Myeloma Cell-Derived Runx2 Promotes Myeloma Progression and Bone-Homing

Background. Multiple myeloma (MM), an incurable plasma-cell malignancy, preferentially grows in bone marrow and frequently metastasizes to new bone sites. The molecular network responsible for progression and bone-metastasis of MM remains largely unknown. Recent work from our laboratory suggests that the Runx2 transcription factor, well-known for its essential role in osteoblast differentiation and bone development, is also required for coordinating the network of molecular signals in MM cells that drive MM progression and bone metastasis. Substantial evidence exists about expression of Runx2 in various cancer cells, including myeloma cells. Moreover, Runx2 is shown to promote tumor-to-bone metastasis, osteolytic bone disease, and tumor progression in solid tumors. However, the role of Runx2 in MM progression and bone metastasis remains unknown. In the present study, we determined the mechanism(s) governing the action of Runx2 in MM cell, especially during tumor growth and bone-homing.

Methods. Molecular, biochemical, and in vivo approaches were used to assess the role of Runx2 in growth and homing of MM to bone tissue. Initially, bone marrow biopsies from 35 MM patients and 14 healthy bone marrow donors were examined for the expression of Runx2. For a mechanistic understanding of the role of Runx2 in MM, we over-expressed and knocked-down Runx2 expression in both murine 5TGM1 and human MM1S myeloma cells. The following methods and strategies were used to examine the Runx2 over-expression (Runx2 k/in), Runx2 knockdown (Runx2 k/d) and control MM cell lines in both ex vivo and in vivo models. (1) Assessment of tumor growth and homing in the 5TGM1 syngenic model of murine myeloma (for 5TGM1 cells) and SCID mice (for MM1S cells). (2) RNA-Sequencing, Real-time PCR and Western blot analyses to determine downstream genes/molecules regulated by Runx2 in MM cells. (3) Cytokine array and ELISA of the conditioned media to identify Runx2 regulated soluble factors secreted by MM cells.

Results. Immunostaining of bone marrow biopsies indicated that Runx2 expression was significantly increased in both cytoplasm and nuclei of MM cells compared to normal plasma cells (p< 0.001). In vivo studies using three different animal models (i.v., subcutaneous and intra-tibial injection) demonstrated that the overexpression of Runx2 in MM cells promoted tumor growth and homing to bone whereas knockdown of Runx2 inhibited tumor progression and bone-homing. Interestingly, Runx2 over-expression in MM cells leads to the increased expression of active β-catenin and phospho-AKT, indicating that Runx2 promotes tumor growth through Wnt/β-catenin and AKT signaling pathway. This regulatory mechanism was further confirmed in 5TGM1 Runx2 k/d cells, in which both the expression of active β-catenin and phospho-AKT was significantly decreased. RNA-sequencing and real-time PCR revealed that, over-expression of Runx2 induced the expression of multiple marker genes of osteoblast (Osteocalcin, osteopontin), the osteocytes (DMP1) as well as the osteoclast (cathepsin K, RANK). Expression of these marker genes was significantly down-regulated in Runx2 k/d 5TGM1 cells. These results imply that Runx2 promotes tumor bone-homing by providing MM cells with the ability to “mimic” bone marrow resident cells and therefore escape immune surveillance in the host bone marrow. Finally, we provided evidence that MM cell-derived Runx2 promote secretion of multiple soluble factors linked with increased metastasis such as DKK1, MMP9, SDF-1, and osteopontin by MM cells.

Conclusions. MM cell-derived Runx2 is a central regulator of MM progression. Runx2 expression promotes MM growth and bone-metastasis via upregulation of (i) Wnt/β-catenin and AKT signaling, (ii) the expression of multiple osteogenic genes, and (iii) the secretion of metastatic soluble factors, in MM cells. This activation could activate a pre-metastatic niche in bone thereby providing MM cells with the ability to avoid immune surveillance in the bone marrow, and supporting survival and growth of tumor cells that arrive in bone. These discoveries provide insights into MM pathology, help to predict the prognosis of MM patients, and identify novel therapeutic targets for MM.