Published OnlineFirst October 19, 2015; DOI: 10.1158/0008-5472.CAN-14-1952 Cancer Molecular and Cellular Pathobiology Research Merlin/NF2 Suppresses Pancreatic Tumor Growth and Metastasis by Attenuating the FOXM1- Mediated Wnt/b-Catenin Signaling Ming Quan1,2, Jiujie Cui1,2, Tian Xia3, Zhiliang Jia2, Dacheng Xie1,DaoyanWei2, Suyun Huang4, Qian Huang1, Shaojiang Zheng5, and Keping Xie2 Abstract Merlin, the protein encoded by the NF2 gene, is a member of the Furthermore, Merlin suppressed the expression of Wnt/b-catenin band 4.1 family of cytoskeleton-associated proteins and functions signaling downstream genes and the nuclear expression of b-cate- as a tumor suppressor for many types of cancer. However, the roles nin protein, and overexpression of Forkhead box M1 (FOXM1) and mechanism of Merlin expression in pancreatic cancer have attenuated the suppressive effect of Merlin on Wnt/b-catenin remained unclear. In this study, we sought to determine the signaling. Mechanistically, Merlin decreased the stability of impact of Merlin expression on pancreatic cancer development FOXM1 protein, which plays critical roles in nuclear translocation and progression using human tissue specimens, cell lines, and of b-catenin. Collectively, these findings demonstrated that Merlin animal models. Decreased expression of Merlin was pronounced critically regulated pancreatic cancer pathogenesis by suppressing in human pancreatic tumors and cancer cell lines. Functional FOXM1/b-catenin signaling, suggesting that targeting novel Mer- analysis revealed that restored expression of Merlin inhibited lin/FOXM1/b-catenin signaling is an effective therapeutic strategy pancreatic tumor growth and metastasis in vitro and in vivo. for pancreatic cancer. Cancer Res; 75(22); 4778–89. Ó2015 AACR. Introduction 5-year survival rate for PDAC is less than 5% (3). Thus, identi- fication of the molecular mechanisms underlying PDAC devel- Pancreatic ductal adenocarcinoma (PDAC) has a dismal prog- opment and progression is urgently needed. nosis and is the seventh leading cause of cancer-related deaths (1). Merlin, the protein encoded by the NF2 gene, is a member of Its incidence is increasing annually, and in the United States, the band 4.1 families of cytoskeleton-associated proteins, which researchers estimated that 46,420 new PDAC cases would be link the integral membrane proteins with the actin cytoskeleton diagnosed and 39,590 patients would die of it in 2014 (2). (4, 5). Investigators first identified Merlin as being associated with Despite improvements in early diagnosis and treatment, more neurofibromatosis type 2 and that it functions as a tumor sup- than 80% of PDAC cases are diagnosed at late stages, and the pressor (6). A number of studies demonstrated that Merlin is a versatile tumor suppressor that can inhibit cancer cell prolifera- 1Department of Oncology, Shanghai Jiaotong University Affiliated tion and motility by modulating a wide range of signaling path- First People's Hospital, Shanghai, PR China. 2Department of Gastro- ways (7). Furthermore, mutation of the NF2 gene and loss of enterology, Hepatology and Nutrition, The University of Texas MD 3 Merlin protein occur in many different types of cancer, suggesting Anderson Cancer Center, Houston, Texas. Department of Gastroen- – terology, Shanghai Changhai Hospital, Second Military Medical a general tumor-suppressive role for Merlin (8 13). Evidently, University, Shanghai, PR China. 4Department of Neurosurgery, The Merlin shuttles between the cell cortex and the nucleus and that University of Texas MD Anderson Cancer Center, Houston, Texas. both cortical Merlin and nuclear Merlin have been implicated in 5 fi Pathology Department of Af liated Hospital, Hainan Provincial Key tumor suppression via interaction with angiomotin and CRL4- Laboratory of Carcinogenesis and Intervention, Hainan Medical Col- lege, Haikou, Hainan, PR China. DCAF1, respectively (11, 13). In a study of ezrin, another member Note: Supplementary data for this article are available at Cancer Research of the band 4.1 protein superfamily, overexpression of Merlin Online (http://cancerres.aacrjournals.org/). inhibited SW1990 PDAC cell proliferation, migration, and adhe- sion (14). However, the roles and mechanism of Merlin expres- M. Quan, J. Cui, and T. Xia contributed equally to this article. sion in PDAC development and progression have remained Corresponding Authors: Keping Xie, Department of Gastroenterology, Hepa- unclear. tology & Nutrition, Unit 1466, The University of Texas MD Anderson Cancer Wnt/b-catenin is one of the most important signaling pathways Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-5073; fl Fax: 713-745-3654; E-mail: [email protected]; Qian