Saminathan Suresh Nathan

The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt‐related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan‐mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin‐1 (Ext1) and heparanase, as well as alters the relative expression of N‐linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O‐linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan‐related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)‐related regulatory feed‐back loop that controls osteoblast proliferation and execution of the osteogenic program. J. Cell. Biochem. 107: 144–154, 2009.

Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility

IntroductionMetastatic breast cancer cells frequently and ectopically express the transcription factor RUNX2, which normally attenuates proliferation and promotes maturation of osteoblasts. RUNX2 expression is inversely regulated with respect to cell growth in osteoblasts and deregulated in osteosarcoma cells.MethodsHere, we addressed whether the functional relationship between cell growth and RUNX2 gene expression is maintained in breast cancer cells. We also investigated whether the aberrant expression of RUNX2 is linked to phenotypic parameters that could provide a selective advantage to cells during breast cancer progression.ResultsWe find that, similar to its regulation in osteoblasts, RUNX2 expression in MDA-MB-231 breast adenocarcinoma cells is enhanced upon growth factor deprivation, as well as upon deactivation of the mitogen-dependent MEK-Erk pathway or EGFR signaling. Reduction of RUNX2 levels by RNAi has only marginal effects on cell growth and expression of proliferation markers in MDA-MB-231 breast cancer cells. Thus, RUNX2 is not a critical regulator of cell proliferation in this cell type. However, siRNA depletion of RUNX2 in MDA-MB-231 cells reduces cell motility, while forced exogenous expression of RUNX2 in MCF7 cells increases cell motility.ConclusionsOur results support the emerging concept that the osteogenic transcription factor RUNX2 functions as a metastasis-related oncoprotein in non-osseous cancer cells.

Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma

To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.

Runx2, p53, and pRB status as diagnostic parameters for deregulation of osteoblast growth and differentiation in a new pre-chemotherapeutic osteosarcoma cell line (OS1).

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS‐2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre‐chemotherapeutic tumor in the femur of a 6‐year‐old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase‐PCR, Western blotting and flow cytometry. OS1 have aberrant G‐banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS‐2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS‐2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin‐E and increased cyclin‐A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS‐2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre‐chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development. J. Cell. Physiol. 221: 778–788, 2009.

Affinity-selected heparan sulfate for bone repair

Bone morphogenetic protein (BMP)-2 is a potent bone healing compound produced at sites of bone trauma. Here we present a therapeutic strategy to harness the activity of endogenously produced BMP-2 by delivery of an affinity-matched heparan sulfate (HS) glycos aminoglycan biomaterial that increases the bioavailability, bioactivity and half-life of this growth factor. We have developed a robust, cost effective, peptide-based affinity platform to isolate a unique BMP-2 binding HS variant from commercially available preparations of HS, so removing the manufacturing bottleneck for their translation into the clinic. This affinity-matched HS enhanced BMP-2-induced osteogenesis through improved BMP-2 kinetics and receptor modulation, prolonged pSMAD signaling and reduced interactions with its antagonist noggin. When co-delivered with a collagen implant, the HS was as potent as exogenous BMP-2 for the healing of critical-sized bone defects in rabbits. This affinity platform can be readily tuned to isolate HS variants targeted ata range of clinically-relevant growth and adhesive factors.

Proximal deep vein thrombosis after hip replacement for oncologic indications

BACKGROUND Patients with cancer who undergo surgery about the hip are at increased risk for the development of deep vein thrombosis. We implemented a program of chemical and mechanical prophylaxis to prevent this problem. This study was performed to assess the effectiveness of that program. METHODS Eighty-seven consecutive patients with an active malignant tumor who underwent hip replacement surgery at our institution over a two-year period were included in the study. All patients were treated with intermittent pneumatic compression devices. Seventy-eight patients received anticoagulants, and nine did not. Postoperative surveillance for proximal deep vein thrombosis was routinely performed on all patients with duplex Doppler ultrasonography. RESULTS Four patients had proximal deep vein thrombosis, and one patient, who did not receive anticoagulation, had a nonfatal pulmonary embolism. The use of prophylactic low-molecular-weight heparin (dalteparin) was associated with a 4% rate of proximal deep vein thrombosis (three of seventy-eight patients). Proximal deep vein thrombosis developed in three of eight patients with pelvic disease, one of nineteen patients with femoral disease, and zero of sixty patients with hip disease (p < 0.00001). The prevalence of proximal deep vein thrombosis was significantly higher (p < 0.02) following replacements in patients with sarcoma (three of twenty-one) than it was after replacements in patients with carcinoma (zero of fifty-seven) or hematologic malignant disease (one of nine). On multivariate analysis, only the location of the disease (the pelvis, femur, or hip) was found to be independently significant for an association with deep vein thrombosis. A wound complication developed in four of twenty-one patients with sarcoma and no patient with carcinoma or hematologic malignant disease (p < 0.001). The pathologic type was the only factor studied that was independently significant for an association with wound complications on multivariate analysis. CONCLUSIONS The rate of proximal deep vein thrombosis in patients who had undergone hip replacement for oncologic indications was low when the use of an intermittent pneumatic compression device was supplemented with prophylaxis with low-molecular-weight heparin.

