Annexin II Interactions with the Annexin II Receptor Enhance Multiple Myeloma Cell Adhesion and Growth in the Bone Marrow Microenvironment,

Multiple myeloma (MM) is an incurable B-cell malignancy that develops in the bone marrow. The marrow microenvironment plays a critical role in supporting homing, lodging, and growth of MM cells by activating signaling pathways in both MM and bone marrow stromal cells (BMSC). We previously showed that annexin II (AXII) is involved in prostate cancer cell lodgment to the bone marrow via the annexin II receptor (AXIIR) expressed on prostate cancer cells. We hypothesized that MM cells use a similar mechanism to lodge and grow in the bone marrow. In support of this hypothesis, we found that MM cell lines and primary MM cells from 8 MM patients express the AXIIR protein, and that MM cells adhered significantly better to BMSC from AXII^+/+ mice than from AXII^−/− mice. Further, knockdown of AXIIR by siRNA in MM1.S and ANBL-6 MM cells decreased AXII binding and decreased adherence of MM cells to human stromal cells and BMSC from AXII^+/+ mice. Furthermore, addition of an anti-AXII antibody to MM1.S cells, did not effect MM cell growth demonstrating that AXII expressed by MM cells does not support MM cell growth. Importantly, soluble AXII was released by osteoclasts into their conditioned media which stimulated the growth of MM cells via ERK1/2 and AKT phosphorylation. In the further study, we further characterized the role of AXIIR in MM-BMSC interactions.

AXIIR expression in MM cells was determined by RT-PCR, Western blotting, and immunocytochemistry. Adhesion and growth assays were performed between MM cells and BMSC or AXII to determine the contribution of the AXII/AXIIR axis in supporting adhesion and growth of MM cells. In addition, MM cells or CD138+ cells from MM patients were treated with AXII to determine AXII-dependent MM cell growth. Further, adhesion and growth assays were performed on MM cells expressing either siAXIIR or shAXIIR. Phosphorylation assays were performed to determine the pathways stimulated by AXII in MM cells. Since OCL secrete large amount of AXII, MM cell growth assays were performed with OCL-CM from AXII^+/+ and AXII^−/− mice in the presence of an AXII antibody.

We now report that in addition to MM1.S and ANBL-6 cells, other MM cell lines, including U266, H929, and OPM2 also express AXIIR, and that AXII stimulated the growth of RPMI8226, ANBL-6 and U266 in addition to MM1.S cells. Finally, an AXIIR antibody prevented adhesion of MM1.S cells to AXII, and that AXII upregulated the adhesion molecule, RhoA in MM cells. Additionally, AXII did not stimulate the proliferation of MM1.S^shAXIIR cells compared to MM1.S^shControl or untreated MM cells, demonstrating that AXII specifically acts through its receptor, AXIIR on MM cells to promote proliferation. More importantly, AXII stimulated the growth of CD138+ cells obtained from MM patients.

Based on our results, we conclude that the interaction between AXII and AXIIR in the bone marrow microenvironment supports adhesion via RhoA and growth of MM cells by stimulating the Erk1/2 and Akt pathways, AXII produced by MM cells does not act in an autocrine manner on MM cell growth. Thus, AXII and AXIIR are key players in MM and targeting the AXII/AXIIR axis may be a novel therapeutic approach for MM.

Roodman:Amgen: Consultancy; Millennium: Consultancy.