Maria Cristina Manara

Expression of P-Glycoprotein in High-Grade Osteosarcomas in Relation to Clinical Outcome

BACKGROUND Increased levels of P-glycoprotein occur in some osteosarcomas. In this study we determined the relation between P-glycoprotein status and outcome in patients with high-grade osteosarcoma. METHODS P-glycoprotein status was determined immunohistochemically in specimens of osteosarcoma of the extremities (stage II) from 92 patients who were treated with surgery and chemotherapy. The P-glycoprotein status was analyzed in relation to the length of event-free survival. RESULTS The presence of increased levels of P-glycoprotein in the osteosarcoma was significantly associated with a decreased probability of remaining event-free after diagnosis (P = 0.002). In a multivariate analysis, P-glycoprotein status (P = 0.001) and the extent of tumor necrosis after preoperative chemotherapy (P = 0.04) were independent predictors of clinical outcome. The risk of adverse events was increased substantially (rate ratio, 3.37; 95 percent confidence interval, 1.60 to 7.10) among patients with increased levels of P-glycoprotein in tumor cells, as compared with patients who did not have increased levels of P-glycoprotein in tumor cells. CONCLUSIONS In patients with high-grade osteosarcoma treated with surgery and chemotherapy, the presence of increased levels of P-glycoprotein in tumor cells is associated with a significantly increased risk of adverse events and is independent of the extent of necrosis after preoperative chemotherapy.

Antitumor Activity of the Insulin-Like Growth Factor-I Receptor Kinase Inhibitor NVP-AEW541 in Musculoskeletal Tumors

Identification of new drugs is strongly needed for sarcomas. Insulin-like growth factor-I receptor (IGF-IR) was found to provide a major contribution to the malignant behavior of these tumors, therefore representing a very promising therapeutic target. In this study, we analyzed the therapeutic potential of a novel kinase inhibitor of IGF-IR, NVP-AEW541, in Ewings sarcoma, osteosarcoma, and rhabdomyosarcoma, the three most frequent solid tumors in children and adolescents. NVP-AEW541 inhibits IGF-I-mediated receptor activation and downstream signaling. Ewings sarcoma cells were generally found to be more sensitive to the effects of this drug compared with rhabdomyosarcoma and osteosarcoma, in agreement with the high dependency of this neoplasm to IGF-IR signaling. NVP-AEW541 induced a G1 cell cycle block in all cells tested, whereas apoptosis was observed only in those cells that show a high level of sensitivity. Concurrent exposure of cells to NVP-AEW541 and other chemotherapeutic agents resulted in positive interactions with vincristine, actinomycin D, and ifosfamide and subadditive effects with doxorubicin and cisplatin. Accordingly, combined treatment with NVP-AEW541 and vincristine significantly inhibited tumor growth of Ewings sarcoma xenografts in nude mice. Therefore, results encourage inclusion of this drug especially in the treatment of patients with Ewings sarcoma. For the broadest applicability and best efficacy in sarcomas, NVP-AEW541 may be combined with vincristine, actinomycin D, and ifosfamide, three major drugs in the treatment of sarcomas.

Preclinical In vivo Study of New Insulin-Like Growth Factor-I Receptor–Specific Inhibitor in Ewing's Sarcoma

Purpose: Small-molecule insulin-like growth factor-I receptor (IGF-IR)-specific tyrosine kinase inhibitors have been recently proposed as clinically viable approaches to impair IGF-IR functions. NVP-AEW541 seems one of the most promising agents. In this article, we point out its effects against migration, metastasis, vasculogenicity, and angiogenesis of Ewings sarcoma cells. Experimental Design:In vivo NVP-AEW541 effectiveness was analyzed against TC-71 Ewings sarcoma growth and bone metastasis after cell inoculation in athymic mice. Activity of the compound against angiogenesis as well as vasculogenesis properties was also considered both in vitro and in xenografts. Serum glucose, urea, transaminase levels, as well as other signs of distress were checked in mice treated with the IGF-IR inhibitor. Results: Significant inhibition of migration, metastasis, vasculogenicity, and angiogenesis was recorded after treatment of Ewings sarcoma cells with NVP-AEW541. In view of its application and the similarity of insulin receptor and IGF-IR, diabetogenic side effects were considered. We observed a significant decrease of glucose blood serum due to increased glucose uptake at cellular level and an increase in urea concentration. Moreover, an initial weight loss was observed in mice bearing tumors. All these side effects were similarly detected in mice treated with vincristine. After the first days of treatment, all the animals started to grow again. Conclusions: Our results globally reinforce the idea that IGF-IR inhibitor NVP-AEW541 could have a role in future combined therapies and suggest to pursue a thorough molecular analysis of the metabolic activity of IGF-IR to avoid possible side effects of these inhibitors.

NVP-BEZ235 as a New Therapeutic Option for Sarcomas

Purpose: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide 3-kinase–mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors (osteosarcoma, Ewings sarcoma, and rhabdomyosarcoma). Experimental Design: Antiproliferative activity as well as the effects on migration and metastasis were evaluated in a panel of osteosarcoma, Ewings sarcoma, as well as rhabdomyosarcoma cell lines. Moreover, simultaneous and sequential treatments were done in association with two of the most important conventional drugs in the treatment of sarcoma, doxorubicin and vincristine. Results: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the experimental conditions tested, the compound induced disease stasis, by arresting cells in G1 phase of cell cycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitation of its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxic agents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phosphoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellular signal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signaling toward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235 with other small signaling inhibitors. Here, we showed synergistic effects when the compound was associated with a anti–insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 also inhibited cell migration and metastasis. Combination with vincristine further potentiated the antimetastatic effects. Conclusions: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advanced cancer patients. Clin Cancer Res; 16(2); 530–40

