Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells

Abstract

The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo-and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Aktregulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected7a dominant-negative mutant Akt, treated7SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/ c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIa inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.

Scatter factor protects tumor cells against apoptosis caused by TRAIL

“…We previously showed that transfection of this vector into DU-145 blocks SF-induced Akt phsophorylation as well as the phosphorylation of several Akt substrates (e.g. GSK3 and mTOR) [20]. As shown in Fig.…”

“…Previously, we reported that Akt is required for SF protection of carcinoma and glioma cells against DNAdamaging agents, such as ADR [18][19][20][21]33,34]. Here, we tested whether Akt signaling is required for protection against TRAIL, by the use of a DN-Akt expression vector that encodes a kinase-dead point mutant Akt (K179A).…”

“…We have been studying the mechanisms by which SF protects carcinoma and glioma cells against DNAdamaging agents as a model for understanding chemoresistance in tumors that overexpress SF and c-Met [18][19][20][21]. In the course of these studies, we observed that SF also protects these cell types against cytotoxicity caused by the tumor necrosis factor-related apoptosisinducing ligand (TRAIL).…”

Immunomodulatory Properties of Mesenchymal Stem Cells: Cytokines and Factors

“…[100][101][102] Transmembrane receptor in combination with tyrosine kinase regulates signal transduction. [109][110][111][112] Moreover, c-Met can bind to Fas and block Fas ⁄ Fas-ligand co-intraction and thus prevents cell death. 103 Many papers have reported that c-Met and HGF are expressed together.…”

“…Survival signal transduction such as MAPK, PI3K-Akt, and NF-jB are responsible for anti-apoptotic characters of c-Met. [109][110][111][112] Moreover, c-Met can bind to Fas and block Fas ⁄ Fas-ligand co-intraction and thus prevents cell death. 113,114 However, apoptotic effects of HGF ⁄ c-Met is not yet fully understood, but it has been observed in number of cell lines such as ovarian carcinoma cell, breast carcinoma cell, mouse sarcoma cell, and mouse hepatocarcinoma cell.…”