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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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Runx2 induces a growth arrest through induction of p27KIP1. (A) Effect of ectopic expression of runx2 on colony suppression assay. (B) Retroviral vectors expressing runx2 27ala or runx2 3ala were used to infect 3T3 or RB−/− 3T3 cells, followed by selection for 3 d in 2 μg/ml puromycin. Cell cycle profile was determined by flow cytometry. (C) IMR90, CCL-211, SAOS2, and U2OS cells were infected with adenoviral constructs expressing FLAG-tagged runx2. Western blot for runx2, p27KIP1, and p21CIP1. The percentage of cells in S-phase, derived from parallel cultures subjected to DNA analysis by flow cytometry, is indicated (bottom). (D) CCL-211 cells were infected with adenoviral constructs expressing runx2. Cyclin A immunoprecipitates were subjected to Western blot for p27KIP1, Cdk2, and cyclin A. The bottom panel is directly blotted for p27KIP1. Asterisks indicate Ig heavy chain bands. (E) Cyclin A immunoprecipitates assayed for kinase activity in the presence of runx2. CCL-211 cells were treated as in C. Direct Western blot for Rb, cyclin A, cyclin E, Cdk2, p27KIP1, p21CIP1, runx2, and GFP to demonstrate equal titers of virus in each culture. (F) Runx2 binds the hypophosphorylated form of pRb. COS-7 cells were transfected with vector, pRb, and HA-tagged pRb. A pulldown was performed using GST-runx2. Input is 5% of that used for the pulldown. ppRb, phosphorylated species of pRb. Black lines indicate that intervening lanes have been spliced out.
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fig2: Runx2 induces a growth arrest through induction of p27KIP1. (A) Effect of ectopic expression of runx2 on colony suppression assay. (B) Retroviral vectors expressing runx2 27ala or runx2 3ala were used to infect 3T3 or RB−/− 3T3 cells, followed by selection for 3 d in 2 μg/ml puromycin. Cell cycle profile was determined by flow cytometry. (C) IMR90, CCL-211, SAOS2, and U2OS cells were infected with adenoviral constructs expressing FLAG-tagged runx2. Western blot for runx2, p27KIP1, and p21CIP1. The percentage of cells in S-phase, derived from parallel cultures subjected to DNA analysis by flow cytometry, is indicated (bottom). (D) CCL-211 cells were infected with adenoviral constructs expressing runx2. Cyclin A immunoprecipitates were subjected to Western blot for p27KIP1, Cdk2, and cyclin A. The bottom panel is directly blotted for p27KIP1. Asterisks indicate Ig heavy chain bands. (E) Cyclin A immunoprecipitates assayed for kinase activity in the presence of runx2. CCL-211 cells were treated as in C. Direct Western blot for Rb, cyclin A, cyclin E, Cdk2, p27KIP1, p21CIP1, runx2, and GFP to demonstrate equal titers of virus in each culture. (F) Runx2 binds the hypophosphorylated form of pRb. COS-7 cells were transfected with vector, pRb, and HA-tagged pRb. A pulldown was performed using GST-runx2. Input is 5% of that used for the pulldown. ppRb, phosphorylated species of pRb. Black lines indicate that intervening lanes have been spliced out.

