PACT-PKR Pathway Transactivates the Tumor Suppressor RhoB through C/EBPbeta in Cancer Cells with High Akt Activity

Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy. H157 cells were plated 2 x 10^6 in T-75 flasks in RPMI 1640 containing 10% FBS and incubated for 24h. The medium was then changed to RPMI 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM of LY294002, OSU03012, PIA23, Perifosine, Miltefosine, API-2, DZ-50, or 100 nM of Wortmannin and Rapamycin for 6 hours, or an equal amount of DMSO as control. Following incubation, total RNA was extracted from cells in T-75 flasks using TRIzol reagent (Invitrogen). Two dye-swapped replicates were performed for each treatment.