Abstract
MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown.
Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin's lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive.
First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity.
To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p < 0.01) compared with MM/ SCR control cells, when co-cultured with BMSCs in particular. Moreover, our preliminary data show that knockdown of JunB overcomes resistance of MM cells against doxorubicin as well as melphalan.
Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p < 0.01) as well as MM cell proliferation. In ongoing experiments, the in vivo relevance of our in vitro data is evaluated in a xenograft mouse model inoculated with MM /JunB-ER-IRES-GFP and MM/ IRES-GFP cells. Finally, gene expression profiles on > 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis.
In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM.
Disclosures
No relevant conflicts of interest to declare.
PHF19 Promotes Drug Resistance through EZH2 Inactivation in Multiple Myeloma
