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Molecular determinants for the outcome in gemcitabine-treated pancreatic cancer

dc.contributor.advisorSchirmer, Markus PD Dr.
dc.contributor.authorLüske, Claudia
dc.titleMolecular determinants for the outcome in gemcitabine-treated pancreatic cancerde
dc.contributor.refereeBrockmöller, Jürgen Prof. Dr.
dc.description.abstractengPancreatic ductal adenocarcinoma (PDAC) is a mostly lethal disease which represents the fourth common cause of all deaths related to cancer. The nucleoside analogue gemcitabine constitutes a currently widely used treatment standard both in a palliative and adjuvant setting. However, variability in response to gemcitabine is high with a substantial impact of genetic variations assumed. Two previously identified single nucleotide polymorphisms (SNPs) associated with the overall survival of gemcitabine treated patients suffering from PDAC were characterized in this thesis concerning the underlying molecular mechanisms: Rs11644322 pertinent to the tumor suppressor gene WWOX, and rs1130609 pertinent to the ribonucleotide reductase RRM2. A panel of 89 lymphoblastoid cell lines (LCLs) with publicly available genotype information was used as model system to study genomic causes of variable gemcitabine sensitivity. Cytotoxicity of gemcitabine was assessed by flow cytometry-based measurement of proliferation inhibition, and gene expression was determined by quantitative real-time PCR. For extended experiments the pancreatic cancer cell lines AsPC1, MiaPaca-II, PaTu8988t, PancI, and L3.6 were used. Modification of gemcitabine response upon WWOX knock-down by siRNA and shRNA (verified by Western Blotting) or upon WWOX overexpression was ascertained. As the transcription factor SP1 bound to the WWOX rs11644322, overexpression of this factor was conducted and the consequences on WWOX transcription with and without gemcitabine, 5-fluorouracil and irinotecan were studied. Whole transcriptome analysis was determined for gemcitabine effects in AsPC1 and MiaPaca-II cells, in PaTu8988t upon shRNA-mediated WWOX knock down, and in pooled LCLs defined by homozygous wild type and variant allele at the WWOX index SNP site, respectively.  Regarding the molecular mechanisms behind the RRM2 polymorphism, electrophoretic mobility shift assays (EMSA) were performed to discern allele-specific transcription factor binding at rs1130609. An in vitro coupled Transcription/Translation system was utilized to study allele-specific differences regarding protein translation.  In LCLs, cytotoxicity of gemcitabine was reduced in dependence of the number of A alleles at WWOX rs11644322, consistent with the worse prognosis of patients with this allele. No transcripts were detected in close vicinity to rs11644322. However, homozygosity for the AA allele at rs11644322 was accompanied by lower WWOX expression of both, the core coding region and of the last exon, separated by 730 kb. A specific gemcitabine-related correlation was identified in LCLs between transcription of WWOX and the growth arrest and DNA damage-inducible gene GADD45A, which was correlated with increased gemcitabine cytotoxicity.  Whole transcriptome analysis in AsPC1 and MiaPaca-II cells revealed that RRM2 expression increased more strongly than any other protein-coding transcript upon gemcitabine exposure. Quantitative relations of the two RRM2 transcripts differing in the noncoding 5' sequence length revealed the major one amounting to 96 to 99 % of the entire transcript numbers, depending on the cell type. This major RRM2 transcript isoform was also increased upon gemcitabine exposure in LCLs and in peripheral blood of patients subjected to gemcitabine-containing chemotherapy. In EMSA experiments stronger protein binding at the RRM2 rs1130609 G allele (the same allele which was associated with worse prognosis) was identified. However, no impact of this SNP on the transcription of the major RRM2 isoform was seen. In contrast, increased expression of the minor isoform with an extended 5'-region was observed in presence of the T variant allele at rs1130609, intensified upon gemcitabine treatment. Preliminary results for cloned RRM2 suggested less translation efficacy for the T compared to the G allele.  Based on previous data and those of my thesis, mechanistic hypotheses for WWOX and RRM2 are suggested: The variant A allele at the WWOX index SNP might bind SP1 to a lesser extent, resulting in decreased expression probably mediated via interaction with the promoter region by looping. By that, epithelial-mesenchymal transition may be increased resulting in reduced cell proliferation and enhanced resistance to gemcitabine, finally providing a mechanistic basis for worse clinical outcome. Regarding RRM2, phosphorylated gemcitabine can block physiological DNA synthesis resulting in RRM2 transcription induction, primarily of the major variant isoform. In case of the T variant allele at the RRM2 index SNP site, RRM2 protein synthesis is presumed to be impaired, what might stimulate transcription of the minor isoform. The obtained data provide new insights in functional mechanisms. By corroborating the clinical associations, these data further supported the two predictive SNPs in WWOX and RRM2 as valid biomarkers for gemcitabine-based chemotherapy in
dc.contributor.coRefereeBurfeind, Peter Prof. Dr.
dc.contributor.thirdRefereeZeisberg, Michael Prof. Dr.
dc.contributor.thirdRefereeMeyer, Thomas Prof. Dr.
dc.contributor.thirdRefereeHahn, Heidi Prof. Dr.
dc.contributor.thirdRefereeKatschinski, Dörthe Prof. Dr.
dc.subject.engPancreatic cancer, gemcitabine, WWOX, RRM2de
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullMedizin (PPN619874732)de

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