RNF135 Promotes Human Osteosarcoma Cell Growth and Inhibits Apoptosis by Upregulating the PI3K/AKT Pathway.

BACKGROUND: Ring finger protein 135 (RNF135) is an E3 ubiquitin ligase that has been implicated in the tumorigenesis of multiple human malignancies. However, whether RNF135 plays a role in the development of human osteosarcoma (OS) remains unknown. METHODS: RNF135 expression in 20 human OS and 20 human osteochondroma specimens were evaluated by means of immunohistochemistry staining. The effects of shRNA-mediated RNF135 knockdown on human OS cell growth and apoptosis were evaluated through a panel of in vitro studies on cell proliferation, colony formation, exposure of phosphatidylserine on the cell surface, and caspase 3/7 activation. The protein levels of PI3K, AKT, and p-AKT were determined by western blot analysis. RESULTS: We detected significantly higher RNF135 levels in human OS tissues than human osteochondroma tissues. In in vitro studies, shRNA-mediated RNF135 knockdown in human OS cells inhibited proliferation and induced apoptosis. In addition, RNF135 knockdown reduced PI3K and p-AKT protein levels and activated caspase 3 and 7. CONCLUSIONS: These results supported that RNF135 contributes to human OS development through PI3K/AKT-dependent mechanisms. Targeting RNF135 may provide a new therapeutic approach for treating this human malignancy.

Analysis of the Actions of RARγ Agonists on Growing Osteochondromas in a Mouse Model.

The actions of the retinoic acid nuclear receptor gamma (RARγ) agonist, palovarotene, on pre-existing osteochondromas were investigated using a mouse multiple osteochondroma model. This approach was based on the knowledge that patients often present to the clinic after realizing the existence of osteochondroma masses, and the findings from preclinical investigations are the effects of drugs on the initial formation of osteochondromas. Systemic administration of palovarotene, with increased doses (from 1.76 to 4.0 mg/kg) over time, fully inhibited tumor growth, keeping the tumor size (0.31 ± 0.049 mm3) similar to the initial size (0.27 ± 0.031 mm3, p = 0.66) while the control group tumor grew (1.03 ± 0.23 mm3, p = 0.023 to the drug-treated group). Nanoparticle (NP)-based local delivery of the RARγ agonist also inhibited the growth of osteochondromas at an early stage (Control: 0.52 ± 0.11 mm3; NP: 0.26 ± 0.10, p = 0.008). Transcriptome analysis revealed that the osteoarthritis pathway was activated in cultured chondrocytes treated with palovarotene (Z-score = 2.29), with the upregulation of matrix catabolic genes and the downregulation of matrix anabolic genes, consistent with the histology of palovarotene-treated osteochondromas. A reporter assay performed in cultured chondrocytes demonstrated that the Stat3 pathway, but not the Stat1/2 pathway, was stimulated by RARγ agonists. The activation of Stat3 by palovarotene was confirmed using immunoblotting and immunohistochemistry. These findings suggest that palovarotene treatment is effective against pre-existing osteochondromas and that the Stat3 pathway is involved in the antitumor actions of palovarotene.

Regulatory role of human fibrocartilage stem cells in condyle osteochondroma.

OBJECTIVE: Osteochondroma is a common benign skeletal disorder for which different molecular and histological features of long bones have been reported. We investigated cell-of-origin and molecular mechanisms of a rare condylar osteochondroma (CO). METHODS: Human fibrocartilage stem cells (hFCSCs) isolated from CO and normal condyle tissue were used for RNA sequencing, real-time PCR, Western Blotting, immunohistology, flowcytometry, as well as for chondrogenic differentiation, proliferation, and apoptosis detection assays. RESULTS: HFCSCs were fewer in number with weaker proliferative capacity and higher apoptosis ratio in the CO group. During the chondrogenic inducing process, hFCSCs from CO were prone to form more mature and hypertrophic cartilage. The result of RNA sequencing of hFCSCs from CO and normal condyle revealed a correlation between the PI3K/AKT signalling pathway and CO. Activated PI3K/AKT signalling might lead to functional changes in hFCSCs by enhancing cell apoptosis in the developmental process of CO. Increased expression of BCL2-like protein 11 (BIM) in CO tissue also supports this conclusion. Furthermore, the activation of the PI3K/AKT pathway in TMJ of mice induced histological disorder and increased apoptosis in condylar cartilage. CONCLUSION: We conclude that the activation of PI3K/AKT signalling in hFCSCs of CO suggests a new hypothesis for the cell-of-origin of human CO and another possible target to treat it.

