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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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Hypoxia-inducible factor (HIF) activity of murine osteosarcoma LM8 cells is increased in the bone microenvironment. (a) HIF-1α protein levels in LM8 in 1% O2. (b) HIF-1α protein levels in LM8 under various O2 concentrations for 16 h. (c) Luciferase activity of LM8/HRE-luc in 1% O2. Relative luciferase activities to the one at 0 h are shown. (d) Luciferase activity of LM8/HRE-luc under various O2 concentrations for 16 h. Relative luciferase activity to the one in 21% O2 are indicated. (e) HIF activity in bone metastasis sites. Bioluminescence (BL) intensity in LM8/HRE-luc or LM8/luc metastases was measured at the indicated times after transplantation. To calculate relative BL intensities, BL intensities at indicated times were divided by those at day 4 (3.17 × 103 photons/s/ROI for LM8/HRE-luc and 3.43 × 103 photons/s/ROI for LM8/luc); n = 4 for LM8/HRE-luc, n = 6 for LM8/luc. *P < 0.01. (f) Inhibition of HIF activity in LM8 cells using acriflavine. HRE-Fluc activity was assessed with indicated concentrations of acriflavine (ACF). *P < 0.05. (g) Antitumor effect of HIF inhibitor. Luciferase activity of LM8/luc bone metastasis was measured at indicated times after transplantation. Relative luciferase activities to the ones on day 2 are indicated; n = 8 for each group. *P < 0.01.
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fig03: Hypoxia-inducible factor (HIF) activity of murine osteosarcoma LM8 cells is increased in the bone microenvironment. (a) HIF-1α protein levels in LM8 in 1% O2. (b) HIF-1α protein levels in LM8 under various O2 concentrations for 16 h. (c) Luciferase activity of LM8/HRE-luc in 1% O2. Relative luciferase activities to the one at 0 h are shown. (d) Luciferase activity of LM8/HRE-luc under various O2 concentrations for 16 h. Relative luciferase activity to the one in 21% O2 are indicated. (e) HIF activity in bone metastasis sites. Bioluminescence (BL) intensity in LM8/HRE-luc or LM8/luc metastases was measured at the indicated times after transplantation. To calculate relative BL intensities, BL intensities at indicated times were divided by those at day 4 (3.17 × 103 photons/s/ROI for LM8/HRE-luc and 3.43 × 103 photons/s/ROI for LM8/luc); n = 4 for LM8/HRE-luc, n = 6 for LM8/luc. *P < 0.01. (f) Inhibition of HIF activity in LM8 cells using acriflavine. HRE-Fluc activity was assessed with indicated concentrations of acriflavine (ACF). *P < 0.05. (g) Antitumor effect of HIF inhibitor. Luciferase activity of LM8/luc bone metastasis was measured at indicated times after transplantation. Relative luciferase activities to the ones on day 2 are indicated; n = 8 for each group. *P < 0.01.

Mentions: The pO2 in bone marrow has been estimated at 23–40 mmHg (3–5% O2),19 which is relatively lower than other organs and tissues. To understand the status of LM8 cells that have migrated to the bone marrow, we examined their hypoxic response in vitro and in vivo. The most abundant HIF, HIF-1, is a central regulator of the hypoxic response in many cell types15 and reported as a key factor in promoting homing, colonization, and progression of bone metastasis.28,29 In LM8 cells, the protein level of HIF-1α, the α subunit of HIF-1, increased as hypoxic treatment was prolonged (Fig.3a) and decreased as the O2 concentration increased (Fig.3b). The HIF transcriptional activity was monitored by using LM8/HRE-luc cells, which express firefly luciferase in a HIF-dependent manner. The activity of HIF in LM8/HRE-luc cells varied in response to hypoxic treatments (Fig.3c,d) in good correlation with HIF-1α expression (Fig.3a,b). In vivo BL signaling from LM8/luc, corresponding to tumor burden, and LM8/HRE-luc, corresponding to HIF activity, was monitored after i.c. injection of LM8 cells (Fig.3e). In s.c. tumors of LM8, HIF activity increased much more slowly than tumor burden (Fig. S4), suggesting that HIF activity in s.c. LM8 tumors reflects a gradual increase in hypoxic regions as tumors grow. However, in the bone metastatic sites, the HIF activity and tumor burden showed a parallel increase during the first week after LM8 injection (Fig.3e), indicating that HIF in LM8 was activated in the hypoxic microenvironment immediately after migration of LM8 to the bone marrow. Furthermore, HIF activity drastically increased in bone metastatic sites during the second week after LM8 injection (Fig.3e) and inhibition of HIF transcriptional activity by acriflavine24 significantly suppressed growth of LM8 bone metastasis (Fig.3f,g). These results support the idea that HIF is activated in LM8 homing to the hypoxic bone marrow and plays an important role in LM8 colonization and the progression of bone metastasis.


