| dc.contributor.advisor | Schirmer, Markus Dr. | |
| dc.contributor.author | Roppel, Sebastian | |
| dc.date.accessioned | 2013-10-31T11:20:14Z | |
| dc.date.available | 2014-05-01T22:50:04Z | |
| dc.date.issued | 2013-10-31 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0001-BC18-5 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-4125 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | |
| dc.subject.ddc | 610 | de |
| dc.title | Functional Assessment of Biomarkers in Gemcitabine-Treated Pancreatic Cancer with Specific Focus on Nucleoside Transporter ENT1 | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Brockmöller, Jürgen Prof. Dr. | |
| dc.date.examination | 2013-10-16 | |
| dc.description.abstracteng | Pancreatic ductal adenocarcinomas are the fourth leading cause of death among all kinds of cancers. The poor prognosis is due to usually late diagnosis and, in most cases, the available therapeutic options lack sustained efficacy. Thus, there is an urgent need to identify new biomarkers, which might help to tailor therapy for patients. At present, the standard chemotherapeutic agent is gemcitabine, a nucleoside analogue and is contained in almost each chemotherapy regimen in pancreatic cancer. A recent clinical study performed in our institute revealed two SNPs in the genetic region of the equilibrative nucleoside transporter 1 (ENT1) associated with the overall survival of patients treated with gemcitabine for pancreatic cancer. The first SNP (rs1057985) of which the variant allele (minor allele frequency 33% in Caucasians) resulted in a significantly prolonged overall survival is located in an ENT1 promoter region. The second (rs45573936) of which the variant allele (2%) was linked to a dramatically reduced overall survival, constitutes an amino acid exchange at codon 216 from isoleucine to threonine. Recently, a genome-wide approach has featured SNPs for the first time in relation to the outcome of gemcitabine-based chemotherapy in pancreatic cancer. The aim of my thesis was to elucidate the molecular mechanisms of the aforementioned ENT1 polymorphisms and to explore new biomarkers for gemcitabine sensitivity in a genome-wide fashion.
ENT1 transcript variant expression was determined via quantitative real time PCR in a panel of various human tissues as well as pancreatic cancer and lymphoblastoid cell lines. These analyses included ascertainment upon gemcitabine exposure. Transcription factor binding was analyzed by electrophoretic mobility shift assay (EMSA) with addressing hypothesized allele-specific disparities. Allele-specific constructs were generated both for the ENT1 promoter region and for Ile216Thr. The functional consequences were assessed by reporter gene assay in a transiently transfected pancreatic cancer cell line and transport kinetics of 3H-labelled gemcitabine in stably transfected HEK293 cells, respectively. Genome-wide dose-response relationships for gemcitabine sensitivity were determined in a training set of 196 and a test set of 95 lymphoblastoid cell lines. Regarding the training set, comprehensive genotypes were available from resequencing. Twenty top hits were examined in the test with primer extension method used for genotyping.
Quantitative real time experiments demonstrated strong expression of an ENT1 transcript variant with the transcription start in vicinity to the index promoter SNP. The variant allele of this SNP was concomitant with higher induction of this transcript variant in 101 LCLs upon gemcitabine exposure. Linkage disequilibrium analysis revealed complete coherence of the so far investigated index SNP (rs1057985) with another SNP (rs507964) located even closer to the start site of the considered ENT1 transcript variant. In silico analysis predicted regulatory elements in the region surrounding rs507964, but not for rs1057985. In EMSA experiments protein binding was detected only in presence of the wild type, but not the variant allele of rs507964. No protein binding was detected when probes for rs1057985 were used regardless of the allele configuration. A bioinformatic screen suggested a member of the SP family to be the observed binding protein on the probe containing rs507964 and was supported by cold competition with respective probes in EMSA. However, reporter gene assays did not reveal any allele-specific impact of rs507964. Regarding Ile216Thr, the two alleles did not alter gemcitabine uptake. One of the findings from a recently published genome wide association study (GWAS) in relation to overall survival of pancreatic cancer patients could be confirmed, i.e. worse outcome of the variant allele of the SNP rs11644322 in the WWOX gene. Since the sequence pattern around this SNP resembled that of rs507964 EMSA experiments were likewise performed. This procedure revealed stronger protein binding for the probe with the wild type than the variant allele of rs11644322 again suggesting a member of the Sp family interacting. The genome-wide screen in LCLs elicited SNP rs6898780 as a potential new candidate for cellular gemcitabine sensitivity.
In conclusion, the ENT1 promoter index SNP appears to act transcript-specifically as corroborated by expression analysis and EMSA. The lack of functionality in reporter gene assay points to so far unknown additional components which may interfere, but not yet understood. With respect to Ile216Thr, because no effect on gemcitabine uptake was noted, the relation to export kinetics should be addressed. In case of rs11644322 in the pro-apoptotic WWOX gene the allele clinically associated with poor survival exhibited reduced protein binding and showed a trend to weaker induction by gemcitabine in vitro. The herein newly identified SNP rs6898780 in relation to gemcitabine sensitivity is currently not attributable to a gene or transcript and should be investigated further. | de |
| dc.contributor.coReferee | Beißbarth, Tim Prof. Dr. | |
| dc.contributor.thirdReferee | Johnsen, Steven Prof. Dr. | |
| dc.contributor.thirdReferee | Meyer, Thomas Prof. Dr. | |
| dc.contributor.thirdReferee | Kehlenbach, Ralph Prof. Dr. | |
| dc.contributor.thirdReferee | Nikolaev, Viacheslav Dr. | |
| dc.subject.eng | Gemcitabine | de |
| dc.subject.eng | SLC29A1 | de |
| dc.subject.eng | ENT1 | de |
| dc.subject.eng | Gemcitabine transport | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0001-BC18-5-5 | |
| dc.affiliation.institute | Medizinische Fakultät | de |
| dc.subject.gokfull | Medizin (PPN619874732) | de |
| dc.description.embargoed | 2014-05-01 | |
| dc.identifier.ppn | 770741193 | |