Abstract A51: Novel Hedgehog co-receptors in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. The Hedgehog (Hh) signaling pathway is upregulated in PDAC. 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is active in early PanIN lesions and persists as the cancer progresses. Interestingly, Hh ligands are secreted by the tumor cells and function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor growth. PDAC is characterized by hypovascularity and a highly desmoplastic stroma. The downstream effects of Hh activation in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdon), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Gas1, Boc, and Cdon are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. During cancer progression, expression of all three co-receptors is prevalent throughout the desmoplastic stroma. Functional studies on Gas1-/-;Boc-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wildtype MEFs, indicating that these co-receptors are important for Hh signal transduction. Despite a reduced Hh-response, we found that Gas1-/-;Boc-/- MEFs promoted the growth of significantly larger, more vascularized tumors than their wildtype counterparts. A more physiologically relevant experiment with pancreatic fibroblasts and primary pancreatic tumor cells yielded the same counterintuitive result: removal of Gas1 and Boc from fibroblasts decreases Hh-response, and yet tumor-promoting potential was significantly increased. Moreover, chick chorioallantoic membrane (CAM) assays with tumor cells and fibroblasts revealed that Gas1-/-;Boc-/- MEFs stimulated blood vessel growth. We repeated these experiments with MEFs in which all three co-receptors were removed. Gas1-/-;Boc-/-;Cdon-/- MEFs displayed a more significantly abrogated Hh-response than both their Gas1-/-;Boc-/- and wildtype counterparts. However subcutaneous co-injection and CAM experiments revealed that these MEFs did not promote tumor growth, behaving similarly to Smo-/- MEFs in previously published findings. These results indicate the importance of Hh dosage in pancreatic cancer, which has important clinical implications. In particular, we found that intermediate levels of Hh signaling, such as that resulting from pharmaceutical inhibition, resulted in a marked angiogenesis response. This finding may partially account for the recent failure of Hh inhibition in a pancreatic cancer clinical trial. Citation Format: Esha Mathew, Yaqing Zhang, Alexander M. Holtz, Kevin T. Kane, Jane Song, Benjamin L. Allen, Marina Pasca di Magliano. Novel Hedgehog co-receptors in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A51.