Contribution of MEK/MAPK and PI3-K signaling pathway to the malignant behavior of Ewing's sarcoma cells: therapeutic prospects

Insulin-like growth factor receptor I (IGF-I)-mediated circuit is a major autocrine loop for Ewing's sarcoma (ES) cells and appears to be particularly important in the pathogenesis of this tumor. In this study, we analyzed the contribution of the 2 major pathways of the intracellular IGF-IR signaling cascade to the overall effects elicited by IGF-I in ES. Both the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K) signaling pathways appeared to be constitutively activated in ES, likely due to the presence of the IGF-IR-mediated autocrine loop. We demonstrated that both MEK/MAPK (PD98059 or U0126) and PI3-K inhibitors (LY294002) profoundly impaired ES cell growth in monolayer and soft agar basal conditions. Both PD98059 and LY294002 inhibited ES cell cycle progression by inducing G1 blockage, whereas only LY294002 significantly affected the survival of ES cells. Exogenous IGF-I completely reverted LY294002-induced growth inhibition by abrogating antiproliferative and proapoptotic effects of the PI3-K inhibitor. By contrast, IGF-I could not rescue cells from growth inhibition induced by PD98059. MEK/MAPK blockade also significantly reduced the migratory ability of ES cells, both in basal and IGF-I-induced conditions, and increased chemosensitivity to doxorubicin, a leader drug in the treatment of ES patients. Our findings therefore identify MAPK pathway as a promising target for pharmacologic intervention in ES.     

Insulin-like growth factor-I, insulin-like growth factor binding protein-3 and the risk of fibrocystic breast conditions among Chinese women

We investigated whether circulating insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) levels are associated with the risk of fibrocystic breast conditions (FBC), in a case-control study nested within a randomized trial of breast self-examination conducted in Shanghai, China. Participants were enrolled during 1989-1991 and were followed over 10 years for the development of breast diseases. Controls (n = 897) were frequency-matched by age to cases (n = 451), who were diagnosed with FBC between 1995 and 2000. Circulating IGF-I and IGFBP-3 levels and their molar ratio were positively associated with risk of FBC. The odds ratios (ORs) and 95% confidence intervals (CI) for the upper fourth of the distribution compared to the lowest fourth for IGF-I, IGFBP3 and their molar ratio were 3.02 (2.02-4.52), 1.92 (1.37-2.71) and 2.26 (1.52-3.36), respectively. The strength of the association between IGF-I levels and FBC was attenuated after adjustment for IGFBP-3 and that for IGFBP-3 was largely eliminated after adjustment for IGF-I. Increasing levels of IGF-I were particularly associated with increasing risk of FBC with proliferative elements (ORs and 95% CIs for the 2nd, 3rd and upper fourth of the distribution of IGF-I: 3.13 (1.50-6.53), 4.57 (2.22-9.39) and 6.30 (3.08-12.89), compared with the lowest fourth. Our results suggest that elevated levels of IGF-I may contribute to the development of FBC.     

Biological indicators of prognosis in Ewing's sarcoma: An emerging role for lectin galactoside‐binding soluble 3 binding protein (LGALS3BP)

Starting from an experimental model that accounts for the 2 most important adverse processes to successful therapy of Ewing's sarcoma (EWS), chemoresistance and the presence of metastasis at the time of diagnosis, we defined a molecular signature of potential prognostic value. Functional annotation of differentially regulated genes revealed 3 major networks related to cell cycle, cell‐to‐cell interactions and cellular development. The prognostic impact of 8 genes, representative of these 3 networks, was validated in 56 EWS patients. High mRNA expression levels of HINT1, IFITM2, LGALS3BP, STOML2 and c‐MYC were associated with reduced risk to death and lower risk to develop metastasis. At multivariate analysis, LGALS3BP, a matricellular protein with a role in tumor progression and metastasis, was the most important predictor of event‐free survival and overall survival. The association between LGALS3BP and prognosis was confirmed at protein level, when expression of the molecule was determined in tumor tissues but not in serum, indicating a role for the protein at local tumor microenvironment. Engineered enhancement of LGALS3BP expression in EWS cells resulted in inhibition of anchorage independent cell growth and reduction of cell migration and metastasis. Silencing of LGALS3BP expression reverted cell behavior with respect to in vitro parameters, thus providing further functional validation of genetic data obtained in clinical samples. Thus, we propose LGALS3BP as a novel reliable indicator of prognosis, and we offer genetic signatures to the scientific communities for cross‐validation and meta‐analysis, which are indispensable tools for a rare tumor such as EWS.     

