ZNF300 may influence the metastatic properties in pancreatic ductal adenocarcinoma

Author(s)Fisher, Amanda
Date Accessioned2015-08-13T13:54:22Z
Date Available2015-08-13T13:54:22Z
Publication Date2014
AbstractAberrant DNA methylation of promoter CpG islands (CGI) is a contributing factor that facilitates the dysregulation of gene expression to promote the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). Moreover, due to the overwhelming number of PDAC patients presented with metastasis at the time of diagnosis, it is necessary to better understand the process of metastasis. To understand the role of DNA methylation may in the metastasis of PDAC, two isogenic cells lines, generated by Dr. Isaiah Fidler at the University of Texas MD Anderson Cancer Center, were used as a cell culture model. These cell lines were generated from orthotopic injections of an established pancreatic cancer cell line, colo357, into the pancreas of nude mice. Upon injection, metastasis formed in the liver. Metastatic cells from the metastases were isolated and grown in culture to yield a lowly metastatic cell line, fast growing (FG). This process was repeated using FG cells, and after three rounds of injection and isolation, the enriched metastatic cells were isolated to yield a highly metastatic variant, L3. 6. Preliminary work, performed by previous lab members, identified genes exhibiting promoter hypermethylation in a high metastatic cell line, L3.6pl, compared to the lowly metastatic isogenic variant cell line, FG. One of those genes, ZNF300, was chosen as a candidate gene for many reasons. First, analysis of percent methylation within the ZNF300 CGI indicated increased methylation among L3.6pl cells compared to FG, specifically sixteen percent to one percent, respectively. In addition to increased methylation, the region of the ZNF300 promoter that was hypermethylated was also shown to affect promoter activity according to luciferase promoter assays. Therefore, it was hypothesized that the observed hypermethylation in L3.6pl cells may lead to decreased ZNF300 expression. Moreover, ZNF300 expression was identified in pancreatic tumor samples from ten PDAC patients via immunohistochemical staining; however, analysis from lymph node metastases from many of these patients showed diminished ZNF300 expression compared to the primary tumor. Taken together, this data supports our overall goal to determine if ZNF300 hypermethylation correlates to ZNF300 expression, and more importantly, if this correlation is related to the metastatic ability of L3.6pl cells. To determine if ZNF300 is epigenetically regulated, methylation levels were quantified via COBRA and bisulfite sequencing in both FG and L3.6pl cells. Additionally, ZNF300 expression was measured via qRT-PCR and western blotting analysis. To determine the functional role of ZNF300 in the metastatic process of L3.6pl, we attempted to overexpress ZNF300 using the p-RetroX-Tre3G Tet-ON inducible expression system. Following induction using doxycycline, both the expression and migratory ability were measured. The data in this study concludes that, contrary to preliminary data, the ZNF300 CpG island is not differentially methylated in L3.6pl cells compared to FG cells. Our expression data is in accordance with the methylation levels, and thus, indicated no significant difference in ZNF300 expression between the two isogenic variant cell lines. Moreover, we were unable to induce ZNF300 expression using the retroviral inducible system, but rather noted potentially off target effects of doxycycline in infected L3.6pl cells. Due to our inability to induce ZNF300 expression, we are unable to identify the role of ZNF300 in the metastatic process of PDAC.en_US
AdvisorLin, Huey-Jen
DepartmentUniversity of Delaware, Department of Biological Sciences
Unique Identifier918185660
PublisherUniversity of Delawareen_US
dc.subject.lcshPancreas -- Cancer.
dc.subject.lcshZinc-finger proteins.
dc.subject.lcshGene expression.
TitleZNF300 may influence the metastatic properties in pancreatic ductal adenocarcinomaen_US
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
4 MB
Adobe Portable Document Format
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
2.22 KB
Item-specific license agreed upon to submission