Estrogen signaling is active in cartilaginous tumors: implications for antiestrogen therapy as treatment option of metastasized or irresectable chondrosarcoma.

PURPOSE: Chondrosarcoma is a malignant cartilaginous matrix-producing tumor that can be lethal in 10% to 50% of the patients. Surgery is the only effective treatment known as these tumors are notorious refractory to all types of conventional chemotherapy or radiotherapy. To identify a target for therapy, we want to determine whether estrogen signaling is active in chondrosarcoma because estrogen is important in the regulation of longitudinal growth that is initiated by chondrocyte proliferation and differentiation in the epiphyseal growth plate of long bones. EXPERIMENTAL DESIGN: We studied protein expression of the estrogen receptor in 35 cartilaginous tumors as well as mRNA levels for the estrogen receptor and for aromatase, an enzyme for estrogen synthesis and another potential therapeutic target. Furthermore, the activity of aromatase was determined in vitro by the tritiated water release assay. Dose-response experiments with chondrosarcoma cultured cells were done with estrogen, androstenedione, and exemestane. RESULTS: All chondrosarcomas tested showed mRNA and nuclear protein expression of the estrogen receptor. Also, aromatase mRNA was detected. The aromatase activity assay showed a functional aromatase enzyme in primary chondrosarcoma cultures and in a cell line. Growth of chondrosarcoma cell cultures can be stimulated by adding estrogen or androstenedione, which can be inhibited by exemestane. CONCLUSIONS: These results show, on the RNA, protein, and cell biological levels, that the ligand and the receptor are active in estrogen-mediated signal transduction. This observation implicates potential use of targeted drugs that interfere with estrogen signaling, such as those applied for treating breast cancer.     

Tyrosine kinase inhibitor SU6668 represses chondrosarcoma growth via antiangiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Background  

As chondrosarcomas are resistant to chemotherapy and ionizing radiation, therapeutic options are limited. Radical surgery often cannot be performed. Therefore, additional therapies such as antiangiogenesis represent a promising strategy for overcoming limitations in chondrosarcoma therapy. There is strong experimental evidence that SU6668, an inhibitor of the angiogenic tyrosine kinases Flk-1/KDR, PDGFRbeta and FGFR1 can induce growth inhibition of various primary tumors. However, the effectiveness of SU6668 on malignant primary bone tumors such as chondrosarcomas has been rarely investigated. Therefore, the aim of this study was to investigate the effects of SU6668 on chondrosarcoma growth, angiogenesis and microcirculation in vivo.     

The effects of histone deacetylase inhibitors on the induction of differentiation in chondrosarcoma cells.

PURPOSE: Histologically, chondrosarcomas represent the degree of chondrogenic differentiation, which is associated with the prognosis of the disease. Histone acetylation and deacetylation play key roles in the regulation of chondrocytic differentiation. Here, we describe the antitumor effects of histone deacetylase (HDAC) inhibitors as differentiating reagents on chondrosarcomas. EXPERIMENTAL DESIGN: We examined the effects of a HDAC inhibitor, depsipeptide, on the growth of chondrosarcoma cell lines. We also investigated the modulation of the expression levels of extracellular matrix genes and the induction of phenotypic change in chondrosarcoma cells treated with depsipeptide. Finally, we examined the antitumor effect of depsipeptide on chondrosarcoma in vivo. RESULTS: Depsipeptide inhibited the growth of chondrosarcoma cells by inducing cell cycle arrest and/or apoptosis. HDAC inhibitors increased the expression of the alpha1 chain of type II collagen (COL2A1) gene due to the enhanced histone acetylation in the promoter and enhancer. Depsipeptide also up-regulated the expressions of aggrecan and the alpha2 chain of type XI collagen (COL11A2) mRNA in a dose-dependent manner. Moreover, long-term treatment with a low dose of depsipeptide resulted in the induction of differentiation into hypertrophic phenotype, as shown by the increment of the alpha1 chain of type X collagen (COL10A1) expression in chondrosarcoma cells. In vivo studies and histologic analyses confirmed that depsipeptide significantly inhibited tumor growth and induced differentiation into the hypertrophic and mineralized state in chondrosarcoma cells. CONCLUSIONS: These results strongly suggest that HDAC inhibitors may be promising reagents for use as a differentiating chemotherapy against chondrosarcomas.     