Adjuvant chemotherapy in Ewing's sarcoma

Adjuvant chemotherapy using vincristine, cyclophosphamide, and adriamycin was used in 28 patients with localized Ewing's sarcoma. Life-table analysis revealed 55% disease-free survival at 3 years. In contrast, our historical control showed less than 10% long-term survival. Adjuvant chemotherapy appears to control both micrometastasis as well as local residual disease. The present study demonstrates the improved survival with adjuvant chemotherapy including adriamycin, vincristine, and cyclophosphamide, in patients with localized Ewing's sarcoma.     

Promoter -202 A/C polymorphism of insulin-like growth factor binding protein-3 gene and non-small cell lung cancer risk

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the mitogenic and antiapoptotic activity of insulin-like growth factor (IGF) by blocking the binding of IGF to its receptor. However, under certain circumstances, IGFBP-3 can enhance the activity of IGF by protecting IGF from degradation. More than half of the interindividual variations in IGFBP-3 levels are known to be genetically determined by the polymorphism at -202 locus of IGFBP-3 gene. Therefore, we attempted to ascertain whether the A-202C polymorphic variation of IGFBP-3 gene constitutes a risk factor for non-small cell lung cancer (NSCLC). Our study included 209 NSCLC patients and 209 age-, gender- and smoking status-matched control subjects. The frequencies of each polymorphic variation in the control population were as follows: AA = 95 (45.5%), AC = 91 (43.5%) and CC = 23 (11.0%). In the NSCLC subjects, the genotypic frequencies were as follows: AA = 131 (62.7%), AC = 73 (34.9%) and CC = 5 (2.4%). We detected statistically significant differences in the genotypic distribution between the NSCLC and the control subjects (p < 0.05, Pearson's chi-square test). The NSCLC risk correlated significantly with AA genotype. Using CC genotype as a reference, the odds ratio for the subjects with AC genotype was 2.45 (95% CI = 1.17-5.40) and that for the ones with AA genotype was 4.58 (95% CI = 2.17-10.30). These results indicate that the dysregulation of IGF axis should now be considered as another important risk factor for NSCLC and a potential target for novel antineoplastic therapies and/or preventative strategies in high-risk groups.     

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 beta subunit, while the extracellular, ligand-binding alpha 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.     

Insulin-like growth factor binding protein-1 over-expression in transgenic mice inhibits hepatic preneoplasia

Insulin-like growth factor binding protein-1 (IGFBP-1) is synthesized in the liver and regulates the mitogenic effects of the insulin-like growth factors (IGFs). The evidence that IGFBP-1 plays a role in hepatocarcinogenesis, however, is equivocal. We have, therefore, investigated the development of preneoplastic hepatic lesions in transgenic mice in which the human IGFBP-1 gene is under the control of the mouse metallothionein promoter. The lesions were induced by treating 15-d-old male mice with a single intraperitoneal injection of 5 mg/kg diethylnitrosamine (DENA). Lesions were scored when the mice were 28 wk of age. Quantitative microscopy of liver sections revealed that significantly fewer transgenic mice treated with zinc to activate the transgene had focal lesions compared to either transgenic mice not treated with zinc or wild-type mice treated with zinc (36.4% versus 85.7% and 83.3%, respectively, P < 0.05 in each case). Zinc-treated transgenic mice also had significantly fewer lesions per liver (11.5 +/- 5.0 versus 74.7 +/- 18.4 and 59.4 +/- 15.6, respectively, P < 0.01 in each case) and a smaller percentage of liver volume occupied by lesions (0.2 +/- 0.1 versus 1.4 +/- 0.3 and 1.1 +/- 0.4 respectively, P < 0.05 in each case). Immunohistochemical staining showed that both IGF-I and IGF-II were overexpressed in most of the lesions. These results show that expression of the IGFBP-1 transgene leads to a marked inhibition of hepatic preneoplasia, possibly by decreasing the mitogenic activity of IGF-I and/or IGF-II. This study adds new evidence to the notion that the IGF axis plays an important role in liver cancer development.     

