Ras pathway activation in mesothelioma

Abstract

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The critical molecular pathways involved in growth and proliferation of malignant pleural mesothelioma (MPM) are largely unknown. Although Ras mutations are uncommon in MPM, silencing of the RASSF1A (a Ras inhibitor) has been described in MPM. Furthermore, the downstream targets of the Ras pathway have not been elucidated in this malignancy. Here we sought to determine the activation status of Ras in MPM cell lines and its downstream effectors in MPM. Furthermore, we studied the potential role of Ras pathway activation on the control of the cellular translational machinery in MPM. Methods: LP9 normal mesothelial cells and human mesothelioma cell lines 2373, 2461, and 2596 were grown in RPMI growth medium supplemented with 10% calf serum to 70% confluence. Cell lysates were prepared and protein concentrations were determined using the Bradford assay. The Ras activation affinity pull-down assay was used to evaluate the amount of Ras-GTP (activated Ras) in 2373, 2461, 2596 in comparison to LP9 cells. Kinase activity of downstream Ras effectors was determined by assessing phosphorylation of related substrates such as Elk-1. Furthermore, crude lysates of MPM cell lines as well as LP9 were subjected to Western analysis for assessing the levels of regulators of translational control, eIF4E, 4EBP-1, and eIF4G. Results: High levels of Ras activation, as determined by the presence of Ras-GTP, was identified in 2 of 3 MPM cell lines compared to LP9 normal mesothelial cells. Furthermore, phosphorylation of Elk-1 by Erk42/44 was greater in all 3 MPM cell lines compared to LP9 cells. Total levels of eIF4E and eIF4G were not different between MPM cell lines and LP9 cells. However, 4EBP-1 was present in its hyper-phosphorylated form in 2 out of 3 MPM cell lines compared to LP9 cells. Conclusions: Ras and its downstream effectors are activated in MPM. One potential downstream target of the Ras pathway is the controlling elements of cap-dependent translation. Therefore, manipulation of such a signaling network via gene therapy or pharmacological methods might play a role in treatment of MPM.