Panel Of Sarcoma Cell Lines Research Articles

Abstract 728: Molecular mechanisms of resistance to anti-IGF-1R therapies in Ewing's sarcoma

Abstract IGF-1R represents a promising therapeutic target, particularly in sarcomas. Identification of patient selection criteria and understanding the potential mechanisms involved in development of resistance will be crucial for an appropriate and successful design of clinical trials. Here we evaluated efficacy of the anti-IGF-IR monoclonal antibody (HAb) AVE1642, in a panel of sarcoma cell lines and we developed a model of resistance to the anti-IGF-1R human antibody AVE1642 or to the small tyrosine kinase inhibitor (TKI) NVP-AEW541 starting from two highly sensitive Ewing's sarcoma cell lines to identify indicators of intrinsic and adaptive resistance to anti-IGF1R therapies. Cells made resistant to AVE1642 showed same growth ability either in monolayer and in anchorage-independent condition respect to sensitive cells. Notably, resistant cells retained the same level of sensitivity to doxorubicin, vincristine, and ifosfamide as the parental cell lines. Annotation analysis highlighted that the insulin pathway seems to be significant in resistant cells. In fact, these cells showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Molecular characterization demonstrate that enhanced IR-A formation and IGF-II production are heavily involved in native and acquired resistance to IGF targeted therapies. This finding was extended to cells resistant to the small tyrosine kinase inhibitor NVP-AEW541. We also show that tumors with low IGF-1R:IR-A 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. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF1R therapies and provide evidences supporting IR-A as an important target in sarcoma therapy. Future trials with these highly selective drugs should involve preselection of patients with tumors that express low levels of IR-A and IGF-2. Studies are supported by grants from the Italian Association for Cancer Research, the Italian Ministry of Health, the Italian Ministry of Research and Instruction and the EU project Eurobonet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2011-728

Abstract 1655: Mechanism of the synergistic antiproliferative effect of gefitinib and interferon-alpha in sarcoma cell lines

Abstract A static 50% 5-year overall survival rate reflects inadequacy of currently available therapies and urgent need to develop treatment strategies for sarcoma. Single epidermal growth factor receptor (EGFR) inhibitor, gefitinib was reported in a phase II trial in synovial sarcoma with a low response rate and short sustained time. Identification of combinations with both a higher proportion of responders and potentially more sustained benefit is needed. One potential partner is interferon-alpha. We have shown in vitro that cell growth signals such as those from an activated EGFR increases resistance to interferon-alpha, and the combination of EGFR inhibitor and interferon-alpha has synergistic antitumor effect in some colon and bladder cancer cell lines. A recent phase II trial of gefitinib and pegylated interferon alfa 2b in previously-treated renal cell carcinoma has demonstrated significant and prolonged disease control benefit with acceptable toxicity. However the effect of the combination therapy in sarcoma is unreported. This study aimed to investigate the effect and mechanisms of this combination therapy in human sarcoma cell lines. Combination treatment using gefitinib and interferon-alpha was investigated in a panel of sarcoma cell lines (6 soft tissue sarcomas (STS) and 3 osteosarcomas (OS)) with synergy analysis based on the Chou-Talalay combination index. Mechanism studies examined a pair of synergistic and antagonistic liposarcoma cell lines using multiple techniques. All 9 cell lines expressed wild-type and/or activated EGFR and interferon-alpha receptors but no EGFR gene mutation was identified. Combination treatment of EGFR inhibition and interferon-alpha achieved synergistic antiproliferative effect (combination index (CI) <1 = synergism) in 6/9 sarcoma cell lines (CI for STS: GCT 0.1-0.3, SW684 0.3-0.7, and 778 0.1-0.7; for OS: MG63 0.3-0.7, U2OS 0.3-0.7, and SJSA 0.1-0.3), accompanied by apoptotic and necrotic cell death. The drug reduction index (DRI) at IC50 measures how much the dose of each drug may be reduced when drugs were combined at the IC50 effect level compared with the dose of drug alone. For the most synergistic GCT cell line, the DRI for gefitinib and interferon-alpha was 4.7 and >100. Characterisation of interferon-alpha and EGFR signaling interaction by both pathway inhibition and western blot detection of downstream factor expression discovered that interferon-alpha enhances gefitinib through affecting the ratio of the signal transducer and activator of transcription (STAT) family members (STAT1 up, STAT3 down) to favour tumor cell death. The activated Akt enhanced by interferon-alpha alone was significantly down regulated by combination therapy. EGFR inhibition combined with interferon-alpha is a worthwhile treatment to pursue in anti-sarcoma therapy. These results provide a rationale for the clinical evaluation of this treatment strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1655.