Ewing's sarcoma

Between 1984–1987, 50 patients with Ewing's sarcoma of the bone were entered on combined modality protocol at Tata Memorial Hospital. Protocol treatment involved induction therapy consisting of 6-week therapy with vincristine, Adriamycin (doxorubicin), and cyclophosphamide (VDC) followed by local radiotherapy 50 Gy to the involved bone. This was followed for six more cycles of VDC. Five patients had metastatic disease at presentation. Seventy-six percent (38/50) of patients had disease either at axial or proximal site. With a median follow-up of 48 months (range 14–87) 21 patients remained alive with disease-free survival of 38.0% ± 2.5% at 5 years and overall survival of 36.0% ± 2.6% at 5 years. Twentyfive patients relapsed with five patients developing local failure and four local and distant metastasis. Using Lee-Desu statistical methods, only response to therapy was a significant factor for survival. We conclude that more aggressive therapy with proper selection of local treatment modality including surgery and/or radiotherapy is required to produce more long-term survival in high-risk Ewing's sarcoma. © 1993 Wiley-Liss, Inc.     

The role of insulin-like growth factor binding protein-3 in the growth inhibitory actions of androgens in LNCaP human prostate cancer cells

Insulin-like growth factor binding protein-3 (IGFBP-3), an antiproliferative and proapoptotic protein, has been shown to be upregulated by growth inhibitory concentrations of androgens in LNCaP human prostate cancer (PCa) cells, but the mechanism of regulation and the role of IGFBP-3 in the modulation of PCa cell proliferation are unknown. In this study, we have examined the effects of a range of concentrations of the synthetic androgen R1881 on IGFBP-3 expression and cell growth in LNCaP cells. We have also investigated the role of androgen-stimulated IGFBP-3 in androgen-induced growth inhibition. We show that low doses of R1881 stimulate LNCaP cell proliferation, but do not induce IGFBP-3 expression, whereas high doses of R1881 that inhibit cell growth, significantly increase expression of IGFBP-3. Importantly, we demonstrate that the combination of calcitriol and androgens not only synergistically upregulates IGFBP-3 expression but also inhibits cell growth better than either hormone alone. siRNA knockdown of IGFBP-3 expression partially reverses the growth inhibition by calcitriol and by androgens. Furthermore, we find that the growth inhibitory dose of R1881 leads to increases in the cyclin dependent kinase inhibitors (CDKIs), p21 and p27 as well as to G1 arrest. These changes can be blocked or partially reversed by IGFBP-3 siRNA, indicating that the induction of CDKIs is downstream of IGFBP-3. Our data suggest, for the first time, that IGFBP-3 is involved in the antiproliferative action of high doses of androgens partly through p21 and p27 pathways and that IGFBP-3 may contribute significantly to androgen-induced changes in LNCaP cell growth.     

Actuarial risk of isolated CNS involvement in Ewing's sarcoma following prophylactic cranial irradiation and intrathecal methotrexate

Records of 154 patients with Ewing's sarcoma treated at the National Cancer Institute were reviewed to assess the incidence and risk of developing isolated central nervous system (CNS) Ewing's sarcoma. Sixty-two of the 154 patients had received CNS irradiation and intrathecal (i.t.) methotrexate as part of their initial therapy to prevent the occurrence of isolated CNS Ewing's sarcoma. The risk of developing isolated CNS Ewing's sarcoma was greatest within the first two years after diagnosis and was approximately 10%. The overall risk of CNS recurrence in the group of patients receiving CNS treatment was similar to the group receiving no therapy directed to the CNS. The occurrence of isolated CNS involvement was not prevented by the use of CNS irradiation and i.t. methotrexate. Because of a lack of efficacy to the CNS irradiation regimen, current treatment regimens do not include therapy directed to the CNS.     

Extraskeletal Ewing's sarcoma presenting with pulmonary embolism

Small round cell tumours with the morphologic characteristics of Ewing's sarcoma arise rarely in soft tissues of the extremities, retroperitoneum, chest and orbit. Patients usually present with symptoms arising from the swelling at the primary site. We report on a patient with a retroperitoneal extraskeletal Ewing's sarcoma who presented with massive tumour embolism to the pulmonary vasculature. To our knowledge, this is the first report of extraskeletal Ewing's sarcoma presenting with pulmonary tumour embolism.     

