we aimed to analyze the unmapped reads. METHODS: CfDNA was extracted from plasma samples which were collected (TUKEB 2009/037) from 50-50 normal, IBD, AD and CRC samples. Fractions were size separated via electrophoresis and DNA fragments were recovered from the gel slices. Intact DNA above 10kb (1st), the fraction is between 200bp to 10kb (2nd) and
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showed differential preference for certain signaling pathways that was dependant on the malignant status of these cell lines. Furthermore, our data uncovered opposing functions for BMP4 in the progression from BE to EAC. Whereas BMP4 inhibited proliferation of all the cell lines, regardless of their differentiation status, it was able to promote metastasis, invasion and esophageal to mesenchymal transition, only in the more differentiated EAC cell lines. Further analyses, revealed that the SMAD pathway was responsible for the BMP4mediated proliferation, and the non-canonical pathways were responsible for the other functions. Conclusion: We have shown that the opposing functions of BMP4 might be a result of differential use of signaling pathways. Because its expression is highly correlated to a differentiated status, and its metastasis-driven functions are mainly predominant in the differentiated cells, it is tempting to speculate that BMP4 might be involved in the progression from early to late stage EAC.
Sa1906
Background: Colorectal cancer (CRC) and adenomatous polyps (AP) are frequently diagnosed at colonoscopy. Recently, some reports have shown the detection of circulating microRNA (miRNA) in plasma of CRC and AP patients. However, the optimal miRNA panels are not clear yet. Since colonoscopy is invasive and the adherence rate is low, detection of circulating miRNA in plasma may significantly improve current approaches of CRC screening and aid in early diagnosis. The focus of this study is to have a better picture of circulating plasma miRNA in detection of CRC and AP. Methods: Patients (matched with age, sex and race) were grouped into three categories. Group A=CRC patients, group B= patients with adenomatous polyps >1cm, and group C=healthy controls with no adenomatous polyps. Plasma from group A (drawn prior to any primary treatment), B and C were used. For the discovery phase, plasma of 5 patients from each group was pooled separately. miRNA sequencing was done to discover genome-wide circulating miRNA profile which might differentiate patients with CRC and AP from patients without polyps. After this phase, quantitative real-time PCR was conducted to validate the expressions of selected plasma miRNAs in group A (n=22), B (n=23) and C (n=52). Results: At the discovery stage, 166 miRNAs were detected that were differentially expressed between groups A, B and C. 170 miRNAs were differentially expressed between groups B and C. Of these 170 miRNAs, 107 miRNAs were differentially expressed in group B and A but not in group C. Of the 107 overlap markers, 60 miRNAs were up-regulated in both group A and in group B, and conversely 47 miRNAs were downregulated in both group A and B. In the validation, we selected 5 miRNAs and confirmed the expression level in the 15 discovery samples by using qPCR. We further observed that miR-152 was down regulated in CRC patients with 3.54-fold change when compared with healthy controls (p value =0.0007), but not in AP patients (1.21-fold change, p value = 0.4). Conclusions: 66 miRNAs were isolated from the plasma which were different between patients with AP and those without AP. Both plasma up-regulated miRNA (N=60) and downregulated miRNA (N=47) can be used to distinguish patients with advanced adenomatous polyps and patients with CRC from matched controls. There is high rate of overlap of miRNA differentially regulated in CRC from healthy controls likewise preferentially regulated in AP versus controls 107/170 (62.9%). mir-152 is preferentially down-regulated in CRC alone but not in healthy controls or those with advanced adenomatous polyps. While prior noninvasive methodologies have been limited in their ability to detect advanced AP, it appears that numerous plasma miRNAs hold promise to differentiate patients with AP as well as CRC from healthy.
