The Pathophysiologic Role of JunB in Multiple Myeloma Pathogenesis: Focus on Bone Marrow Angiogenesis

Angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and is correlated with adverse prognosis. The increase in vascular density within the bone marrow (BM) microenvironment is triggered by oncogene-mediated expression and secretion of pro-angiogenic growth factors and cytokines, most prominently including VEGF. Members of the AP-1 family of transcription factors (TFs) have emerged as actively pursued therapeutic targets over the past years. Our previous studies demonstrated a critical role for the AP-1 family member JunB in MM cell proliferation, survival and drug resistance. Whether JunB also contributes to MM BM angiogenesis is currently unknown. We first sought to identify correlative expression patterns of JUNB and angiogenic factors using the Oncomine software. Indeed, similar to JUNB our data identified significant induction of VEGF, VEGFB, IGF-1 and PlGF, progressing from normal plasma cells to cells from patients with monoclonal gammopathy of undetermined significance (MGUS) and MM in two independent gene expression profiling data sets. In contrast, no correlation was observed between expression of JUNB and ANGPT1, ANGPT2, SDF-1 and FGF4. Besides BM stromal cells (BMSCs) also osteoblasts (OBs) upregulate JunB protein levels in MM: stromal cell co-cultures. This effect is, at least in part, mediated by the humoral milieu, IL-6 in particular. Whether BMSC- and OB- mediated production and secretion of angiogenic factors is mediated via JunB in MM cells was investigated next. Our data show that doxycyclin- induced inhibition of BMSC: OB- mediated JunB upregulation in TetR-shJUNB/ MM.1S cells abrogated production and secretion of VEGF, VEGFB, IGF-1 and PlGF as evidenced by qPCR and ELISA assay. Similar effects were observed in MM: stromal cell co-cultures using the IL-6R inhibitor tocilizumab. Consequently, supernatant of both doxycycline- treated BMSC: Tet-shJUNB/ MM.1S and OB: Tet-shJUNB/ MM.1S co-cultures as well as tocilizumab significantly inhibited angiogenesis, as evidenced by matrigel- based tube formation as well as wound healing assays. Conversely, tamoxifen- induced JunB activity in JunB-ER/MM cells triggered the expression and secretion of angiogenic factors and angiogenesis. Importantly, the functional role of JunB on BM angiogenesis was also verified in vivo using a MM xenograft mouse model. To this purpose, immunodeficient NSG mice were inoculated subcutaneously with Tet-SCR/ MM.1S or Tet-shJUNB/ MM.1S together with BMSCs into the left and right flanks of mice, respectively, and fed with doxycycline in their drinking water for 5 weeks. Treatment with doxycycline inhibited JunB protein levels in Tet-shJUNB/ MM.1S, but not in Tet-SCR/ MM.1S and induced a significant reduction in growth and angiogenesis in tumors formed by Tet-shJUNB/ MM.1S versus control cells, as evidenced by Ki-67 and anti-CD31 staining. In summary, our findings demonstrate for the first time a role for JunB in MM bone marrow angiogenesis, thereby strongly supporting that this TF is a promising new therapeutic target in MM.