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Correspondence to David Thomas: david.thomas{at}petermac.org
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Abstract |
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Abbreviations used in this paper: BMP, bone morphogenetic protein; MEF, murine embryonic fibroblast; PCNA, proliferating cell nuclear antigen.
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Introduction |
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The biology of osteoblast differentiation has recently been mapped in considerable detail. Runx2 (runt-related transcription factor 2) is a key transcriptional regulator of osteogenesis (Ogawa et al., 1993; Levanon et al., 1994; Ducy et al., 1997, 1999). Mice nullizygous for RUNX2 exhibit a complete lack of ossification (Komori et al., 1997; Otto et al., 1997), whereas heterozygotes exhibit skeletal abnormalities comparable to cleidocranial dysplasia (Mundlos et al., 1997). The runt family, to which runx2 belongs, is strongly linked to human cancer (Lund and van Lohuizen, 2002). RUNX1 (AML1) is mutated in human leukemia, and mice expressing loss-of-function runx1 mutants are prone to leukemia (Perry et al., 2002). RUNX3 is subject to inactivating mutations or promoter hypermethylation in gastric cancers (Li et al., 2002). Runx2 physically interacts with the retinoblastoma tumor suppressor protein (pRb; Thomas et al., 2001), which is mutated in up to 60% of osteosarcomas (Toguchida et al., 1989). Finally, runx2 expression varies with cell cycle status and may regulate osteoblast proliferation by unknown mechanisms (Pratap et al., 2003).
In this study, we investigated the effects of osteogenic differentiation on proliferation and growth arrest, and their disruption in osteosarcomas. Consistent with a role in suppression of proliferation, runx2 protein was absent or nonfunctional in six out of seven osteosarcoma cell lines. Both spontaneous and induced osteoblast differentiation are associated with increased p27KIP1 mRNA and protein expression. Ectopic expression of runx2 induced an Rb- and p27KIP1-dependent growth arrest. This was due in part to increased expression of p27KIP1 protein, which inhibited S-phase Cdk complexes and the dephosphorylation of pRb. Interestingly, runx2 is shown to interact preferentially with the hypophosphorylated form of pRb, a known coactivator of runx2. Although p27KIP1 expression is associated with osteoblast differentiation, loss of p27KIP1 had only a minor effect on osteoblast differentiation in vitro and in vivo. Notably, the irreversibility of both the osteogenic phenotype and terminal cell cycle exit in vitro is dependent on expression of p27KIP1. Immunohistochemical analysis of human osteosarcomas confirmed that expression of p27KIP1 was lost as tumors lost evidence of osteogenic differentiation. Together, these data suggest that runx2 establishes a terminally differentiated state through Rb- and p27KIP1-dependent mechanisms, and that these processes are disrupted in osteosarcomas.
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Results |
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Lack of correlation between runx2 protein levels and transcriptional activity
There was little or no correlation between runx2 protein levels and transcriptional activity. Immunoblot analysis of endogenous runx2 protein demonstrated low levels in five out of seven cell lines (Fig. 1 D, top and middle). The upper band represents the MRIPV isoform of runx2, whereas the faster migrating band in SAOS2 represents the osteoblast-specific MASN isoform (unpublished data). The MRIPV isoform strongly induces AP activity but not osteocalcin, whereas the MASN isoform more potently activates osteocalcin but not AP (Harada et al., 1999). Consistent with the results of the transcriptional assays, no runx2 protein was detectable by Western blot in G292 cells, which showed the greatest induction of transcription by ectopic runx2 (Fig. 1 D). In contrast, abundant endogenous runx2 protein was present in SAOS2 cells. The striking contrast between protein levels and intrinsic activity of runx2 in SAOS2 cells is highlighted by comparison with protein levels in the osteoblastic control, although osteoblast gene expression is 5.6 ± 2.9fold higher in the reference than in SAOS2 cells. We have confirmed that no mutations exist in the genomic sequences of runx2 in SAOS2 cells or any other cell line in this study. Thus, some osteosarcomas, such as G292, appear to lack functional runx2, whereas others, such as SAOS2, appear unable to activate transcription of runx2-dependent genes even when ectopic runx2 is supplied. Interestingly, we observed that all lines responsive to ectopic runx2 express pRb, whereas those that fail to respond express no or low levels of this known runx2 coactivator. Furthermore, some pRb-positive cell lines show only limited activation in response to runx2 expression, suggesting that additional factors influence the activity of runx2 in osteosarcoma cell lines (Fig. 1 D). Together, these data confirm gathering evidence that runx2 activity is critically dependent on cofactors or posttranslational modifications, and that oncogenic transformation results in consistent dysregulation of runx2 activity by multiple mechanisms.