Rb-loss is associated with high malignancy in chondrosarcoma

Loss of function of the human retinoblastoma gene (Rb) is a frequent genetic abnormality in human malignancies and causes a disturbance in the cell cycle and loss of normal proliferation and differentiation. We studied the loss of heterozygosity (LOH) of the Rb gene in 31 formalin-fixed, paraffin-embedded cartilaginous tumors using polymerase chain reaction. The tumors were subdivided into 8 cases of dedifferentiated (DD) chondrosarcoma, 17 cases of conventional chondrosarcoma (nine grade 1, seven grade 2 and one grade 3), 4 enchondromas and 2 chondroblastomas. Both components of DD chondrosarcoma, the low-grade and anaplastic components, were separated by a microdissection approach. The genetic data were correlated with the expression of the Rb protein examined by Rb immunohistochemistry. We found Rb-LOH in one grade 3 chondrosarcoma, and in the anaplastic component in 7 of 8 cases of DD chondrosarcoma (89% of all high-grade chondrosarcomas). All tumors with Rb-LOH were immunohistochemically Rb-negative. The only case of DD chondrosarcoma negative for Rb-LOH in both components of the tumor also showed weak expression of the Rb protein in the anaplastic component. All benign cartilaginous tumors, low-grade chondrosarcomas and low-grade tumor components of DD chondrosarcomas were negative regarding Rb-LOH but positive in Rb immunohistostaining. We concluded that Rb-LOH predominantly occurs in high-grade chondrosarcomas. However, it is not a marker for identifying low-grade tumors with a tendency towards progression or local recurrence.     

Inhibition of EGFR-induced glucose metabolism sensitizes chondrosarcoma cells to cisplatin

Chondrosarcomas are malignant cartilage-forming tumors which are resistant to conventional chemotherapy and radiotherapy. By searching in Oncomine which is a cancer microarray database and web-based data mining platform, we found Glut1 and LDHA were upregulated in human chondrosarcoma patient samples. In this study, we reported total epidermal growth factor receptor (EGFR) expression and phosphorylated EGFR were highly activated in human chondrosarcoma cell lines. In addition, overexpression of EGFR contributed to cisplatin resistance. EGFR promoted glucose metabolism of chondrosarcoma cells through the upregulation of glycolysis key enzymes. Interestingly, cisplatin-resistant chondrosarcoma cells showed upregulated glucose metabolism and EGFR signaling pathway. Finally, we demonstrated that the combination of either EGFR inhibitor or anaerobic glycolysis inhibitor with cisplatin showed synergistically inhibitory effects on cisplatin-resistant chondrosarcoma cells through the inducements of apoptosis and cell cycle arrest. Our project proposed a novel function of EGFR in the regulation of glucose metabolism in chondrosarcoma cells and contributed to the development of therapeutic strategies for the clinical treatment of chondrosarcoma patient.     

MYC amplification and polysomy 8 in chondrosarcoma: array comparative genomic hybridization, fluorescent in situ hybridization, and association with outcome.

