Hedgehog Pathway Inhibition in Liver Cancer Model Figure 1. Increased hepatic Hedgehog (Hh) pathway activity in Mdr22/2 mice. A. Western blot analysis for Sonic Hedgehog (SHh) and Indian Hedgehog (IHh) in whole liver extracts pooled from young Mdr22/2 mice (2–16 weeks old), old (36–64 weeks old) Mdr22/2 mice and age- matched controls (n = 3–5 mice/group/time point). A representative Western blot demonstrating results from young mice is shown. Mean6SEM data from older mice are graphed. B. Representative liver sections stained to demonstrate Gli2 from young (4 wk old), middle aged (36 wk old) and old (52 wk old) Mdr22/2 mice. Little Gli2 expression was noted in wildtype mice at any age, so results from a representative 36 wk old wild type mouse is shown. (106) C. Quantitative Gli2 immunohistochemical data. The number of nuclear Gli2(+) cells was counted in at least 5 high power fields (HPF) per liver section in Mdr22/2 mice and wild type controls at 4, 36 and 64 weeks of age (n = 3–4 mice/group/time point) and mean6SEM are graphed. *p,0.05, **p,0.01 in Mdr22/2 groups vs. respective controls. doi:10.1371/journal.pone.0023943.g001 (Figure 3B). Treatment with GDC-0449 decreased a-sma- expressing myofibroblastic cells, hepatic expression of TGF-b and PDGF-b, Sirius red staining, and hydroxyproline content, demon- strating that Hh pathway inhibition reduced liver fibrosis (Figures 3C–D and Figures S3C–D). Hedgehog signaling inhibition with GDC-0449 decreases accumulation of liver progenitor cells In response to injury, progenitor populations in the liver proliferate. Hence, immunohistochemical analysis for progenitor markers, such as cytokeratin-19 (CK-19) and a-fetoprotein (AFP), showed age-related increases in Mdr22/2 mice (Figure 4A). Co- staining for CK19 and Gli2 confirmed that the progenitor population was Hh responsive (Figure 4B). Upon treatment with GDC-0449, progenitor markers decreased (Figure 4C), indicat- ing that Hh signaling was required to maintain progenitor populations during tumorigenesis, and that Hh inhibition was sufficient to shrink the size of progenitor populations even in mice with advanced HCC. Decreasing Hedgehog signaling reduces expression of osteopontin and prevents accumulation of osteopontin- responsive (CD44-positive) cells Stem/progenitor cell populations for many types of cancer, including HCC, are thought to be enriched with cells that express CD44, a receptor for the stem cell growth factor, osteopontin [35,36]. Osteopontin expression is regulated by Hh signaling [37]. Therefore, we examined the effects of GDC-0449 on osteopontin and its receptor, CD44. Immunohistochemistry and quantitative morphometry demonstrated that inhibition of the Hh pathway with GDC-0449 significantly decreased osteopontin staining within primary liver tumors (Figure 5A). Similar treatment-related decreases in CD44+ tumor cells were also noted (Figure 5B). Gene expression analysis showed that expression of both osteopontin and CD44 mRNAs also tended to decrease in GDC-0449-treated mice (Figure 5C). Together, these results suggest that Hh signaling may regulate putative liver cancer stem/progenitor cells by modulating availability of osteopontin. PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e23943