A chemical biology screen identifies glucocorticoids that regulate c-maf expression by increasing its proteasomal degradation through up-regulation of ubiquitin

Abstract

IntroductionMultiple myeloma appears to arise from initiating chromosomal translocations and duplications in postgerminal center plasma cells with subsequent secondary mutations contributing to disease progression. 1,2 Six unique myeloma variants can be identified by gene expression profiling with 3 of the 6 groups anchored by an initiating chromosome translocation. It is noteworthy that a unifying event of cyclin D dysregulation is identified in all subgroups. 1,2 One of the initiating chromosomal abnormalities in myeloma involves members of the Maf family of oncogenes. 1 Maf proteins are basic leucine zipper transcription factors in the AP1 family and regulate gene transcription by binding DNA sequences known as MAF responsive elements (MAREs). 3,4 Of the Maf family, c-maf was the first endogenous member identified and is probably the best characterized with respect to function. 5 Genes up-regulated by c-maf include cyclin D2, ␤-integrin 7, ARK 5 and CCR1, all of which are important in the pathogenesis of multiple myeloma. [6][7][8] c-maf is dysregulated in multiple myeloma. For example, approximately 25% of myeloma cell lines have a t(14;16) translocation. 6 In patients with myeloma, approximately 6% have a t(14;16) or t(14;20), translocations that juxtapose IgH with c-maf and its homolog mafB, respectively. 9,10 The frequency of c-maf overexpression in patients who lack the t(14;16) has varied from study to study, depending on the method to assess its overexpression and ranges from 5% to 50%. 6,11,12 In malignant cell lines, including multiple myeloma, overexpression of c-maf augments cell proliferation and increases tumor formation in xenograft models. 6,7 Conversely, inhibition of c-maf with dominant-negative constructs decreases cell proliferation, impairs adhesion to marrow stroma, and delays tumor growth. 6 Overexpression of c-maf is also clinically relevant in that patients with myeloma and the t(14;16) c-maf translocation have a shorter overall survival. 13 Although the functional importance of c-maf has been described, the mechanisms that govern its regulation have not been fully elucidated. Therefore, molecules that decrease c-maf and subsequently its downstream targets, particularly cyclin D2, could further our understanding of the regulation of c-maf. Improved understanding of c-maf will help develop therapies that target this protein.To this end, we developed a high-throughput chemical genomics screen to identify compounds that inhibit c-maf-dependent transactivation of the cyclin D2 promoter. With this assay, we screened libraries of off-patent drugs and chemicals and were surprised to identify glucocorticoids as inhibitors of c-mafdependent cyclin D2 transactivation. Subsequent studies demonstrated that glucocorticoids decrease c-maf protein by promoting its ubiquitination through the up-regulation of ubiquitin C mRNA. Thus, this chemical biology approach has provided insights into a novel mechanism of c-maf regulation. The publication costs of this article were defrayed in part by page charge ...

The Tricyclic Antidepressant Amitriptyline Inhibits d-Cyclin Transactivation and Induces Myeloma Cell Apoptosis by Inhibiting Histone Deacetylases: In Vitro and In Silico Evidence

“…To identify cyclin D2 inhibitors with psychoactive activity, we conducted a chemical screen for inhibitors of the D-cyclin promoter similar to the method described previously (Mao et al, 2007). In brief, NIH3T3 cells stably expressing the cyclin D2 promoter driving firefly luciferase (10,000 cells/ well) were plated in 96-well plates by a Biomek FX liquid handler (Beckman Coulter, Fullerton, CA).…”

Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical antimyeloma activity

“…From screening the Spectrum compound library (Figure 2A), we identified 38 hits, of which 29 were also glucocorticosteroids. The mechanisms by which corticosteroids decrease cyclin D gene expression has been the subject of parallel investigation and is described elsewhere (12). Notably, 9 nonsteroid compounds were identified as putative suppressors of CCND2; these included kinetin riboside, dihydrogambogic acid, monensin, patulin, gentian violet, pararosaniline pamoate, pristimerin, aklavine, and camptothecin.…”

The ubiquitin ligase HERC4 mediates c-Maf ubiquitination and delays the growth of multiple myeloma xenografts in nude mice

“…9 This reporter and c-Maf were cotransfected and stably expressed in NIH3T3 cells. These cells were then transfected with individual E3 ligase plasmids from a subset collection of E3 ligases (the SPARC BioCentre, Toronto, ON, Canada).…”

“…Luciferase activity was analyzed with the Bright-Glo substrate (Promega) as described previously. 8,9 Immunoblotting assay Immunoblotting (IB) assay was performed against specific antibodies as described previously.…”

“…7 Previous studies demonstrated that c-Maf protein stability is modulated by the ubiquitin-proteasome system. 8 c-Maf can be degraded in the proteasome after polyubiquitination, 4,9 which is mediated by 3 types of sequential enzymes, including ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). In humans there are 2 E1s, 35 known E2s, and ;300 to 600 E3s; these enzymes coordinately modulate ubiquitination of a vast diversity of substrate proteins.…”