Microenvironment and Immunology Therapeutic Efficacy of Bifunctional siRNA Combining TGF-b1 Silencing with RIG-I Activation in Pancreatic Cancer

Deregulated TGF-b signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-b by siRNA. By introducing a triphosphate group at the 50 end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9–mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-I activation with gene silencing of TGF-b1 (ppp-TGF-b) and studied its therapeutic efficacy in the orthotopic Panc02 mouse model of pancreatic cancer. Intravenous injection of ppp-TGF-b reduced systemic and tumorassociated TGF-b levels. In addition, it induced high levels of type I IFN and CXCL10 in serum and tumor tissue, systemic immune cell activation, and profound tumor cell apoptosis in vivo. Treatment of mice with established tumors with ppp-TGF-b significantly prolonged survival as compared with ppp-RNA or TGF-b siRNA alone. Furthermore, we observed the recruitment of activated CD8þ T cells to the tumor and a reduced frequency of CD11bþ Gr-1þ myeloid cells. Therapeutic efficacy was dependent on CD8þ T cells, whereas natural killer cells were dispensable. In conclusion, combing TGF-b gene silencing with RIG-I signaling confers potent antitumor efficacy against pancreatic cancer by breaking tumor-induced CD8þ T cell suppression. Cancer Res; 73(6); 1709–20. 2013 AACR. Introduction Pancreatic cancer is the fourth leading cause of cancerrelated death and is characterized by early metastasis and resistance to chemotherapy and irradiation. The identification of deregulated molecular pathways in pancreatic cancer and the development of novel targeted therapies had so far little impact on clinical outcome (1). Prognosis of patients with pancreatic cancer has remained extremely poor with a 5-year survival rate of less than 5%. A key event in tumor progression of pancreatic cancer is deregulation of TGF-b signaling (2). Under normal conditions, TGF-bmaintains tissue homeostasis by controlling cellular proliferation, differentiation, survival, and cell adhesion. Deregulated TGF-b signaling allows tumors to usurp homeostatic effects for promoting tumor growth, invasion, metastasis, and tumor angiogenesis (2). Moreover, TGF-b has immunosuppressive effects such as inhibition of cytotoxic T cells and natural killer (NK) cells, induction of FoxP3þ regulatory T cells, and shifting antigen-presenting cell function toward tolerance (3, 4). Both tumor cells and immune cells, such as regulatory T cells and myeloid-derived suppressor cells (MDSC), contribute to enhanced TGF-b production in patients with cancer. Elevated TGF-b levels in serum and tumors correlate with poor prognosis in patients with tumor (5, 6). Thus, TGF-b has generated interest as a target for novel anticancer agents. Anti-TGF-b compounds have shown efficacy in preclinical studies, and some of these have moved into clinical investigation for melanoma, brain tumors, colorectal, renal, and pancreatic cancer (7–11). Tumor infiltration with T cells represents a positive prognostic factor for pancreatic carcinoma, indicating that immune surveillance may occur despite locally active immunosuppressive mechanisms (12). However, tumor-infiltrating T cells frequently lack effector function due to the hostile tumor microenvironment, which is enriched with immunosuppressive Authors' Affiliations: Medizinische Klinik und Poliklinik IV, Klinikum der Universit€ at M€ unchen; Center for Integrated Protein Sciences Munich and Division of Clinical Pharmacology, Medizinische Klinik und Poliklinik IV, Klinikum der Universit€ at M€ unchen; Pathologisches Institut der LMU M€ unchen; MedizinischeKlinik, Universit€ atsklinikumRechts der Isar, Technische Universit€ at, M€ unchen; Institut f€ ur Klinische Chemie und Klinische Pharmakologie, Universit€ atsklinikum, Bonn, Germany; and Medical School of Nanjing University, Nanjing, China. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Ellermeier, J. Wei, and P. Duewell contributed equally to this work. Corresponding Author: Max M. Schnurr, Medizinische Klinik und Poliklinik IV, Klinikum der LMU, Ziemssenstr. 1, M€ unchen 80336, Germany. Phone: 49-89-5160-5300; Fax: 49-89-5160-4568; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-11-385