Runx2 inhibits cell growth through p27KIP1 and pRb
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 6090%, 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 ACdk2 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.
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Permanent cell cycle withdrawal is a feature of both senescence and terminal differentiation (Goldstein, 1990; Sellers et al., 1998). Postdifferentiated wild-type MEFs assumed a binucleated, enlarged, flattened morphology reminiscent of replicative senescence. Whether differentiated or not, significant numbers of postconfluent wild-type MEFs stained for senescence-associated ß-galactosidase activity (Dimri et al., 1995). This effect was greater in cultures that had been treated with BMP2 (Fig. 6 D). In contrast, MEFs lacking p27KIP1 did not stain for senescence-associated ß-galactosidase and morphologically resembled undifferentiated cultures. Although the effects of both BMP2 and p27KIP1 were independently statistically significant, the interaction between these factors failed to reach significance, perhaps because of the powerful effect of confluence on accumulation of p27KIP1 in both treated and control cultures. Together, these data suggest that culture conditions required for differentiation of MEFs (including both BMP2 treatment and prolonged confluence), as well as expression of p27KIP1, contribute to the entry of MEFs into a senescence-like state.
Expression of p27KIP1 is lost in osteosarcoma
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 pRbrunx2 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.
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Discussion |
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The induction of p27KIP1 leads to more than a simple proliferative arrest. Although the proliferation of early passage MEFs was not strikingly affected by loss of p27KIP1 (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996; and unpublished data), we found that p27KIP1 was required to maintain a growth-arrested state in differentiated cells. p27KIP1 has been suggested previously to be a part of the normal timer that determines the cessation of proliferation and commitment to differentiation of oligodendrocyte precursors (Casaccia-Bonnefil et al., 1997; Durand et al., 1998). The Drosophila melanogaster p27KIP1 homologue, dacapo, initiates terminal cessation of cell division and differentiation, an effect that interacts genetically with pRb (de Nooij et al., 1996; Lane et al., 1996). These data suggest that p27KIP1 may act as a "fate switch" that, once expressed at sufficient levels, commits the osteoblast to a postmitotic state. Irreversible cell cycle exit is a feature of senescence, in which p27KIP1 plays a role and which may represent a defense to oncogenic transformation (Serrano et al., 1997; Sellers et al., 1998; Alexander and Hinds, 2001; Thomas et al., 2001). Clearly, terminal cell cycle exit, whether in response to differentiation or to oncogenic events, is fundamentally inconsistent with oncogenic transformation.
Does p27KIP1 act as a tumor suppressor in bone? In animal models, loss of p27KIP1 is associated with infrequent spontaneous pituitary tumors and intestinal adenomas but accelerates the rate of tumor formation when combined with carcinogen exposure (Fero et al., 1998) or mutations to TP53 (Philipp-Staheli et al., 2004). Interestingly, osteosarcomas were observed in this latter study, albeit at low frequency. Consistent with a role for p27KIP1 in osteosarcoma, the protooncogene c-Fos, which causes osteosarcoma in mice (Grigoriadis et al., 1993), induces cyclin ACdk2 activity and represses p27KIP1 in osteoblasts (Sunters et al., 2004). Unusually, p27KIP1 appears to act as a haploinsufficient tumor suppressor in the mouse (Fero et al., 1998), and, where human tumors have undergone a loss-of-heterozygosity event, silencing of the remaining allele is rare (Kawamata et al., 1995; Ponce-Castaneda et al., 1995). This may be due to the dual role of p27KIP1 as an assembly factor for G1-phase cyclin complexes as well as a stoichiometric inhibitor of S-phase cyclin complexes (Sherr and Roberts, 1999). Importantly, decreases in p27KIP1 protein expression have been found in 60% of human carcinomas (Slingerland and Pagano, 2000), and are associated in breast cancer with poor prognosis (Fredersdorf et al., 1997). Our clinical studies reveal an association between p27KIP1 expression and loss of differentiation in human osteosarcomas, independent of rates of proliferation per se (Fig. 7). Loss of differentiation in sarcomas in general is a marker of high-grade status, which in turn is associated with worse prognosis.