PURPOSE: To identify recurrent regions of genomic gain or loss in chondrosarcoma in a clinically relevant and statistically valid fashion. Materials and METHODS: Array comparative genomic hybridization (CGH) results of 15 frozen tumor samples of high-grade chondrosarcoma for chromosome 8 are presented. A separate subset of 116 cartilaginous tumors with outcome data was used for validation. RESULTS: Array CGH identified gain at 8q24.12-q24.13, the region of the MYC (c-Myc) oncogene, as a frequent change in high-grade chondrosarcoma. In the validation arm of 116 cartilaginous tumors, MYC was frequently amplified in G2 (15%), G3 (20%), and dedifferentiated (21%) chondrosarcomas. No amplification was identified in samples of enchondroma and grade 1 chondrosarcoma. In samples without MYC amplification, polysomy 8 was a frequent finding in grade 1 (18%), grade 2 (31%), grade 3 (80%), and dedifferentiated (29%) chondrosarcomas, but was not found in any samples of enchondroma. MYC protein expression was identified in all samples with amplification, but was also frequent in the remaining samples without amplification or polysomy 8. Kaplan-Meier survival curves for overall survival showed a statistically significant difference for patients with MYC amplification or polysomy 8 (P = .034). Univariate analysis involving Cox proportional hazards models showed that grade (P = .003), polysomy 8 (P = .045), and MYC amplification (P = .053) correlated with shorter overall survival. By multivariate analysis, grade of chondrosarcoma (P = .026) was the only factor to reach statistical significance. CONCLUSION: MYC amplification and polysomy 8 can be used as markers of prognostic importance in chondrosarcoma. Molecular targeting of MYC expression may have therapeutic potential in the future for subsets of chondrosarcoma.     

Endogenous secretory receptor for advanced glycation endproducts as a novel prognostic marker in chondrosarcoma

BACKGROUND: Chondrosarcoma, the second most frequent primary malignant bone tumor, is classified into 3 grades according to histologic criteria of malignancy. However, a low-grade lesion can be difficult to distinguish from a benign enchondroma, whereas some histologically low-grade lesions may carry a poor prognosis. The receptor for advanced glycation endproducts (RAGE) and its ligand, high-mobility group box-1 (HMGB1), was quantified in enchondromas and chondrosarcomas to determine whether these markers were associated with histological malignancy and prognosis. METHODS: Enchondromas (n = 20) and typical chondrosarcomas (n = 39) were evaluated for RAGE, endogenous secretory RAGE (esRAGE, a splice variant form), and HMGB1 protein expression by immunohistochemistry including laser confocal microscopy. The content of esRAGE in resected specimens was measured with an enzyme-linked immunosorbent assay. Associations of these molecules with histology and clinical behavior of tumors were analyzed. RESULTS: Expression of esRAGE and HMGB1 was observed in all specimens. The numbers of cells positive for esRAGE and HMGB1 expression were positively associated with histologic grade. Expression of esRAGE was significantly higher in chondrosarcomas than in enchondromas (P < .001). Tissue esRAGE content was also significantly higher in grade 1 and 2 chondrosarcomas than enchondromas (P = .0255 and P = .008, respectively). High expression of esRAGE in grade 1 chondrosarcoma was associated with subsequent recurrence (P = .0013), lung metastasis (P = .0071), and poor survival (P < .001). CONCLUSIONS: Assessment of esRAGE expression should aid in diagnostic and prognostic determinations in chondrosarcoma.     

Genetic alterations in chondrosarcomas – keys to targeted therapies?

Background

Chondrosarcomas are malignant tumors of chondrocytes and represent the second most common type of primary bone tumors. Within the context of normal chondrogenesis, this review summarizes results from recent research outlining the key molecular changes that occur during the development of this sarcoma type.

Results

Current data support the notion that a two-hit scenario, common to many tumors, also underlies chondrosarcoma formation. First, early-stage mutations alter the normal proliferation and differentiation of chondrocytes, thereby predisposing them to malignant transformation. These early-stage mutations, found in both benign cartilaginous lesions and chondrosarcomas, include alterations affecting the IHH/PTHrP and IDH1/IDH2 pathways. As they are not observed in malignant cells, mutations in the EXT1 and EXT2 genes are considered early-stage events providing an environment that alters IHH/PTHrP signaling, thereby inducing mutations in adjacent cells. Due to normal cell cycle control that remains active, a low rate of malignant transformation is seen in benign cartilaginous lesions with early-stage mutations. In contrast, late-stage mutations, seen in most malignant chondrosarcomas, appear to induce malignant transformation as they are not found in benign cartilaginous lesions. These late-stage mutations primarily involve cell cycle pathway regulators including p53 and pRB, two genes that are also known to be implicated in numerous other human tumor types.