Mentions: It appears that normal runx2 function is incompatible with malignant transformation of osteoblastic cells. To determine why, we examined the effect of reexpression of runx2 in G292 cells. As shown in Fig. 1 (B and C), in G292 cells runx2 levels appeared to be rate limiting for transcriptional activity. This suggests that the molecular apparatus for full runx2 activity, including any potential tumor suppressor functions, exists in G292 cells. Consistent with this idea, ectopic expression of runx2 suppressed cell growth (Fig. 2 A). In contrast, this effect was not seen in SAOS2 cells, in which forced expression of runx2 had little effect on transcriptional activity. Because SAOS2 lacks functional pRb, whereas G292 has wild-type pRb, we hypothesized that the lack of pRb may account for the inability of runx2 to reduce the proliferative capacity of these cells. Indeed, when overexpressed in wild-type and RB−/− 3T3 cell lines, runx2 suppressed colony numbers of 3T3 cells by 60–90%, an effect dependent on pRb (unpublished data). To study this effect further, we used two runx2 constructs containing transactivation domain mutations. One of these (27ala) lacks transcriptional activity, whereas the other (3ala) possesses wild-type activity (Thirunavukkarasu et al., 1998). Introduction of these constructs into 3T3 cells showed that the colony suppression activity of runx2 is due to a G1 cell cycle arrest dependent on transcriptional activity and pRb (Fig. 2 B). Runx2 was ectopically expressed in primary human fibroblasts (CCL-211 and IMR90) and osteosarcoma cell lines (U2OS and SAOS2), using adenoviral vectors. As expected, runx2 inhibited the S-phase fraction of fibroblastic but not osteosarcoma cells. Initial studies of cell cycle protein expression in these transfected cells revealed a specific induction of p27KIP1 protein but no effect on p21CIP1 (Fig. 2 C). Coimmunoprecipitation from CCL-211 cells showed that p27KIP1 was strongly associated with cyclin A and Cdk2 (Fig. 2 D), suppressed in vitro kinase activity of cyclin A–Cdk2 complexes (Fig. 2 E), and was accompanied by dephosphorylation of endogenous pRb (Fig. 2 E). We have shown previously that pRb binds and coactivates runx2 (Thomas et al., 2001). We hypothesized that a feed-forward loop, integrating progressive cell cycle withdrawal and differentiation, would be completed if runx2 specifically interacted with the hypophosphorylated form of pRb. This is the case (Fig. 2 F). Collectively, these data are consistent with the transcriptional induction of growth arrest by runx2 through an Rb- and p27KIP1-dependent mechanism that is reinforced by coactivation of runx2 by direct interactions with the hypophosphorylated form of pRb.


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)

Runx2 induces a growth arrest through induction of p27KIP1. (A) Effect of ectopic expression of runx2 on colony suppression assay. (B) Retroviral vectors expressing runx2 27ala or runx2 3ala were used to infect 3T3 or RB−/− 3T3 cells, followed by selection for 3 d in 2 μg/ml puromycin. Cell cycle profile was determined by flow cytometry. (C) IMR90, CCL-211, SAOS2, and U2OS cells were infected with adenoviral constructs expressing FLAG-tagged runx2. Western blot for runx2, p27KIP1, and p21CIP1. The percentage of cells in S-phase, derived from parallel cultures subjected to DNA analysis by flow cytometry, is indicated (bottom). (D) CCL-211 cells were infected with adenoviral constructs expressing runx2. Cyclin A immunoprecipitates were subjected to Western blot for p27KIP1, Cdk2, and cyclin A. The bottom panel is directly blotted for p27KIP1. Asterisks indicate Ig heavy chain bands. (E) Cyclin A immunoprecipitates assayed for kinase activity in the presence of runx2. CCL-211 cells were treated as in C. Direct Western blot for Rb, cyclin A, cyclin E, Cdk2, p27KIP1, p21CIP1, runx2, and GFP to demonstrate equal titers of virus in each culture. (F) Runx2 binds the hypophosphorylated form of pRb. COS-7 cells were transfected with vector, pRb, and HA-tagged pRb. A pulldown was performed using GST-runx2. Input is 5% of that used for the pulldown. ppRb, phosphorylated species of pRb. Black lines indicate that intervening lanes have been spliced out.
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Related In: Results  -  Collection