Impaired Cell Cycle Regulation of the Osteoblast-Related Heterodimeric Transcription Factor Runx2-Cbfβ in Osteosarcoma Cells

Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfβ heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G1. Runx2 levels are modulated during the cell cycle, which are maximal in G1 and minimal beyond the G1/S phase transition (S, G2, and M phases). It is not known whether Cbfβ gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfβ are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfβ gene expression during cell cycle progression in MC3T3‐E1 osteoblasts, as well as ROS17/2.8 and SaOS‐2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3‐E1 cells arrested in late G1 (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfβ protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3‐E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfβ levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G1 and S in MC3T3‐E1 cells, but not in ROS17/2.8 and SaOS‐2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma. J. Cell. Physiol. 221: 560–571, 2009.

Treatment algorithm for locally recurrent osteosarcoma based on local disease-free interval and the presence of lung metastasis.

Local recurrence in osteosarcoma is clinically distinct from metastasis, although associated with a similar reduction in survival. The prognostic factors in locally recurrent osteosarcoma were investigated and these factors were translated into a management strategy.

Three-dimensional porous silk tumor constructs in the approximation of in vivo osteosarcoma physiology.

The lack of good preclinical models has hampered anticancer drug discovery. Standard preclinical protocols require the growth of cells in high throughput two-dimensional (2D) culture systems. However, such in vitro drug testing methods yield drug efficacy results that differ greatly from animal models. Conversely, it is much more difficult and expensive to use animal models for large-scale molecular biology research. It is conceivable that three-dimensional (3D) growth may be responsible for some of these changes. Porous silk sponges were fabricated through freeze drying and seeded with 143.98.2 osteosarcoma cells. Molecular profiles were obtained by carrying out real-time polymerase chain reaction for angiogenic growth factors and proliferation markers for osteosarcoma cells grown under 2D, 3D, and SCID mouse xenograft conditions. The angiogenic factor expression profiles for cells grown in 2D differed greatly from the 3D silk scaffold model (P < 0.05 for bFGF, HIF-1α, IL-8, and VEGF-A), whereas 3D tumor model profiles were found to be able to approximate that for the in vivo tumor better with no statistically different expression of HIF-1α and VEGF-A between the two. Immunohistochemistry staining for HIF-1α, VEGF-A, and VEGF receptor on osteosarcoma cells grown on the scaffolds validated the results obtained with the gene expression profiles. The results suggest that 3D tumor models could be used to bridge the gap between in vitro and in vivo tumor studies, and aid in the study of mechanisms activated during tumorigenesis for the development of novel targeted chemotherapy.

Restoring the anatomical tibial slope and limb axis may maximise post-operative flexion in posterior-stabilised total knee replacements.

The optimal management of the tibial slope in achieving a high flexion angle in posterior-stabilised (PS) total knee replacement (TKR) is not well understood, and most studies evaluating the posterior tibial slope have been conducted on cruciate-retaining TKRs. We analysed pre- and post-operative tibial slope differences, pre- and post-operative coronal knee alignment and post-operative maximum flexion angle in 167 patients undergoing 209 TKRs. The mean pre-operative posterior tibial slope was 8.6° (1.3° to 17°) and post-operatively it was 8.0° (0.1° to 16.7°). Multiple linear regression analysis showed that the absolute difference between pre- and post-operative tibial slope (p < 0.001), post-operative coronal alignment (p = 0.02) and pre-operative range of movement (p < 0.001) predicted post-operative flexion. The variance of change in tibial slope became larger as the post-operative maximum flexion angle decreased. The odds ratio of having a post-operative flexion angle < 100° was 17.6 if the slope change was > 2°. Our data suggest that recreation of the anatomical tibial slope appears to improve maximum flexion after posterior-stabilised TKR, provided coronal alignment has been restored.