K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3.

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.

Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells.

K-Ras is the most prominent driver of oncogenesis and no effective K-Ras inhibitors have been established despite decades of intensive research. Identifying new K-Ras-binding proteins and their interaction domains offers the opportunity for defining new approaches in tackling oncogenic K-Ras. We have identified Galectin-8 as a novel, direct binding protein for K-Ras4B by mass spectrometry analyses and protein interaction studies. Galectin-8 is a tandem-repeat Galectin and it is widely expressed in lung and pancreatic carcinoma cells. siRNA-mediated depletion of Galectin-8 resulted in increased K-Ras4B content and ERK1/2 activity in lung and pancreatic carcinoma cells. Moreover, cell migration and cell proliferation were inhibited by the depletion of Galectin-8. The K-Ras4B-Galectin-8 interaction is indispensably associated with the farnesylation of K-Ras4B. The lysine-rich polybasic domain (PBD), a region that is unique for K-Ras4B as compared to H- and N-Ras, stabilizes the interaction and accounts for the specificity. Binding assays with the deletion mutants of Galectin-8, comprising either of the two carbohydrate recognition domains (CRD), revealed that K-Ras4B only interacts with the N-CRD, but not with the C-CRD. Structural modeling uncovers a potential binding pocket for the hydrophobic farnesyl chain of K-Ras4B and a cluster of negatively charged amino acids for interaction with the positively charged lysine residues in the N-CRD. Our results demonstrate that Galectin-8 is a new binding partner for K-Ras4B and it interacts via the N-CRD with the farnesylated PBD of K-Ras, thereby modulating the K-Ras effector pathways as well as cell proliferation and migration.

Oncogenic K-Ras down-regulates Rac1 and RhoA activity and enhances migration and invasion of pancreatic carcinoma cells through activation of p38.

Activating mutations in the K-ras gene are genetic alterations frequently found in human carcinomas, particularly in pancreatic adenocarcinomas. Mutation of the K-ras gene is thought to be an early and important event in pancreatic tumor initiation, but the precise role of the mutant K-Ras proteins in neoplastic progression is still unknown. In the present study, we have characterized the influence of oncogenic K-Ras on the phenotype and on the signal transduction of epitheloid PANC-1 pancreatic carcinoma cells by generating PANC-1 cell clones, which stably express EGFP(enhanced green fluorescent protein)-K-Ras (V12). EGFP-K-Ras (V12)-expressing cells exhibited a more fibroblastoid cellular phenotype with irregular cell shape and disorganized cytokeratin filaments. Moreover, these cells showed a marked enhancement of their migratory and invasive properties. Stable expression of EGFP-K-Ras (V12) down-regulated the activity of Rac1 and RhoA, resulting in reduced subcortical actin filaments and stress fibers, which might contribute to the epithelial dedifferentiation. Characterization of the activity of mitogen-activated protein kinases revealed that EGFP-K-Ras (V12) enhanced the activity of p38, but did not affect the activities of the Raf/MEK/ERK cascade and JNK. While inhibition of either MEK or JNK activity had no effect on EGFP-K-Ras (V12)-induced migration, inhibition of p38 activity markedly reduced EGFP-K-Ras (V12)-induced migration. Collectively, the results suggest that oncogenic K-Ras enhances the malignant phenotype and identify the mitogen-activated protein kinase p38 as a target to inhibit oncogenic K-Ras-induced pancreatic tumor cell migration.