Hedgehog Pathway Inhibition in Liver Cancer Model Figure 5. Inhibition of Hh signaling decreases osteopontin and osteopontin-responsive (CD44) positive cells in tumors and peri- tumoral tissues of aged Mdr22/2 mice. A. Tumor sections from representative DMSO- and GDC-0449 treated Mdr22/2 mice were stained to demonstrate osteopontin (OPN) Representative sections are displayed (Right panel). OPN staining was quantified by morphometric analysis of at least 5 HPF per tumor section using 206 magnification (n = 5 mice/group). Results in the GDC-0449-treated group were normalized to that of the group treated with DMSO vehicle and graphed as fold change. Data are displayed as Mean6SEM (**p,0.01). B. Immunohistochemical staining for the osteopontin receptor, CD44, in peri-tumoral tissues of representative DMSO- and GDC-0449- treated Mdr22/2 mice. (Right panel) CD44 staining was quantified by morphometric analysis as described in A. Results in GDC-0449-treated mice were normalized to those of vehicle-treated controls and graphed as Mean6SEM (**p,0.01). C. QRT-PCR analysis of liver tumor RNA from DMSO- (open bar) and GDC-0449- (closed bar) treated Mdr22/2 mice for OPN (left) and CD44 (right). After normalization to results in the DMSO-treated group, Mean6SEM were graphed (*p,0.05). doi:10.1371/journal.pone.0023943.g005 during chronic fibrosing liver injury. Mdr2-deficient mice lack a phospholipid flippase that is required for normal bile formation, and consequently exhibit liver injury and ductular proliferation from a young age. All afflicted mice eventually develop significant liver fibrosis and metastatic HCC. Our results demonstrate that this pathology is accompanied by progressive activation of the Hh pathway. Introduction of a specific inhibitor of the key Hh signaling intermediate, Smoothened, reduced pathway activity and proved that sustained Hh signaling was required to maintain the expanded populations of liver myofibroblasts and progenitors that had accumulated in the damaged livers. Moreover, treating aged Mdr2-deficient mice (which already had advanced liver fibrosis and HCC) with the Hh pathway inhibitor significantly reduced liver fibrosis and tumor burden, demonstrating for the first time that inhibiting Hh signaling has clinically-relevant, therapeutic value for both liver fibrosis and HCC. Even more exciting is evidence that the prohibitive effect on hepatic tumor growth in vivo pertains to both intra-hepatic HCC and distant metastasis. In addition, our results provide insight into some of the underlying mechanisms involved. Hepatic production of Hh ligands was increased in mice that developed progressive liver fibrosis and invasive HCC. These mice also demonstrated consistently higher serum aminotransferase levels, in keeping with other evidence that Mdr2 deficiency provokes chronic hepatocyte injury [38,39]. Various stressors that reduce hepatocyte viability have been shown to induce production and release of Hh ligands by wounded hepatocytes [40,41]. Thus, hepatocyte injury is likely one of the factors that increases Hh ligand generation in mdr2- deficient livers. Myofibroblastic cells and ductular-type progenitors that progressively accumulate in chronically injured livers also produce Hh ligands, as well as other factors, such as PDGF-b, that PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e23943