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 Ewing's 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. Ewing's 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 Ewing's sarcoma xenografts in nude mice. Therefore, results encourage inclusion of this drug especially in the treatment of patients with Ewing's 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.     

The molecular mechanisms responsible for resistance to ET-743 (Trabectidin; Yondelis) in the Ewing's sarcoma cell line, TC-71

Identification of new active agents against sarcoma is considered an important challenge in medical oncology. ET-743 (Trabectidin; Yondelis) has recently emerged as the first active drug developed against sarcoma in the last two decades, with promising results especially against soft-tissue sarcoma and Ewing's sarcoma (ES). In this study, we analyzed the molecular mechanisms responsible for resistance to ET-743 in ES cells. Three resistant cell variants (TC/ET 3 nM, TC/ET 6 nM and TC/ET 12 nM) were obtained, showing 28-, 47- and 102-fold increase in ET-743 resistance. Cross-resistance to other drugs was analyzed. Comparative genomic hybridization and cDNA microarray technology were employed to characterize and compare the gene expression profile of two TC/ET variants with the parental cell line. TC/ET cells show a conventional multidrug resistance phenotype and P-glycoprotein overexpression was found to significantly contribute to ET-743 resistance. However, functional studies with the cyclosporine analogue, PSC-833, indicate that other mechanisms are involved in resistance to ET-743. The gene expression profile of TC/ET cells indicated, among up-regulated genes, an increase in expression of insulin-like growth factor receptor-I (IGF-IR) and one of its major intracellular mediators, insulin receptor substrate-1. Functional studies using a neutralizing antibody anti-IGF-IR confirmed involvement of this signaling pathway in resistance to ET-743. Simultaneous blockage of both P-glycoprotein and IGF-IR completely restored sensitivity to ET-743 in ES cells. Overall, these findings provide impetus for future studies testing the therapeutic value of new specific inhibitors of P-glycoprotein and IGF-IR, which could represent a concrete therapeutic option for ES patients refractory to conventional agents.     

Sequence-dependent enhancement of cytotoxicity produced by ecteinascidin 743 (ET-743) with doxorubicin or paclitaxel in soft tissue sarcoma cells.

Ecteinascidin 743 (ET-743) is a potent antitumor agent from the Caribbean tunicate Ecteinascidin turbinata and is presently in clinical trials for human cancers. To better understand how ET-743 might be used clinically, the present study used SRB assays to examine the cytotoxicity resulting from combining ET-743 with three other antineoplastic agents: doxorubicin (DXR), trimetrexate, and paclitaxel in different administration schedules in two soft tissue sarcoma cell lines, HT-1080 and HS-18, in vitro. Concurrent exposure of ET-743 with DXR resulted in synergistic interactions in both cell lines. Addition of ET-743 for 24 h before DXR was the most effective cytotoxic regimen against both cell lines. Morphological study by fluorescence microscopy revealed that combination treatment of both cells with ET-743 and DXR induced apoptosis. Exposure to paclitaxel before ET-743 was also an effective regimen. These results encourage studies of the combination of ET-743 and DXR in the treatment of soft tissue sarcoma, because each of these agents have activity in this disease.     

Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival

Sarcomas are rare malignant mesenchymal tumors for which there are limited treatment options. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Dasatinib (BMS-354825), a small-molecule inhibitor of Src kinase activity, is a promising cancer therapeutic agent with p.o. bioavailability. Dasatinib exhibits antitumor effects in cultured human cell lines derived from epithelial tumors, including prostate and lung carcinomas. However, the action of dasatinib in mesenchymally derived tumors has yet to be shown. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in 12 cultured human sarcoma cell lines derived from bone and soft tissue sarcomas. Dasatinib inhibited Src kinase activity at nanomolar concentrations in these sarcoma cell lines. Downstream components of Src signaling, including focal adhesion kinase and Crk-associated substrate (p130(CAS)), were also inhibited at similar concentrations. This inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in the osteosarcoma and Ewing's subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by small interfering RNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results show that dasatinib inhibits migration and invasion of diverse sarcoma cell types and selectively blocks the survival of bone sarcoma cells. Therefore, dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas in patients.     