Osteochondroma formation is independent of heparanase expression as revealed in a mouse model of hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a rare, pediatric disorder characterized by osteochondromas that form along growth plates and provoke significant musculoskeletal problems. HME is caused by mutations in heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. Seemingly paradoxically, osteochondromas were found to contain excessive extracellular heparanase (Hpse) that could further reduce HS levels and exacerbate pathogenesis. To test Hpse roles, we asked whether its ablation would protect against osteochondroma formation in a conditional HME model consisting of mice bearing floxed Ext1 alleles in Agr-CreER background (Ext1f/f ;Agr-CreER mice). Mice were crossed with a new global Hpse-null (Hpse-/- ) mice to produce compound Hpse-/- ;Ext1f/f ;Agr-CreER mice. Tamoxifen injection of standard juvenile Ext1f/f ;Agr-CreER mice elicited stochastic Ext1 ablation in growth plate and perichondrium, followed by osteochondroma formation, as revealed by microcomputed tomography and histochemistry. When we examined companion conditional Ext1-deficient mice lacking Hpse also, we detected no major decreases in osteochondroma number, skeletal distribution, and overall structure by the analytical criteria above. The Ext1 mutants used here closely mimic human HME pathogenesis, but have not been previously tested for responsiveness to treatments. To exclude some innate therapeutic resistance in this stochastic model, tamoxifen-injected Ext1f/f ;Agr-CreER mice were administered daily doses of the retinoid Palovarotene, previously shown to prevent ectopic cartilage and bone formation in other mouse disease models. This treatment did inhibit osteochondroma formation compared with vehicle-treated mice. Our data indicate that heparanase is not a major factor in osteochondroma initiation and accumulation in mice. Possible roles of heparanase upregulation in disease severity in patients are discussed.

Understanding the Action of RARγ Agonists on Human Osteochondroma Explants.

Osteochondromas are cartilage-capped growths located proximate to the physis that can cause skeletal deformities, pain, limited motion, and neurovascular impingement. Previous studies have demonstrated retinoic acid receptor gamma (RARγ) agonists to inhibit ectopic endochondral ossification, therefore we hypothesize that RARγ agonists can target on established osteochondromas. The purpose of this study was to examine the action of RARγ agonist in human osteochondromas. Osteochondroma specimens were obtained during surgery, subjected to explant culture and were treated with RARγ agonists or vehicles. Gene expression analysis confirmed the up-regulation of RARγ target genes in the explants treated with NRX 204647 and Palovarotene and revealed strong inhibition of cartilage matrix and increased extracellular matrix proteases gene expression. In addition, immunohistochemical staining for the neoepitope of protease-cleaved aggrecan indicated that RARγ agonist treatment stimulated cartilage matrix degradation. Interestingly, cell survival studies demonstrated that RARγ agonist treatment stimulated cell death. Moreover, RNA sequencing analysis indicates changes in multiple molecular pathways due to RARγ agonists treatment, showing similarly to human growth plate chondrocytes. Together, these findings suggest that RARγ agonist may exert anti-tumor function on osteochondromas by inhibiting matrix synthesis, promoting cartilage matrix degradation and stimulating cell death.

Knockdown of Ski decreases osteosarcoma cell proliferation and migration by suppressing the PI3K/Akt signaling pathway.