Disruption of runx2-dependent transcriptional activity is common in osteosarcoma cell lines and leads in a clinically measurable fashion to loss of both differentiation and expression of p27KIP1 in human osteosarcomas. Although the specific mechanisms contributing to the loss of function of runx2 are not understood, global demethylation of osteosarcoma cell lines results in reactivation of differentiation concomitant with reversion of transformation (unpublished data). Methylation is a well-described, common method of silencing tumor suppressor pathways (Baylin and Herman, 2000), and the restoration of differentiation by demethylation further supports the notion that tumors gain a selective survival advantage by silencing differentiation-related growth inhibitory processes. We hope that identifying targets of methylation-induced silencing will shed additional light on the interactions between differentiation and cell cycle exit.
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Materials and methods |
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SV-Rb and SV-HARb were used for the expression of full-length pRb (Hinds et al., 1992). Expression constructs for runx2 were cloned into pBABEpuro (Morgenstern and Land, 1990). The retrovirus was amplified and purified in amphotropic packaging cell line Phoenix 293 (courtesy of G. Nolan, Stanford University, Palo Alto, CA), according to the method of Pear et al. (1993). Plasmids were transfected into cells with the use of Fugene, according to the manufacturer's instructions (Roche Pharmaceuticals). Adenoviral constructs expressing pRb and runx2-FLAG were generated as reported previously (Thomas et al., 2001).
Cell-based assays
Luciferase assays were performed according to manufacturer's instructions (Promega). Where indicated (Fig. 1, B and C), results were normalized for transfection efficiency with ß-galactosidase activity or protein content. AP activity was assayed as described previously (Sellers et al., 1998). Assays for mineralization were performed as described previously (Thomas et al., 2001). Quantitation was performed by dissolving stained mineralized cultures in 10% cetylpyridinium chloride, followed by spectrophotometric analysis at 540 nm. Both AP activity and mineralization were normalized to protein content (Bio-Rad Laboratories). Flow cytometry for DNA content was performed as described previously (Thomas et al., 2001).
RT-PCR analysis of gene expression
RNA was extracted with the use of TRIzol (Invitrogen), according to the manufacturer's instructions. cDNA was produced from 1 µg of total RNA with the use of a commercially available kit (SUPERSCRIPT Choice system for cDNA synthesis; GIBCO BRL). Semiquantitative PCR analysis was performed after optimization. Primer sequences and PCR conditions are available on request. For quantitative RT-PCR, expression of each target gene was normalized to expression of ARPP0 with the use of an ABI-Prism 7700 Light Cycler and SYBR Green. Optimal PCR conditions were established for each gene in preliminary experiments.
Analysis of protein expression and kinase assays
Nuclear extracts and immunoblot analyses were performed as described previously (Thomas et al., 2001). The following antibodies were used: anti-FLAG antibody (M2; Sigma-Aldrich); human pRb: monoclonal antibody 245 (BD Biosciences); runx2: M-70 (Santa Cruz Biotechnology, Inc.); p27KIP1: K25020 (Transduction Laboratories); and cyclin E: HE12, cyclin A: H432, and Cdk2: M2 (Santa Cruz Biotechnology, Inc.). Horseradish peroxidaseconjugated secondary antibodies were used (Jackson ImmunoResearch Laboratories) and signal was detected by ECL (NEN Life Science Products). The GST-runx2 pulldown studies shown in Fig. 4 D were performed as described previously (Thomas et al., 2001). Kinase assays were performed as described previously (Alexander and Hinds, 2001). Cyclin A was immunoprecipitated using agarose-conjugated antibody BF683 (Upstate Biotechnology).
