Table 1. Effect of GDC-0449 treatment on intrahepatic HCC and HCC metastases. Surgical Resection MRI Imaging Vehicle Treatment Vehicle Treatment Intrahepatic Tumor Nodules Metastases 80% 20% 56% 0% doi:10.1371/journal.pone.0023943.t001 78% 50% 50% 20% suppress all of these responses, consistent with published evidence that activation of Smoothened in Hh-responsive cells promotes nuclear localization of Gli2, and consequent induction of Gli1 and osteopontin transcription [43,44]. Osteopontin, in turn, has been shown to promote myofibroblast accumulation and liver fibrosis in mice [37,45]. It also acts via its receptor, CD44, to enhance the viability and growth of certain types of liver progenitors, including liver cancer stem/progenitor cells [35,36,46,47]. Hence, the Hh pathway might be modulating both liver fibrosis and HCC outgrowth by regulating the autocrine/paracrine availability of osteopontin. Myofibroblasts and ductular progenitor cells are also important sources of other pro-fibrogenic factors (e.g., PDGF-b and TGF-b) that are capable of activating Gli2 via mechanisms that do not require Smoothened [48,49,50]. We observed increased hepatic expression of both PDGF-b and TGF-b in Mdr2-deficient mice. This suggests that Smoothened-independent mechanisms that re- enforce the effects of the canonical Hh pathway may evolve during fibrogenic repair. In mdr2-deficient mice, however, GDC-0449 significantly reduced expression of TGF-b and tended to suppress expression of PDGFb, demonstrating that canonical Hh signaling with resultant Smoothened activation is ultimately required to fully engage noncanonical pathways that rely on interaction of TGF-b and PDGF-b with their respective receptors to activate Gli2 in injured, fibrotic livers. Finally, the fact that liver fibrosis/cirrhosis is a major risk factor for the development of HCC should not be misconstrued to imply that fibrosis per se causes cancer. It is conceivable that fibrosis and HCC each result from some adaptive response that occurs during chronic liver injury. Our results demonstrate that injury-related activation of the Hh pathway typifies an adaptive response that is both pro-fibrogenic and pro-carcinogenic. This discovery, in turn, provides novel evidence that helps to explain why fibrosis and HCC often develop in the same livers. As such, it has immediate clinical relevance to the many cirrhotic patients with HCC. Additional research will be required, however to delineate the precise down-stream mechanism(s) involved and to determine which (if any) of those subsequent fibrogenic and carcinogenic processes are inter-dependent, and which are totally independent of each other. Proof that inhibition of Hh signaling substantially reduced the hepatic content of myofibroblasts and progenitors suggests that these cell types promote and/or maintain the outgrowth of malignant hepatocytes. This finding, in turn, provides a starting point for further research. Indeed, others have reported that Smoothened antagonists lead to the involution of pancreatic cancers by influencing tumor angiogenesis [51]. Myofibroblasts are known to be an important source of vascular growth factors, such as VEGF [52,53,54], and we found increased hepatic expression of VEGF and its receptor, VEGFR1, in the diseased livers of Mdr2-deficient mice compared to the healthy livers of Hedgehog Pathway Inhibition in Liver Cancer Model age/gender-matched controls (data not shown). Hence, it is possible that myofibroblast depletion exerted a negative impact on the hepatic vasculature that ultimately resulted in HCC involution. This concept is supported by evidence that GDC-0449 tended to suppress hepatic expression of VEGF/VEGFR1 (data not shown) and increased necrosis in the HCC of GDC-0449-treated mice. Hh signaling is also known to maintain various progenitor populations [55,56,57]. Previously, we reported that Hh ligands function as autocrine and paracrine survival signals for liver progenitors and showed that liver myofibroblasts are an important paracrine source of Hh ligands that serve this purpose [40,42,58,59,60]. Liver progenitors, in turn, generate Hh ligands that provide paracrine signals that re-enforce the growth of liver myofibroblasts [61,62]. Given this background, it is not surprising that blocking Hh signaling with a Smoothened antagonist resulted in the mutual depletion of both cell types. To our knowledge, however, by demonstrating marked treatment-related decreases in CD44, cytokeratin-19, and a-fetoprotein within HCC tumors, the current data provide the first evidence that Smoothened antagonists reduce populations of cells that exhibit features of tumor stem/progenitor cells. Further investigation is required to ascertain how aberrant Hedgehog signaling promotes these cancer stem cell compartments. In conclusion, our findings in the Mdr22/2 mouse model of progressive liver fibrosis and spontaneous hepatocarcinogenesis demonstrate that increased production of Hh ligands and progressive accumulation of Hh-responsive cell types, such as myofibroblasts and liver progenitor cells, precede the emergence of HCC, and persist after the development of HCC. Despite advanced liver fibrosis and HCC, a short course of treatment with a highly-specific and clinically available Hh signaling inhibitor is well-tolerated and demonstrates appreciable anti- tumor effects. Thus, Hh pathway inhibition with GDC-0449 merits evaluation as a potential treatment for HCC arising in cirrhotic patients, providing a novel treatment option for an emerging disease with a poor prognosis and limited therapeutics. Supporting Information Figure S1 Evidence of ongoing liver injury in Mdr22/2 mice. AST and ALT measurements from Mdr22/2 mice and their age-matched wild type counterparts at various time points. Each data point represents n = 2–7 animals and Mean6SD is graphed. (*p,0.05 vs the age-matched wild-type control). (TIFF) Figure S2 Systemic treatment of GDC-0449 treatment is well tolerated in Mdr22/2 mice with advanced liver disease and HCC. A. Daily body weight measurements were obtained for all animals in each of the three treatment groups and graphed over time. Data expressed as Mean6SD. B. Liver to body weight ratios of animals in each of the three treatment groups. Data expressed as Mean6SD. C. Western blot analysis for Shh, Gli1, Gli2 and actin (loading control) in whole liver extracts from Mdr22/2 mice treated with vehicle, 20 mg/kg GDC-0449, and 40 mg/kg GDC-0449. Liver extracts from each treated mouse were loaded individually. (TIFF) Figure S3 Increased hepatic expression of TGFb and PDGFb in Mdr22/2 mice is reversed by GDC-0449 treatment. Whole liver RNA was isolated from Mdr22/2 mice and age/gender-matched wild type controls (WT) (n = 3 mice/ group) and QRT PCR was done to compare expression of (A) TGFb and (B) PDGFb. Similar approaches were used to assess the PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e23943