Ski, an evolutionary conserved protein, is involved in the development of a number of tumors, such as Barrett's esophagus, leukemia, colorectal cancer, gastric cancer, pancreatic cancer, hemangiomas and melanoma. However, studies on the functions of Ski in osteosarcoma (OS) are limited. In this study, firstly the differential expression of Ski in OS tissues and osteochondroma tissues was detected, and the expression of Ski in both human OS cell lines (MG63 and U2OS) and normal osteoblasts (hFoB1.19) was then detected. The results demonstrated that Ski expression was significantly upregulated in both human OS tissues and cell lines. The results led us to hypothesize that Ski may play an essential role in the pathological process of OS. Thus, Ski specific small interfere RNA (Ski­siRNA) was used. The results revealed that OS cell proliferation was markedly inhibited following the knockdown of Ski, which was identified by CCK8 assay, EdU staining and cell cycle analysis. In addition, OS cell migration was significantly suppressed following Ski knockdown, which was identified by wound healing assay. Moreover, the protein levels of p­PI3K and p­Akt in OS cells declined prominently following Ski knockdown. On the whole, the findings of this study revealed that Ski expression was significantly upregulated in OS tissue and OS cells. The knockdown of Ski decreased OS cell proliferation and migration, which was mediated by blocking the PI3K/Akt signaling pathway. Thus, Ski may act as a tumor promoter gene in tumorigenesis, and Ski may prove to be a potential therapeutic target for the treatment of OS.

Histopathological features of condylar hyperplasia and condylar Osteochondroma: a comparison study.

BACKGROUND: Both mandibular condylar hyperplasia and condylar osteochondroma can lead to maxillofacial skeletal asymmetry and malocclusion, although they exhibit different biological behavior. This study attempted to compare the histological features of mandibular condylar hyperplasia and condylar osteochondroma using hematoxylin-and-eosin (H&E) staining, and immunohistochemistry staining of PCNA and EXT1 with quantitative analysis method. RESULTS: The H&E staining showed that condylar hyperplasia and condylar osteochondroma could be divided into four histological types and exhibited features of different endochondral ossification stages. There was evidence of a thicker cartilage cap in condylar osteochondroma as compared condylar hyperplasia (P = 0.018). The percentage of bone formation in condylar osteochondroma was larger than was found in condylar hyperplasia (P = 0.04). Immunohistochemical staining showed that PCNA was mainly located in the undifferentiated mesenchymal layer and the hypertrophic cartilage layer, and there were more PCNA positive cells in the condylar osteochondroma (P = 0.007). EXT1 was mainly expressed in the cartilage layer, and there was also a higher positive rate of EXT1 in condylar osteochondroma (P = 0.0366). The thicker cartilage cap, higher bone formation rate and higher PCNA positive rate indicated a higher rate of proliferative activity in condylar osteochondroma. The more significant positive rate of EXT1 in condylar osteochondroma implied differential biological characteristic as compared to condylar hyperplasia. CONCLUSIONS: These features might be useful in histopathologically distinguishing condylar hyperplasia and osteochondroma.

Insights into the molecular regulatory network of pathomechanisms in osteochondroma.

Osteochondroma is a benign autosomal dominant hereditary disease characterized by abnormal proliferation of cartilage in the long bone. It is divided into solitary osteochondroma and hereditary multiple exostoses (HMEs). The exostosin-1 (EXT-1) and exostosin-2 (EXT-2) gene mutations are well-defined molecular mechanisms in the pathogenesis of HME. EXT-1 and EXT-2 encode glycosyltransferases that are necessary for the synthesis of heparin sulfate. Accumulating evidence suggests that mutations in the EXT family induce changes in isolated hypogonadotropic hypogonadism-parathyroid hormone-related protein, bone morphogenetic protein, and fibroblast growth factor signaling pathways. Studies have also found that a large number of microRNAs (miRNAs) are abnormally expressed in osteochondroma tissues, and some of them also participate in several major signaling pathways. The regulation of miRNA expression could be another breakthrough in the treatment of osteochondroma. Although the pathogenesis of osteochondroma is very complicated, significant progress has been made in recent years. It is hoped that the pathogenesis of osteochondroma will be clearly understood and the most effective methods for the prevention and treatment of osteochondroma will be determined. This review provides an update on the recent progress in the interpretation of the underlying molecular mechanisms of osteochondroma.

Differences in molecular regulation between osteochondroma and bizarre parosteal osteochondromatous proliferation.

