Abstract C142: Mortalin/HSPA9 regulates p21CIP1 expression in Raf/MEK/ERK-activated cancer cells.

Abstract

Abstract Dysregulated Raf/MEK/ERK signaling is a common hallmark of tumorigenesis. However intriguingly, aberrant Raf/MEK/ERK activation triggers senescence-like growth arrest as a primary response in many cell types. This response is recognized as an innate tumor suppressive mechanism, which should be bypassed for Raf/MEK/ERK to drive carcinogenesis. Therefore, understanding of the bypass mechanisms is likely to provide an opportunity to design a novel therapeutic strategy. In this study, we report that mortalin, a member of the heat shock protein 70 (HSP70) family, has a role in bypassing the Raf/MEK/ERK-induced growth inhibition in cancer. Using tandem affinity purification and proteomic mass spectrometry, we found that mortalin is present in the MEK1/2 proteome. A specific physical interaction between mortalin and MEK was validated by co-immunoprecipitation analyses and in vitro binding assays of recombinant proteins. Immunohistochemical analysis revealed that mortalin is upregulated in human melanoma biopsies in correlation with tumor malignancy. Of note, mortalin levels were inversely correlated with p21CIP1 levels in a number of Raf/MEK/ERK-activated cancer cell lines, suggesting a role for mortalin in p21CIP1 regulation. Indeed, mortalin depletion elicited increased p21CIP1 transcription and MEK/ERK activation which were accompanied by G2/M phase cell cycle arrest and cell death in different B-Raf or K-Ras mutated cancer cell lines. Remarkably, blocking MEK/ERK activation, using the MEK1/2 inhibitor, AZD6244, dominant negative mutants of MEK or ERK, or RNA interference, abrogated p21CIP1 induction by mortalin depletion in cancer cells harboring B-RafV600E regardless of their p53 status. In contrast, mortalin overexpression suppressed B-RafV600E or ΔRaf-1:ER-induced MEK/ERK activation, p21CIP1 expression, and cell cycle arrest in cell types exhibiting normal MEK/ERK status. Other HSP70 family chaperones could not effectively replace mortalin for the observed effects on p21CIP1 regulation, suggesting a unique role for mortalin. These findings suggest that mortalin upregulation is a mechanism that underlies p21CIP1 silencing in Raf/MEK/ERK-activated cancer, identifying mortalin as a novel negative regulator of the pathway signaling. Mortalin may provide a target to reactivate the tumor suppressive signaling of Raf/MEK/ERK in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C142. Citation Format: Pui Kei Wu, Seung-Keun Hong, Sudhakar Veeranki, Mansi Karkhanis, Dmytro Starenki, Jose A. Plaza, Jong-In Park. Mortalin/HSPA9 regulates p21CIP1 expression in Raf/MEK/ERK-activated cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C142.