K-Ras Promotes Growth Transformation and Invasion of Immortalized Human Pancreatic Cells by Raf and Phosphatidylinositol 3-Kinase Signaling

Abstract

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomeraseimmortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced

Recombinant expression of different mutant K-ras gene in pancreatic cancer Bxpc-3 cells and its effects on chemotherapy sensitivity

“…Similarly, Minjgee et al [17] reported that transfection of G12V mutant K-ras not only increased the expression of p-Akt, but also enhanced Erk phosphorylation in head-and-neck cancer FaDu cells. Moreover, expression of G12D mutant K-ras was reported to be able to promote activating of both PI3K and Erk activities in human pancreatic E6/E7/st cells [18]. Further, the present study also revealed the differences in the downstream activation by different types of K-ras mutation in human pancreatic Bxpc-3 cells.…”

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

“…The cancer alleles that were knocked-in in human cells have been previously described to display distinct biochemical and biological properties (10)(11)(12)(13)(14). Indeed, we found that introduction of oncogenic mutations in the EGFR, KRAS, BRAF and PIK3CA genes in hTERT-HME1 breast cells resulted in activation of the corresponding proteins and triggered specific signaling pathways (Fig.…”

“…Using this approach, we generated isogenic cell lines carrying mutations frequently found in human tumors, including KRAS G13D (33), BRAF V600E (34), EGFR delE746-A750 (28), and PIK3CA H1047R (11). Several studies have shown that single cancer alleles, when ectopically expressed, can transform human cells (9,10,13,18,19). In contrast, we found that the introduction of cancer alleles in the genome of immortalized human cells of epithelial origin was generally not sufficient to confer transforming properties.…”

“…The in vitro measurable property that more closely correlates with the tumorigenic potential of cancer cells is their ability to grow in anchorage-independent fashion. Accordingly, ectopic expression of the cDNAs corresponding to the four cancer alleles had been previously shown to promote transformation of epithelial cells, such as those used in the present study (9,10,13,18,19). We evaluated the oncogenic properties of all KI cells by a conventional colony-formation assay in soft agar.…”

Response to MLN8237 in Pancreatic Cancer Is Not Dependent on RalA Phosphorylation

“…No authentication was performed on the HuPT3 cell line by the authors. The immortalized human pancreatic duct-derived (HPNE) cells were described previously (30, 31). …”