Bone resorption facilitates osteoblastic bone metastatic colonization by cooperation of insulin-like growth factor and hypoxia.
Bottom Line: We found that treatment with receptor activator of factor-κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL-induced bone resorption facilitates growth of cancer cells colonized in the bone.We show that insulin-like growth factor-1 released from the bone during bone resorption and hypoxia-inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow.These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.
Affiliation: Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology, Tokyo, Japan.Show MeSH
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Mentions: Treatment with RANKL induced significant bone resorption (Fig.2b) that triggers the release of growth factors stored in the bone. Therefore, the contribution of growth factors in cooperation with HIF activity to LM8 metastatic progression was examined. As TGF-β and IGF-1 are the major growth factors stored in the bone matrix, we first examined their effects on LM8 proliferation. Transforming growth factor-β as well as RANKL did not show any proliferative effects on LM8/luc under normoxia or hypoxia (Fig.4a), which was explained by the results of the qRT-PCR analysis indicating that LM8 did not express the TGF-β2 receptor (Fig. S5a). We further confirmed that the TGF-β/Smad pathway was not activated in LM8 after TGF-β treatment (Fig. S5b, Data S1). Thus, it was concluded that TGF-β did not have a direct effect on LM8 for RANKL-induced promotion of LM8 metastasis. In contrast, IGF/IGFR signaling seemed important because IGF-1 was able to promote proliferation of LM8 cells under both normoxia and hypoxia (Fig.4a). Furthermore, IGF-1 promoted anchorage-independent growth of LM8 cells in hypoxia (Fig.4b). Because the effect of IGF-1 on LM8 proliferation was more significant under hypoxia than normoxia, the activity of IGF/IGFR signals in LM8 cells was further examined in hypoxia. Remarkably, the Igf1r expression and IGF1R phosphorylation after IGF stimulation were significantly increased under hypoxic conditions (Fig.4c,d). We then assessed the involvement of IGF-1 in HIF activation in LM8 cells and found that IGF-1 treatment significantly enhanced the protein stability of HIF-1α and transcriptional activity of HIF in LM8 under hypoxic conditions (Fig.4e,f). Furthermore, we examined the expression level of downstream genes of the IGF/IGFR signal and HIF (Fig.4g). Under hypoxic conditions, Igf1 expression significantly increased (Fig.4g), suggesting that autocrine IGF-1 made the proliferation effect of exogenous IGF-1 on LM8 less significant in hypoxia (Fig.4a).
Affiliation: Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology, Tokyo, Japan.