Pancreatic ductal adenocarcinoma is characterized by a high rate of activating mutations involving codon 12 of the K-ras protooncogene. As a means of ras-targeted intervention, the effects of enhanced Krev-1 gene expression on the growth and tumorigenicity of the hamster pancreatic adenocarcinoma cell line PC-I were evaluated. Overexpression of the Krev-1 gene product resulted in morphologic reversion to a less transformed phenotype, as well as retarded growth kinetics and diminished potential for anchorage-independent growth.Among six transfected cell lines, the magnitude of these changes correlated with the degree of Krev-1 overexpression as assessed by Western blot. When PC-1 cells overexpressing high levels of the Krev-1 gene product were assessed for tumorigenicity in syngeneic animals, an increased latency to tumor growth and a decreased tumor size were noted. The results confirm that overexpression of the Krev-1 gene may provide a useful strategy for ras-targeted intervention in this disease. Key Words: Adenocarcinoma-K-ras-Krev-1-Hamster.Pancreatic ductal adenocarcinoma remains one of the most lethal human malignancies, representing the fifth leading cause of cancer death in the United States (1). More than 90% of pancreatic cancers exhibit activating mutations in codon 12 of the K-rus protooncogene (2), leading to significant interest regarding rus-targeted intervention in the treatment of these tumors. Several strategies for rus inhibition were evaluated previously in pancreatic cancer cells, including antisense oligonucleotides (3), the expression of dominant negative rus mutants (4), and the pharmacologic inhibition of posttranslational modification (5,6). While the availability of specific farnesyl-transferase inhibitors represents an exciting new development in ras-targeted therapy, many pancreatic adenocarcinoma cell lines are resistant to these agents (6). This resistance may result from the ability of K-rus, but not H-rus, to undergo compensatory geranylgeranylation in the absence of farnesylation (7). As an alternative approach, we investigated the ability of enhanced Manuscript Krev-1 expression to reverse rus-dependent transformation in pancreatic ductal adenocarcinoma cells. The Krev-1 gene, also known as rap-IA and smg-p21, was initially isolated because of its ability to reverse rusdependent transformation (8). Subsequent biochemical studies confirmed the ability of Krev-1 to inhibit rus function effectively, both in vivo and in vitro (reviewed in Ref. 9). In this report, we demonstrate that induced overexpression of Krev-1 alters the morphology, growth kinetics, in vitro anchorage-independent growth, and in vivo tumorigenicity of pancreatic ductal adenocarcinoma cells.
We investigated tumor-cell-intrinsic chromatin accessibility patterns of pancreatic ductal adenocarcinoma (PDAC) by ATAC-seq on EpCAM+ PDAC malignant epithelial cells, sorted from 54 freshly resected human tumors, and discovered a signature of 1092 chromatin loci displaying differential accessibility between patients with disease free survival (DFS) < 1 year and patients with DFS > 1 year. Analyzing transcription factor (TF) binding motifs within these loci, we identified two TFs (ZKSCAN1 and HNF1b) displaying differential nuclear localization between patients with short vs. long DFS. We further developed a novel chromatin accessibility microarray methodology termed ATAC-Array, an easy-to-use platform obviating the time and cost of next generation sequencing. Applying this novel methodology to the original ATAC-seq libraries as well as independent libraries generated from patient-derived organoids, we validated ATAC-array technology in both the original ATAC-Seq cohort as well as in an independent validation cohort. We conclude that PDAC prognosis can be predicted by ATAC-array, which represents a novel, low-cost, clinically feasible technology for assessing chromatin accessibility profiles.
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