Huang, Department in PDAC development and progression (15). Brie y, this pathway of Oncology, Shanghai Jiaotong University Affiliated First People's Hospital, is initiated by binding of Wnt ligands to receptors of the Frizzled Shanghai, PR China. Phone: 86-21-63240090-3121; E-mail: family and coreceptors on the cell surface. With ligand binding, [email protected]; and Shaojiang Zheng, Department of Pathology, the cytoplasmic degradation complex, which consists of Axin, Hainan Medical College Affiliated Hospital, Haikou, PR China. Phone: 86-898- adenomatous polyposis coli, glycogen synthase kinase-3b, and 66723333; E-mail: [email protected] casein kinase 1, is inhibited, leading to nuclear localization of doi: 10.1158/0008-5472.CAN-14-1952 b-catenin. In the nucleus, b-catenin binds to T-cell factor/lym- Ó2015 American Association for Cancer Research. phoid enhancer factor to activate Wnt downstream target genes, 4778 Cancer Res; 75(22) November 15, 2015 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 19, 2015; DOI: 10.1158/0008-5472.CAN-14-1952 Regulation of Pancreatic Cancer Progression by Merlin including cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9, (25). The use of human specimens was approved by the relevant and VEGF. Although studies of mouse models demonstrated that institutional review boards. The staining results were scored by activation of Wnt/b-catenin signaling was insufficient to initiate two investigators blinded to the clinical data as described previ- PDAC, this pathway played key roles in PDAC stem cell mainte- ously (26). nance, progression, and drug resistance (15, 16). Recent studies indicated that Merlin suppressed the Wnt/b-catenin signaling Plasmids and siRNAs pathway (9, 17, 18); however, the molecular mechanism of this The plasmids pcDNA3.1-FOXM1B (pFOXM1), Flag-tagged suppression and the relevance of the interaction between Merlin FOXM1B (Flag-FOXM1), and a control vector were described and b-catenin remain to be elucidated. previously (22, 27). The plasmid pcDNA3.0-Flag-tagged Merlin Forkhead box M1 (FOXM1) is a transcription factor in the FOX was the full-length Merlin isoform I and was obtained from superfamily characterized by a conserved winged helix DNA- Addgene. Full-length Merlin was cloned into the pcDNA3.0 binding domain (19). FOXM1 is a key regulator of cell-cycle (pMerlin) and pcDNA3.0-hemagglutinin (HA; HA-Merlin) vec- progression, and series of studies suggested that it plays critical tors as a BamHI-EcoRI fragment. Mutated Merlin-S518A was roles in PDAC growth, angiogenesis, invasion, and metastasis generated via overlapping PCR and inserted into pcDNA3.0 (20, 21). Recently, we found that FOXM1 bound directly to (pM-S518A) and pcDNA3.0-HA (HA-M-S518A) vectors (ampli- 0 b-catenin and had a key role in mediation of nuclear accumula- fied via two-step PCR using the following primers: 5 -atgcggatc- 0 0 tion of b-catenin and downstream target gene expression (22, 23). catggccggggccatcgcttcccg-3 and 5 -ctttttctttctctatctccatggcaagccgct- 0 0 In the present study, we sought to determine the roles and tcatgtcagtatctttg-3 plus 5 -caaagatactgacatgaagcggcttgccatggagata- 0 0 0 mechanism of Merlin expression in PDAC growth and metastasis. gagaaagaaaaag-3 and 5 -atgcgatatcctagagctcttcaaagaaggccact-3 0 0 We discovered that expression of Merlin was suppressed in PDAC for the first reaction and 5 -atgcggatccatggccggggccatcgcttcccg-3 0 0 cells and that restored expression of Merlin inhibited PDAC cell and 5 -atgcgatatcctagagctcttcaaagaaggccact-3 for the second reac- growth and metastasis in vitro and in vivo. Further studies dem- tion). Mutated Merlin-S518D was obtained using the same meth- 0 0 0 onstrated that re-expression of Merlin decreased the nuclear od (primers: 5 -atgcggatccatggccggggccatcgcttcccg-3 and 5 -ctt- 0 0 translocation of b-catenin by inducing instability of FOXM1 tttctttctctatctccatgtcaagccgcttcatgtcagtatctttg-3 plus 5 -caaagatact- 0 0 protein. gacatgaagcggcttgacatggagatagagaaagaaaaag-3 and 5 -atgcgatat- cctagagctcttcaaagaaggccact-30 for the first reaction and 50- atgcggatccatggccggggccatcgcttcccg-30 and 50-atgcgatatcctagagctctt- Materials and Methods caaagaaggccact-30 for the second reaction). An HA-ubiquitin Cell culture and treatment vector described previously was used as a control (28). Full-length Human embryonic kidney 293 (HEK293) cells and the Merlin was cloned into the retroviral expression vector pBABE- human PDAC cell lines PANC-1, MiaPaCa-2, AsPC-1, BxPC- puro as a BamHI-EcoRI fragment (pBABE-Merlin). Retroviruses 3, CaPan-1, CaPan-2, and PA-TU-8902 were purchased from were produced by transfecting packaging cells (HEK293) with a theATCC.ThePDACcelllineMDAPanc-28wasagiftfromDr. three-plasmid system