Expression of an IGF-I receptor dominant negative mutant induces apoptosis, inhibits tumorigenesis and enhances chemosensitivity in Ewing's sarcoma cells

IGF‐IR plays an essential role in the establishment and maintenance of the transformed phenotype of ES cells and interference with the IGF‐IR pathways causes reversal of the malignant potential of this neoplasm. In this report, we stably transfected a dominant negative IGF‐IR expression plasmid in an ES cell line to determine the effectiveness of this strategy against the in vitro and in vivo growth of ES cells. DXR sensitivity of TC‐71 cells expressing dominant negative mutants of IGF‐IR was also examined. The mutated IGF‐IR that we used carries a mutation in the ATP‐binding domain of the intracellular β subunit, while the extracellular, ligand‐binding α subunit remains unchanged. Cells carrying the dominant mutant IGF‐IR had a marked decrease in proliferation, a significant increase in anoikis‐induced apoptosis and a severely reduced ability to form colonies in soft agar. In vivo, when cells carrying dominant negative IGF‐IR were injected into nude mice, the tumor formation and metastatic abilities of ES cells were reduced and survival increased. Furthermore, transfected clones showed significantly higher sensitivity to DXR, a major drug in the treatment of ES. These results indicate that the IGF/IGF‐IR stimulation of ES cells may be inhibited by expression of mutated IGF‐IR on their surfaces and that this strategy may be considered a possible alternative to impair this important target of ES cells, whose therapeutic potential was further confirmed.

The Expression of ccn3(nov) Gene in Musculoskeletal Tumors

The CCN3(NOV) protein belongs to the CCN [cysteine-rich CYR61, connective tissue growth factor (CTGF), nephroblastoma overexpressed gene (Nov)] family of growth regulators, sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Although previous studies have revealed an expression of CCN3 protein in several normal tissues, including kidney, nervous system, lung, muscle, and cartilage, less is known about its expression in tumors. In this study, we analyzed the expression of CCN3 in musculoskeletal tumors, using a panel of human cell lines and tissue samples. An association between CCN3 expression and tumor differentiation was observed in rhabdomyosarcoma and cartilage tumors, whereas, in Ewings sarcoma, the expression of this protein seemed to be associated with a higher risk to develop metastases. CCN3 expression was found in 15 of 45 Ewings sarcoma tissue samples. In particular, we did not observe any expression of CCN3 in the 15 primary tumors that did not develop metastases. In contrast, 15 of the 30 primary tumors that developed lung and/or bone metachronous metastases showed a high expression of the protein (P < 0.001, Fishers test). Our studies indicate that CCN3 is generally expressed in the cells of the musculoskeletal system. This protein may play a role both in normal and pathological conditions. However, the regulation of CCN3 expression varies in the different neoplasms and depends on the type of cells. Thus, as reported for other CCN genes, the biological properties and regulation of expression of CCN3 are dependent on the cellular context and the nature of the cells in which it is produced. Further studies will help to clarify the biological role of this protein in musculoskeletal neoplasms.

Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling

Identification of patient selection criteria and understanding of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin-like growth factor (IGF)-1R therapies. Few Ewings sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF-1R:IR ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy.

Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: Actin and zyxin as key intracellular mediators

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewings sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell–cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

Effectiveness of insulin-like growth factor I receptor antisense strategy against Ewing's sarcoma cells

The insulin-like growth factor I receptor (IGF-IR) plays an essential role in the establishment and maintenance of transformed phenotype of Ewings sarcoma (ES) cells, and interference with the IGF-IR pathways by a neutralizing antibody causes reversal of the malignant potential of this neoplasm. In this paper, we stably transfected an IGF-IR antisense mRNA expression plasmid in an ES cell line to determine the effectiveness of antisense strategies against the in vitro and in vivo growth of ES cells. Doxorubicin sensitivity of TC-71 cells expressing antisense targeted to IGF-IR mRNA was also examined. Cells carrying antisense IGF-IR had a reduced expression of the receptor, a modest decrease in cell proliferation, a significant increase in anoikis-induced apoptosis, and a severely reduced ability to form colonies in soft agar. Moreover, TC/AS cells showed a marked reduction in their motility. In vivo, when cells carrying antisense IGF-IR were injected subcutaneously in nude mice, tumor formation was delayed and survival increased. Metastatic ability of ES cells was also significantly reduced. Furthermore, TC/AS clones showed a significantly higher sensitivity to doxorubicin — a major drug in the treatment of ES. These results indicate that inhibiting IGF-IR by antisense strategies may be relevant to the clinical treatment of ES patients by reducing the malignant potential of these cells and enhancing the effectiveness of chemotherapy.

CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis.

Ewing sarcoma (EWS) is an aggressive bone tumor of uncertain cellular origin. CD99 is a membrane protein that is expressed in most cases of EWS, although its function in the disease is unknown. Here we have shown that endogenous CD99 expression modulates EWS tumor differentiation and malignancy. We determined that knocking down CD99 expression in human EWS cell lines reduced their ability to form tumors and bone metastases when xenografted into immunodeficient mice and diminished their tumorigenic characteristics in vitro. Further, reduction of CD99 expression resulted in neurite outgrowth and increased expression of beta-III tubulin and markers of neural differentiation. Analysis of a panel of human EWS cells revealed an inverse correlation between CD99 and H-neurofilament expression, as well as an inverse correlation between neural differentiation and oncogenic transformation. As knockdown of CD99 also led to an increase in phosphorylation of ERK1/2, we suggest that the CD99-mediated prevention of neural differentiation of EWS occurs through MAPK pathway modulation. Together, these data indicate a new role for CD99 in preventing neural differentiation of EWS cells and suggest that blockade of CD99 or its downstream molecular pathway may be a new therapeutic approach for EWS.