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Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW - J. Cell Biol. (2004)

Bottom Line: Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts.Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas.Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Ian Potter Foundation Centre for Cancer Genomics and Predictive Medicine, and Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Center, Victoria, Melbourne, Australia. david.thomas@petermac.org

ABSTRACT
The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

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Expression of p27KIP1, osteocalcin, and proliferating cell nuclear antigen (PCNA) in human osteosarcoma samples. (A–I) High-power photomicrographs of parallel sections from two high-grade (A–C and G–I) and one low-grade (G–I) human osteosarcomas were stained for p27KIP1 (A, D, and G), osteocalcin (B, E, and H), and PCNA (C, F, and I). Arrows in D–F indicate multinucleated osteoclast; arrows in G–I indicate osteocytes. Bar, 50 μm. (J) Blinded quantitation of staining for p27KIP1 and PCNA in tumors with evidence of osteoblast differentiation (osteoid production) compared with dedifferentiated tumors. Error bars represent SEM. *, P < 0.05.
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fig7: Expression of p27KIP1, osteocalcin, and proliferating cell nuclear antigen (PCNA) in human osteosarcoma samples. (A–I) High-power photomicrographs of parallel sections from two high-grade (A–C and G–I) and one low-grade (G–I) human osteosarcomas were stained for p27KIP1 (A, D, and G), osteocalcin (B, E, and H), and PCNA (C, F, and I). Arrows in D–F indicate multinucleated osteoclast; arrows in G–I indicate osteocytes. Bar, 50 μm. (J) Blinded quantitation of staining for p27KIP1 and PCNA in tumors with evidence of osteoblast differentiation (osteoid production) compared with dedifferentiated tumors. Error bars represent SEM. *, P < 0.05.

Mentions: We finally wished to determine whether these observations have relevance to human osteosarcoma. p27KIP1 expression appears to be key for cell cycle withdrawal and terminal differentiation in osteoblasts, and integrates the functions of BMPs, pRb, and runx2 in these processes. Regardless of the nature of the defect in the pRb–runx2 pathway in osteosarcoma cells, the net effect will be loss of growth restraint due to diminished expression of p27KIP1. Consistent with this, we found negligible expression of p27KIP1 protein in high-grade osteosarcoma cells, although p27KIP1 was clearly seen in osteoclasts as reported previously (Okahashi et al., 2001; Fig. 7, A and D), correlating inversely with expression of proliferating cell nuclear antigen (PCNA; Fig. 7, D and F, arrows). These high-grade osteosarcomas demonstrated frequent mitotic figures and little differentiation, as evidenced by osteoid production and osteocalcin expression (Fig. 7, B and E). High-grade tumor cells expressed high levels of PCNA, consistent with a high S-phase fraction (Fig. 7, C and F). In contrast, in lower grade tumors with mineralizing osteoid and lower cellularity with more normal osteoblastic morphology, expression of both p27KIP1 and osteocalcin is evident, especially in terminally differentiated (PCNA negative) osteocytes embedded within bone (Fig. 7, G and H, arrows). Critically, there was a significant relationship between expression of p27KIP1 protein and osteoblast differentiation scored by osteoid production in a panel of 100 osteosarcomas (Fig. 7 J, P < 0.05). This effect was independent of proliferative rate, because there was no significant relationship between PCNA expression and p27KIP1. These data support the view that the loss of differentiation of osteosarcomas, which conveys adverse prognostic significance, is associated with loss of expression of p27KIP1.


Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW - J. Cell Biol. (2004)

Expression of p27KIP1, osteocalcin, and proliferating cell nuclear antigen (PCNA) in human osteosarcoma samples. (A–I) High-power photomicrographs of parallel sections from two high-grade (A–C and G–I) and one low-grade (G–I) human osteosarcomas were stained for p27KIP1 (A, D, and G), osteocalcin (B, E, and H), and PCNA (C, F, and I). Arrows in D–F indicate multinucleated osteoclast; arrows in G–I indicate osteocytes. Bar, 50 μm. (J) Blinded quantitation of staining for p27KIP1 and PCNA in tumors with evidence of osteoblast differentiation (osteoid production) compared with dedifferentiated tumors. Error bars represent SEM. *, P < 0.05.
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Related In: Results  -  Collection

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fig7: Expression of p27KIP1, osteocalcin, and proliferating cell nuclear antigen (PCNA) in human osteosarcoma samples. (A–I) High-power photomicrographs of parallel sections from two high-grade (A–C and G–I) and one low-grade (G–I) human osteosarcomas were stained for p27KIP1 (A, D, and G), osteocalcin (B, E, and H), and PCNA (C, F, and I). Arrows in D–F indicate multinucleated osteoclast; arrows in G–I indicate osteocytes. Bar, 50 μm. (J) Blinded quantitation of staining for p27KIP1 and PCNA in tumors with evidence of osteoblast differentiation (osteoid production) compared with dedifferentiated tumors. Error bars represent SEM. *, P < 0.05.
Mentions: We finally wished to determine whether these observations have relevance to human osteosarcoma. p27KIP1 expression appears to be key for cell cycle withdrawal and terminal differentiation in osteoblasts, and integrates the functions of BMPs, pRb, and runx2 in these processes. Regardless of the nature of the defect in the pRb–runx2 pathway in osteosarcoma cells, the net effect will be loss of growth restraint due to diminished expression of p27KIP1. Consistent with this, we found negligible expression of p27KIP1 protein in high-grade osteosarcoma cells, although p27KIP1 was clearly seen in osteoclasts as reported previously (Okahashi et al., 2001; Fig. 7, A and D), correlating inversely with expression of proliferating cell nuclear antigen (PCNA; Fig. 7, D and F, arrows). These high-grade osteosarcomas demonstrated frequent mitotic figures and little differentiation, as evidenced by osteoid production and osteocalcin expression (Fig. 7, B and E). High-grade tumor cells expressed high levels of PCNA, consistent with a high S-phase fraction (Fig. 7, C and F). In contrast, in lower grade tumors with mineralizing osteoid and lower cellularity with more normal osteoblastic morphology, expression of both p27KIP1 and osteocalcin is evident, especially in terminally differentiated (PCNA negative) osteocytes embedded within bone (Fig. 7, G and H, arrows). Critically, there was a significant relationship between expression of p27KIP1 protein and osteoblast differentiation scored by osteoid production in a panel of 100 osteosarcomas (Fig. 7 J, P < 0.05). This effect was independent of proliferative rate, because there was no significant relationship between PCNA expression and p27KIP1. These data support the view that the loss of differentiation of osteosarcomas, which conveys adverse prognostic significance, is associated with loss of expression of p27KIP1.

Bottom Line: Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts.Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas.Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Ian Potter Foundation Centre for Cancer Genomics and Predictive Medicine, and Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Center, Victoria, Melbourne, Australia. david.thomas@petermac.org

ABSTRACT
The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

Show MeSH
Related in: MedlinePlus