Conclusions

Now the key genetic alterations involved in both early and late stages of chondrosarcoma development have been identified, focus should be shifted to the identification of druggable molecular targets for the design of novel chondrosarcoma-specific therapies.     

By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility,invasion and lung colonization

Interstitial fluid flow and associated shear stress are relevant mechanical signals in cartilage and bone (patho)physiology. However, their effects on chondrosarcoma cell motility, invasion and metastasis have yet to be delineated. Using human SW1353, HS.819.T and CH2879 chondrosarcoma cell lines as model systems, we found that fluid shear stress induces the accumulation of cyclic AMP (cAMP) and interleukin-1β (IL-1β), which in turn markedly enhance chondrosarcoma cell motility and invasion via the induction of matrix metalloproteinase-7 (MMP-7). Specifically, shear-induced cAMP and IL-1β activate PI3-K, ERK1/2 and p38 signaling pathways, which lead to the synthesis of MMP-7 via transactivating NF-κB and c-Jun in human chondrosarcoma cells. Importantly, MMP-7 upregulation in response to shear stress exposure has the ability to promote lung colonization of chondrosarcomas in vivo. These findings offer a better understanding of the mechanisms underlying MMP-7 activation in shear-stimulated chondrosarcoma cells, and provide insights on designing new therapeutic strategies to interfere with chondrosarcoma invasion and metastasis.     

Sox9 在人普通型与去分化型软骨肉瘤差异表达*

目的:探讨Sox9 在人普通软骨肉瘤,去分化软骨肉瘤和正常软骨中的表达。方法:取12例2003年1 月至2007年1 月在北京大学人民医院经病理切片确诊为软骨肉瘤（普通软骨肉瘤6 例,去分化软骨肉瘤6 例）及正常软骨组织（6 例）的新鲜冰冻标本,利用基因芯片的方法分析后,将Sox9 作为候选基因,而后利用Real-time PCR,Western blot和免疫组化的方法比较其在人普通软骨肉瘤,去分化软骨肉瘤和正常软骨组织中的差异表达。结果:基因芯片结果显示:与正常软骨组织相比,Sox9 在普通软骨肉瘤中上调约1.6 倍,在去分化软骨肉瘤中,其表达水平为正常软骨组织表达水平的0.082 倍;Real-time PCR检测结果显示:Sox9mRNA 在普通软骨肉瘤和去分化软骨肉瘤中表达水平分别为1.68± 0.119 和0.088 ± 0.017;Western blot分析表明Sox9 蛋白在人普通软骨肉瘤的表达量明显高于正常软骨组织,在去分化软骨肉瘤中的表达量显著低于正常软骨组织。免疫组化实验结果表明与正常软骨组织相比,6 例普通软骨肉瘤均有Sox9 表达,细胞染色呈较强的阳性,而去分化软骨肉瘤未发现较强阳性细胞出现,只有可疑的阳性细胞散在出现。结论:Sox9 在普通软骨肉瘤中的表达水平明显高于正常软骨组织,在去分化软骨肉瘤中的表达水平显著低于正常软骨组织。Sox9 在去分化软骨肉瘤中低水平表达与去分化软骨肉瘤疾病进展快,预后差呈正向相关。      

Constitutive activation of insulin receptor substrate 1 is a frequent event in human tumors: therapeutic implications

Insulin receptor substrate 1 (IRS-1) is a major substrate of insulin, insulin-like growth factors, and cytokine signaling and plays an important role in mediating apoptosis, cell differentiation, and cell transformation. We found that IRS-1 is constitutively activated in a variety of solid tumors, including breast cancers, leiomyomas, Wilms' tumors, rhabdomyosarcomas, liposarcomas, leiomyosarcomas, and adrenal cortical carcinomas. Blocking the constitutively activated IRS-1 signaling in breast cancer cells with a dominant-negative IRS-1, an IRS-1 with all 18 potential tyrosine-phosphorylation sites replaced by phenylalanines (F18), dramatically reduced cancer cell growth. Breast cancer cells that expressed F18 also formed smaller and far fewer colonies in soft agar culture than did the cells that did not express F18. These studies suggest that constitutive IRS-1 activation is a common phenomenon in tumors and that activated IRS-1 may present an attractive therapeutic target.     

