Hedgehog Promotes Neovascularization through the Regulation of Bone-Marrow Derived Progenitors.

Abstract 3048

Poster Board II-1024

The hedgehog (Hh) pathway has been implicated in the development of embryonic blood vessels and pathogenesis of cancer. Smoothened (Smo), one of the receptors in Hh signaling, is a promising molecular target for the treatment of malignancies. Pancreatic ductal adenocarcinoma (PDAC) is one of the tumors in which sonic hedgehog (Shh) is misexpressed. Although there are cell-autonomous effects of Hh on the proliferation of tumor cells, recent studies have demonstrated an oncogenic function of Hh in stromal cells. Cyclopamine antagonizes Smo and can attenuate PDAC growth in mice, resulting in regression of the tumor vasculature with reduced pericyte coverage. However, the inhibitory effect of cyclopamine on proliferation of KP-1N cells, a human PDAC line highly expressing Shh, was modest, indicating additional effects of Hh signaling on tumor progression. Here, we have identified novel molecular mechanisms by which Hh regulates tumor angiogenesis. Expression of Gli2 protein in the stroma, but not in cancer cells, was attenuated markedly by cyclopamine administration, consistent with the general absence of autocrine Hh signaling in PDAC cells. Cyclopamine significantly attenuated the homing of bone marrow (BM)-derived cells into KP-1N xenografts and their interaction with the tumor vasculature, suggesting that Hh signaling may play a role during migration and differentiation of BM-derived progenitors to participate in neovascularization. Host derived Ang-1 and IGF-1 mRNA levels in xenografts were strongly downregulated by cyclopamine, which may contribute to the maintenance and maturation of tumor vasculature. In vitro co-culture experiments demonstrated that KP-1N cells induced Ang-1/IGF-1 production in BM-progenitors (c-Kit+ fraction of BM mononuclear cell), and this induction was significantly attenuated either by cyclopamine or lentiviral shRNA targeting Smo. In addition, in vitro tube formation assay with the mouse endothelial line MS-1 and a matrigel plug assay supports the role of Shh secreted from PDAC cells to induce migration and capillary formation of BM-derived progenitors. IGF-1 is a crucial target of Hh signaling in BM-derived cells during neovascularization, since anti-IGF-1 neutralizing antibody blocked the induction of the capillary morphogenesis by BM-progenitors. Finally, this “paracrine” effect of Hh seems to be a late event during pancreatic tumorigenesis, as stromal Patch1/Gli2 expression was detected within PDAC lesions in Pdx1-Cre;LSL-KrasG12D;p53lox/+ mice, but not in PanIN lesion, a potential precursor of PDAC, in Pdx1-Cre;LSL-KrasG12D mice. We also observed upregulation of VE-cadherin and Ptch1 mRNA in lineage–/c-Kit+ fraction of BM mononuclear cells (primitive BM-derived progenitors) from PDAC mice as compared to wild-type/PanIN mice, suggesting that pro-angiogenic conditions are prepared at the level of the BM in cancer-bearing hosts. The primitive progenitors derived from ‘activated BM’ are imported to the tumor microenvironment where they become fully activated. Hh-ligand from cancer cells can therefore have a profound effect on neovascularization through the regulation of the progenitors during late stages of tumorigenesis. This work was supported by New Energy and Industrial Technology Development Organization (NEDO) of Japan.