Immunohistochemistry
25 paraffin-embedded osteosarcoma samples were obtained from the pathology archives at St. Vincent's Hospital Melbourne, with approval from the Human Research Ethics Committee. 2-mm cores were punched and then assembled into a tissue microarray. Sections were cut at 3 µm and mounted onto Superfrost Plus slides. Primary antibodies were incubated for 30 min. For p27KIP1, clone SX53G8 (DakoCytomation) was used at 1:50. A predilute monoclonal antibody to human osteocalcin (OC-1; Biogenex) was used at 1:4. For PCNA, clone PC10 (DakoCytomation) was used at 1:400. The primary antibody was detected with the mouse Envision+ system (DakoCytomation). Immunoreactivity was visualized with AEC+ chromogen (DakoCytomation), using hematoxylin as a counterstain. Samples for p27KIP1 and osteocalcin were incubated in 10 mM boiling sodium citrate buffer, pH 6.0, for 2 min before staining.
Slides were imaged using a microscope (Axioskop 2; Carl Zeiss MicroImaging, Inc.) with a Plan-Neofluar objective (40x, 0.75 NA), and a cooled color digital camera (RT Slider SPOT; Diagnostic Instruments) and software (SPOT V4.0.2 for Windows). Subsequent processing of TIFF files was undertaken in Adobe Photoshop (V7.0.1). Images were cropped, labeled as indicated in Fig. 7, and assembled into composites for figures after minor adjustments for contrast and color balance were applied to all parts of each image.
Microarray analysis
Total RNA from osteosarcoma and common reference cell lines was isolated using phenol-chloroform extraction (TRIzol; Invitrogen) and purified by column chromatography (RNeasy; QIAGEN). The common reference RNA, containing pooled RNA from 11 human tumor cell lines, was prepared as described previously (Pollack, 2002). Total RNA (4050 µg) was reverse transcribed with Moloney Murine Leukemia Virus Reverse transcriptase (Promega), in the presence of amino-allyl (AA)modified dUTP (Sigma-Aldrich). AA-dUTP cDNA was labeled by coupling to Cy3 and Cy5 (reference and sample, respectively) monoreactive dyes (Amersham Biosciences). cDNA arrays containing 10.5 K elements representing 9,381 unique cDNA (Unigene build 172) were produced at Peter MacCallum Cancer Centre Microarray Core facility on superamine slides (Telechem), with a robotic arrayer (Virtek/Bio-Rad Laboratories). Labeled probe was hybridized to the array in 3.1 x SSC and 50% formamide at 42°C for 1416 h in a humidified and temperature-controlled chamber (HyPro20; Thermo Hybaid). Slides were washed at room temperature with 0.5 x SSC/0.01% SDS (for 1 min), then with 0.5 x SSC (for 3 min), and finally with 0.06 x SSC (for 3 min). Scanning was performed with an Agilent G2565AA Microarray Scanner and data was extracted with GenePix Pro 4.1 software (Axon Instruments, Inc.). All array experiments, including cell culture, were performed independently twice. A complete list of genes is available from the authors on request. Data from each independent experiment were averaged, and then the median obtained from all six cell lines was used in the data shown in Fig. 1. Data were analyzed with GeneSpring software (Silicon Genetics), and samples were normalized with LOWESS (Yang et al., 2002).
Histomorphometry
Tibiae were collected from male p27KIP1/ and wild-type littermates at 16 wk of age, fixed in cold 4% paraformaldehyde in PBS overnight, and embedded in methylmethacrylate (Sims et al., 2000). Double fluorochrome labeling to quantitate mineral appositional rates was performed as described previously (Sims et al., 2000). 5-µm sections were stained with toluidine blue or analyzed unstained for fluorochrome labels according to standard procedures in the proximal tibia using the Osteomeasure system (Osteometrics, Inc.). Tibial cortical thickness and periosteal mineral appositional rates were measured as described previously (Sims et al., 2000).
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Acknowledgments |
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D.M. Thomas is the recipient of a National Health and Medical Research Council R.D. Wright fellowship (Regkey 251752) and is supported by the Cancer Council of Victoria. P.W. Hinds and G. Gutierrez are supported by National Institutes of Health grant AG20208.
Submitted: 30 September 2004
Accepted: 28 October 2004
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