The differences in molecular mechanisms between osteochondroma and bizarre parosteal osteochondromatous proliferation (BPOP) remain to be fully elucidated. In the present study, the differentially expressed genes between BPOP and osteochondroma were obtained from the Gene Expression Omnibus online database, and the associations among these genes were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online bioinformatics software. The results revealed several differentially expressed genes between human BPOP and osteochondroma. These differentially expressed genes were also enriched in different subgroups based on the analysis using DAVID online software, including 'transforming growth factor ß receptor signaling pathway', 'BMP signaling pathway', 'Wnt receptor signaling pathway', 'response to chemical stimulus', 'regulation of inflammatory response', 'response to stress', 'glycosaminoglycan binding', 'polysaccharide binding', 'extracellular matrix structural constituent' and 'growth factors binding'. Taken together, these findings led to the conclusion that different gene regulatory mechanisms exist between BPOP and osteochondroma. Environmental stimulation and inflammation may contribute to BPOP or osteochondroma, and differences in extracellular matrix may contribute to differences in biological characteristics between BPOP and osteochondroma.

Correlations Between Hector Battifora Mesothelial-1 (HBME-1) Expression and Clinical Pathological Characteristics and Prognosis of Osteosarcoma Patients.

BACKGROUND The aim of this study was to investigate the correlation between Hector Battifora mesothelial-1 (HBME-1) expression and the clinical pathological characteristics and prognosis of osteosarcoma (OS). MATERIAL AND METHODS HBME-1 expression was assessed using immunohistochemistry in OS tissues (n=152), osteochondroma tissues (n=91), and normal bone tissues (n=74). We carried out a follow-up lasting 8-60 months to investigate HBME-1 expression and its correlations with the clinical pathological characteristics and prognosis of OS. RESULTS HBME-1 was highly expressed in OS tissues compared with osteochondroma tissues and normal bone tissues, and was highly expressed in osteochondroma tissues compared with normal bone tissues (all P<0.05). HBME-1 expression was correlated with clinical stages, postoperative recurrence, metastasis, and 5-year survival (all P<0.05). The area under the receiver operating characteristics curve of HBME-1 expression was 0.864, with sensitivity of 80.92%, specificity of 91.89%, and accuracy of 84.51%. The survival rate was lower in the HBME-1 positive expression group than the HBME-1 negative expression group (P<0.05). Clinical stages, metastasis, and HBME-1 expression were independent risk factors for the survival of patients with OS (all P<0.05). CONCLUSIONS HBME-1 expression was correlated with the occurrence and development of OS. HBME-1 positive expression was a risk factor for the prognosis of OS.

Correlation between TRAIL and caspase-8 expression and their relationship with cell proliferation and apoptosis in human osteosarcoma.

Osteosarcoma is a common malignant bone tumor that mainly affects children and adolescents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily. Caspase-8 appears in the upstream of apoptosis signaling pathway among caspases. We investigated TRAIL and caspase-8 levels in osteosarcoma patients to determine their correlation with cell proliferation and apoptosis. Osteosarcoma and osteochondroma patients receiving surgery in our hospital were selected. TRAIL and caspase-8 expression levels in tissue were determined by immunohistochemistry, and protein levels in cells were evaluated by western blotting. Human osteosarcoma cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The osteosarcoma and osteochondroma cell cycles and apoptosis were investigated by flow cytometry. Correlation analysis was applied to TRAIL and caspase-8 levels during cell apoptosis. Positive TRAIL and caspase-8 expression rates in osteosarcoma tissue were significantly lower than in the controls (P < 0.05). TRAIL (0.114 ± 0.002) and caspase-8 (0.352 ± 0.124) levels in experimental cells were obviously lower than in the controls (P < 0.05). Osteosarcoma cells in the experimental group demonstrated higher proliferation and lower apoptosis at 24, 48, and 72 h (P < 0.05). The experimental cell number increased in the G1 stage and decreased in the S stage (P < 0.05). TRAIL and caspase-8 proteins showed positive correlation with apoptosis in osteosarcoma (P < 0.05). Human osteosarcoma presented reduced TRAIL and caspase-8 levels with enhanced cell proliferation and reduced apoptosis. TRAIL and caspase-8 expression levels were positively correlated with apoptosis in osteosarcoma.

The clinical pathological significance of Thy1 and CD49f expression in chondrosarcomas.

