RalA signaling pathway as a therapeutic target in hepatocellular carcinoma (HCC).

Ral (Ras like) leads an important proto-oncogenic signaling pathway down-stream of Ras. In this work, RalA was found to be significantly overactivated in hepatocellular carcinoma (HCC) cells and tissues as compared to non-malignant samples. Other elements of RalA pathway such as RalBP1 and RalGDS were also expressed at higher levels in malignant samples. Inhibition of RalA by gene-specific silencing caused a robust decrease in the viability and invasiveness of HCC cells. Additionally, the use of geranyl-geranyl transferase inhibitor (GGTI, an inhibitor of Ral activation) and Aurora kinase inhibitor II resulted in a significant decrease in the proliferation of HCC cells. Furthermore, RalA activation was found to be at a higher level of activation in HCC stem cells that express CD133. Transgenic mouse model for HCC (FXR-Knockout) also revealed an elevated level of RalA-GTP in the liver tumors as compared to background animals. Finally, subcutaneous mouse model for HCC confirmed effectiveness of inhibition of aurora kinase/RalA pathway in reducing the tumorigenesis of HCC cells in vivo. In conclusion, RalA overactivation is an important determinant of malignant phenotype in differentiated and stem cells of HCC and can be considered as a target for therapeutic intervention.

Overactivation of Ras signaling pathway in CD133+ MPNST cells.

Cancer stem cells (CSCs) are believed to be the regenerative pool of cells responsible for repopulating tumors. Gaining knowledge about the signaling characteristics of CSCs is important for understanding the biology of tumors and developing novel anti-cancer therapies. We have identified a subpopulation of cells positive for CD133 (a CSC marker) from human primary malignant peripheral nerve sheath tumor (MPNST) cells which were absent in non-malignant Schwann cells. CD133 was also found to be expressed in human tissue samples and mouse MPNST cells. CD133+ cells were capable of forming spheres in non-adherent/serum-free conditions. The activation levels of Ras and its downstream effectors such as ERK, JNK, PI3K, p38K, and RalA were significantly increased in this population. Moreover, the CD133+ cells showed enhanced invasiveness which was linked to the increased expression of ß-Catenin and Snail, two important proteins involved in the epithelial to mesenchymal transition, and Paxilin, a focal adhesion protein. Among other important characteristics of the CD133+ population, endoplasmic reticulum stress marker IRE1α was decreased, implying the potential sensitivity of CD133+ to the accumulation of unfolded proteins. Apoptotic indicators seemed to be unchanged in CD133+ cells when compared to the wild (unsorted) cells. Finally, in order to test the possibility of targeting CD133+ MPNST cells with Ras pathway pharmacological inhibitors, we exposed these cells to an ERK inhibitor. The wild population was more sensitive to inhibition of proliferation by this inhibitor as compared with the CD133+ cells supporting previous studies observing enhanced chemoresistance of these cells.