Histone deacetylase inhibitor-mediated sensitization to TRAIL-induced apoptosis in childhood malignancies is not associated with upregulation of TRAIL receptor expression, but with potentiated caspase-8 activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for the treatment of cancer because it targets tumor cells while sparing normal cells. Several cancers, however, fail to respond to TRAIL's antineoplastic effects. These resistant tumors require cotreatment with sensitizing agents in order for TRAIL to exert anticancer activity. Histone deacetylase inhibitors (HDACi) have been recognized as potent TRAIL sensitizers. In searching for the determinants of TRAIL responsiveness, HDACi-mediated TRAIL sensitization has been predominantly attributed to TRAIL receptor upregulation. This explanation, however, has been challenged by a few studies. The aim of the present study was to explore the relevance of TRAIL receptor expression for HDACi-mediated TRAIL sensitization in childhood tumors, i.e., in medulloblastoma, Ewing's sarcoma and osteosarcoma. In previous studies, we had shown that TRAIL and HDACi were synergistic in inducing apoptosis in medulloblastoma and Ewing's sarcoma. In the present study, we demonstrate that HDACi cooperated with TRAIL in eliciting cell death in osteosarcoma. However, HDACi treatment did not alter or even reduced cell surface expression of TRAIL receptors in the three childhood tumors. In gaining insight into the apoptotic pathway involved in TRAIL sensitization, HDACi were found to potentiate TRAIL-induced caspase-8 activation. Taken together, our findings suggest that HDACi-mediated TRAIL sensitization is not the result of TRAIL receptor upregulation, but the result of a receptor-proximal event in childhood tumor cells.     

In vitro studies with melphalan and pediatric neoplastic and normal bone marrow cells

Chemotherapeutic agents are used in increasingly high dosages to treat patients with refractory cancers. An in vitro clonogenic assay was used to investigate the effects of melphalan on cultures of human neuroblastoma, Ewing's sarcoma, and osteosarcoma cells, as well as on normal bone marrow cells. A 1-hr incubation with 10(-5) M melphalan significantly inhibited tumor colony growth of both fresh neuroblastoma and osteosarcoma cells (p less than 0.01) while Ewing's sarcoma cells and normal bone marrow appeared resistant to melphalan even at a 100-fold higher concentration. Incubation with melphalan for 8 hr did not significantly increase the sensitivity of neuroblastoma and osteosarcoma or the relative resistance of Ewing's sarcoma cells; however, normal bone marrow which had remained resistant to melphalan after 1 hr of incubation, showed significant inhibition of colony growth after an 8-hr incubation with the agent. Repeated exposure to melphalan increased the degree of inhibition of both tumor and normal marrow colonies. Fresh neuroblastoma cells were significantly more sensitive than long-term cultured neuroblastoma cells at all drug doses tested. HPLC studies demonstrated that the loss of melphalan followed first-order kinetics with a half-life of 69 min, and that in addition to the 2 known breakdown products, mono- and dihydroxy-melphalan, several other peaks were present which were not attributable to the tissue culture medium or the buffer solutions.     

Ecteinascidin-743: a marine-derived compound in advanced, pretreated sarcoma patients--preliminary evidence of activity.

PURPOSE: To report the activity of the chemotherapeutic agent ecteinascidin-743 (ET-743) in advanced pretreated sarcoma patients observed during a phase I study and a named-patient basis, compassionate use program. PATIENTS AND METHODS: Twenty-nine pretreated, advanced soft tissue sarcoma (STS) and bone sarcoma patients consecutively seen in our centers were included, 12 from a phase I trial and 17 from a compassionate use program cohort. Patients were treated every 3 weeks at either 1,200 microg/m(2) (six patients), 1,500 microg/m(2) (the recommended dose, 22 patients), or 1,800 microg/m(2) (the maximum-tolerated dose, one patient), given as a 24-hour infusion every 3 to 4 weeks. RESULTS: Fifteen men and 14 women were treated. The median patient age was 46 years (range, 16 to 71 years), with a median World Health Organization performance status of 1 (range, 0 to 2). Twenty-five patients had STS, three had osteosarcoma, and one had Ewing's sarcoma, and all had progressive disease at accrual. Fifteen patients had bulky disease, and 14 had clinical resistance to anthracyclines. A total of 136 treatment cycles were administered (median per patient, five cycles; range, one to 12 cycles). Transient grade 3 and 4 transaminitis was reported in 24% and 5% of cycles, respectively, grade 3 to 4 neutropenia occurred in 32% of cycles, with concomitant sporadic grade 3 to 4 thrombocytopenia in 5.1% of cycles. Grade 2 to 3 asthenia occurred in 21% of cycles. There were two partial responses (PRs) in STS patients and two PRs in osteosarcoma patients. Two minor responses and 10 disease stabilizations were seen. Median duration of response was 10.5 months (range, 2.8 to 15 months), and mean duration of stabilization was 5.2 months. CONCLUSION: ET-743 has activity in advanced, highly pretreated STS and osteosarcoma patients and warrants further trials to establish the extent of its activity in this setting.     

