Miriam Benezra

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification

CONTEXT Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

BRAFV600E mutation is associated with preferential sensitivity to mitogen-activated protein kinase kinase inhibition in thyroid cancer cell lines.

CONTEXT Mutually exclusive mutations of RET, RAS, or BRAF are present in about 70% of papillary thyroid carcinomas, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. Although the signal output common to these oncoproteins is ERK, a recent report showed that only BRAF mutations consistently predicted responsiveness to MAPK kinase (MEK) inhibitors. OBJECTIVES Here we investigated whether sensitivity to MEK inhibition was determined by oncogene status in 13 human thyroid cancer cell lines: four with BRAF mutations, four RAS, one RET/PTC1, and four wild type. RESULTS Growth of BRAF (+) cells was inhibited by the MEK antagonist PD0325901 with an IC(50) of less than 5 nm. By contrast, RAS, RET/PTC1, or wild-type cells had IC(50) of 4 nm to greater than 1000 nm. Sensitivity was not predicted by coexisting mutations in PIK3CA or by PTEN status. Similar effects were obtained with the MEK inhibitor AZD6244. PD0325901 induced a sustained G1/S arrest in BRAF (+) but not BRAF (-) lines. PD0325901 was equipotent at inhibiting pERK1/2 after 2 h, regardless of genetic background, but pERK rebounded at 24 h in most lines. MEK inhibitor resistance was associated with partial refractoriness of pERK to further inhibition by the compounds. AZD6244 was more potent at inhibiting growth of NPA (BRAF +) than Cal62 (KRAS +) xenografts. CONCLUSION Thyroid cancers with BRAF mutation are preferentially sensitive to MEK inhibitors, whereas tumors with other MEK-ERK effector pathway gene mutations have variable responses, either because they are only partially dependent on ERK and/or because feedback responses elicit partial refractoriness to MEK inhibition.

Antiproliferative activity to vascular smooth muscle cells and receptor binding of heparin-mimicking polyaromatic anionic compounds.

Proliferation of bovine aortic smooth muscle cells (SMCs) induced by thrombin, basic fibroblast growth factor, or serum is inhibited by anionic, nonsulfated aromatic compounds that mimic many of the effects of heparin. Among these compounds are aurintricarboxylic acid (ATA) and a newly synthesized polymer of 4-hydroxyphenoxy acetic acid (compound RG-13577). Iodinated- or 14C-labeled compound RG-13577 binds to cultured SMCs in a highly specific and saturable manner. Scatchard analysis of the binding data revealed the presence of an estimated 1 x 10(7) binding sites per cell with an apparent dissociation constant of 3 x 10(-6) mol/L. Binding of radiolabeled RG-13577 was efficiently competed for by related aromatic anionic compounds and by apolipoprotein E, but not by heparin, heparan sulfate, suramin, or various purified growth factors and extracellular matrix proteins. Receptor cross-linking of SMC-bound 125I-RG-13577 revealed a single species of high M(r) (approximately 280 kD) cell surface receptors detected in the absence but not the presence of excess unlabeled compound RG-13577. Binding was susceptible to downregulation and restoration of receptor levels in a manner similar to that of hormone and growth factor receptors. We suggest that the antiproliferative activity of compound RG-13577 and related compounds is initiated by binding to specific growth-inhibiting cell surface receptors. Heparin-mimicking compounds may be applied to inhibit SMC proliferation associated with atherosclerosis and restenosis.

Thrombin enhances degradation of heparan sulfate in the extracellular matrix by tumor cell heparanase.

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation.

A synthetic heparin‐mimicking polyaromatic anionic compound RG‐13577 (polymer of 4‐hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr∼5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of 14C‐RG‐13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor‐related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG‐13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild‐type and heparan sulfate‐deficient Chinese hamster ovary (CHO) cells, as well as normal‐ and LDL receptor negative‐ human skin fibroblasts bind RG‐13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor‐related protein (LRP) expressed a markedly reduced binding of RG‐13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG‐13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG‐13577, suggesting that this inhibition is mediated by RG‐13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and therby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis. J. Cell. Biochem. 81:114–127, 2001.