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)

Hypoxia-inducible factor (HIF) activity of murine osteosarcoma LM8 cells is increased in the bone microenvironment. (a) HIF-1α protein levels in LM8 in 1% O2. (b) HIF-1α protein levels in LM8 under various O2 concentrations for 16 h. (c) Luciferase activity of LM8/HRE-luc in 1% O2. Relative luciferase activities to the one at 0 h are shown. (d) Luciferase activity of LM8/HRE-luc under various O2 concentrations for 16 h. Relative luciferase activity to the one in 21% O2 are indicated. (e) HIF activity in bone metastasis sites. Bioluminescence (BL) intensity in LM8/HRE-luc or LM8/luc metastases was measured at the indicated times after transplantation. To calculate relative BL intensities, BL intensities at indicated times were divided by those at day 4 (3.17 × 103 photons/s/ROI for LM8/HRE-luc and 3.43 × 103 photons/s/ROI for LM8/luc); n = 4 for LM8/HRE-luc, n = 6 for LM8/luc. *P < 0.01. (f) Inhibition of HIF activity in LM8 cells using acriflavine. HRE-Fluc activity was assessed with indicated concentrations of acriflavine (ACF). *P < 0.05. (g) Antitumor effect of HIF inhibitor. Luciferase activity of LM8/luc bone metastasis was measured at indicated times after transplantation. Relative luciferase activities to the ones on day 2 are indicated; n = 8 for each group. *P < 0.01.
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fig03: Hypoxia-inducible factor (HIF) activity of murine osteosarcoma LM8 cells is increased in the bone microenvironment. (a) HIF-1α protein levels in LM8 in 1% O2. (b) HIF-1α protein levels in LM8 under various O2 concentrations for 16 h. (c) Luciferase activity of LM8/HRE-luc in 1% O2. Relative luciferase activities to the one at 0 h are shown. (d) Luciferase activity of LM8/HRE-luc under various O2 concentrations for 16 h. Relative luciferase activity to the one in 21% O2 are indicated. (e) HIF activity in bone metastasis sites. Bioluminescence (BL) intensity in LM8/HRE-luc or LM8/luc metastases was measured at the indicated times after transplantation. To calculate relative BL intensities, BL intensities at indicated times were divided by those at day 4 (3.17 × 103 photons/s/ROI for LM8/HRE-luc and 3.43 × 103 photons/s/ROI for LM8/luc); n = 4 for LM8/HRE-luc, n = 6 for LM8/luc. *P < 0.01. (f) Inhibition of HIF activity in LM8 cells using acriflavine. HRE-Fluc activity was assessed with indicated concentrations of acriflavine (ACF). *P < 0.05. (g) Antitumor effect of HIF inhibitor. Luciferase activity of LM8/luc bone metastasis was measured at indicated times after transplantation. Relative luciferase activities to the ones on day 2 are indicated; n = 8 for each group. *P < 0.01.
Mentions: The pO2 in bone marrow has been estimated at 23–40 mmHg (3–5% O2),19 which is relatively lower than other organs and tissues. To understand the status of LM8 cells that have migrated to the bone marrow, we examined their hypoxic response in vitro and in vivo. The most abundant HIF, HIF-1, is a central regulator of the hypoxic response in many cell types15 and reported as a key factor in promoting homing, colonization, and progression of bone metastasis.28,29 In LM8 cells, the protein level of HIF-1α, the α subunit of HIF-1, increased as hypoxic treatment was prolonged (Fig.3a) and decreased as the O2 concentration increased (Fig.3b). The HIF transcriptional activity was monitored by using LM8/HRE-luc cells, which express firefly luciferase in a HIF-dependent manner. The activity of HIF in LM8/HRE-luc cells varied in response to hypoxic treatments (Fig.3c,d) in good correlation with HIF-1α expression (Fig.3a,b). In vivo BL signaling from LM8/luc, corresponding to tumor burden, and LM8/HRE-luc, corresponding to HIF activity, was monitored after i.c. injection of LM8 cells (Fig.3e). In s.c. tumors of LM8, HIF activity increased much more slowly than tumor burden (Fig. S4), suggesting that HIF activity in s.c. LM8 tumors reflects a gradual increase in hypoxic regions as tumors grow. However, in the bone metastatic sites, the HIF activity and tumor burden showed a parallel increase during the first week after LM8 injection (Fig.3e), indicating that HIF in LM8 was activated in the hypoxic microenvironment immediately after migration of LM8 to the bone marrow. Furthermore, HIF activity drastically increased in bone metastatic sites during the second week after LM8 injection (Fig.3e) and inhibition of HIF transcriptional activity by acriflavine24 significantly suppressed growth of LM8 bone metastasis (Fig.3f,g). These results support the idea that HIF is activated in LM8 homing to the hypoxic bone marrow and plays an important role in LM8 colonization and the progression of bone metastasis.

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