In vitro and in vivo characterization of a novel hedgehog signaling antagonist in human glioblastoma cell lines Pietro Ferruzzi 1 * , Federica Mennillo 1 * , Antonella De Rosa 1 , Cinzia Giordano 1 , Marco Rossi 1 , Giovanni Benedetti 2 , Roberta Magrini 1 , Gal.la Pericot Mohr 3 , Vincenzo Miragliotta 4 , Letizia Magnoni 5 , Elisa Mori 5 , Russell Thomas 6 , Patrizia Tunici 1 and Annette Bakker 1 1 Department of Oncology, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, Siena, Italy 2 MET profiling, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, Siena, Italy 3 Medicinal Chemistry, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, Siena, Italy 4 Toxicology, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, Siena, Italy 5 Data analysis, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, Siena, Italy 6 Director of Project operations, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, Siena, Italy Glioblastoma multiforme (GBM) is composed of heterogeneous and genetically different cells, which are highly invasive and motile. The standard chemotherapeutic agent, temozolomide, affects GBM cell proliferation but is generally unable to prevent tumor recurrence. Hedgehog pathway activation has been reported to be relevant in GBM and different pharmacological pathway modulators have been identified. We report that by growing a commercially available recurrent GBM cell line (DBTRG- 05MG) without serum and in the presence of defined growth factors; we obtained a less differentiated cell population, growing in suspension as neurospheres, in which the Hedgehog pathway is activated. Furthermore, the expression profile of Hedgehog pathway components found in DBTRG-05MG neurospheres is similar to primary stem-like cells derived from recurrent GBM patients. We report the effect of our novel specific Smoothened receptor antagonist (SEN450) on neurosphere growing cells and compared its effect to that of well known benchmark compounds. Finally, we showed that SEN450 is both antiproliferative on its own and further reduces tumor volume after temozolomide pretreatment in a mouse xenograft model using DBTRG-05MG neurosphere cells. Altogether our data indicate that the Hedgehog pathway is not irreversibly switched off in adherent cells but can be reactivated when exposed to well-defined culture conditions, thus restoring the condition observed in primary tumor-derived material, and that pharmacological modulation of this pathway can have profound influences on tumor proliferation. Therefore, pharmacological inhibition of the Hedgehog pathway is a potentially useful therapeutic approach in GBM. Glioblastoma (GBM) represents the most malignant and le- thal among brain tumors because of its high infiltration capacity and invasion into the normal brain, resulting in high rates of tumor recurrence following surgery and chemo- therapic/radiotherapic treatments. 1 Despite new therapeutic approaches developed in the last decade, the average survival of GBM patients is around 1 year. The identification of new relevant therapeutic targets in brain tumors can provide new ways to approach and treat these tumors. 2 The increasing evidence that cancers might contain and arise from stem cells, named cancer stem-like cells (CSC), has led to a poten- tial understanding of the origin of the tumor bulk and the capability to reinitiate tumor development after standard of care treatment. 3 Although initially the cancer stem cell hypothesis asserted that solid tumors were exclusively main- tained and initiated by a rare fraction of Prominin-1 (CD133) positive cancer cells with stem cell properties, 4 there is an ongoing debate about the exact identity and behavior of these cells. 5,6 Different putative neural and/or cancer stem markers used for the identification of this tumor cell subpo- pulation include Nestin, CD133, SOX2, Nanog, BMI1 and Musashi1. 4,7,8 In glioblastoma, the alkylating agent, and standard of care chemotherapy, temozolomide (TMZ) has been demonstrated to reduce the proliferation and survival of glioma cells but was often shown to be unable to prevent tu- mor recurrence. Moreover, the recurrent tumors are very of- ten completely insensitive to temozolomide. It has been sug- gested that it is the CSC cell population that remains after standard of care treatment, and being less sensitive to the standard chemotherapies and radiotherapies, this population ultimately gives rise to recurrent tumors. 9,10 On the basis of Key words: glioblastoma, Hedgehog, gli, patched, smoothened, temozolomide Additional Supporting Information may be found in the online version of this article. *P.F. and F.M. contributed equally to this work DOI: 10.1002/ijc.27349 History: Received 9 Dec 2010; Accepted 21 Oct 2011; Online 9 Nov 2011 Correspondence to: Pietro Ferruzzi, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy, Tel.: þ39-0577-381259, Fax: þ39-0577-381303, E-mail: pferruzzi@sienabiotech.it Cancer Cell Biology Int. J. Cancer: 000, 000–000 (2011) V C 2011 UICC International Journal of Cancer IJC