Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0112 Cancer Molecular and Cellular Pathobiology Research Autocrine BMP-4 Signaling Is a Therapeutic Target in Colorectal Cancer Yuichiro Yokoyama1,2, Toshiaki Watanabe2, Yusuke Tamura1, Yoshinobu Hashizume3, Kohei Miyazono1, and Shogo Ehata1 Abstract Poor prognoses for colorectal cancer patients with meta- LDN-193189 elevated expression of the phosphatase DUSP5 static lesions have driven demand for the development of in colorectal cancer cells, inducing apoptosis via dephosphor- novel targeted therapies. Here, we demonstrate that expres- ylation of Erk MAPK. Administering LDN-193189 to mice sion of bone morphogenetic protein 4 (BMP-4) is universally diminished tumor formation of colorectal cancer cells. Our upregulated in human colorectal cancer cells and tissues, findings suggest inhibition of autocrine BMP-4 as a candidate resulting in activated BMP signaling. Inhibition of endoge- treatment strategy for colorectal cancer. Cancer Res; 77(15); nous BMP signaling by the BMP type I receptor inhibitor 4026–38. Ó2017 AACR. Introduction their ability to bind certain type I receptors. Upon binding to type I and type II receptors, BMPs form heterotetrameric com- Colorectal cancer is the third most common cancer and the plexes; the protein kinase of the type II receptor activates the fourth most common cause of cancer-related death worldwide protein kinase of the type I receptor, which in turn phosphor- (1). Although surgical resection can cure early-stage colorectal ylates the BMP-specific receptor-regulated Smads (R-Smads), cancer, a combination of surgery and chemotherapeutic agents is Smad1 and Smad5. Phosphorylated R-Smads induce a hetero- recommended in advanced stages of colorectal cancer (1). In meric assembly with common-partner Smad (Co-Smad; addition to conventional cytotoxic agents, new agents targeting Smad4) and translocate into the nucleus, regulating the tran- VEGF signaling and EGFR signaling have been introduced during scription of target genes. BMPs can also activate non-Smad the last decade (2). Although colorectal cancer prognoses have signaling pathways, including the MAPK pathway (3). steadily improved, the 5-year survival rate remains low, especially Divergent roles of BMPs have been reported during cancer in patients with metastatic lesions (1). Thus, the development of progression (4–6). BMPs inhibit proliferation of gastric cancer, new molecular targets for treatment of advanced colorectal cancer breast cancer, and prostate cancer cells, induce differentiation of is critical for improving patient outcomes. glioma-initiating cells, and inhibit glioblastoma tumor formation Bone morphogenetic proteins (BMP) are members of the (5), indicating a tumor-suppressive role of BMPs. In contrast, TGF-b family and are multifunctional cytokines (3–5). BMPs BMPs have been reported to enhance the motility and invasive- recognize two distinct receptors, termed type I and type II ness of various types of cancer cells, such as breast cancer, prostate receptors, with serine/threonine and tyrosine kinase activities. cancer, and malignant melanoma cells, suggesting that BMPs also Type I BMP receptors include activin receptor-like kinase (ALK)- function as tumor-promoting factors (4). 1, -2, -3, and -6, and type II receptors include BMP type II In this study, the role of BMP-4 produced by colorectal cancer receptor (BMPR-II), activin type II receptor (ActR-II), and cells in cancer progression was investigated. We reveal for the first activin type IIB receptor (ActR-IIB). BMPs are classified into time that inhibition of BMP-4 induces the apoptosis of colorectal several subgroups, including the BMP-2/4 group, BMP-5/6/7/8 cancer cells through the attenuation of MAPK activity in culture group, BMP-9/10 group, and growth and differentiation factor and that the small-molecule BMP inhibitor LDN-193189 (GDF)-5/6/7 group, according to structural similarities and diminishes colorectal cancer formation in vivo. 1Department of Molecular Pathology, Graduate School of Medicine, The Univer- Materials and Methods sity of Tokyo, Tokyo, Japan. 