Hedgehog signaling and desmoplasia are regulated by hypoxia in pancreatic cancer
Authors: Spivak-Kroizman Taly , Hostetter Galen , Hoff Daniel Von , Posner Richard , Abdelmelek Mena , James Brian , Demeure Michael , Powis Garth 
Published: May 2015

Abstract
Pancreatic cancer is one of the most aggressive solid tumors, with less than 5% of patients surviving 5 years. Chemotherapy treatments have modest improvement in survival rates and surgical resection in most cases is not an option. New strategies for therapy are needed to improve overall survival in this deadly disease. Pancreatic tumors are notoriously hypoxic and characterized by a dense fibroinflammatory stromal reaction (desmoplasia). We hypothesized that signals emanating from tumor cells exposed to hypoxia enable pancreatic tumors to thrive. To explore this we conducted a transcriptome microarray study comparing genes transcribed in normoxia and hypoxia (1% O2 for 24 hrs). We have found that hypoxia significantly increases the levels of the hedgehog (Hh) pathway ligand, sonic hedgehog (sHh), and its receptor, patched (PTCH), in two pancreatic cancer cell lines, MiaPaca 2 and Panc-1. In addition, siRNA to HIF-1α showed that this increase is HIF-1α dependent. Western blotting confirmed a hypoxia/HIF-1α-dependent increase in sHh protein levels in both MiaPaca-2 and Panc-1 cells. To further explore the hypoxia-induced increase in expression of hedgehog pathway components we used a specific pathway array profiling the expression of 84 key genes involved in the hedgehog signaling. REN, (retinoic acid, EGF and NGF induced gene), was the only other hedgehog pathway gene to increase in hypoxia. Panc-1 cells transfected with a GLI-luciferase reporter of hedgehog activation, showed no increase in GLI activity in either normoxia, hypoxia or upon addition of conditioned media containing sHh, suggesting there is not an autocrine stimulation of the cancer cells themselves by the secreted sHh. This may be explained by the increase in PTCH and REN, both inhibitors of the hedgehog pathway. sHh is a known key factor promoting stromal desmoplasia in pancreatic cancer and therefore we tested whether sHh produced by cancer cells can activate fibroblasts. Indeed, NIH-3T3 fibroblasts stably transfected with GLI-luciferase (NIH-3T3/GLI-luc) showed an increase in GLI activity when co-cultured with MiaPaca-2 cells in hypoxia, but not normoxia, or when treated with conditioned media from Panc-1 or MiaPaca-2 cells grown in hypoxia. A 3-D system simulating conditions of tumor growth and demonstrating a hypoxic core within the cancer cells mass showed GLI transactivation in NIH-3T3/GLI-luc cells only when cells were co-cultured with pancreatic cancer cells (Panc-1, MiaPaca-2, Capan-2 or Su-86), but not when cells were cultured alone. Using siRNA this GLI activation was shown to be dependent on both HIF-1α and sHh. Finally, an immunohistochemical study in pancreatic cancer tumor samples showed a significant positive correlation between tumor and stromal HIF-1α staining and stromal sHh. Thus, we propose a new mechanism of the tumor's response to hypoxia in which pancreatic tumor cells secrete sHh that acts on stroma to increase fibrosis which in turn leads to decreased blood flow, and thus a continuous cycle of more hypoxia and Hh signaling. [Supported by CA109552-05]