OBJECTIVE: This study investigated the protein expression and clinicopathological significance of Thy1 and CD49f in chondrosarcomas. METHODS: Thy1 and CD49f protein expression in 59 chondrosarcomas and 33 osteochondromas were measured by immunohistochemical staining. RESULTS: The percentage of positive Thy1 and CD49f expression was significantly higher in patients with chondrosarcoma than in patients with osteochondroma (P<0.01). The percentage of positive Thy1 and CD49f expression was significantly lower in patients with histological grade I, Enneking stage I, AJCC stage I/II stage, non-metastatic and non-invasive chondrosarcoma than in patients with histological grade III, Enneking stage II+III, AJCC stage III/IV, metastatic and invasive chondrosarcoma (P<0.05 or P<0.01). Thy1 expression was positively correlated with CD49f expression in chondrosarcoma. Kaplan-Meier survival analysis showed that histological grade, AJCC stage, Enneking stage, metastasis, invasion, and Thy1 and CD49f expression significantly correlated with shorter mean survival time in chondrosarcoma patients (P<0.05 or P<0.01). Cox multivariate analysis showed that positive Thy1 and CD49f expression was an independent prognostic factor that negatively correlated with overall postoperative survival. CONCLUSION: Positive Thy1 and CD49f expression is significantly associated with the progression and poor prognosis of chondrosarcoma.

Bizarre parosteal osteochondromatous proliferation in the lingual area of the mandibular body versus osteochondroma at the mandibular condyle.

BACKGROUND: Bizarre parosteal osteochondromatous proliferation (BPOP) is benign and usually occurs in the small tubular bones of the hands and feet, but it is extremely rare in the oral and maxillofacial region. METHODS: The present study compares a case of BPOP occurring in the lingual area of the right mandibular body with a representative case of osteochondroma occurring in the left mandibular condyle using immunohistochemical methods. RESULTS: BPOP showed no continuity to the cortical bone of the mandible on X-ray and was histologically composed of immature cartilage and bone tissues, whereas osteochondroma showed overgrowth of hypertrophic chondrocytes accompanied by mature bone with endochondral ossification. Although BPOP showed no features of cellular atypia or malignant transformation, it expressed more osteogenic proteins, including BMP-2, BMP-4, RUNX2, OC, AP, OPG, RANKL, CTGF, and bFGF, than osteochondroma. Furthermore, the perichondral spindle cells and marrow osteoblasts/fibroblasts of BPOP showed stronger immunoreaction of PCNA, p53, ß-catenin, BCL2, pAKT, survivin, 14-3-3, CEA, EMA, pan-K, and S-100 than the tumor cells of osteochondroma. CONCLUSIONS: Therefore, it was presumed that similar to embryonal osteochondroid tissue, BPOP might be activated by osteogenic and oncogenic signaling and that this increased signaling may explain the rapid growth and high recurrence of BPOP.

The prognostic significance of Src and p-Src expression in patients with osteosarcoma.

BACKGROUND: The expression of Src and phospho-Src (p-Src) is closely related to tumor invasion and metastasis. The aim of the present study was to investigate the expression of these molecules in osteosarcoma and their relationship with each other, to provide a theoretical basis to understand the prognosis of osteosarcoma. MATERIAL AND METHODS: We selected surgically resected osteosarcoma specimens from 116 patients of Zhongnan Hospital of Wuhan University and Hubei Cancer Hospital, Hubei, China, between January 2000 and January 2010 with detailed follow-up data. Twenty osteochondroma specimens from the corresponding period were used as controls. Expression of Src and p-Src was detected in osteosarcoma and osteochondroma by immunohistochemistry. We analyzed the relationship of the 2 proteins and osteosarcoma patient prognosis. RESULTS: The expression of Src and p-Src in osteosarcoma was significantly higher than the expression level in osteochondroma (P<0.05). The expression levels of the 2 proteins, clinical stage, and tumor metastasis were significantly associated with survival time (P<0.05), but there was no correlation between age or sex and survival time. The expression of Src and p-Src in osteosarcoma was positively correlated. CONCLUSIONS: Src and p-Src can be used as an auxiliary indicator to determine a malignant phenotype of bone tumors, and the combined detection of Src and p-Src may indicate the prognosis of osteosarcoma.

The type 2 diabetes associated rs7903146 T allele within TCF7L2 is significantly under-represented in Hereditary Multiple Exostoses: insights into pathogenesis.