Effective combination of ET-743 and doxorubicin in sarcoma: preclinical studies

PURPOSE: To investigate the cytotoxic and antitumor effects of the combination of the novel anticancer drug ET-743 and doxorubicin (Dx) and to determine whether any pharmacokinetic interaction occurs in sarcoma-bearing mice. METHODS: The cytotoxicity of each drug and of their combinations was assessed in the rhabdomyosarcoma cell line TE-671 by a clonogenic assay, and isobologram analysis was performed to detect any synergistic, additive or antagonistic effects. The antitumor activities of each drug and of the combinations were also evaluated in nude mice transplanted subcutaneously with human TE-671 rhabdomyosarcoma and in C3H female mice injected intravenously with UV2237 M fibrosarcoma or with the Dx-resistant subline UV2237 M-ADM which overexpresses Pgp. Antitumor activity was evaluated by monitoring the TE-671 tumor volume over time and, in the case of the murine fibrosarcomas, by evaluation of lung deposits at autopsy quantified by determining lung weight. Pharmacokinetic studies were performed in TE-671-bearing mice. ET-743 was determined in plasma by an HPLC-MS method and Dx in plasma and tissue by an HPLC method with fluorescence detection. RESULTS: The combination of ET-743 and Dx was found to be additive with the average combination index slightly lower than 1 at all survival levels, suggesting weak synergism. In TE-671 tumors in vivo the activity of ET-743 or Dx given alone was marginal, whereas the combination produced a significant antitumor effect. The log cell kill (LCK) values were 0.13 and 0.33 for ET-743 and Dx alone, whereas they ranged from 0.85 to 1.12 for the combination. Giving ET-743 1 h before Dx slightly enhanced the effect (LCK 1.12) compared with giving the drugs simultaneously (LCK 0.85) or in the opposite sequence (LCK 0.92). In UV2237 M fibrosarcoma, both Dx and ET-743 showed an effect in reducing the weight of lung metastases, although the combination of the two drugs was not superior to each drug alone. In UV2237 M-ADM tumors neither of the two drugs was active, whereas the combination, particularly when the two drugs were given simultaneously, produced a significant effect. Plasma levels of ET-743 and Dx were not significantly different when the drugs were given alone or in combination. The concentrations of Dx in tissues including tumor, liver, heart and kidney were found to be the same whether the drug was given alone or in combination with ET-743. CONCLUSIONS: These results indicate that ET-743 and Dx in combination produce an additive effect against human sarcoma cells, reinforcing the idea that they act by a different mechanism of action. In mice no pharmacokinetic interaction between the two drugs was found. The observed activity in UV2237 M-ADM and in human TE-671 sarcoma suggests that the combination of the two drugs could be effective for tumors displaying low sensitivity to each drug given alone. Based on these findings a phase I study on the combination of the two drugs was recently initiated.     

