Enhanced Krev-1 Expression Inhibits the Growth of Pancreatic Adenocarcinoma Cells

Abstract

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.

Farnesyltransferase inhibitor (L-744,832) restores TGF-β type II receptor expression and enhances radiation sensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2

“…These inhibitors mimic the prenyl group substrate or the tetrapeptide recognition sequence on ras that is targeted by farnesyltransferase (known as CAAX motif; cysteine, aliphatic, aliphatic, X). Several studies have shown that inhibition of ras signaling alter the growth of pancreatic cancer cell lines (Leach et al, 1998;Shichinohe et al, 1996;Song et al, 2000). In this study, we selected two pancreatic cancer cell lines: one with wild type K-ras and the other with mutant K-ras.…”

Rap1 and its effector KRIT1/CCM1 regulate β-catenin signaling

“…5C), but not an increase in progression to adenocarcinoma. As Rap1 was originally identified as Krev (Kirsten ras revertant 1), a GTPase whose activity reversed Ras-induced transformation (Sakoda et al, 1992) and inhibited the development of multiple forms of adenocarcinoma (Burney et al, 1994;Damak et al, 1996;Leach et al, 1998), and because KRIT1 is upregulated during cAMPstimulated reversal of Ras-mediated cell transformation (Bachrati et al, 1999), it will be of interest in future studies to examine whether the increase in adenoma number is the result of an effect on tumor initiation/transformation downstream of nuclear -catenin signaling.…”

TIEG proteins join the Smads as TGF-β-regulated transcription factors that control pancreatic cell growth

“…Other useful models for studying the growth-regulatory effects of TGF-␤ in pancreas are provided by several transformed cell lines derived from human pancreatic cancers or chemically induced tumors from experimental animals. For example, several of these exocrine pancreatic cell lines are responsive to TGF-␤ peptides and can be used for in vitro anchoragedependent and -independent cell growth assays (5,20,33,41,43,45,46,51,55,72,83). In addition, because these cells often carry complex genetic alterations, including mutations in several mediators of TGF-␤ signaling, the results obtained from using these lines can be informative when dissecting specific defects in this pathway (5,23,33,83,85).…”