Serum insulin‐like,growth factor binding protein‐related protein 1 (IGFBP‐rP1) and endometrial cancer risk in Chinese women

Hyperinsulinemia and the metabolic syndrome confer increased risks of endometrial carcinoma. The roles of insulin, and, insulin‐like growth factor‐binding proteins (IGFBPs) in the etiology of endometrial carcinoma, remain unclear. We recruited 206 patients with endometrial carcinoma and 350 healthy women to a case–control study of fasting insulin and IGFBP‐related protein 1 (IGFBP‐rP1) in a Chinese tertiary centre. Patients with endometrial carcinoma had higher insulin concentrations (14.8 ± 16.7 vs. 8.1 ± 9.4 μU/mL; p < 0.001) and lower IGFBP‐rP1 levels (17.5 ± 17.2 vs. 22.4 ± 22.8 μg/L; p = 0.018) than controls. High insulin and IGFBP‐rP1 levels were both positively and negatively associated with endometrial cancer (odds ratio for the highest tertile versus the lowest tertile: insulin: 4.11; 95% CI = 2.61–6.47; IGFBP‐rP1: 0.38; 95% CI = 0.24–0.60). Logistic regression analysis confirmed the associations between endometrial carcinoma and fasting insulin or IGFBP‐rP1 after adjustments for age, BMI, serum glucose, cholesterol, triglycerides and high‐density lipoprotein cholesterol (odds ratio for the highest tertile versus the lowest tertile: insulin: 2.13; 95% CI = 1.30–3.49; IGFBP‐rP1: 0.57; 95% CI = 0.34–0.94). Hyperinsulinemia and high IGFBP‐rP1 levels confer altered risks for endometrial carcinoma.     

类胰岛素生长因子结合蛋白相关蛋白-1及其病理作用

类胰岛素生长因子结合蛋白相关蛋白-1(IGFBP-rP1)是类胰岛素生长因子结合蛋白(IGFBP)超家族中唯一具有高胰岛素亲和力的成员,其命名、配体、调节均与肿瘤相关.研究表明,IGFBP-rP1在人类肿瘤组织中的表达呈上调或下调的不同趋势,具有致癌或抑癌的双向作用,正成为抗肿瘤治疗研究的新靶点.     

Ewing's sarcoma of the phalangeal bone

A rare case of Ewing's sarcoma of the toe bone is described and compared with three reported cases of phalangeal Ewing's sarcoma. The lesion in two cases was initially treated as infection and the diagnosis was delayed for several months. Roentgenographic and aspiration cytology examinations in cases with clinically unresolving infection of the hands and feet may prevent delayed diagnosis. Radical surgery alone may be curative if no metastases are found preoperatively in careful clinical investigations.     

Stromal cell-derived factor-1 stimulates vasculogenesis and enhances Ewing's sarcoma tumor growth in the absence of vascular endothelial growth factor

Stromal cell-derived Factor-1alpha (SDF-1alpha) stimulates the migration of bone marrow (BM) cells, similar to vascular endothelial growth factor (VEGF). We previously demonstrated that inhibition of VEGF(165) by small interfering RNA inhibited Ewing's sarcoma tumor growth, tumor vessel formation and recruitment of BM cells to the tumor. To determine the importance of BM cells in tumor vessel development, we investigated the effects of SDF-1alpha on VEGF-inhibited TC/siVEGF(7-1) Ewing's tumor neovasculature formation and growth. The effect of SDF-1alpha on CD34(+) progenitor cell chemotaxis was determined in vivo. Using a BM transplantation model with GFP(+) transgenic mice as BM donors and nude mice as recipients, we evaluated the effect of SDF-1alpha on the recruitment of BM-derived cells to VEGF(165)-inhibited TC/siVEGF(7-1) tumors, as well as its effect on neovasculature development, vessel morphology and tumor growth. SDF-1alpha stimulated the migration of CD34(+) progenitor cells to Matrigel plugs in vivo and promoted the retainment of BM-derived pericytes in close association with perfused, functional tumor vessels. Intratumor inoculation of Ad-SDF-1alpha into TC/siVEGF(7-1) tumors resulted in increased SDF-1 and PDGF-BB expression, augmented tumor growth, an increase in the number of large, lumen-bearing vascular structures, and enhanced vessel pericyte coverage, with no change in VEGF(165). SDF-1alpha stimulates BM cell chemotaxis and the association of these cells with functional tumor vessels. Furthermore, SDF-1alpha enhances tumor neovascularization and growth with no alteration in VEGF(165). Our work suggests that SDF-1-mediated vasculogenesis may represent an alternate pathway that could potentially be utilized by tumors to sustain growth and neovasculature expansion after anti-VEGF therapy.     

