Molecular biological analysis of the role of placenta-specific 8 in the context of tumour progression in pancreatic ductal adenocarcinoma

Despite intensive research, the prognosis for patients suffering from pancreatic cancer remains poor with a 5‑year survival rate of only 9‑10 %. Causes for this include the lack of specific symptoms and suitable biomarkers for early detection, the high aggressiveness of this cancer, the high resi...

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Main Author: Stark, Maren
Contributors: Buchholz, Malte (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2022
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Summary:Despite intensive research, the prognosis for patients suffering from pancreatic cancer remains poor with a 5‑year survival rate of only 9‑10 %. Causes for this include the lack of specific symptoms and suitable biomarkers for early detection, the high aggressiveness of this cancer, the high resistance to the currently available forms of therapy and the high heterogeneity between the individual tumours. For the reasons mentioned, it is mandatory to find biomarkers that enable earlier diagnosis and to develop more effective forms of therapy tailored to the respective tumour. Since our group was able to identify PLAC8 as a strongly overexpressed gene in a subgroup of pancreatic ductal adenocarcinoma tumours, a targeted therapy directed against PLAC8 or a gene downstream regulated by PLAC8 could represent a new form of therapy for patients with tumours of this subgroup. Since this therapy would act more specifically on the tumour than conventional cancer therapies, such a therapy could improve the prognosis for these patients. Analyses regarding the mode of action of PLAC8 showed that PLAC8 promotes proliferation of pancreatic ductal adenocarcinoma cells by interfering with cell cycle. Further analyses of CRISPR/Cas9 clones suggested that this is not mediated via the PLAC8 protein, but via the PLAC8 RNA. Based on that, it was assumed that the PLAC8 RNA could act similar to a long non‑coding RNA on proliferation of these cells. If this were indeed true, it would be reflected, among other things, in the fact that it should not be possible to generate a PLAC8 RNA‑deficient pancreatic ductal adenocarcinoma cell line, as such a cell line would no longer be capable of proliferation and would thus no longer be viable. One aim of this work was to examine this thesis. Another aim was to also unravel the exact mode of action of PLAC8 on the molecular level. To approach these goals, it was first aimed to be tested whether the selection against a PLAC8 RNA‑deficient cell line would be further confirmed in the analysis of further CRISPR/Cas9 clones derived from the pancreatic ductal adenocarcinoma cell line S2‑ 007. If this was shown to be the case, further CRISPR/Cas9 approaches were to be carried out to investigate whether it would be possible to switch off the endogenous PLAC8 gene locus completely using CRISPR/Cas9, provided that a recombinant, non‑coding PLAC8 construct would be introduced into the cell at the same time. The idea was that ‑ after genomic integration ‑ the recombinant construct could take over the essential function of the endogenously expressed PLAC8 transcripts and would thus allow the silencing of the endogenous PLAC8 gene locus. If this were possible, it would be a strong indication that the initial hypothesis is correct and would also show which of the PLAC8 transcript variants, expressed in the S2‑007 cell line, is/are the one/s being essential for sustained proliferation of this cell line. To obtain a more accurate picture of the mode of action of PLAC8 on the molecular level, RNA sequencing was aimed to be performed with cDNA samples of S2‑007 cells with and without PLAC8 knockdown. If altered expression patterns in genes were found after PLAC8 knockdown, further analyses would then be carried out to test for a functional relationship between these genes and PLAC8. The previously observed selection against a PLAC8 RNA‑deficient S2‑007 cell line could further be strengthened in the analyses presented in this work. Initially, it had been shown that despite the analysis of 38 CRISPR/Cas9 clones derived from the PLAC8 protein‑deficient S2‑ 007 clone G2#1, it had not been possible to switch off the endogenous PLAC8 gene locus and thus PLAC8 RNA expression. As this clone only had one remaining „wild type“‑allele at the PLAC8 gene locus due to a preceding CRISPR/Cas9 editing (using a donor cassette encoding for a transcriptional stop signal) and the CRISPR/Cas9 techology in general is very effective, this strongly suggests that the expression of the PLAC8 RNA seems to be essential for the viability of the S2‑007 cell line. Together with the fact that for PLAC8 protein‑deficient S2‑007 clones a growth‑inhibitory phenotype could still be detected after treatment with PLAC8‑specific siRNAs, this result can be traced back to the fact that the PLAC8 RNA seems to be essential for sustained proliferation of these cells, preventing a PLAC8 RNA‑deficient cell line from being created. This thesis is further supported by the fact that it had neither been possible to switch of the endogenous PLAC8 gene locus in 248 other clones that originated from clone G2#1 or the parental cell line S2‑007. However, the actual aim of generating these clones, to switch off the endogenous PLAC8 gene locus and to replace the essential function of this locus by introducing a recombinant PLAC8 construct, has not yet been successfully realised. For this reason, it is still not possible to draw a conclusion as to whether one or more of the PLAC8 transcript variants, expressed in S2‑007 cells, are essential for sustained proliferation of these cells. Since 6 different PLAC8 transcript variants had been identified via long‑read sequencing in cDNA samples of S2‑007 cells, it is reasonable to assume that the proliferation of these cells is regulated by one or more of these PLAC8 transcript variants. In the framework of this work it had not yet been possible to determine a specific mode of action of PLAC8 on the molecular level. Although, by performing RNA sequencing analyses, a downregulation in the RNA expression had been shown for the genes E2F1, E2F2 and E2F5, as well as HYOU1, CHPT1, TGOLN2 and B3GALT5 after PLAC8 knockdown, no functional relationship between PLAC8 and one of these genes could yet be confirmed in further analyses. Therefore, it is still an open question whether PLAC8 mediates its pro‑proliferative effect in its role as a long non‑coding RNA via the regulation of one or more of these or any, so far unidentified, genes in S2‑007 cells.
Physical Description:117 Pages
DOI:10.17192/z2022.0196