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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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Receptor activator of factor-κB ligand (RANKL) promotes metastatic colonization of murine osteosarcoma LM8 cells in the bone. (a) Experimental protocol. PBS (Control) or RANKL (1 mg/kg) was injected i.p. at the indicated days after intracardiac (i.c.) transplantation of LM8/luc. Two treatment schedules, RANKL1 and RANKL2, are indicated. Mice were dissected 14 days after i.c. injection of LM8. (b) Representative micro X-ray computed tomography cross-section images of mouse femur 3 days after daily injection of PBS (control) or RANKL (1 mg/kg) (n = 5). *P < 0.05. (c) Representative bioluminescence (BL) in vivo images and quantitative analysis of bone metastasis in hind limbs. RANKL1 (left panel): n = 7 for Control; n = 19 for RANKL1); RANKL2 (right panel): n = 6 for both Control and RANKL2. *P < 0.05. (d) Bone metastasis frequency. RANKL1: n = 7 for Control; n = 11 for RANKL1; RANKL2: n = 4 for Control and RANKL2. *P < 0.05. N.S., not significant.
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fig02: Receptor activator of factor-κB ligand (RANKL) promotes metastatic colonization of murine osteosarcoma LM8 cells in the bone. (a) Experimental protocol. PBS (Control) or RANKL (1 mg/kg) was injected i.p. at the indicated days after intracardiac (i.c.) transplantation of LM8/luc. Two treatment schedules, RANKL1 and RANKL2, are indicated. Mice were dissected 14 days after i.c. injection of LM8. (b) Representative micro X-ray computed tomography cross-section images of mouse femur 3 days after daily injection of PBS (control) or RANKL (1 mg/kg) (n = 5). *P < 0.05. (c) Representative bioluminescence (BL) in vivo images and quantitative analysis of bone metastasis in hind limbs. RANKL1 (left panel): n = 7 for Control; n = 19 for RANKL1); RANKL2 (right panel): n = 6 for both Control and RANKL2. *P < 0.05. (d) Bone metastasis frequency. RANKL1: n = 7 for Control; n = 11 for RANKL1; RANKL2: n = 4 for Control and RANKL2. *P < 0.05. N.S., not significant.

Mentions: Although osteoblastic metastasis lesions often contain osteolytic lesions in clinical patients,1,11 the role of bone resorption in osteoblastic bone metastasis has not been extensively studied. A clinical study showed that inhibition of RANKL increased bone metastasis-free survival of prostate cancer patients,14 implying that bone resorption might play important roles in establishment of osteoblastic bone metastasis. Our previous study revealed that RANKL-induced bone resorption started within 24 h of inoculation, and obvious bone loss was observed after 50 h.23 Therefore, in order to elucidate the role of bone resorption in establishment of osteoblastic bone metastasis, we used two different schedules of i.p. administration of RANKL: 5 days of consecutive administration initiated 1 h (RANKL1) or 4 days (RANKL2) after i.c. injection of LM8/luc (Fig.2a). An analysis using multimodal imaging revealed that treatment with RANKL clearly reduced bone mass 3 days after inoculation (Fig.2b), and that significantly higher BL signals (Fig.2c) and a significantly increased number of metastatic sites (Fig.2d) were observed in the bone of early RANKL-treated (RANKL1) mice than in those of untreated and late RANKL-treated (RANKL2) mice. Circulating cancer cells were dramatically reduced 24 h after cancer cell injection and undetectable on day 4, the day RANKL was given in the RANKL2 schedule (data not shown), suggesting that the chance of cancer cells entering the bone after day 4 was very low. These results strongly suggest that RANKL influences the fate of cancer cells that homed in the bone during the first 3 days after i.c. injection. The enhanced metastatic progression of LM8/luc cells by RANKL was not a direct effect of RANKL on LM8/luc cells, as LM8 cells do not express RANK, a receptor of RANKL (Fig. S3). These results indicate that RANKL-induced bone resorption promotes the colonization process of LM8 metastasis by influencing the microenvironment in the bone marrow.