Hereditary Multiple Exostoses (HME) is an autosomal-dominant disorder characterized by benign cartilage tumors (exostoses) forming near the growth plates, leading to severe health problems. EXT1 and EXT2 are the two genes known to harbor heterozygous loss-of-function mutations that account for the vast majority of the primary genetic component of HME. However, patients present with wide clinical heterogeneity, suggesting that modifier genes play a role in determining severity. Our previous work has pointed to an imbalance of ß-catenin signaling being involved in the pathogenesis of osteochondroma formation. TCF7L2 is one of the key 'gate-keeper' TCF family members for Wnt/ß-catenin signaling pathway, and TCF7L2 and EXT2 are among the earliest associated loci reported in genome wide appraisals of type 2 diabetes (T2D). Thus we investigated if the key T allele of single nucleotide polymorphism (SNP) rs7903146 within the TCF7L2 locus, which is strongly over-represented among T2D cases, was also associated with HME. We leveraged genotype data available from ongoing GWAS efforts from genomics and orthopedic centers in the US, Canada and Italy. Collectively 213 cases and 1890 controls were analyzed and, surprisingly, the T allele was in fact significantly under-represented in the HME patient group [P = 0.009; odds ratio = 0.737 (95% C.I. 0.587-0.926)]; in addition, the direction of effect was consistent within each individual cohort. Immunohistochemical analyses revealed that TCF7L2 is differentially expressed and distributed in normal human growth plate zones, and exhibits substantial variability in human exostoses in terms of staining intensity and distribution. In summary, the data indicate that there is a putative genetic connection between TCF7L2 and EXT in the context of HME. Given this observation, we suggest that these loci could possibly modulate shared pathways, in particular with respect to ß-catenin, and their respective variants interplay to influence HME pathogenesis as well as T2D.

The clinical pathological significance of FRAT1 and ROR2 expression in cartilage tumors.

PURPOSE: Chondrosarcoma is a malignant bone tumor with poor prognosis. Surgical treatment is the first choice for chondrosarcomas. Chondrosarcoma is not sensitive to chemotherapy and radiotherapy. Identification of biological markers is important for the early diagnosis and targeted treatment of chondrosarcoma. This study investigated the protein expression and clinicopathological significance of ROR2 and FRAT1 in 59 chondrosarcomas and 33 osteochondromas. METHODS: ROR2 and FRAT1 protein expression in tissues was measured by immunohistochemistry. RESULTS: The percentage of positive ROR2 and FRAT1 expression was significantly higher in patients with chondrosarcoma than in patients with osteochondroma (P < 0.01). The percentage of positive ROR2 and FRAT1 expression was significantly lower in patients with histological grade I, AJCC stage I/II stage, Enneking stage I, non-metastatic and invasive chondrosarcoma than patients with histological grade III, AJCC stage III/IV, Enneking stage II + III, metastatic and invasive chondrosarcoma (P < 0.05 or P < 0.01). ROR2 expression was positively correlated with FRAT1 expression in chondrosarcoma. Kaplan-Meier survival analysis demonstrated that histological grade, AJCC stage, Enneking stage, metastasis, invasion, and ROR2 and FRAT1 expression significantly correlated with a short mean survival time of patients with chondrosarcoma (P < 0.05 or P < 0.01). Cox multivariate analysis showed that positive ROR2 and FRAT1 expression was an independent prognostic factor that negatively correlated with postoperative survival and positively correlated with mortality. CONCLUSION: Positive ROR2 and FRAT1 expression is associated with the progression and poor prognosis of chondrosarcoma.

High expression of periostin is dramatically associated with metastatic potential and poor prognosis of patients with osteosarcoma.

BACKGROUND: Recent studies have found that periostin (PN), as a kind of secreted glycoprotein, is closely related to the metastatic potential and prognosis of many kinds of tumors. This study aimed to examine the expression of PN in patients with osteosarcoma and explore the relationship of PN expression with clinicopathologic factors and prognosis. METHODS: PN was detected by histopathological and immunohistochemical methods in 62 cases of osteosarcoma and 62 of osteochondroma. Detailed pathological and clinical data were collected by reviewing medical records. RESULTS: The results showed that increased PN protein expression was prevalent in osteosarcoma and was significantly associated with pathologic subtype (P =0.000), tumor size (P =0.016) and Enneking stage (P =0.047). Additionally, expression of PN was found to be an independent prognostic factor in osteosarcoma patients. High expression of PN protein is closely correlated to the tumor progression and poor survival of osteosarcoma. CONCLUSIONS: Our data suggest that PN is a promising biomarker for identifying individuals with poor prognostic potential and suggests its possible use as a prognostic marker in patients with osteosarcoma.