Central nervous system mesenchymal chondrosarcoma

Central nervous system mesenchymal chondrosarcomas are rare malignant tumors that constitute a separate entity from the classical chondrosarcoma and myxoid variant. Clinical behaviour of central nervous system chondrosarcomas is still unknown. We describe two rare examples of intracranial mesenchymal chondrosarcoma with a review of the literature, in an attempt to clarify the clinical characteristics, prognosis and treatment of choice of these unusual tumors. Among the 55 reported cases, 23 had postoperative radiotherapy. Although there is no statistical significance according to the Log-Rank test (p=0.7), the patients treated with radiation therapy seem to have a better chance of survival. Patients who had adjuvant chemotherapy (only 5) showed survival times similar to those patients who had none. Although clinical behaviour of central nervous system chondrosarcomas remains to be defined, data from our series as well as literature show that radical removal is the best therapeutic choice. In addition, patients treated with postoperative radiotherapy seem to show a trend toward increased survival.     

Clear-cell chondrosarcoma: a report of five cases including ultrastructural study.

Five cases of clear-cell variant of chondrosarcoma (Unni et al.) are reported. The tumors occurred in the epiphyseal region of long bones; three in the femoral head. Roentgenographically, the lesion was usually a well-defined and benign appearing one, either purely lytic (3 cases) or with central radiodensity (2 cases). Histologically, all five cases had areas of conventional chondrosarcoma; however, the greater portion of the tumor was made up of sheets of clear-cells intermixed with nonneoplastic bone trabeculae but devoid of chondroid matrix. Electron microscopic studies showed that these clear-cells possess cytoplasmic microvilli, abundant glycogen particles and prominent golgi complexes, like normal or tumorous chondroid cells usually have. In our experience, the best treatment seemed to be en bloc resection with joint replacement; indeed, despite the fact that they are true chondrosarcomas, these tumors usually have a very slow rate of growth.     

siRNA-based targeting of antiapoptotic genes can reverse chemoresistance in P-glycoprotein expressing chondrosarcoma cells

Background  

High expression of P-glycoprotein is one of the well-known mechanisms of chemoresistance in chondrosarcomas. However, the role of antiapoptotic proteins, a common mechanism responsible for chemoresistance in other tumors, has not been well studied in chondrosarcomas. We examined the importance of P-glycoprotein and antiapoptotic proteins in the chemoresistance to doxorubicin of two Grade II chondrosarcoma cell lines, JJ012 and SW1353.     

Versican V1 isoform regulates cell-associated matrix formation and cell behavior differentially from aggrecan in Swarm rat chondrosarcoma cells

Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and is composed of large extracellular matrix aggregates, has been shown to correlate with tumor progression. No studies have examined the roles of versican in chondrosarcoma nor compared them to those of aggrecan. In clinical specimens of human chondromatous tumors, versican expression was significantly increased in malignant tumors, moreover, as the tumor grade increased. To clarify the roles of versican in chondrosarcoma, versican splicing variant 1, variant 3 or only GFP was stably transfected to Swarm rat chondrosarcoma cells with Trap-In System. Forced expression of versican V1 isoform in Swarm rat chondrosarcoma cells induced a marked increase of cell-associated matrix compared to V3-, GFP- transfected or RCS cells. Versican was immunolocalized in a fashion similar to that of hyaluronan and more diffusively than aggrecan. Anchor-dependent and -independent growth was not affected by versican isoform expression, whereas cell motility and migration were significantly enhanced by V1 isoform transfection. Tumors formed in vivo with V1-transfected cells exhibited more myxomatous area and included more spindle shaped cells. These results support the concept that versican has the capacity to form more extensive cell-associated matrix than aggrecan, and the prominent matrix formation alters the cell behavior of chondrosarcoma more aggressively. These observations suggest that versican expression may serve as a marker of tumor grade determination in chondrosarcoma and possibly help to decide on therapeutic targets in higher grades of chondrosarcoma.     