Absence of stimulatory effect of g-csf on the growth of human sarcoma-cells

The Colony-Stimulating Factors (CSFs) are undergoing clinical trials for their ability to stimulate the regeneration of bone marrow in patients receiving anticancer chemotherapy. However, the reported effects on the growth of tumor cell lines of different derivations, including osteosarcoma, raise the possibility that the use of these cytokines may induce proliferative effects also in residual tumor cells. In this study, we have used a panel of 12 human osteosarcoma (2 cell lines and 10 primary cultures) and 7 Ewing's sarcoma cell lines (5 cell lines and 2 primary cultures) to evaluate the presence of the G-CSF receptor by RT-PCR and the effects of recombinant Human (rHu) G-CSF on their in vitro growth ability. RT-PCR did not reveal the presence of G-CSF receptor band in any of the osteosarcoma or Ewing's cell lines examined. Moreover, after exposure to rHuG-CSF, no significant stimulatory or inhibitory effects were observed in any of the cell lines. Therefore, G-CSF may be safely used to stimulate marrow regeneration after high-dose chemotherapy both in osteosarcoma and in Ewing's sarcoma.     

Periosteal Ewing's sarcoma

Ewing's sarcoma is a small cell malignant tumor that usually arises in the medullary cavity of bone. Less frequently, it originates in soft tissue and may secondarily invade underlying bone. The origin of Ewing's sarcoma in a periosteal location without extension into either the bone or adjacent soft tissue has not been clearly documented. Other malignant tumors of bone (e.g., osteosarcoma) appear to have a somewhat better prognosis when confined between periosteum and bone. The case of a patient with a periosteal Ewing's sarcoma who received a radical excision and postoperative chemotherapy and who is without evidence of disease with over 2 years follow-up is reported.     

Lovastatin induces apoptosis in a primitive neuroectodermal tumor cell line in association with RB down-regulation and loss of the G1 checkpoint

To develop a new approach to the treatment of primitive neuroectodermal tumors we evaluated the effect of the HMG-CoA reductase inhibitor lovastatin on the Ewing's sarcoma cell line CHP-100. Lovastatin induced neural morphology and markers including neuron-specific enolase and neurofilament protein. The acquisition of neural morphology required new mRNA synthesis, and cDNA microarray analysis confirmed that lovastatin altered the program of gene expression. After morphologic differentiation the cells underwent rapidly progressive apoptosis. In normal development of neuronal progenitors, differentiation signals trigger p21WAF1 accumulation, RB hypophosphorylation, enhanced RB-E2F-1 association, and G1 arrest, and these events have been shown to protect from apoptosis. In contrast, in the Ewing's sarcoma cells lovastatin triggered differentiation without causing cell cycle arrest: p21WAF1 was not induced, RB remained hyperphosphorylated, and RB protein expression and RB-E2F-1 association were markedly downregulated, suggesting that loss of an RB-regulated G1 checkpoint promoted apoptosis. Consistent with this hypothesis, adenoviral p21WAF1 decreased DNA synthesis and partially protected from lovastatin-induced cytotoxicity. The data demonstrate a new model for examining the genetic regulation of cell fate in a neural progenitor tumor and suggest a new approach to the treatment of this neoplasm.     

Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability.     

Ewing's sarcoma and extracranial primitive neuroectodermal tumors.

Ewing's sarcoma and primitive neuroectodermal tumor of bone and soft tissue are rare tumors, considered with the small round blue cell tumors of childhood. Recent advances link Ewing's sarcoma and primitive neuroectodermal tumor, and support the concept that these tumors represent stages of differentiation in a neoplasm of neuroectodermal origin. Advances in chemotherapy have resulted in the survival of the majority of patients presenting with these tumors. This review briefly outlines the year's literature regarding the histogenesis, biology, and treatment of this tumor.     

Induction of cell death by basic fibroblast growth factor in Ewing's sarcoma

Ewing's sarcoma is thought to arise after developmental arrest of primitive neural cells during embryogenesis. Because basic fibroblast growth factor (bFGF) has a critical role in the regulation of cell survival, proliferation, and differentiation during embryogenesis, we have tested the hypothesis that bFGF and FGF receptors may contribute to the development of Ewing's sarcoma and may provide a mechanism for the modulation of their behavior. All four of the Ewing's sarcoma cell lines examined expressed bFGF and FGF receptors, which were detected by immunofluorescence and Western blotting. bFGF-induced a significant dose-dependent decrease in Ewing's sarcoma cell proliferation on plastic and reduced anchorage-independent growth in soft agar. Unexpectedly, this decrease in cell number reflected bFGF-induced apoptosis and necrosis, as demonstrated by electron microscopy, binding of annexin V, and staining with acridine orange. Induction of cell death was dependent on dosage of, and period of exposure to, bFGF. bFGF did not induce differentiation of Ewing's sarcoma cells in either the presence or the absence of serum or nerve growth factor. Treatment of NuNu mice with bFGF decreased growth of the highly tumorigenic Ewing's sarcoma cell lines. Histologically tumors grown in the NuNu mice treated with bFGF were less cellular than those in control mice, and showed an increased level of apoptotic nuclei. This is in contrast to the mitogenic effect bFGF has in most other cancer cells. In summary, bFGF decreases Ewing's sarcoma growth in vitro and in vivo by the induction of cell death. This novel observation may provide a new therapeutic strategy for Ewing's sarcomas.     

