This data supports the premise that Hh ligand originates within the tumor cells and that pathway activation also occurs within tumor cells (either the same cells or neighboring cells). Several authors remain unconvinced that Type Vandetanib ZD6474 II signaling actually exists in vivo because much of this data is based on studies with higher doses of cyclopamine which exhibit some non-specific cytotoxicity.25,26,46,55 However, in our group��s report of Hh signaling in acute lymphocytic leukemia (ALL), we demonstrated findings of increased Hh pathway expression in human ALL cell lines and clinical samples. Using a luceriferase reporter assay, we observed decreased Gli1 expression in ALL cell lines following treatment with 5E1, antagonist to Hh ligand, cyclopamine, or IPI-926 (Infinity Pharmaceuticals, Cambridge, MA), a semi-synthetic Smo inhibitor at doses which did not result in apoptosis or growth inhibition.

Treatment with these Hh inhibitors resulted in decreased self-renewal when cells were treated alone without the presence of stromal cells both in in vitro clonogenic assays, as well as in serial transplantation models in mice. Although there is likely a contributory effect of stromally-mediated Hh signaling in ALL, we believe that our data also supports a role for autocrine, Type II Hh signaling in ALL.15 Tumors characterized by Type II signaling may be susceptible to Hh inhibition at either the level of Hh ligand binding or further downstream. A growing body of data confirms the importance of Type III Hh signaling which is ligand dependent and paracrine; that is, ligand is secreted by one type of cell (either tumor or stroma) and Hh pathway activation occurs in another (tumor or stroma).

Ligand secretion by tumor cells resulting in Hh signaling in supportive stromal cells is termed Type IIIa signaling, whereas ligand secretion by stromal cells resulting in Hh signaling in the tumor cells is termed reverse paracrine signaling or Type IIIb. Tumor types in which paracrine signaling has been described include prostate,9 pancreas, and metastatic colon.25 Human prostate cancer cell lines showed enhanced growth in vivo with addition of Hh ligand while no differences were seen when cells were grown alone in vitro in absence of stroma, suggesting a role for stromally mediated Hh signaling in promoting tumor growth.

RT-PCR and in situ hybridization confirmed that increased tumor ligand expression correlated with increased mouse Gli1, Gli2, and Ptch1 from stromal cells.9 Yauch et al demonstrated similar findings of increased mouse Gli1 expression Cilengitide in response to human Hh ligand expression in pancreatic cancer and metastatic colon cancer in xenografts from human cell lines and primary tumors.25 Importantly, these findings from mouse models were also seen upon examination of human clinical samples comprised of tumor cells and infiltrating stromal cells in prostate, pancreatic, and metastatic colon cancer.