Decreased FPN1 in Myeloma Promotes Malignant Cell Growth and Osteoclast Differentiation

Background: Many patients with multiple myeloma (MM) suffer from anemia due to systematic alteration in iron metabolism, as is commonly seen in many cancers. Decreased iron availability resulting in impaired erythropoiesis is the main cause of myeloma-associated anemia. Bone lytic lesion is a hallmark of MM. Recent study have shown that the activation of osteoclast demands high iron uptake to facilitate mitochondrial biogenesis. Export of intracelluar iron is regulated by Ferroportin (FPN1), but its role in MM remains unclear. Here we provided evidence that FPN1 is critical regulator of progression in MM.

Materials and methods: Gene expression profiling (GEP) analysis was applied to identify the iron signature and the association of FPN1 with MM outcome. MM cells with enhanced expression of FPN1 by lentiviral transduction were tested in clonogenic assays in vitro and xenograft model in vivo. The intracellular labile iron pool was detected by the calcein-AM assay. The C57BL/KaLwRij model was employed to test the role of FPN1 and iron in vivoby inducible expression of FPN1 in 5TGM1 cells and administration of iron-dextran. Quantitive real-time PCR (qPCR) was used to detect gene expression in MM cells and macrophage-osteoclast cells. The MM cell induced formation of osteoclast (OCL) in the macrophage cell line RAW264.7 or primary bone marrow macrophages (BMM) was detected by TRAP staining. BMM with a dominant negative mutation of FPN1 were used to elucidate the downstream signaling of FPN1.

Result: Patient GEP data showed that MM cells had an aberrant iron gene expression signature and MM cells were susceptible to iron chelation. Expression of FPN1 was significantly lower in plasma cells (PCs) from MM patients than in PCs from MGUS or healthy subjects (p < 0.05). In 351 newly diagnosed MM patients, expression of FPN1 was also significantly lower in the high-risk compared with the low-risk group (p < 0.001). Downregulation of FPN1 was linked to decreased survival in the Total Therapy 2 (TT2) (p < 0.001) and APEX (p < 0.001) clinical trials. Restoring expression of FPN1 suppressed MM cell growth both in vitro and in vivo, presumably by down-regulating IL-6-STAT3 signaling and the intracellular labile iron pool. FPN1 was rapidly down-regulated in MM cell- and RANKL- induced osteoclast differentiation, which happened earlier than other key regulators such as PGC1β, TFRC and NFATc, indicating its driving role in the osteoclastogenesis. Indeed, overexpression of FPN1 significantly suppressed myeloma cell- and RANKL-induced osteoclast differentiation. Mechanistically, loss of function mutation of FPN activated TFRC, NFκB and JNK signaling in BMM derived from mice, which facilitated osteoclast differentiation.

Conclusion: We demonstrated that the FPN1 axis plays a critical role in MM progression. At the mRNA level, reduced expression of FPN1 in MM cells correlated positively with cell growth while inhibition of FPN1 in OCL precursor enhanced OCL differentiation. Loss of FPN1 in both MM cells and macrophage-osteoclast promoted MM progression. Mechanistic studies revealed that FPN1 suppressed IL-6-STAT3 signaling in MM cells and functional inhibition of FPN1 in OCL precursor upregulated TFRC and activated JNK and NFκB signaling. Our data provide evidence that the expression of FPN1 can serve as a prognostic marker and may be a valuable therapeutic target in MM.