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)

Receptor activator of factor-κB ligand (RANKL) promotes metastatic colonization of murine osteosarcoma LM8 cells in the bone. (a) Experimental protocol. PBS (Control) or RANKL (1 mg/kg) was injected i.p. at the indicated days after intracardiac (i.c.) transplantation of LM8/luc. Two treatment schedules, RANKL1 and RANKL2, are indicated. Mice were dissected 14 days after i.c. injection of LM8. (b) Representative micro X-ray computed tomography cross-section images of mouse femur 3 days after daily injection of PBS (control) or RANKL (1 mg/kg) (n = 5). *P < 0.05. (c) Representative bioluminescence (BL) in vivo images and quantitative analysis of bone metastasis in hind limbs. RANKL1 (left panel): n = 7 for Control; n = 19 for RANKL1); RANKL2 (right panel): n = 6 for both Control and RANKL2. *P < 0.05. (d) Bone metastasis frequency. RANKL1: n = 7 for Control; n = 11 for RANKL1; RANKL2: n = 4 for Control and RANKL2. *P < 0.05. N.S., not significant.
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fig02: Receptor activator of factor-κB ligand (RANKL) promotes metastatic colonization of murine osteosarcoma LM8 cells in the bone. (a) Experimental protocol. PBS (Control) or RANKL (1 mg/kg) was injected i.p. at the indicated days after intracardiac (i.c.) transplantation of LM8/luc. Two treatment schedules, RANKL1 and RANKL2, are indicated. Mice were dissected 14 days after i.c. injection of LM8. (b) Representative micro X-ray computed tomography cross-section images of mouse femur 3 days after daily injection of PBS (control) or RANKL (1 mg/kg) (n = 5). *P < 0.05. (c) Representative bioluminescence (BL) in vivo images and quantitative analysis of bone metastasis in hind limbs. RANKL1 (left panel): n = 7 for Control; n = 19 for RANKL1); RANKL2 (right panel): n = 6 for both Control and RANKL2. *P < 0.05. (d) Bone metastasis frequency. RANKL1: n = 7 for Control; n = 11 for RANKL1; RANKL2: n = 4 for Control and RANKL2. *P < 0.05. N.S., not significant.
Mentions: Although osteoblastic metastasis lesions often contain osteolytic lesions in clinical patients,1,11 the role of bone resorption in osteoblastic bone metastasis has not been extensively studied. A clinical study showed that inhibition of RANKL increased bone metastasis-free survival of prostate cancer patients,14 implying that bone resorption might play important roles in establishment of osteoblastic bone metastasis. Our previous study revealed that RANKL-induced bone resorption started within 24 h of inoculation, and obvious bone loss was observed after 50 h.23 Therefore, in order to elucidate the role of bone resorption in establishment of osteoblastic bone metastasis, we used two different schedules of i.p. administration of RANKL: 5 days of consecutive administration initiated 1 h (RANKL1) or 4 days (RANKL2) after i.c. injection of LM8/luc (Fig.2a). An analysis using multimodal imaging revealed that treatment with RANKL clearly reduced bone mass 3 days after inoculation (Fig.2b), and that significantly higher BL signals (Fig.2c) and a significantly increased number of metastatic sites (Fig.2d) were observed in the bone of early RANKL-treated (RANKL1) mice than in those of untreated and late RANKL-treated (RANKL2) mice. Circulating cancer cells were dramatically reduced 24 h after cancer cell injection and undetectable on day 4, the day RANKL was given in the RANKL2 schedule (data not shown), suggesting that the chance of cancer cells entering the bone after day 4 was very low. These results strongly suggest that RANKL influences the fate of cancer cells that homed in the bone during the first 3 days after i.c. injection. The enhanced metastatic progression of LM8/luc cells by RANKL was not a direct effect of RANKL on LM8/luc cells, as LM8 cells do not express RANK, a receptor of RANKL (Fig. S3). These results indicate that RANKL-induced bone resorption promotes the colonization process of LM8 metastasis by influencing the microenvironment in the bone marrow.

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