Although in in vitro analyses GANT61 sensitized Daoy Coumarin mg to RITA treatment, this was not fully reflected in the in vivo xenograft studies in nude mice, as the combinatorial treatment elicited a comparable reduction of tumor growth. However, the dual drug administration reduced within-group variation, seen in volume measurements throughout the experiment [Fig. S7A], and downregulated tumor cell proliferation [Fig. 5D]. In fact, in the Rh36 subcutaneous xenografts, the observed tumor suppressive effects were correlated with HH signaling downregulation, primarily with RITA and to a lesser extent with GANT61 administration. Moreover, in the combinatorial treatment, GANT61 apparently blocked the suppressive effects of RITA on HH targets [Fig. 5E,G; Fig. S7B]. Consequently, our findings pinpoint to variable modes of RITA and GANT61 action, with the combinatorial treatment blocking the RITA effects and eliciting a broader response, exemplified by the GO and KEGG pathway enrichment analyses [Fig. S8]. In conclusion, our data demonstrate that RITA reduces HH signaling irrespective of p53 in medulloblastoma and rhabdomyosarcoma cells. Surprisingly, GANT61 is not as effective as RITA in downregulating HH signaling, with the dual drug administration almost completely blocking the HH signaling response in vivo, suggesting a certain antagonism of the two drugs.
Acknowledgements This study was funded by the Swedish Cancer Society and the Swedish Childhood Cancer Foundation. A.A. was supported by the Karolinska Institutet Doctoral (KID) funding program and Y.D. by the China Scholarship Council. Rune Toftgård and Vladimir Bykov are acknowledged for helpful discussions. Raoul Kuiper and Agneta B. Andersson are acknowledged for their help in immunohistochemistry analyses. The authors also would like to thank the core facility at NEO, BEA, Bioinformatics and Expression analysis, which is supported by the board of research at the Karolinska Institute and the research committee at the Karolinska Hospital.
Introduction The molecular dissection of the Hedgehog (Hh)-Gli signal transduction pathway in insects (e.g. [1], [2], [3], [4], [5], [6], [7]) and vertebrates (e.g. [8], [9], [10], [11], [12], [13], [14], [15], [16]), has revealed it to be complex and context-dependent with a surprising number of distinct cellular outputs. We are just beginning to understand the meaning of species-specific differences in Hh signaling but what is clear is that a single-species (e.g., mouse)-centric view is not universally informative. How or why organisms would have evolved multiple Ptc receptors (as in worms) for instance, increase the number of Hh ligands or of Gli proteins (as in zebrafish), or constraint HH signaling to primary clia in some species and tissues is unclear but likely to have important clues to speciation and the evolution of the morphogenetic plan (reviewed in [15]). The outputs are numerous since the HH pathway controls aspects of cell proliferation, survival, migration and stemness. How these are orchestrated over time in developing tissues remains unclear. The GLI proteins also regulate and are regulated by tumor suppressors, such as p53 and this reveals yet another important aspect of HH-GLI signaling: its major role in human cancer (reviewed in [16]). But perhaps the most intriguing aspect of this and other pathways is their context-dependency. How is it that the same extracellular input can be interpreted differently by responding cells? How is it that reception of a HH ligand can lead to diverse responses in time, space and in different cell types? While the complexity of the pathway makes a complete discussion for a review chapter not feasible, we focus here on the GLI zinc finger transcription factors, which represent the terminal station of the canonical HH signaling path. Whereas other reviews and papers address key aspects of the morphogenetic function of HH ligands (e.g. [17], [18], [19], [20]) we elect to focus this review on 3 key points of the highly context-dependent nature of the HH-GLI pathway, where the history and the molecular make-up of the receiving cell determines the qualitative and quantitative output and biological effect: 1 – The GLI code; 2 – Regulation of the GLI code by non-HH signals and by the oncogenic load; and 3 – Mechanisms of GLI regulation. In choosing to do so, here we wish to emphasize the fact that the GLI transcription factors act as key determinants in the interpretation of context- and concentration-dependent canonical HH-GLI signaling in development and disease, and that the GLI code is a signaling integration node.