Functional Domains in Thrombin Outside the Catalytic Site

Thrombin is known to be sequestered within the matrix of the fibrin gel, where it may remain active and intact for extended periods of time (Wilner et al., 1981; Mann, 1987). Since deposition of fibrin accompanies wound healing, thrombin may participate in modulation of this process. Inflammation is a localized protective response elicited by injury or destruction of tissues, which serves to destroy both the injurious agent and the injured tissue. Most forms of acute and chronic inflammation are amplified and propagated as a result of the recruitment of humoral and cellular components of the immune system. Immunologically mediated elimination of foreign material proceeds through a series of integrated steps. The actual destruction of antigens by immune mechanisms is mediated by cells with phagocytic capability. Such cells may migrate freely or may exist at fixed sites as components of the mononuclear phagocyte system. Since inflammation in its early stages is characterized by an influx of inflammatory cells, we wondered whether thrombin might participate in recruitment of such cells.

Abstract LB-299: F-dasatinib inhibits glioma cell proliferation and alters expression of PDGFR signaling pathway intermediates in PDGFR-overexpressing glioma models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Current knowledge in tumor biology has enhanced our understanding of the role of kinase receptors, such as platelet-derived growth factor receptor (PDGFR), and associated downstream signal transduction pathways in tumor growth and maintenance. New tyrosine kinase (TK) inhibitors to these receptors and signaling pathways have demonstrated efficacy in certain clinical settings and offer the potential to revolutionize disease treatment. Imatinib mesylate (Gleevec) and PTK787/ZK222684, inhibitors of PDGFR, have failed to show significant clinical benefit as monotherapies. Dasatinib (BMS-354825), a more potent second-generation inhibitor of multiple TK receptors, including PDGFR, has demonstrated promising therapeutic potential in managing intracranial leukemic disease. Here, we assayed the in vitro dose-dependent uptake and inhibitory response of brain tumor cells derived from PDGFR-overexpressing genetically-engineered gliomas, to a novel PDGFR inhibitor and dasatinib analogue (fluorinated dasatinib). In addition, we determined the relative potency of F-dasatinib in vitro, as well as non-invasively assessed PDGFR receptor status and tumor-specific targeting in vivo using radiolabeled dasatinib (18F-dasatinib) and positron emission tomography (PET) imaging. Uptake and inhibitory response of fluorinated dasatinib in vitro was found to be dose-dependent, resulting in cell cycle arrest and alterations in tumor cell proliferation and apoptosis. No difference was seen between BMS-354825 and its fluorinated analogue in terms of tumor cell proliferative or inhibitory responses. Tumor cell proliferation was examined over a range of concentrations (10-200 nM), with significant inhibition seen from 50-150 nM over a 96-h interval; 80% inhibition was found at 150 nM (IC50=10 nM). Serum-starved glioma cells demonstrated total inhibition of cell proliferation in the presence of PDGF (25 ng/ml) and 150 nM F-dasatinib. By contrast, glioma cell proliferation was not significantly inhibited by imatinib or PTK787/ZK222684 over similar concentration ranges. In addition to F-dasatinib being a potent inhibitor of glioma cell proliferation, expression of p-PDGFRalpha and key downstream signaling pathway intermediates were observed; p-ERK, p-Akt and p-Src were inhibited within 15 min of treatment with 150 nM F-dasatinib and in the presence of 10% FBS or 25 ng/ml PDGF. Further, dose-dependent alterations in the percentage of cells in the S phase of the cell cycle (∼100% reductions at 150 nM) and apoptosis were found, the latter effects enhanced up to concentrations of 150 nM and accompanied by increased PARP protein expression at 250 nM F-dasatinib. In vivo studies using GEM glioma models revealed tumor-specific uptake of 18F-dasatinib probe using dynamic PET imaging. These results suggest that, in GEM gliomas, dasatinib analogues may serve as diagnostic or therapeutic biomarkers. 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 LB-299.