Vascular endothelial growth factor (VEGF) induces proliferation of MM cells and induces interleukin-6 (IL-6) secretion in a paracrine loop involving MM cells and bone marrow stromal cells. In turn, IL-6 triggers multiple myeloma (MM) cell proliferation and also protects against apoptosis by upregulating Myeloid-cell-leukemia 1 (Mcl-1), a critical survival protein in MM cells. The goal of our study was to investigate the role of Mcl-1 in VEGF induced-proliferation and protection against apoptosis. Using two murine embryonic fibroblast cell lines as a model (a Mcl-1 deleted cell line and its wild type: Mcl-1Δ/null and Mcl-1wt/wt MEFs, respectively), we here demonstrate that deletion of Mcl-1 reduces fetal bovine serum (FBS), VEGF, and IL-6 induced-proliferation. In addition, we demonstrate that the percentage of cells in S phase is lower in Mcl-1Δ/null compared to Mcl-1wt/wt MEFs (21% (+/−1) versus 30% (+/− 3), respectively). Taken together, these results demonstrate that Mcl-1 is required to mediate VEGF, Il-6 and FBS-induced-proliferation and cell cycle progression.

To highlight the key anti-apoptotic role of Mcl-1 in MM cells, humans MM1s cells were transfected with Mcl-1 siRNA. Specific inhibition of Mcl-1 was associated with decreased proliferation (42% and 61% decreases at 24 and 48 h, respectively) and induction of apoptosis (subG1 peak: 22% and 41% in Mcl-1 siRNA transfected cells versus 15% and 15 % in non-transfected cells at 24 and 48 h, respectively), confirming that Mcl-1 is critical for both proliferation and protection against apoptosis in MM cells. In 3 human MM cell lines (MM1s, U266 and MM1R) and MM patient cells we next showed that Mcl-1 protein expression, but not other bcl-2 family members, is upregulated by VEGF in a time and dose manner; and conversely that the pan-VEGF inhibitor GW654652, blocks VEGF induced-upregulation of Mcl-1. Furthermore using flow cytometry with a double staining (CD38-FITC and Apo 2.7-PE), we demonstrate that VEGF protects MM patient cells from FBS-starvation-induced-apoptosis: the percentage of apoptotic MM patient cells (CD38++ and Apo 2.7+) in non starved medium (RPMI 1640 supplemented with 10% FBS) was 15% versus 93% in starved medium (RPMI 1640 supplemented with FBS 2%), and 48% in starved medium supplemented with 25ng/ml VEGF. In conclusion, our study demonstrates that VEGF protects MM cells against apoptosis, and that VEGF-induced MM cell proliferation and survival is mediated via Mcl-1. these studies provide the preclinical framework for novel therapeutics targeting both Mcl-1 and/or VEGF to improve patient outcome in MM.

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