Current management of advanced non-small cell lung cancer: targeted therapy

Lung cancer is one of the most frequent causes of cancer-related death in the United States. For patients with advanced non-small cell lung cancer (NSCLC), chemotherapy, alone or in combination with radiation therapy, is considered the standard treatment. Although this treatment may result in a modest improvement in patient survival, overall prognosis of these patients remains dismal, and the treatment is nonspecific, nonselective, and toxic. Therefore, new therapeutic strategies are needed. During the past decade, several molecules that contribute to lung cancer progression and metastasis have been identified. Growth factors and proangiogenic factors have been the focus of intense research in cancer since therapeutic approaches for their inhibition do exist. The role of these factors was studied in different organs and tumors and was found to be phenotypically distinct. Several molecular targeted therapies have shown efficacy and had been approved for treatment of specific cancers. Most advanced in clinical research for lung cancer are targeted therapies that inhibit the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) signaling pathways. Others are signaling pathway inhibitors. The first targeted therapy for lung cancer is gefitinib, an EGFR inhibitor, which was approved in several countries in 2003. Goals of molecular targeted therapy studies include the following: better understanding of the exact role of particular growth factors in specific tumors; establishment of new clinical study designs for biological agents; and tailoring appropriate combinations of conventional chemotherapy and/or radiotherapy with biological therapy for specific patients. Achievement of these goals will hopefully lead to incorporation of biological therapy into the current anticancer arsenal, for the benefit of lung cancer patients.     

Histochemical characterization of mucosubstances in bone and soft tissue-tumors.

The present investigation endeavors to characterize the mucosubstance content of 170 myxoid and chondromatous tumors and chordomas by histochemical methods. The results obtained using the critical electrolyte concentration (CEC) method as introduced by Scott and co-workers23,24 were compared with those obtained by staining with alcian blue and toluidine blue at different pH's with and without pretreatment with bovine testicular hyaluronidase. Tissues known biochemically to contain different heteroglycans were used as controls: synovial fluid and cock's comb (hyaluronic acid) stained with alcian blue up to a MgCl2 concentration of 0.1 M; fetal cartilage (chondroitin 4- and 6-sulphate) pulposus with notochordal remnants (keratan sulphate) up 10 1.0 M. The staining reaction of intramuscular myxoma and myxoid liposarcoma corresponded to that of synovial fluid and cock's comb (containing hyaluronic acid). Benign chondromatous tumors (osteochondroma, enchondroma, extraskeletal chondroma, chondromatosis in bursae, synovia, and tendon) as well as well-differentiated chondrosarcomas had a similar staining reaction to that of adult cartilage (containing keratan sulphate). However, the intensity of the reaction was lower in these tumors than in the adult cartilage, indicating that the keratan sulphate content of the tumors is lower. Most of the moderately well-differentiated chondrosarcomas, the poorly differentiated chondrosarcomas, and pulmonary metastases of chondrosarcoma, as well as mesenchymal chondrosarcoma and extra-skeletal chondrosarcoma possessed the same staining properties as fetal cartilage, known to contain chondroitin 4- and 6-sulphate but not keratan sulphate. A few of the moderately well-differentiated chondrosarcomas stained up to a MgCl2 concentration of 1.0 M. Three cases of poorly differentiated chondrosarcomas stained with alcian blue up to 0.35-0.45 M in the lowest differentiated areas, indicating the presence of sulphated heteroglycans, as chondroitin 4- and 6-sulphate. Most chordomas possessed the same staining properties as fetal cartilage; however, a few chordomas stained in the same way as notochordal remnants of nucleus pulposus (containing keratan sulphate), which are thought to be the origin of these tumors. The results of staining of the tumors in the present series with the Scott technique corresponds well with toluidine blue and alcian blue at different pH's with and without pretreatment of the sections with testicular hyaluronidase. Since bone and soft tissue tumors may contain varying mucosubstances depending on the tissue of origin and on differentiation, histochemical investigation of the heteroglycan content of these tumors may be a valuable diagnostic aid.