Immunohistochemical Localization of Bone Morphogenetic Proteins (BMPs) and their Receptors in Solitary and Multiple Human Osteochondromas.

The expression of bone morphogenetic proteins (BMPs) and their cognate receptors (BMPRs) in osteochondromas has not been investigated. We determined the immunohistochemical localization and distribution of BMP-2/4, -6 and -7; BMP receptors BMPR-1A, BMPR-1B and BMPR-2; signal transducing proteins phosphorylated Smad1/5/8; and BMP antagonist noggin in the cartilaginous cap of solitary (SO) and multiple (MO) human osteochondromas and compared these with bovine growth plate and articular cartilage. The distribution and localization patterns for BMP-6, BMP-7, BMPR-1A and BMPR-2 were similar between the cartilaginous cap and the growth plate. BMP-2/4 and BMPR-1B were present throughout the growth plate. However, BMP-2/4 and phosphorylated Smad1/5/8 were mainly detected in proliferating chondrocytes of the cartilaginous cap. Also, BMPR-1B was found in hypertrophic chondrocytes of SO and proliferating chondrocytes of MO. Noggin was observed in resting chondrocytes and, to a lesser extent, in clustered proliferating chondrocytes in SO. On the other hand, noggin in MO was observed in proliferating chondrocytes. Since BMPs can stimulate proliferation and hypertrophic differentiation of chondrocytes, these findings suggest that there is an imbalance of BMP-2/4 and noggin interactions that may lead to abnormal regulation of chondrocyte proliferation and differentiation in the cartilaginous cap of human osteochondromas.

Heparan sulfate as a regulator of endochondral ossification and osteochondroma development.

Most elements of the vertebrate skeleton are formed by endochondral ossification. This process is initiated with mesenchymal cells that condense and differentiate into chondrocytes. These undergo several steps of differentiation from proliferating into hypertrophic chondrocytes, which are subsequently replaced by bone. Chondrocyte proliferation and differentiation are tightly controlled by a complex network of signaling molecules. During recent years, it has become increasingly clear that heparan sulfate (HS) carrying proteoglycans play a critical role in controlling the distribution and activity of these secreted factors. In this review we summarize the current understanding of the role of HS in regulating bone formation. In human, mutations in the HS synthetizing enzymes Ext1 and Ext2 induce the Multiple Osteochondroma syndrome, a skeletal disorder characterized by short stature and the formation of benign cartilage-capped tumors. We review the current insight into the origin of the disease and discuss its possible molecular basis. In addition, we summarize the existing insight into the role of HS as a regulator of signal propagation and signaling strength in the developing skeleton.

Loss of ß-catenin induces multifocal periosteal chondroma-like masses in mice.

Osteochondromas and enchondromas are the most common tumors affecting the skeleton. Osteochondromas can occur as multiple lesions, such as those in patients with hereditary multiple exostoses. Unexpectedly, while studying the role of ß-catenin in cartilage development, we found that its conditional deletion induces ectopic chondroma-like cartilage formation in mice. Postnatal ablation of ß-catenin in cartilage induced lateral outgrowth of the growth plate within 2 weeks after ablation. The chondroma-like masses were present in the flanking periosteum by 5 weeks and persisted for more than 6 months after ß-catenin ablation. These long-lasting ectopic masses rarely contained apoptotic cells. In good correlation, transplants of ß-catenin-deficient chondrocytes into athymic mice persisted for a longer period of time and resisted replacement by bone compared to control wild-type chondrocytes. In contrast, a ß-catenin signaling stimulator increased cell death in control chondrocytes. Immunohistochemical analysis revealed that the amount of detectable ß-catenin in cartilage cells of osteochondromas obtained from hereditary multiple exostoses patients was much lower than that in hypertrophic chondrocytes in normal human growth plates. The findings in our study indicate that loss of ß-catenin expression in chondrocytes induces periosteal chondroma-like masses and may be linked to, and cause, the persistence of cartilage caps in osteochondromas.