Evidence-based chemotherapy for patients with bone and soft part sarcoma

In the present paper, we review the evidence for chemotherapy in patients with bone and soft part sarcoma and discuss the contributions and improvements made by chemotherapy to the treatment of patients with bone and soft part sarcoma. In the osteosarcoma and Ewing's sarcoma family, neoadjuvant and adjuvant chemotherapy have improved the 5-year disease-free survival to 60%, and limb-salvage operations have improved this to 70-80% in cases of non-metastatic malignant bone tumor. Several trials were conducted in order to overcome rate relapses and metastatic bone sarcoma. With osteosarcoma, thoracotomy improved the survival of lung metastatic patients, but CDDP-ADM branch switched according to the neoadjuvant chemotherapy and failed to elevate the continuous disease-free survival of patients. Dose intensive use of cytotoxic drugs with G-CSF or autologous bone marrow transplantation and multidrug programs were conducted in preliminary studies and achieved favorable results in a high risk factors group for tumors of the Ewing's sarcoma family. Surgical techniques have brought improvements in the treatment of soft tissue sarcoma, but there has been no impact by chemotherapy. Ifosfamide and adriamycin combination is being evaluated in the treatment of local advanced and metastatic soft part sarcoma by local control rate or survival from relapse.     

Regulation of apoptosis and proliferation in Ewing's sarcoma--opportunities for targeted therapy

The Ewing's sarcoma family of tumors are malignant tumors of bone and soft tissue which occur predominantely in children and adolescents. Whereas cure rates for patients with localized tumors are around 70%, survival rates for patients with metastases or relapse are poor in spite of intensive chemo- and radiation therapy, demonstrating a clear need for new, more effective therapies. Insights into the biology of the tumors of the Ewing's sarcoma family with identification of the EWS/ETS gene rearrangement as the key event in malignant transformation and its influence on the regulation of various pathways involved in proliferation, differentiation and apoptosis has led to the identification of potential targets for the development of new molecular therapeutics. This review will focus on the regulation of major pathways of proliferation and apoptosis in tumors of the Ewing's sarcoma family and point out how modulation of these pathways might be of potential use for future therapy.     

Small-cell osteosarcoma: correlation of in vitro and clinical radiation response

Small-cell osteosarcoma is an entity which shares some clinical and pathological features with both classic osteosarcoma and Ewing's sarcoma of bone. While noted to be "not radiosensitive" when first described, a retrospective review the National Cancer Institute experience of five patients with small-cell osteosarcoma treated with radiation therapy following biopsy (three pts) or limited excision (two pts) showed local control in all five patients with two long-term disease-free survivors (12, 18 years). This compares to three patients treated with surgery alone where one patient failed locally and one patient is a long-term disease-free survivor (7 years). We have studied the in vitro radiation response of a recently established small-cell osteosarcoma cell line (TC-252) and compared its response with that of a classic osteosarcoma cell line (U2-OS) and an Ewing's sarcoma cell line (5838). The small-cell osteosarcoma line responded with a similar Do and extent of PLDR compared to the Ewing's line and was different from the in vitro radiation response of classic osteosarcoma. Based on this small clinical series and the in vitro radiation studies, we conclude that small-cell osteosarcoma is a radioresponsive tumor. Definitive radiation therapy or conservative surgery plus radiation therapy are effective alternative therapeutic options, compared to ablative surgery, for the local treatment of this uncommon bone tumor of children and young adults.