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Bone resorption facilitates osteoblastic bone metastatic colonization by cooperation of insulin-like growth factor and hypoxia.

Kuchimaru T, Hoshino T, Aikawa T, Yasuda H, Kobayashi T, Kadonosono T, Kizaka-Kondoh S - Cancer Sci. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology, Tokyo, Japan.

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Insulin-like growth factor (IGF)/IGF receptor (IGFR) signaling and hypoxia cooperatively stimulate progression of murine osteosarcoma LM8 cells. (a) Proliferation assay of LM8/luc with IGF-1 (100 ng/mL), transforming growth factor-β1 (TGF-β1; 10 ng/mL), and receptor activator of factor-κB ligand (RANKL; 100 ng/mL) in 21% or 1% O2. (b) Colony formation assay of LM8/luc with IGF-1 (100 ng/mL) for 14 days in 21% or 1% O2. (c) Quantitative RT-PCR (qRT-PCR) analysis of Igf1r expression in LM8/luc cultured for 16 h in 21% and 1% O2. (d) Protein expression and phosphorylation levels of IGF1R. The cells were precultured for 16 h in 21% or 1% O2 then treated with IGF-1 (100 ng/mL) for 1 h. (e) Effect of IGF-1 on hypoxia-inducible factor-1α (HIF-1α) protein level. LM8 cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. (f) Effect of IGF-1 on HIF transcriptional activity. LM8/HRE-luc cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. *P < 0.05. (g) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF. LM8/luc cells were treated with IGF-1 (IF) (100 ng/mL) for 6 h in 21% and 1% O2. *P < 0.05. (h) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF in bone metastasis at 14 days after LM8 injection. *P < 0.05.
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fig04: Insulin-like growth factor (IGF)/IGF receptor (IGFR) signaling and hypoxia cooperatively stimulate progression of murine osteosarcoma LM8 cells. (a) Proliferation assay of LM8/luc with IGF-1 (100 ng/mL), transforming growth factor-β1 (TGF-β1; 10 ng/mL), and receptor activator of factor-κB ligand (RANKL; 100 ng/mL) in 21% or 1% O2. (b) Colony formation assay of LM8/luc with IGF-1 (100 ng/mL) for 14 days in 21% or 1% O2. (c) Quantitative RT-PCR (qRT-PCR) analysis of Igf1r expression in LM8/luc cultured for 16 h in 21% and 1% O2. (d) Protein expression and phosphorylation levels of IGF1R. The cells were precultured for 16 h in 21% or 1% O2 then treated with IGF-1 (100 ng/mL) for 1 h. (e) Effect of IGF-1 on hypoxia-inducible factor-1α (HIF-1α) protein level. LM8 cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. (f) Effect of IGF-1 on HIF transcriptional activity. LM8/HRE-luc cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. *P < 0.05. (g) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF. LM8/luc cells were treated with IGF-1 (IF) (100 ng/mL) for 6 h in 21% and 1% O2. *P < 0.05. (h) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF in bone metastasis at 14 days after LM8 injection. *P < 0.05.

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).


Bone resorption facilitates osteoblastic bone metastatic colonization by cooperation of insulin-like growth factor and hypoxia.

Kuchimaru T, Hoshino T, Aikawa T, Yasuda H, Kobayashi T, Kadonosono T, Kizaka-Kondoh S - Cancer Sci. (2014)

Insulin-like growth factor (IGF)/IGF receptor (IGFR) signaling and hypoxia cooperatively stimulate progression of murine osteosarcoma LM8 cells. (a) Proliferation assay of LM8/luc with IGF-1 (100 ng/mL), transforming growth factor-β1 (TGF-β1; 10 ng/mL), and receptor activator of factor-κB ligand (RANKL; 100 ng/mL) in 21% or 1% O2. (b) Colony formation assay of LM8/luc with IGF-1 (100 ng/mL) for 14 days in 21% or 1% O2. (c) Quantitative RT-PCR (qRT-PCR) analysis of Igf1r expression in LM8/luc cultured for 16 h in 21% and 1% O2. (d) Protein expression and phosphorylation levels of IGF1R. The cells were precultured for 16 h in 21% or 1% O2 then treated with IGF-1 (100 ng/mL) for 1 h. (e) Effect of IGF-1 on hypoxia-inducible factor-1α (HIF-1α) protein level. LM8 cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. (f) Effect of IGF-1 on HIF transcriptional activity. LM8/HRE-luc cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. *P < 0.05. (g) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF. LM8/luc cells were treated with IGF-1 (IF) (100 ng/mL) for 6 h in 21% and 1% O2. *P < 0.05. (h) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF in bone metastasis at 14 days after LM8 injection. *P < 0.05.
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fig04: Insulin-like growth factor (IGF)/IGF receptor (IGFR) signaling and hypoxia cooperatively stimulate progression of murine osteosarcoma LM8 cells. (a) Proliferation assay of LM8/luc with IGF-1 (100 ng/mL), transforming growth factor-β1 (TGF-β1; 10 ng/mL), and receptor activator of factor-κB ligand (RANKL; 100 ng/mL) in 21% or 1% O2. (b) Colony formation assay of LM8/luc with IGF-1 (100 ng/mL) for 14 days in 21% or 1% O2. (c) Quantitative RT-PCR (qRT-PCR) analysis of Igf1r expression in LM8/luc cultured for 16 h in 21% and 1% O2. (d) Protein expression and phosphorylation levels of IGF1R. The cells were precultured for 16 h in 21% or 1% O2 then treated with IGF-1 (100 ng/mL) for 1 h. (e) Effect of IGF-1 on hypoxia-inducible factor-1α (HIF-1α) protein level. LM8 cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. (f) Effect of IGF-1 on HIF transcriptional activity. LM8/HRE-luc cells were treated with IGF-1 (100 ng/mL) for 12 h in 21% and 1% O2. *P < 0.05. (g) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF. LM8/luc cells were treated with IGF-1 (IF) (100 ng/mL) for 6 h in 21% and 1% O2. *P < 0.05. (h) qRT-PCR analysis of downstream genes of IGF/IGFR signaling and HIF in bone metastasis at 14 days after LM8 injection. *P < 0.05.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus