IL-6, IGF-1 and IL-21 Act in Synergy with HGF in Myeloma Cells by Inducing the HGF-Receptor c-Met.

HGF is known to be a prognostic factor in multiple myeloma, however the proliferative effect of HGF alone has been moderate. We investigated the effects of the myeloma growth factors IL-6, IGF-1 and IL-21 in combination with HGF in the myeloma cell lines IH-1, INA-6 and OH-2. In proliferation experiments the cells were grown in serum free media with 3 different HGF-concentrations together with various concentrations of IL-6, IGF-1 or IL-21. HGF alone had low effect. However, 200 ng/ml HGF in combination with IL-6 doubled the effect of IL-6 and tripled the effect of IGF-1 in INA-6. Similar results were obtained in OH-2 combining IL-6, IGF-1 and IL-21 with HGF and in IH-1 combining IL-6 with HGF. A combination of IL-6 and IGF-1 in INA-6 gave no synergistic effects, so the synergism seemed to be a specific effect for HGF in combination with the other cytokines.The myeloma cell line ANBL-6 harbours an autocrine growth promoting HGF-loop. We inhibited this autocrine HGF loop with a specific c-Met receptor tyrosine kinase inhibitor PHA-665752 in the presence of IL-6 and IGF-1. IL-6 and IGF-1 potentiated the decrease in proliferation caused by PHA-665752. These results demonstrate synergistic effects between the three cytokines (IL6, IGF-1, IL-21) and HGF. We also combined PHA-665752 and IL-6 in INA-6 cells, and 200 nM of the c-Met inhibitor halved the effect of IL-6. We have not seen any unspecific effects of PHA-665752 at 200 nM. This result raises the very interesting possibility that some of the effects of IL-6 can be due to c-Met signalling. At the protein level the cell lines responding to IL-6, IGF-1 or IL-21 also increased the level of c-Met expression in response to the individual cytokines. In OH-2 there was also an increase in the HGF transcript after IL-6, IGF-1 and IL-21 stimulation, as investigated by RT-PCR, indicating another mechanism for synergy between the HGF-c-Met pathway and the other cytokines in this cell line. In conclusion our results demonstrates the induction of c-Met by IL-6, IGF-1 and IL-21, and proliferative synergy between these cytokines and HGF in myeloma cell lines. Inhibition of c-Met with a specific tyrosine kinase inhibitor decreased the proliferative effect of IL-6. Together these results indicate that inhibition of c-Met signalling would target several pathways and could therefore be a promising treatment strategy in multiple myeloma.