Preclinical activity of palifosfamide lysine (ZIO-201) in pediatric sarcomas including oxazaphosphorine-resistant osteosarcoma

Oxazaphosphorines, such as ifosfamide (IFA), are frequently used in the treatment of pediatric sarcomas. They are pro-drugs and undergo hepatic metabolism into the active moiety and potentially toxic by-products such as acrolein and chloracetaldehyde, which may cause hemorrhagic cystitis and encephalopathy, respectively. In addition, resistance to oxazaphosphorines can be mediated by overexpression of enzymes involved in their catabolism. Isophosphoramide mustard (IPM, palifosfamide) is the active moiety of IFA. In the current study, the activity of palifosfamide lysine (ZIO-201), a stable form of palifosfamide, was evaluated in a panel of sarcoma cell lines and tumor xenografts including oxazaphosphorine-resistant xenografts. The cytotoxic effect of palifosfamide lysine was studied in osteosarcoma (OS), Ewing's sarcoma (ES) and rhabdomyosarcoma (RMS) cell lines using the MTT assay. In vivo, the maximum tolerated dose (MTD) of palifosfamide lysine was determined in SCID mice based on a 3-day intravenous (IV) administration schedule. The effect on tumor growth and event-free survival was assessed at the MTD in all three sarcoma xenografts. In OS, cyclophosphamide (CPA)-resistant and -sensitive xenografts (OS31 and OS33, respectively) were evaluated for palifosfamide lysine activity. ALDH1A1 and ALDH3A1 gene expression data for the OS xenografts were mined from the Pediatric Preclinical Testing Program gene expression data. ALDH3A1 enzyme levels were compared between the CPA-resistant and -sensitive xenografts. Palifosfamide lysine was cytotoxic against all the cell lines tested with the IC(50) ranging from 0.5 to 1.5 microg/ml except for OS222, which had an IC(50) of 7 microg/ml. The IV MTD of palifosfamide lysine in mice was 100 mg/kg per day for three consecutive days. Tumor growth inhibition was seen in both OS31 and OS33 xenografts and the RMS xenograft resulting in a significant difference in event-free survival between the control and the treated groups. Differential gene expression of ALDH3A1 but not ALDH1A1 was noted in the OS31 xenograft. This was confirmed by RT-PCR and the ALDH3A1 enzyme assay. ALDH3A1 enzyme activity was measured at 100 mIU/mg of protein in OS31 xenograft but no significant activity was seen in the OS33 xenograft. We conclude that palifosfamide lysine has broad activity in a panel of sarcoma cell lines. It inhibits tumor growth in OS and RMS xenografts. Furthermore, it is active against the CPA-resistant, ALDH3A1 overexpressing, OS xenograft suggesting that it might have the potential of overcoming this resistance mechanism against oxazaphosphorines and may be an active agent in resistant/relapsed sarcomas in patients.

Extreme sensitivity to Yondelis® (Trabectedin, ET‐743) in low passaged sarcoma cell lines correlates with mutated p53

Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.

Expression of growth factor receptors, the focal adhesion kinase, and other tyrosine kinases in human soft tissue tumors.

The tyrosine kinases are a family of genes that includes many growth factor receptors and protooncogenes. They appear to have a role in many cancers, but have not been systematically studied in the pathogenesis and progression of human sarcomas. To characterize the protein tyrosine kinases that are expressed in human sarcomas, we used a polymerase chain reaction (PCR)-based method to construct kinase-specific cDNA libraries from low-grade and high-grade primary tumors. Thereafter, individual tyrosine kinase gene expression was assessed in a panel of sarcoma cell lines and primary tumors using Northern blotting and PCR. We identified 19 species of tyrosine kinase genes, including many growth factor receptors, the human homolog of the focal adhesion kinase (FAK) gene, and a novel trk-related kinase designated HGK2. Messenger RNA expression analyses showed relative overexpression of the two forms of the platelet-derived growth factor receptors (PDGFRs) with expression of the alpha form restricted to a subgroup of high-grad and metastatic sarcomas. We were unable to demonstrate coexpression of the PDGF isoforms in primary tumors that overexpressed the receptors, suggesting that a PDGF/PDGFR autocrine pathway may not be a central mechanism in the malignant transformation of sarcomas in vivo. FAK expression was observed in a variety of sarcomas, with increased levels in several high-grade and metastatic leiomyosarcomas. When grouped together by histologic cell type and grade, the expression data of the 19 kinases in primary tumors described a greater degree of heterogeneity than is generally appreciated by clinicopathologic classification schemes. This diversity suggests that sarcomas, even those that appear to be clinically similar, arise through a variety of molecular pathways involving tyrosine kinases.