Biology of Human Tumors Sonic Hedgehog Paracrine Signaling Activates Stromal Cells to Promote Perineural Invasion in Pancreatic Cancer

Purpose: Pancreatic cancer is characterized by stromal desmoplasia and perineural invasion (PNI). We sought to explore the contribution of pancreatic stellate cells (PSC) activated by paracrine Sonic Hedgehog (SHH) in pancreatic cancer PNI and progression. Experimental Design: In this study, the expression dynamics of SHH were examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of carcinomatous and nonneoplastic pancreatic tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of PSCs activated by paracrine SHH signaling in pancreatic cancer PNI and progression. Results:Weshow that SHHoverexpression in tumor cells is involved in PNI inpancreatic cancer and is an important marker of biologic activity of pancreatic cancer. Moreover, the overexpression of SHH in tumor cells activates the hedgehog pathway in PSCs in the stroma instead of activating tumor cells. These activated PSCs are essential for the promotion of pancreatic cancer cell migration along nerve axons and nerve outgrowth to pancreatic cancer cell colonies in an in vitro three-dimensional model of nerve invasion in cancer. Furthermore, the coimplantation of PSCs activated by paracrine SHH induced tumor cell invasion of the trunk and nerve dysfunction along sciatic nerves and also promoted orthotropic xenograft tumor growth, metastasis, and PNI in in vivo models. Conclusions:These results establish that stromal PSCs activatedbySHHparacrine signaling inpancreatic cancer cells secrete high levels of PNI-associatedmolecules to promote PNI in pancreatic cancer.Clin Cancer Res; 20(16); 4326–38. 2014 AACR. Introduction Perineural invasion (PNI) in pancreatic cancer is a common pathologic characteristic in which tumor cells invade the perineural space of local peripheral nerves and intimately disseminate along nerve fascicles (1). PNI is thought to contribute to bothpain (2, 3) and local disease recurrence (2, 4, 5), and be an important prognostic factor for pancreatic cancer (6). In addition, pancreatic tumors are surrounded by a dense desmoplastic reaction (7), which is involved in pancreatic stellate cells (PSC; refs. 8 and 9) or fibroblasts (10) in the stroma, and exhibit neural alterations (11), such as hypertrophy and degeneration of intrapancreatic nerve fibers (12, 13). A positive correlation between fibrosis in the pancreatic tumor microenvironment and intrapancreatic neuropathy or PNI has been reported (14, 15). Recently, awide range of studies (16, 17) have shown that paracrine SonicHedgehog (SHH) protein, which is a hedgehog (Hh) pathway ligand and derived from pancreatic cancer epithelial cells, is the pivotal factor in both the regulation of the pancreatic tumor microenvironment and the promotion of tumor development and metastasis. Tumor-derived SHH protein acts on PSCs (18), whose activation, in turn, promotes the malignant behavior of pancreatic cancer cells, including reduced patient survival rates (19), uncontrolled growth (20), invasion (21), and therapeutic resistance (22). As active stromal PSCs in the desmoplasia reaction are potentially associated with PNI in pancreatic cancer tissues (14, 15), we sought to explore the contribution of PSCs activated by paracrine SHH in tumor PNI in pancreatic cancer. We show that SHH overexpression in tumor cells was involved in the PNI of pancreatic cancer. The SHH Authors' Affiliations: Departments of Hepatobiliary Surgery, General Surgery, and Oncology, First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi, China; Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota; and Department of Gastroenterology, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). X. Li and Z. Wang contributed equally to this work. Corresponding Authors: Qingyong Ma, Department of Hepatobiliary Surgery, First Affiliated Hospital of Medical College, Xi'an Jiaotong University, 277 West Yanta Road, Xi'an 710061, China. Phone: 86-2985323899; Fax: 86-29-85323899; E-mail: [email protected]; and Keping Xie, Department of Gastroenterology, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. Phone: 713-834-6685; Fax: 713-745-1163; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-3426 2014 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 20(16) August 15, 2014 4326 on April 15, 2017. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst June 19, 2014; DOI: 10.1158/1078-0432.CCR-13-3426