Pancreatic adenocarcinoma is a rapidly lethal cancer, with less than 3-4% patient survival at five years and no known effective therapy. Previously, we have demonstrated that the Hedgehog (Hh) pathway plays a central role in the initiation and maintenance of pancreatic cancer. Inhibition of the Hh pathway by cyclopamine, a Smoothened (Smo) inhibitor, has been shown to suppress the growth of cell lines in vitro and in vivo, suggesting that the Hedgehog pathway may be an important target for new therapies. While these results are promising, the biologic response of cell line-derived tumors may bear no relation to the biologic response of patient tumors. Here we report the use of a mouse xenograft model based on patient-derived pancreatic adenocarcinoma specimens to determine 1) whether inhibition of the pathway may have similar anti-tumor effects, and 2) whether tumor-specific variables such as differentiation may modify response to Hh pathway inhibition. Twelve mice were generated carrying the tumors from five different patients. Three patients' tumors were well to moderately differentiated; the other two were poorly differentiated. All xenograft tumors retain the histologic appearance seen in the original patient specimen and have an active Hh pathway. After tumors reached 125 mm3, mice were treated with one of three agents targeting different levels of the pathway: 5E1, an anti-Shh antibody, cyclopamine, or forskolin, a Gli antagonist. In response to treatment, all tumors showed a significant decrease in pathway activity as assessed by the decreased expression of at least two of four target genes (PTCH-1, -2, GLI-1, -2). Despite pathway inhibition, only 8 of 12 tumors had a response to treatment. The biologic response of tumor xenografts correlates strongly with the degree of differentiation. Well- to moderately-differentiated tumors show a decrease in glandular density, a decrease in proliferation, and an increase in apoptosis in response to treatment. No changes were seen in poorly-differentiated tumors. These data suggest that while most pancreatic tumors upregulate the Hh pathway, their dependence on the pathway for survival diminishes as they progress to a less differentiated phenotype. This could have important implications for tailoring anti-Hh therapies to patients with specific tumor characteristics in the future.

The Hedgehog (Hh) signaling pathway has been implicated in pancreatic tumorigenesis, where it is believed to play a critical role as both an initiator and maintenance factor. However, the prevalence and magnitude of Hh pathway aberration in sporadic human pancreatic adenocarcinomas remains unknown. Here we present quantitative real-time PCR data from 20 human adenocarcinomas and show increased mRNA expression for 7 representative Hedgehog pathway genes. Methods: Individual samples were obtained from 20 human pancreatic adenocarcinomas freshly resected at our institution. Total RNA was extracted, genomic DNA was removed, and the RNA was subjected to quantitative real-time PCR analysis, using algorithmically generated primers for the genes SHH, IHH, DHH, PTCH, SMO, GLI1, and GLI2. All samples in a given run were normalized to a standardized sample of 18S rRNA, and gene expression levels in tumors were compared to those in an averaged expression level of 10 normal human pancreata. Results: All human tumors showed marked upregulation of the Hedgehog pathway when compared to normal pancreatic controls. All tumors misexpress Hh ligands, principally SHH and IHH (median 168- and 165-fold upregulation, respectively). Furthermore, this pathway is active, as indicated by an impressive upregulation of downstream mediators of the Hh pathway, GLI1 and GLI2 (median 71- and 96-fold). DHH, PTCH, and SMO are identified but demonstrate comparatively less upregulation (median 9-, 2-, and 5-fold). Conclusions: Misexpression of Hh ligands and activation of the Hh pathway is a prevalent finding in human pancreatic adenocarcinoma. The finding that all adenocarcinomas tested to date have an impressive increase in activity suggests that Hh pathway aberration is an important fundamental mechanism in pancreatic tumorigenesis. Thus, inhibition of the Hh pathway may represent a powerful potential treatment modality in pancreatic adenocarcinoma.

Sixty percent of pancreatic adenocarcinomas arise in the pancreatic head; the reasons remain unclear. The developmental signaling factor Sonic hedgehog (Shh) has been recently identified as an early initiator of pancreatic cancer and is also believed to be an important factor in GI mucosal renewal and maintenance. Pancreatic ductal adenocarcinoma and its precursor PanIN lesions are known to express GI mucins, which are thought to be a marker for neoplastic progression and malignant transformation. We find, both in normal mice and in humans, small branches (pancreatic duct glands) of the proximal pancreatic ducts, which end blindly in the ductal mesenchyme. These glands are found in association with the major ducts of the pancreas but are predominantly located in the proximal ducts of the pancreatic head. The epithelia of these glands/branches stain positively for PAS and Alcian blue, suggesting that they retain some GI epithelial characteristics. Interestingly in normal controls, Shh expression is identified at a low level in these branches/glands. In response to repetitive injury (mice) and chronic inflammation (humans) these glands become hypertrophic, with increased expression of Shh and mucins. BrdU incorporation (mice) and Ki67 expression (humans) show high proliferative activity in these duct glands. Furthermore, in a subset of hypertrophic and proliferating duct glands that express Shh, nuclear atypia and enhanced mitotic activity, consistent with early PanIN lesions, are found. These results suggest that duct glands of the pancreas (1) retain a transitional character with expression of GI markers, (2) play an important role in mucosal protection (mucins) and maintenance (proliferation) in response to injury, and (3) may, through upregulation of the Hedgehog pathway in states of chronic stimulation, be the site of origin of adenocarcinoma in the pancreatic head.