Published OnlineFirst September 22, 2016; DOI: 10.1158/0008-5472.CAN-16-0438 Cancer Molecular and Cellular Pathobiology Research IGFBP2 Activates the NF-kB Pathway to Drive Epithelial–Mesenchymal Transition and Invasive Character in Pancreatic Ductal Adenocarcinoma Song Gao1,2, Yan Sun3, Xuebin Zhang4, Limei Hu1, Yuexin Liu1, Corrine Yingxuan Chua1, Lynette M. Phillips1, He Ren2, Jason B. Fleming5, Huamin Wang1, Paul J. Chiao6, Jihui Hao2, and Wei Zhang1,6,7 Abstract The molecular basis underlying the particularly aggressive tion (EMT). Mechanistic investigations revealed that IGFBP2 nature of pancreatic ductal adenocarcinoma (PDAC) still induced the nuclear translocation and phosphorylation of remains unclear. Here we report evidence that the insulin-like the p65 NF-kB subunit through the PI3K/Akt/IKKb pathway. growth factor–binding protein IGFBP2 acts as a potent onco- Conversely, enforced expression of PTEN blunted this signaling gene to drive its extremely malignant character. We found pathway and restored an epithelial phenotype to PDAC cells that elevated IGFBP2 expression in primary tumors was asso- in the presence of overexpressed IGFBP2. Overall, our results ciated with lymph node metastasis and shorter survival in identify IGFBP2 as a pivotal regulator of an EMT axis in patients with PDAC. Enforced expression of IGFBP2 promoted PDAC, the activation of which is sufficient to confer the invasion and metastasis of PDAC cells in vitro and in vivo by characteristically aggressive clinical features of this disease. inducing NF-kB–dependent epithelial–mesenchymal transi- Cancer Res; 76(22); 6543–54. Ó2016 AACR. Introduction long-term prognosis remains dismal because of metastasis and recurrence (3). Thus, tracking and treatment of metastatic tumors Pancreatic ductal adenocarcinoma (PDAC) is estimated to have remain the greatest challenges in the clinical management of caused 39,590 deaths in the United States in 2014, ranking as the PDAC. fourth leading cause of cancer-related death (1). Lack of an Experimentally, metastasis can be closely modeled by aug- effective early diagnostic modality renders 80% of cases not mented cellular migration and invasion, traits acquired by amenable to radical tumor resection because of local and distant epithelial cells that are undergoing transition to the mesen- tumor spread (2). Even among patients who undergo surgery, the chymal lineage. Epithelial–mesenchymal transition (EMT) was first identified as a developmental process that allows 1Department of Pathology, The University of Texas MD Anderson cells that are a part of a rigid architecture to escape and Cancer Center, Houston,Texas. 2Department of Pancreatic Carcinoma, spread (4), which plays a central role in metastasis of PDAC Tianjin Medical University Cancer Institute and Hospital, National (5, 6). Clinical Research Center for Cancer, Key Laboratory of Cancer Pre- vention and Therapy, Tianjin, P.R. China. 3Department of Pathology, The insulin-like growth factor (IGF) system has recently been Tianjin Medical University Cancer Institute and Hospital, National implicated in the progression of PDAC (7). IGF-binding pro- Clinical Research Center for Cancer, Key Laboratory of Cancer Pre- tein 2 (IGFBP2) is one of six proteins in the IGFBP family that 4 vention and Therapy, Tianjin, P.R. China. Department of Pathology, bind IGFs with high affinity (8). IGFBP2 expression has been Tianjin Huanhu Hospital, Tianjin, P.R. China. 5Department of Surgical Oncology,The University of Texas MD Anderson Cancer Center, Hous- shown to be elevated in many cancer types in both tumor cells ton, Texas. 6Department of Molecular and Cellular Oncology, The (9–13) and in the plasma (14–16). Published studies showed University of Texas MD Anderson Cancer Center, Houston, Texas. that IGFBP2 was upregulated in pancreatic juice (17) and 7Department of Cancer Biology, Comprehensive Cancer Center of Wake Forest Baptist Medical Center, Winston-Salem, North Carolina. plasma (18) of patients with PDAC. Whether IGFBP2 exhibits oncogenic activities in PDAC has not been elucidated. Of Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). particular interest is the nding that IGFBP2 drives glioma progression through the integrin/integrin-linked kinase/NF-kB Corresponding Authors: Wei Zhang, Department of Cancer Biology, Compre- network (19), and NF-B activation has been shown to be a key hensive Cancer Center of Wake Forest Baptist Medical Center, 1 Medical Center fl Blvd, Winston-Salem, NC 27157. Phone: 336-713-7508; Fax: 336-713-7566; event in PDAC progression (20). As a vital in ammation- E-mail: [email protected]; and Jihui Hao, Department of Pancreatic related transcription factor, NF-kB has been identified as a Carcinoma, Tianjin Medical University Cancer Institute and Hospital, National potent regulator of EMT (21–24). Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and In this study, we hypothesized that IGFBP2 is a key EMT inducer Therapy, Tianjin 300060, P.R. China. Phone: 86-22-2334-0123; E-mail: for PDAC and that this EMT induction is mediated through [email protected] activation of the NF-kB–associated pathway. We tested the phys- doi: 10.1158/0008-5472.CAN-16-0438 iologic relevance of this pathway in an in vivo model as well as in Ó2016 American Association for Cancer Research. clinical samples. www.aacrjournals.org 6543 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 22, 2016; DOI: 10.1158/0008-5472.CAN-16-0438 Gao et al. Materials and Methods Western blot analysis Whole-cell extracts were prepared by subjecting cells to lysis Human tissue specimens and immunohistochemical analysis in RIPA buffer supplemented with a proteinase inhibitor cock- Through a protocol approved by the Ethics Committee of the tail. The nuclear and cytoplasmic components were fraction- Tianjin Cancer Institute and Hospital (Tianjin, P.R. China), tumor ated by using the NE-PER Nuclear and Cytoplasmic Extraction samples were obtained from 80 randomly selected patients with Reagents Kit (Thermo Fisher Scientific) according to the man- PDAC treated at the hospital between 2000 and 2011. All 80 ufacturer's protocol. Protein lysates (20 mg) were separated by patients underwent radical pancreaticoduodenectomy with R0 SDS-PAGE, and target proteins were detected by Western blot margins confirmed by two pathologists. Station 1 and 2 lymph analysis. nodes around the pancreas (6, 8a, 8p, 12a, 12b, 12p, 13, 14p, 14d, and 17 group) were dissected routinely and assessed by two pathologists. No chemotherapy was administered before surgery. Animal experiments and measurement of metastasis in an After the surgery, all the patients received standard chemotherapy orthotopic model (single-agent gemcitabine) for six cycles. No radiotherapy was Female 4-week-old nude nu/nu mice were maintained in a given before or after surgery. barrier facility on high-efficiency particulate air-filtered racks. Consecutive sections of formalin-fixed, paraffin-embedded All animal studies were conducted under a protocol approved (FFPE) tumors were subjected to IHC analysis for IGFBP2, PTEN, by Institutional Animal Care and Use Committee in accor- RELA (NF-kB subunit p65), E-cadherin, and vimentin (Supple- dance with the principles and procedures outlined in the NIH mentary Table S1) using a DAB substrate kit (Maxin) according to Guide for the Care and Use of Laboratory Animals. Thirty mice the manufacturer's instructions. The results were scored as were divided into two groups (15 mice/group): one group was described previously (25) by two pathologists blinded to the injected with mixed populations from IGFBP2-overexpressing clinicopathologic data. AsPc-1 cells and the other group with AsPc-1 cells transfected with empty vector (EV). As described previously (27), cells were harvested by trypsinization, washed in PBS, and resus- Cell culture and reagents pended at 107 cells/mL in a 1:1 solution of PBS/Matrigel. For Human PDAC cell lines AsPc-1 and PANC-1 were purchased À À each mouse, the pancreas was exposed and injected with 50 mL from ATCC. IKKb / mouse embryonic fibroblasts (MEF) were a of cells in suspension. Six weeks later, all of the mice were kind gift from Dr. Michael Karin (University of California, Los À À sacrificed and necropsied for the observation of visible met- Angeles, Los Angeles, CA). IKKb / MEFs and PANC-1 cells were astatic lesions in the mesentery. The primary and metastatic cultured in DMEM. AsPc-1 cells were cultured in RPMI1640 pancreatic tumors were excised, weighed, fixed in formalin, medium. The media were supplemented with 10% FBS. Cultures and embedded in paraffin. Half of the tissues were subjected to were incubated at 37 C in a humidified chamber with 5% CO . 2 hematoxylin and eosin and IHC staining as described for The PDAC cell lines were characterized or authenticated by ATCC human PDAC tumors. The other half was subjected to Western using short tandem repeat profiling and passaged in our labora- blot analysis. tory for fewer than 6 months before use. The following reagents were used: protease and phosphatase inhibitor cocktails (Roche); PI3K inhibitor LY294002 and IKK Gene expression profiling and pathway analyses inhibitor Bay117082 (Sigma-Aldrich); and active full-length Microarray analysis for the mixed population of IGFBP2-over- human IGFBP2 protein (Abcam). Before treatment, the cells expressing