Abstract 4961: Functional analysis of prostate cancer-specific MED12 mutation
Authors: Kämpjärvi Kati , Kim Nam Hee , Park Min Ju , Turunen Mikko , Mäkinen Netta , Heikkinen Tuomas , Keskitalo Salla , Varjosalo Markku , Boyer Thomas G. , Vahteristo Pia 
Published: August 2015

Mediator is a highly conserved protein complex regulating RNA polymerase II-dependent transcription. It also mediates transcriptional signals from several gene-specific transcription factors. In higher eukaryotes the core Mediator comprises 26 subunits organized into three distinct modules; head, middle, and tail. Separate kinase module regulating the activity of the Mediator is formed by MED12, MED13, Cyclin C, and CDK8/19. Mediator complex subunit 12 (MED12) gene on Xq13.1 encodes MED12 protein which links the kinase module to the Mediator core and is required for Cyclin C-dependent CDK8 activity. Somatic mutations in 5′-terminus of MED12 are highly frequent in uterine leiomyomas and in breast fibroadenomas. Similar mutations occur recurrently, although with lower frequencies, also in uterine leiomyosarcomas and in chronic lymphocytic leukemia. All the observed mutations have been missense mutations or in-frame insertions and deletions in exons 1 and 2. Functional analyses have indicated that mutations in both exon 1 and exon 2 lead to dissociation of Cyclin C-CDK8/19 from the core Mediator, loss of Mediator-associated CDK kinase activity, and similar global gene expression profiles. Following the initial observation of MED12 mutations in uterine leiomyomas, exome sequencing study by Barbieri and co-workers identified a recurrent MED12 mutation in prostate cancer. Seven mutations were observed in total and strikingly five of these affected the same codon in exon 26 resulting in substitution of leucine 1224 with phenylalanine. The same L1224F mutation has been observed in prostate cancer in sequencing efforts by The Cancer Genome Atlas. The aim of this study was to elucidate the molecular mechanisms by which the mutation causes tumorigenesis in prostate cancer. To identify the possible effects on protein-protein interactions, we generated stable and inducible HEK293 Flp-In cell lines expressing MED12 wild type and L1224F mutant protein and performed affinity purification followed by liquid chromatography-mass spectrometry. Interactions with other subunits of the Mediator core and the kinase module were further analyzed with immunoprecipitations accompanied with Western blotting. In addition, Mediator-associated CDK kinase activity of the kinase modules with respective protein constructs were measured. Based on these analyzes, L1224F mutation does not interfere the interaction between MED12 and Cyclin C-CDK8/19, and thus does not lead to reduced Mediator-associated CDK kinase activity. Significant decrease was, however, observed in the binding of L1224F mutant versus wild type protein with several other Mediator subunits. These results indicate that prostate cancer-specific MED12 mutation is promoting tumorigenesis through clearly distinct mechanisms than the ones seen in myomagenesis.

Citation Format: Kati Kämpjärvi, Nam Hee Kim, Min Ju Park, Mikko Turunen, Netta Mäkinen, Tuomas Heikkinen, Salla Keskitalo, Markku Varjosalo, Thomas G. Boyer, Pia Vahteristo. Functional analysis of prostate cancer-specific MED12 mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4961. doi:10.1158/1538-7445.AM2015-4961