Purpose: Recent studies have revealed that the majority of pediatric low-grade astrocytomas (PLGA) harbor the BRAF-KIAA1549 (B-K) fusion gene resulting in constitutive activation of the RAS/MAPK pathway. However, the clinical significance of this genetic alteration is yet to be determined. We aimed to test the prognostic role of the B-K fusion in progression of incompletely resected PLGA.Experimental Design: We retrospectively identified 70 consecutive patients with incompletely resected "clinically relevant" PLGA. We added 76 tumors diagnosed at our institution between 1985 and 2010 as controls. We examined BRAF alterations by reverse transcriptase PCR, FISH, and single-nucleotide polymorphism array analysis and correlated that with progression-free survival (PFS).Results: Overall, 60% of tumors were B-K fusion positive. All patients with B-K fused PLGA are still alive. Five-year PFS was 61% AE 8% and 18% AE 8% for fusion positive and negative patients, respectively (P ¼ 0.0004). B-K fusion resulted in similarly significant favorable PFS for patients who received chemotherapy. Multivariate analysis revealed that B-K fusion was the most significant favorable prognostic factor in incompletely resected PLGA and was independent of location, pathology, and age. In vitro, BRAF overexpression resulted in growth arrest associated with DNA damage (gH2AX expression). Five-year PFS was 68% AE 15% and 0% for patients with B

Differential regulation of telomerase activity in normal and tumor cells provides a rationale for the design of new classes of telomerase inhibitors. The telomerase enzyme complex presents multiple potential sites for the development of inhibitors. GRN163L, a telomerase enzyme antagonist, is a lipid-modified 13-mer oligonucleotide N3V ! P5V -thiophosphoramidate, complementary to the template region of telomerase RNA (hTR). We evaluated both the in vitro and in vivo effects of GRN163L using A549-luciferase (A549-Luc) human lung cancer cells expressing a luciferase reporter. GRN163L (1 Mmol/L) effectively inhibits telomerase activity of A549-Luc cells, resulting in progressive telomere shortening. GRN163L treatment also reduces colony formation in soft agar assays. Surprisingly, after only 1 week of treatment with GRN163L, A549-Luc cells were unable to form robust colonies in the clonal efficiency assay, whereas the mismatch control compound had no effect. Finally, we show that in vivo treatment with GRN163L is effective in preventing lung metastases in xenograft animal models. These in vitro and in vivo data support the development of GRN163L as a therapeutic for the treatment of cancer. (Cancer Res 2005; 65(17): 7866-73)

Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.

Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after <4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice, concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy.

Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29-35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissues in vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cells in vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.

Using fixed microvessel endothelial cell monolayers the molecules involved in the adhesion of liver-preferring murine RAW117 large cell lymphoma cells to murine liver-derived microvessel endothelial cells were identified by affinity isolation. Detergent lysates obtained from poorly (P) or highly (H10) liver-metastatic cells inhibited RAW117-H10 cell adhesion to hepatic sinusoidal endothelial (HSE) cell monolayers. Allowing detergent lysates of cell surface-labeled RAW117 cells to bind to fixed HSE cell monolayers and eluting the bound components indicated that several tumor cell surface molecules (approximately 70, approximately 35, approximately 32, approximately 22, and approximately 14 kDa) might be involved in RAW117 cell-HSE cell adhesion. The approximately 35 kDa component was cation dependent in its binding to target HSE cells. Increasing detergent concentration had no effect on binding of the approximately 35 kDa component to HSE cell monolayers, whereas treatment with 0.5 M NaCl resulted in its selective elution from HSE cells. Incubation of the HSE cell monolayers with detergent lysates from cell surface-labeled RAW117-H10 cells resulted in selective depletion of the approximately 35 kDa component, suggesting that the binding is saturable. This divalent cation-dependent molecule is one of the major tumor cell surface components bound by several types of endothelial cells and murine hepatocytes, whereas there was poor binding of this component to unfixed or fixed human red blood cells. The purified, partially (approximately 40%) sequenced molecule had amino acid sequence identity with murine but not bovine annexin II, indicating that it was not bound from the bovine serum used to grow RAW117 cells. Using antibodies specific for annexin II flow cytometry indicated equivalent amounts of annexin II are expressed on RAW117 cell surfaces in the absence or presence of excess EDTA, whereas annexin I was only found in low amounts on the surfaces of RAW117 cells. Annexin II antibodies inhibited by approximately 40-50% the adhesion of RAW117 tumor cells to live or fixed endothelial cells, and purified tumor cell surface fractions containing the approximately 35 kDa component partially inhibited (approximately 35%) RAW117 cell-HSE cell adhesion. The data indicate that annexin II is expressed on the extracellular surface of RAW117 cells, and cell surface-annexin II mediates a portion of the Ca(2+)-dependent RAW117 cell adhesion to liver microvessel endothelial cells.

BackgroundPlasma cells constitute the majority of tumor cells in multiple myeloma (MM) but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC). These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities.Methodology/Principal FindingsHuman MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1) as revealed by quantitative real-time PCR.ConclusionsTelomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms.

Cultured tumor cell lines, tumor xenografts grown in athymic nude mice, and a murine experimental metastasis model were used to assess the in vitro and in vivo anti-tumor activity of the potent IMP dehydrogenase (IMPDH) inhibitor, mycophenolic acid (MPA), and its morpholinoethyl ester pro-drug, mycophenolate mofetil (MM). The growth of all the cell lines tested was inhibited by MPA in vitro, with EC50 values ranging from less than 0.1 microM to 3.9 microM. Mice were monitored for s.c. tumor outgrowth in the case of human tumor xenograft models or survival time for the murine experimental metastasis model. Treatment with MM p.o. was started 24 hr after tumor challenge or after tumors became palpable. Treatment of athymic nude mice bearing A3.01 (T-lymphoblast), Molt-4 (T-cell leukemia), CaPan-2 (pancreatic adenocarcinoma), CaLu-3 (non-small-cell lung adenocarcinoma), LS174T and T84 (colon adenocarcinoma), and Daudi (B-cell lymphoma) human tumor xenografts with MM significantly inhibited s.c. tumor growth. Treatment of BALB/c mice with MM after i.v. injection of murine RAW117-H10 lymphoma cells in an experimental metastasis assay resulted in increased survival time for treated animals. No significant inhibitory effect on s.c. tumor outgrowth was seen with MM treatment of SK-Hep-1, a human hepatic endothelioma, or Hep-3B, a liver adenocarcinoma, at any of the doses tested.