2Department of Surgical Oncology, The University of Cell culture and reagents 3 Tokyo, Bunkyo-ku, Tokyo, Japan. RIKEN Program for Drug Discovery and Human colon adenocarcinoma cells HT29 and DLD-1 (Japa- Medical Technology Platforms, Wako, Saitama, Japan. nese Cancer Research Resource Bank) were cultured in RPMI Note: Supplementary data for this article are available at Cancer Research containing 10% FBS, penicillin, and streptomycin. Human colon Online (http://cancerres.aacrjournals.org/). adenocarcinoma SW480 cells (ATCC) were cultured in DMEM Corresponding Authors: Shogo Ehata, Graduate School of Medicine, The containing 10% FBS, penicillin, and streptomycin. Routine Myco- University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: plasma testing was performed by PCR regularly on these cells. The 813-5841-3356; Fax: 813-5841-3354; E-mail: [email protected]; and Kohei cells were bought in 2002 and have been stocked as cryopreserved Miyazono, [email protected] aliquots in liquid N2. The cells were used within 8 passages after doi: 10.1158/0008-5472.CAN-17-0112 thawing and reauthenticated by short tandem repeat profiling in Ó2017 American Association for Cancer Research. 2017. LDN-193189 was obtained from Wako or RIKEN. 4026 Cancer Res; 77(15) August 1, 2017 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0112 BMP-4 as a New Therapeutic Target in Colorectal Cancer Quantitative real-time RT-PCR analyses and chromatin NCI-60 cell line panel indicated that, among various BMPs, immunoprecipitation–qRT-PCR analyses expression of BMP4 was commonly elevated in colon cancer cells Quantitative real-time RT-PCR (qRT-PCR) analysis and chro- (Fig. 1A). NCBI GEO database GSE14258 revealed that expression matin immunoprecipitation (ChIP)–qRT-PCR analysis were of BMP4, but not of other BMPs, was significantly higher in performed as previously described (7, 8). Primer sequences colon cancer tissues than in normal colon tissues (Fig. 1B). Next, are described in Supplementary Table S1. Anti-TCF4 antibody the correlation between BMP4 expression and colorectal cancer (sc-8631) was purchased from Santa Cruz Biotechnology. patient prognosis was examined. GSE14333 showed that elevated expression of BMP4 was associated with poor prognosis in Immunoblotting patients with stage II colorectal cancer (Fig. 1C). Furthermore, Immunoblotting was performed as previously described (7). multivariate analysis demonstrated that BMP4 expression was an Antibodies are described in Supplementary Materials and Meth- independent prognostic factor in stage II colorectal cancer (Sup- ods. ImageJ (NIH) was used to quantify blot band intensities. plementary Table S2). Expression of BMP-4 and activation of Smad-dependent BMP signaling was then examined using human Apoptosis assay colorectal cancer tissues and cells. IHC analysis revealed that Terminal deoxynucleotidyl transferase-mediated dUTP expression levels of BMP-4 and phosphorylated Smad1/5 were nick end labeling (TUNEL) assay was performed as previously upregulated in colorectal cancer tissues compared with those in described (7). Fluorescence was examined using a Leica corresponding normal tissues (Fig. 1D). ELISAs demonstrated DMI6000 B. that these colorectal cancer cells produced BMP-4, whereas, with siRNA the exception of SUIT-2 pancreatic cancer cells, other cancer cells fi Stealth RNAi Pre-Designed siRNAs targeting CTNNB1, BMP4 examined did not (Fig. 1E). These ndings suggest that colorectal and DUSP5 were synthesized by Thermo Fisher Scientific. Cells cancer cells produce BMP-4, which may act in an autocrine were transfected in the presence of 30 nmol/L siRNA or control manner, and that BMP-4 expression may be related to colorectal siRNA in a 500 mL volume with 3 mL RNAiMAX reagent (Thermo cancer progression. BMP4 Fisher Scientific) per well in a 6-well plate. Next, we sought to clarify the mechanism by which mRNA was elevated in colorectal cancer. Mutations in the APC Lentiviral production and infection gene occur in the early phase of colorectal cancer progression, We used a lentiviral vector system to induce specific gene which in turn increases the stability of b-catenin (15). Because introduction and knockdown as previously described (9, 10). BMP4 expression is reported to be regulated by the Wnt/b-catenin The target sequences for shRNA are described in the Supplemen- pathway (16), the involvement of Wnt/b-catenin in the induction tary Materials and Methods. of BMP4 expression in colorectal cancer cells was assessed. b-Cate- nin protein levels were elevated in colorectal cancer cells but not in RNA-sequence analyses non-colorectal cancer cells, such as pancreatic cancer cells (SUIT- RNA-sequence (RNA-seq) analysis was performed as described 2) and breast cancer cells (MDA-231-D; Fig. 2A). Knockdown of previously (11). Raw and processed data are available at GEO the CTNNB1 gene (encoding b-catenin) in colorectal cancer cells (GSE96914). Gene Ontology analysis was performed using CLC by siRNAs suppressed BMP4 mRNA expression and BMP-4 pro- Genomics Workbench (Qiagen Bioinformatics). tein production, as well as expression of a direct downstream target of the Wnt/b-catenin pathway, AXIN2 (Fig. 2B and C). Subcutaneous xenograft model Similar to colorectal cancer cells, introduction