Sa1904 HER-2 Amplifications Correlate With Genetic Instability and Appear Late During Malignant Progression of Barrett's Esophagus Silvia Calpe, Akueni Davelaar, Chiu Ting Lau, Sybren L. Meijer, Paul Fockens, Kausilia K. Krishnadath Objective: In Barrett's associated esophageal adenocarcinoma (EAC), amplification of the Her-2 proto-oncogene correlates with poor prognosis. The exact mechanisms leading to Her-2 gene abnormalities in these cancers are unknown. Aneuploidy and p53 abnormalities are major and frequent events in Barrett's esophagus (BE), and are indicative for genetic instability. In several cancers genetic instability is associated with Her-2 amplification, yet this has not been studied in the Barrett's associated esophageal adenocarcinomas. The aim of this study was to gain more insight in genetic events related to genetic instability that frequently occur during BE progression by evaluating the correlation of p53 abnormalities, aneuploidy and Her-2 amplifications. Methods: Her-2 locus (17q11.2) amplification, TP53 loss and aneuploidy (CEP7/CEP17 gains) were evaluated by DNA fluorescent in situ hybridization (FISH) in a population of 174 BE patients with different degrees of dysplasia and 28 EAC patients. Results were validated by analysing the p53 mutational and FISH profiles of a panel of nine well characterized esophageal cell lines representing the Barrett's carcinogenesis sequence, including: the normal esophageal (EPC-htert), the Barrett-derived non-dysplastic (CPA) and dysplastic (CPB, CPC, CPD) and EAC cell lines with different degrees of differentiation (Flo-1, SKGT4, OE33, OE19). Results: We found that p53 losses and gain of chromosome 17 are earlier events, already detectable at early stages of metaplasia, significantly increasing with dysplasia stage, and reaching a high frequency in EAC. Her-2 locus amplification, however, was absent in BE and only detectable in High Grade Dysplasia (HGD) and EAC cases. Concordantly, a relatively small number of abnormalities (p53 mutations and CEP17/ 7 gains) were observed in the normal squamous (EPC-htert) or early BE cell line (CPA). The frequency of these abnormalities progressively increased throughout the BE-EAC sequence reaching almost 100% in the undifferentiated malignant EAC cell lines. In contrast, and in keeping with the human data, Her-2 amplifications were only observed in cell lines characterized by a malignant phenotype (OE33, OE19). Conclusion: Our studies have unravelled a sequence of genetic abnormalities that associates with malignancy. They support that gains of chromosome 17 may to Her-2 amplifications but only in case of genetic instability manifested by p53 abnormalities or/and aneuploidy. The concordance of the genetic abnormality sequence between our panel of cell lines and the human samples renders this panel of cell lines as a unique in vitro model to study molecular events in the progression and development of Barrett's cancers. These abnormalities could be used as prognostic clinical tools for evaluation of the biological behaviour of BE and EAC and for patient outcome.
Sa1907 Cohesin Overexpression Is a Frequent Event in Hepatocellular Carcinogenesis: Implications for Chemoprevention Richard Kalman, Mart DeLaCruz, Ramesh K. Wali, Lisa I. Jepeal, David Nunes, Hemant K. Roy Background: Hepatocellular Carcinoma (HCC) is the third most common cause of cancer related mortality worldwide and a feared complication of chronic liver disease. One challenge in HCC diagnosis and prevention has been its genetic complexity, with an average of 39 mutations per cancer (Vogelstein et al., Science 2013). A hallmark of HCC is dysregulation of proliferation, metastasis and angiogenesis. These changes are driven predominantly by genetic and more frequently epigenetic events altering gene expression. These include microRNA methylation which can be potentially targeted by chemopreventive agents. In other malignancies, a recently discovered modality of gene regulation has been through chromatin looping controlled by the cohesin family of proteins (e.g. Solomon et al., Nature Gen 2013). The cohesin multimer includes structural maintenance proteins (SMC1A and SMC3) which interact with the zinc-finger DNA binding protein CTCF. The role of cohesins in HCC has been unexplored. Our goal was to elucidate the role of cohesins in HCC development and chemoprevention For the latter, we used metformin given it's established risk reduction in HCC (~50%, Singh et al., Am J Gas 2013). Methods: We performed immunohistochemical (IHC) analysis on HCC and