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fig2: Runx2 induces a growth arrest through induction of p27KIP1. (A) Effect of ectopic expression of runx2 on colony suppression assay. (B) Retroviral vectors expressing runx2 27ala or runx2 3ala were used to infect 3T3 or RB−/− 3T3 cells, followed by selection for 3 d in 2 μg/ml puromycin. Cell cycle profile was determined by flow cytometry. (C) IMR90, CCL-211, SAOS2, and U2OS cells were infected with adenoviral constructs expressing FLAG-tagged runx2. Western blot for runx2, p27KIP1, and p21CIP1. The percentage of cells in S-phase, derived from parallel cultures subjected to DNA analysis by flow cytometry, is indicated (bottom). (D) CCL-211 cells were infected with adenoviral constructs expressing runx2. Cyclin A immunoprecipitates were subjected to Western blot for p27KIP1, Cdk2, and cyclin A. The bottom panel is directly blotted for p27KIP1. Asterisks indicate Ig heavy chain bands. (E) Cyclin A immunoprecipitates assayed for kinase activity in the presence of runx2. CCL-211 cells were treated as in C. Direct Western blot for Rb, cyclin A, cyclin E, Cdk2, p27KIP1, p21CIP1, runx2, and GFP to demonstrate equal titers of virus in each culture. (F) Runx2 binds the hypophosphorylated form of pRb. COS-7 cells were transfected with vector, pRb, and HA-tagged pRb. A pulldown was performed using GST-runx2. Input is 5% of that used for the pulldown. ppRb, phosphorylated species of pRb. Black lines indicate that intervening lanes have been spliced out.
Mentions: It appears that normal runx2 function is incompatible with malignant transformation of osteoblastic cells. To determine why, we examined the effect of reexpression of runx2 in G292 cells. As shown in Fig. 1 (B and C), in G292 cells runx2 levels appeared to be rate limiting for transcriptional activity. This suggests that the molecular apparatus for full runx2 activity, including any potential tumor suppressor functions, exists in G292 cells. Consistent with this idea, ectopic expression of runx2 suppressed cell growth (Fig. 2 A). In contrast, this effect was not seen in SAOS2 cells, in which forced expression of runx2 had little effect on transcriptional activity. Because SAOS2 lacks functional pRb, whereas G292 has wild-type pRb, we hypothesized that the lack of pRb may account for the inability of runx2 to reduce the proliferative capacity of these cells. Indeed, when overexpressed in wild-type and RB−/− 3T3 cell lines, runx2 suppressed colony numbers of 3T3 cells by 60–90%, an effect dependent on pRb (unpublished data). To study this effect further, we used two runx2 constructs containing transactivation domain mutations. One of these (27ala) lacks transcriptional activity, whereas the other (3ala) possesses wild-type activity (Thirunavukkarasu et al., 1998). Introduction of these constructs into 3T3 cells showed that the colony suppression activity of runx2 is due to a G1 cell cycle arrest dependent on transcriptional activity and pRb (Fig. 2 B). Runx2 was ectopically expressed in primary human fibroblasts (CCL-211 and IMR90) and osteosarcoma cell lines (U2OS and SAOS2), using adenoviral vectors. As expected, runx2 inhibited the S-phase fraction of fibroblastic but not osteosarcoma cells. Initial studies of cell cycle protein expression in these transfected cells revealed a specific induction of p27KIP1 protein but no effect on p21CIP1 (Fig. 2 C). Coimmunoprecipitation from CCL-211 cells showed that p27KIP1 was strongly associated with cyclin A and Cdk2 (Fig. 2 D), suppressed in vitro kinase activity of cyclin A–Cdk2 complexes (Fig. 2 E), and was accompanied by dephosphorylation of endogenous pRb (Fig. 2 E). We have shown previously that pRb binds and coactivates runx2 (Thomas et al., 2001). We hypothesized that a feed-forward loop, integrating progressive cell cycle withdrawal and differentiation, would be completed if runx2 specifically interacted with the hypophosphorylated form of pRb. This is the case (Fig. 2 F). Collectively, these data are consistent with the transcriptional induction of growth arrest by runx2 through an Rb- and p27KIP1-dependent mechanism that is reinforced by coactivation of runx2 by direct interactions with the hypophosphorylated form of pRb.

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