Myeloma Cells Inhibit the Non-Canonical Wnt Co-Receptor Ror2 in Human Mesenchymal/Osteoprogenitor Cells: Effect of Wnt5a/Ror2 Pathway Activation On MM-Induced Impairment of the Osteogenic Differentiation Process.

Abstract 741

Osteogenic differentiation of human mesenchymal stem cells (hMSC) is typically impaired in multiple myeloma (MM) patients leading to osteoblast deficiency. Canonical Wnt signal pathway is critical in the regulation of bone formation process and its activation in osteoblastic cells improves bone mass reducing the development of osteolytic lesions in MM mouse model. Together to canonical Wnt signaling, a non-canonical Wnt pathway, independently to β-catenin activation, has been identified. Non-canonical Wnt signaling is transduced through FZD receptor and Ror2 co-receptor to several cascades either disheveled pathways involving Rho family small GTPase and JNK or Ca⁺⁺ dependent pathways involving the nuclear factor of activated T cells (NFAT). Interestingly, recent evidences suggest that non-canonical Wnt pathway activation by Wnt5a or Wnt4, rather than canonical one by Wnt3a, stimulates the osteogenic properties of hMSC through Ror2 activation. The effect of MM cells on non-canonical Wnt pathway as well as the role of the activation of this pathway in hMSC on the osteogenic differentiation impairment induced by MM cells are not known and have been investigated in the present study.

First we checked the expression of non-canonical Wnt related molecules by bone marrow (BM) hMSC and osteoprogenitor cells (PreOB) at the first passage by oligonucleotide arrays. We found that both cells expressed the activator of non-canonical Wnt pathways Wnt5a but lack of express the main activators of canonical Wnt signaling as Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptor FZD2 and FZD5 was also detected in both cells as well as of Ror2. Interestingly we found that osteogenic differentiation of hMSC towards preOB significantly increased Ror2 but not Wnt5a expression. Secondly, we performed a series of co-culture between PreOB and MM cells using either the human myeloma cell lines (JJN3, XG-1, XG-6, KMS12, KMS27) or purified CD138⁺ cells obtained from MM patients finding that MM cells inhibit Ror2 protein expression by PreOB and consistently the activity of NFATc1 at nuclear level. Following activation of non-canonical Wnt signaling pathway either by Wnt5a treatment or by the induction of both Wnt5a and Ror2 overexpression by lentivirus vectors have been performed in primary hMSC obtained from MM patients. The efficiency of Wnt5a and Ror2 trasduction was checked by GFP expression using flow cytometry whereas the efficacy was evaluated by the level of Wnt5a and Ror2 mRNA and protein expression as well as by the intracytoplasmatic increase of Ca⁺⁺ influx, phospho-PKC expression and NFATc1 activity. We found that Wnt5a treatment as well as Wnt5a or Ror2 overexpression significantly increased osteogenic differentiation and the expression of alkaline phosphatase in hMSC. Consistently, in the co-culture system with MM cells, Wnt5a and Ror2 overexpression by hMSC blunted the inhibitory effect of MM cells on alkaline phosphatase expression and osteogenic differentiation. Finally, these observations were further confirmed showing that Wnt5a or Ror2 silencing in PreOB by siRNA or shRNA trasfection, respectively inhibited the expression of osteogenic markers alkaline phosphatase, osteocalcin and collagen I.

In conclusion our data indicate that activation of non-canonical Wnt5a/Ror2/Ca⁺⁺ signal pathway in hMSC increases osteogenic differentiation and counterbalance the inhibitory effect of MM cell suggesting that this pathway could represent a potential target in MM microenvironment.