normal liver (n=96) with slides scored on a 4 point scale. We measured the expression of cohesin SMC3, and SA-1 and SA-2 (two homologs of Scc3, a cohesin protein), as well as CTCF. Cell culture studies were performed on the human HCC cell line HepG2. These cells were grown in 6-well plates and treated with 20mM of metformin or vehicle for 48hrs and subjected to Western blot analysis. Results: IHC revealed expression of SA1, SMC-3 and CTCF. As demonstrated in table 1, there was a marked upregulation in all three proteins in HCC when compared with nonneoplastic hepatocytes. There was no difference between staining intensity among tumor grades, suggesting that cohesin overexpression may be an early event. In HepG2 cells, Western blot analysis revealed a robust expression of SA1, SMC-3 and CTCF, mirroring the IHC data. As noted in figure 2, metformin treatment led to a statistically significant decrease in SA-1, SMC-3 and CTCF when compared to vehicle-treated controls (64%, p<4.5x10-4 and 44%, p<-4.84x10-5 and 51%, p<1.78x10-5 respectively). Conclusion: We demonstrated that SA-1 and SMC-3, along with CTCF were dramatically upregulated in HCC. Thus, all facets of the cohesin complex are comparably altered, supporting their role in high order chromatin alterations and hence transcriptional activation in HCC. Importantly, this appears to be a therapeutic target as indicated by the profound decrease with the chemopreventive agent metformin. This supports the significant role of cohesin as both a biomarker for HCC and a putative "druggable" target. Furthermore, this may give novel insights into HCC biology. Immunohistochemistry Staining Intensity
Sa1905 Psoriasin Expression in Human Colorectal Cancer Lucy Satherley, Lin Ye, Rachel Hargest, Wen G. Jiang Introduction Psoriasin (S100A7) is a calcium binding protein initially identified in psoriatic keratinocytes. Elevated psoriasin expression has been found in a variety of human cancers including bladder, breast, prostate and lung cancer. Overexpression of psoriasin has been associated with increased cancer cell growth and invasion but studies of cell adhesion have shown conflicting results. The aim of the present study was to investigate the expression of psoriasin in colorectal cancer tissue and to determine whether there is any association between psoriasin expression and stage of disease or prognosis. Methods Primary colorectal cancer tissue (n=90) and normal colorectal tissue (n=67) were collected at operation and examined by a consultant pathologist. Frozen sections of each tissue sample were prepared from which RNA was extracted. Reverse transcription was then used to generate cDNA which was analysed using quantitative transcript analysis (qPCR) to determine psoriasin expression. Patients were routinely followed up clinically and radiologically after surgery and the median follow up period was 65 months. The expression profile was then analysed against the clinical, pathological and outcome data. Results Psoriasin expression was significantly higher in colorectal cancer tissue compared with normal colorectal tissue, both for the whole cohort (p<0.005) and for paired samples (p<0.005). Median transcript number increased significantly with grade of differentiation and there was a trend towards increasing psoriasin expression with nodal status, although this did not reach significance. Psoriasin expression was significantly higher in tumour tissue across all TNM stages, Dukes' stages and T stages compared with normal colorectal tissue. Although psoriasin expression appeared to be higher in patients with distant metastases and those who subsequently developed local recurrence or who died of their disease, this was not significant. Conclusions To our knowledge, this is the first study examining psoriasin expression in colorectal cancer. Psoriasin expression is significantly higher in colorectal cancer tissue compared with normal colorectal tissue in keeping with previous studies which show increased levels of the protein in a range of malignancies. Our results suggest that psoriasin expression may be associated with distant disease and poorer prognosis but further work is required to confirm this. Future studies are required to determine the functional role of psoriasin in colorectal cancer cells.
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AGA Abstracts
AGA Abstracts
Circulating Plasma MicroRNA As Markers in the Early Detection of Colorectal Cancer Hankui Chen, Sandeep S. Nayak, Helu Liu, Phillip Engen, Junmei Ai, Yan Li, Joshua E. Melson, Youping Deng