C3a and C5a Facilitates the Metastasis of Myeloma Cells By Activating Nrf2

Background and objective: Multiple myeloma (MM) is still an incurable hematological malignancy, with even poorer prognosis in MM patients with distant invasion. Accumulative evidence has indicated the role of complements in tumor activation and progression. During complement activation, soluble multifunctional pro-inflammatory peptide fragments C3a and C5a are released from C3 and C5, respectively, which exert diverse biological activities in the immune response. Nuclear factor erythroid derived 2-like 2(Nrf2) plays a vital role in the main stages of tumorigenesis and tumor progression.Previous studies have confirmed that the high expression of Nrf2 in solid tumors is associated with tumor cell survival, growth, angiogenesis, invasion and metastasis, blocking Nrf2 can inhibit tumor growth and there might be association between the activation of complement genes and the increase of C3a expression and Nrf2 activation. Heme oxygenase (HO) is an enzyme involved in antioxidant stress, which is also a target gene of Nrf2. Studies have confirmed that HO-1 is involved in regulating the migration of acute myelocytic leukemia cells in the peripheral blood and bone marrow. Upregulating the expression of HO-1 can increase the homing of leukemia stem cells. Other studies have found that HO-1 negatively regulates activation of the complement system. Hence, in this study, we aim to investigate whether human MM cell lines and primary MM cells from patient samples express functional C3 and C5 cleavage fragment receptors and whether activation of the complement system and release of C3a and C5a anaphylatoxin could affect the migration, invasion, and adhesion of MM cells by regulating Nrf2.

Methods: ELISA and RT-qPCR was used to detect the expression of C3aR/C5aR and Nrf2 in clinical samples. Transwell migration and invasion assay, adhesion of MM cells to fibronectin assay were used to exam the metastasis ability of MM cells. Western blot analysis and immunofluorescence staining were used to test the expression of metastasis related molecules. 4-week-old NOD/SCID mice were housed under specific pathogen-free conditions and allowed to acclimate for two weeks before experiments . The mice were randomly divided into six groups. Human Nrf2 knockdown U266 cell lines and their control counterparts were constructed for in vivo transplantation . Before transplantation, non-transfected U266 cells were treated ex vivo for 6h with C3a (1 μg/ml), C5a (140 ng/ml) or normal saline only as a control. All treated cells were subsequently washed and transplanted into SCID-beige inbred mice , which were initially irradiated with 250 cGy 24 h before transplantation. At 24 h post transplantation, BMs, livers and lungs were collected, followed by evaluation of the presence of metastatic cancer cells (murine–human chimeras).

Results: As a result, the plasma concentrations of C3a and C5a was higher in MM patients than those of healthy donors. And similar findings were also observed in the mRNA expression of Nrf2. Consequently, the expression of C3aR1, C5aR1 and C5L2 on sorted myeloma cells from MM patients was higher than sorted plasma cells from healthy donors, which was not associated with the disease stage. Moreover, Pearman correlation analysis showed that the mRNA levels of both C3aR1 and C5aR1 were significantly associated with the mRNA expression level of Nrf2. C3a and C5a have been confirmed to increase the migration, invasion and adhesion of MM cell lines by activating the MEK / ERK pathway and increasing the nuclear transfer of Nrf2 in vitro. Furthermore, the MM cell line U266 with Nrf2 down-regulation was incubated with C3a and C5a, followed by injection into the tail vein of NOD-SCID mice. We found that Nrf2 down-regulation attenuated the migration of anaphylatoxin C3a and C5a to MM tumor cells in bone marrow, liver and lung in vivo.

Conclusion: Our results suggested that activation of the complement cascade in MM patients may facilitate the migration, invasion and adhesion of MM cells. In addition, this enhanced motility may contribute to the systemic dissemination of MM cells. Furthermore, this kind of tumor cell spread in MM is, at least partially, regulated by Nrf2. Therefore, complement suppression or Nrf2 down-regulation offer a novel therapeutic opportunity for MM patients.