Transforming growth factor beta (TGF-beta), a cytokine with potent promalignant properties, is overexpressed in different tumors. Therefore, the development of TGF-beta inhibitory molecules is of paramount importance to improve the efficacy of antitumor therapies. Several strategies using neutralizing antibodies, TGF-beta soluble binding proteins, antisense compounds, or small molecule inhibitors targeting TGF-beta signaling, have shown that TGF-beta inhibition helps in antitumor therapy. Some of these compounds have shown their value by directly inhibiting tumor growth and metastasis, whereas others have been able to enhance antitumor immune responses. The aim of our study was to investigate the effect of two peptide inhibitors of TGF-beta, namely P144 (1) and P17 (2), in 6 human tumor cell lines derived from medulloblastoma (DAOY), rhabdomyosarcoma (TE671), neuroblastoma (SK-NSH and SH-SY5Y), and glioblastoma (A172 and U87-MG). P144 and P17 are derived from human TGF-beta type III receptor sequence (1) and phage display library technology (2) respectively. Both peptides have high affinity for soluble TGF-beta and potent inhibitory effects on TGF-beta binding to its receptors. MTT assay and an apoptosis test (Cell Death Detection ELISA PLUS, Roche) were performed before and after treatment with the peptides, which were added to the media at a final concentration of 100 ug/ml. P17 did not produce any effect on the cell lines, while P144 induced growth inhibition and apoptosis in three brain tumor cell lines (DAOY, A172, and U87-MG). The level of phosphorylated Smad2 (that initiates the activation of the TGF-beta intracelular signaling pathway) was decreased in presence of P144 in all cases, as determined by Western blot, confirming that P144 inhibits TGF-beta signaling. qRT-PCR was performed in order to analyze different genes related with the regulation of the TGF-beta signaling, such as Smad7, Ski, SnoN, FoxG1, which are inhibitors of the pathway, and p21CIP. Our preliminary data show that P144 affects the transcription of Smad dependent genes, e.g. Smad7, SnoN and Ski, although confirmatory experiments are still required. Our data suggest that inhibition of the TGF-beta pathway by P144 inhibits cell growth and increases apoptosis in brain tumor cell lines, possibly in a Smad-dependent manner. Therefore, P144 should be further tested as a therapeutic agent against brain tumors. 1. Ezquerro IJ, et al. A synthetic peptide from transforming growth factor beta type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury. Cytokine 22:12-20, 2003. 2. Dotor J, et al. Identification of peptide inhibitors of transforming growth factor beta 1 using a phage-displayed peptide library. Cytokine. 39:106-115, 2007. 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 5044. doi:10.1158/1538-7445.AM2011-5044

In this work, we aimed at analyzing genome-wide gene expression and DNA gains and losses in neuroblastoma cell lines and in neurospheres containing stem-like cells. SKNDZ and SIMA neuroblastoma cell lines were grown in DMEM cell culture medium, and in DMEM-F12 selective serum free medium (with EGF, bFGF and B27; to induce the neurosphere-forming phenotype). After RNA and genomic DNA extractions from the 4 cell lines (2 standard, 2 neurosphere-forming cell lines), expression microarrays and array-CGH analyses were performed (Aligent Technologies). Array-CGH did not show any significant differences between standard and neurosphere-forming cell lines, both in SKNDZ and in SIMA. Microarray expression analysis demonstrated a total of 425 upregulated and 170 downregulated genes in neurosphere-forming cell lines. The differentially expressed genes were classified using the PANTHER classification system (www.pantherdb.org). As a result, apoptosis, cell adhesion, cell communication, cell cycle, and immune system processes appeared upregulated and downregulated in neurospheres. Some of those genes participate in pathways related with cancer (Table 1). In conclusion, the stem-like phenotype does not seem to be linked to anatomic changes at the level of deletions/gains of DNA, but to changes in expression of genes, like those of the TGF-beta, Notch and Sonic Hedgehog pathways. Those pathways, specially TGF-beta, widely described as an important therapeutic target against cancer, should be further studied to determine their real implication in the neurosphere-formation process in neuroblastoma, and therefore, as candidate targets for the treatment of neuroblastoma stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 413. doi:1538-7445.AM2012-413

Neuroblastoma, the most common extracranial malignant solid tumor in childhood has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to molecular differences in tumor cell subpopulations. The cancer stem cells (CSC) model proposes the existence of a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the stem-like cell population content of two commercial neuroblastoma cell lines (SKNDZ and SIMA) by the use of conditioned cell culture media for neurospheres, and compared genome expression and genomic gains and losses appearing in neurospheres versus the alterations appearing in standard tumor cells, by microarray analysis and array-CGH, respectively. Array-CGH did not show any significant differences between standard and neurosphere-forming cell lines, both in SKNDZ and in SIMA. The microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the stem-cell like phenotype. As expected, some signalling pathways detected were involved in self-renewal of normal tissues (Wnt, Notch, Hedgehog and TGF-β) and seem to contribute to CSC phenotype when deregulated. Among them, we focused on the aberrant activation of Hedgehog and TGF-β signalling pathways. We confirmed the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by quantitative PCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We analyzed two CSC surface markers, and found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk of different pathways in neuroblastoma, and the importance of it in stem-like cells, as confirmed by the overexpression observed in JAG1, one of the targets of TGF-β and also the main activator of Notch signalling pathway. Further analysis based on this work could identify possible new targets for molecular CSC therapies against neuroblastoma, highlighting the importance of redirecting current cancer treatments towards CSC to achieve total elimination of tumor cell population and improve treatment effectiveness. Citation Format: Raquel Ordoñez, Gabriel Gallo, Soledad Martínez, Sheila Legarra, Noémie Pata-Merci, Justine Guegan, Giselle Danglot, Xing Fan, Juan A. Rey, Alain Bernheim, Javier S. Castresana. Genome-wide microarray expression and genomic alteration by array-CGH analysis in neuroblastoma stem-like cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3051. doi:10.1158/1538-7445.AM2014-3051