Establishing long-term survival and cure in young patients with Ewing's sarcoma

This paper investigates the potential for long-term survivorship for young patients diagnosed with Ewing's sarcoma. Data are examined from two successive UKCCSG Ewing's Tumour studies (ET-1 and ET-2). Patients have been followed for up to 20 years. These studies had suggested that better 5-year survival with ET-2 over the earlier ET-1 was achieved by replacing cyclophosphamide by ifosfamide and increasing the dose of doxorubicin in a four-drug chemotherapy regimen. The updated hazard ratio, stratified for metastatic status at diagnosis, of 0.39 (95% confidence interval 0.12-0.61) confirmed the advantage of the ET-2 regimen in terms of overall survival. Cure models, based on the Weibull distribution, suggested that factors for long-term survival in addition to presence of metastases were age, primary site of tumour and study. Modelling identified the proportion cured with the ET-2 protocol as best at 70% in those who are under 10 years with a nonpelvic primary site and without metastatic disease. This contrasts to only 13% cure in those with the corresponding adverse prognostic indicators. Additionally, the risk of death remains greatest but relatively constant over the first 2 years postdiagnosis, and then declines to a lower but constant value for the next 3 years before reaching the 'cure plateau' at about 5 years. This investigation suggests that 'cure' is possible for patients with Ewing's sarcoma. This is established at approximately 5 years post diagnosis and the proportion cured depends on the presence of metastases, pelvic site and age at diagnosis.     

Local and distant control in non-metastatic pelvic Ewing's sarcoma patients

BACKGROUND AND OBJECTIVES: Due to possible complication and loss of function, surgery is not often indicated in pelvic Ewing's sarcoma (ES). The purpose of this study was to review our experience and evaluate the role of different local treatment in non-metastatic pelvic ES patients. METHODS: One hundred twenty-nine patients with pelvic ES were treated at our institution between 1975 and 1999. We excluded patients presenting metastases, patients who had died of other causes, or those with incomplete clinical documentation. Among the 73 eligible patients, 17 (23%) with progression of tumor growth during induction chemotherapy eventually died. The analysis was focused on the remaining 56 patients with good or stable clinical response to the chemotherapy. RESULTS: Patients treated with surgery, with or without radiation therapy, had a better local control (82.6% vs. 66.7%) and a significantly higher rate of 5-year EFS (73.9% vs. 30.3%, P = 0.036) than those who were only treated with local radiation therapy. CONCLUSION: Chemotherapy is the key factor in the treatment of pelvic ES. In our series, surgical treatment was associated with good prognosis for pelvic ES. The use of radiotherapy alone was less effective and should be only used in non-operable patients. Radiotherapy after surgery as a rescue method might not act effectively, while preoperative radiotherapy was associated with good clinical response and should be recommended.     

Diagnostic difficulties in extraosseous Ewing's sarcoma: A proposal for diagnostic criteria

Five cases diagnosed as extraosseous Ewing's sarcoma (EES) during a 15-year period, and the relevant literature, were reviewed. The diagnosis in these cases was difficult to confirm, mainly because the distinction between the osseous form of Ewing's sarcoma (OES) and either periosteal reactions or direct tumour invasion into adjacent bone by EES was often unclear. The literature suggests that other authors have also encountered difficulties. The authors believe that many cases reported as EES are likely to have been OES. This distinction has some importance, as the two conditions are usually treated in differing ways. The following criteria are proposed for the diagnosis of primary EES: (i) no evidence of bony involvement on magnetic resonance imaging; (ii) no evidence of increased uptake in bone or periosteum adjacent to the tumour on static isotope bone scan images; (iii) a small round cell tumour with no differentiating features on light microscopy, immunochemistry or electron microscopy; and (iv) demonstration of cytoplasmic glycogen.     

Unifocal Langerhans’cell histiocytosis (eosinophilic granuloma) resembling Ewing's sarcoma

In a 9 year old boy, a destructive lesion in the diaphysis of the right femur was wrongly diagnosed as a Ewing's sarcoma on the basis of the radiologic findings and fine needle aspiration cytology report. The clinical and radiologic picture was suggestive of Ewing's sarcoma, but an open biopsy of the lesion revealed a histopathological picture of eosinophilic granuloma. A brief review of the literature is given, together with discussion on the differential diagnosis of a mid shaft femoral lesion in young patients.