ALOS4 Modulates Cellular Signaling Through The MAPK Pathway

Introduction

ALOS4, developed in our laboratory, is a novel cyclic peptide mediating its activity through integrin signaling transduction. One of these pathways is the MAPK cascade (Fyn/Ras/Raf/MEK/ERK), which is involved in various types of cancers. MAPK activation is followed by the expression of downstream immediate early genes, regulating further gene expression. Activated ERK kinase induces c-Fos and c-Jun expression who together form the AP-1 complex reported to be involved in cancer development. Additionally, ERK activation mediates p53 phosphorylation resulting in cell cycle arrest or apoptosis. We previously demonstrated that ALOS4 significantly reduced lung tumor growth and solid tumor volume, and significantly extended the life span of mice in malignant melanoma and subcutaneous melanoma models. Furthermore, ALOS4 significantly inhibited migration of B16F10 melanoma cells.

Materials and methods

In this study, we investigate the intracellular effect of ALOS4. C57BL6 mice were inoculated subcutaneously into flank side body with B16F10 cells and treated with ALOS4 at 0.3 mg/kg, i.p. 1 or 7 days post-inoculation of cancer cells. Tumors were measured and collected at day 13 for post-treatment RNA extraction. qRT-PCR and western blot analysis were performed to quantify key gene expression downstream to MAPK in in vitro and ex vivo tumor tissue. For in vitro studies, B16F10 cells were treated with ALOS4, at concentrations of 0.01, 0.03, 0.1, 0.3, 1 and 3 µM, for 48 or 72 hours.

Results

We found that ALOS4 affects expression of ERK, leading to modulations in transcription levels of genes downstream to the MAKP pathway both in vitro and ex vivo. Ex vivo tumors exhibit a significant decrease in the expression of c-Fos and c-Jun and suppression of Trp53 levels in the group treated with ALOS4 at day 7 post-inoculation as compared to control. These results correlate with significant inhibition of tumor growth in mice treated with ALOS4. In B16F10 cells, we observed results similar to ex vivo experiments, a dramatic reduction in c-Fos transcription at 48 hours, and significant suppression of c-Jun and Trp53 levels. ALOS4, at low concentrations of 0.03 µM, induced significant reduction in ERK activation at 48 hours post-treatment in vitro.

Discussion and conclusions

Based on our results, we suggest that ALOS4 modulates cellular signaling through the MAPK pathway and suppresses downstream expression of genes involved in cancer progression. Suppression of the proto-oncogenes c-Fos, c-Jun and Trp53 may support the suggested mechanism, due to their involvement in proliferation and transformation in cancer cells, further explaining the anti-tumorigenic effects of ALOS4.