Interleukin-6 Drives Multiple Myeloma Progression through Upregulating of CD147/Emmprin Expression and Its Sialylation

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. During malignant transformation, many glycoproteins undergo a wide range of glycosylation alterations, especially increased sialylation, have been associated with malignant transformation and metastasis. However, it is still unclear the mechanisms of regulating CD147 and its glycosylation in MM. In this study, we found that CD147 and its sialylation can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCI-H929 were stimulated with 20ng/mL of IL-6, we found that the expression of CD147 on MM cell membrane was significantly upregulated, as determined by both flow cytometric analysis and Western blotting. Importantly, the results of Western blotting also clearly show that CD147 is a glycoprotein with higher molecular weight bands than core CD147. By using PNGase F and sialidase to digest the protein samples, we further demonstrated that CD147 is typical N-linked glycoprotein with high level of sialylation. Moreover, we found that the expression of α-2,3 and α-2,6 sialic acid on the cell surface were significantly upregulated by IL-6 analyzed by flow cytometry using MAA and SNA lectin binding assay. Next, we investigated the signaling pathway involved in the IL-6 mediated upregulation of CD147 and its sialylation in MM cells. By using Real-Time PCR, we confirmed that CD147 was transcriptionally upregulated by IL-6. Simultaneously, several sialyltransferases like ST3GAL3, ST3GAL6 and ST6GAL1 were also transcriptionally upregulated by IL-6. By using Western blotting, we further confirmed that IL-6 can activate the JAK/STAT3 signaling pathway by inducing the phosphorylation of STAT3 at Tyrosine 705 in MM cells. Further bioinformatics and ChIP analysis demonstrated that the existing of transcription factor STAT3 binding sites in the promoter of CD147 and sialyltransferase (ST3GAL3, ST3GAL6 and ST6GAL1) genes.To further validate the role of JAK/STAT3 in regulating CD147 and its sialylation, we used a specific STAT3 inhibitor Cryptotanshinone to treat the MM cells and found that the expression of CD147 and its sialylation in MM cells was accordingly decreased. Furthermore, we validated the roles of high level of CD147 and its sialylation in MM biology by knocking-down CD147 or inhibiting its sialylation using 3Fax-Peracetyl Neu5Ac, which is a specific sialyltransferase inhibitor. We found that both siRNA mediated knock-down of CD147 and sialylation inhibition did not affect the proliferation in RPMI8226 and MM1.S cells, measured by EDU incorporation assay using flow cytometry. However, knock-down of CD147 and sialylation inhibition were found to significantly reduce the adhesion to BMSCs and HUVECs, and the migration to BMSC-conditioned media in vitro. Finally, to further validate the influence of IL-6 and the related sialyltransferase genes in MM patients, we assessed the effect of expression on survival of patients using GEP data from the CoMMpass trial (n=664), and noted a significantly reduced PFS (progression-free survival) for patients with high levels of expression of IL-6 and ST3GAL6 (P <0.0001). Taken together, our data provide evidence that IL-6 can up-regulate CD147 expression and its sialylation in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM.