Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-Like Receptors That Bind Lipopolysaccharide
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Published OnlineFirst June 21, 2016; DOI: 10.1158/0008-5472.CAN-16-0172 Cancer Microenvironment and Immunology Research Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide Wei-Ting Kuo1, Tsung-Chun Lee1,2, and Linda Chia-Hui Yu1 Abstract Colorectal carcinogenesis is affected by overexpression of the cell lines displayed increased cell proliferation and cell-cycle lipopolysaccharide (LPS) receptors CD14 and TLR4, which antag- progression following LPS challenge. This effect was inhibited onize each other by affecting epithelial cell proliferation and by eritoran and by silencing CD14 or TLR4. In contrast, apoptosis apoptosis. Eritoran is an investigational drug for sepsis treatment induced by eritoran was eliminated by silencing CD14 or protein that resembles the lipid A moiety of LPS and therefore acts as a kinase Cz (PKCz) but not TLR4. Lastly, LPS and eritoran caused TLR4 inhibitor. In the present study, we explored the potential hyperphosphorylation of PKCz in a CD14-dependent and TLR4- therapeutic uses and mechanisms of action of eritoran in reducing independent manner. Blocking PKCz activation by a Src kinase colon cancer progression. Eritoran administration via intracolo- inhibitor and a PKCz-pseudosubstrate prevented eritoran- nic, intragastric, or intravenous routes significantly reduced tumor induced apoptosis. In summary, our work offers a preclinical burden in a chemically induced mouse model of colorectal proof of concept for the exploration of eritoran as a clinical carcinoma. Decreased proliferation and increased apoptosis were treatment, with a mechanistic rationale to reposition this drug observed in mouse tumor cells after eritoran treatment. In vitro to improve the management of colorectal cancer. Cancer Res; 76(16); cultures of mouse primary tumor spheroids and human cancer 4684–95. Ó2016 AACR. Introduction plasmic signaling, resulting in the production of proinflamma- tory cytokines, such as myeloid differentiation factor (MyD88), Colorectal carcinoma is characterized by unlimited cell pro- MAPK, and inhibitor of kB(IkB)/nuclear factor-kB(NFkB; liferation and resistance to cell death. This is in contrast to refs. 11, 12). Accumulating evidence suggests that LPS receptors normal intestinal tissue, which has a balanced epithelial turn- are involved in the dysregulation of epithelial apoptosis and over that is maintained by crypt renewal and surface/villus proliferation that predisposes the tissue of the colon to tumor- apoptosis (1, 2). The development of colon cancer has been igenesis. Previous animal studies have indicated that TLR4/ linked to the aberrant recognition of enteric bacterial bypro- MyD88 signaling causes tumor cell hyperproliferation via path- ducts through both myeloid and epithelial immune receptor ways dependent on cyclooxygenase (COX), EGFR, and b-cate- signaling (3, 4). The surface of the intestinal mucosa is con- nin (5, 13–15). In vitro studieshaveshownadirecteffectofLPS stantly exposed to a large amount of bacterial lipopolysaccha- via TLR4/MD2 signaling on the stimulation of intestinal epi- ride (LPS). Upregulated expression of the LPS receptor com- thelial cell proliferation and a resistance to TNF-related apo- plex, CD14/Toll-like receptor (TLR4)/MD2, has been identified ptosis-inducing ligand (TRAIL)–induced apoptosis (16–18). in the intestinal mucosa of animal models and patients with Our recent study demonstrated a functional antagonism inflammatory bowel disease and colorectal cancers (5–8). between CD14 and TLR4 in the regulation of epithelial apo- The binding of LPS to CD14 on the lipid raft domain ptosis and intestinal carcinogenesis (19). TLR4 counteracted activates a cascade of lipid messengers and protein kinase Cz CD14-dependent epithelial apoptosis and promoted survival (PKCz) to recruit TLR4 to form a complex with CD14 (9, 10). in developing cancer cells (19–21). The transfer of LPS to TLR4/MD2 subsequently initiates cyto- Eritoran (E5564) is an investigational drug for the treatment of severe sepsis that functions as a TLR4 inhibitor because of its structural similarity to the LPS lipid A moiety, which is composed 1Graduate Institute of Physiology, National Taiwan University College of 6 acyl chains (22, 23). Recent advances have been made in 2 of Medicine,Taipei,Taiwan. Department of Internal Medicine, National understanding the interaction between LPS and its receptor based Taiwan University Hospital, Taipei, Taiwan. on studying the crystal structures of TLR4/MD2 complexed with Note: Supplementary data for this article are available at Cancer Research LPS or eritoran (24). It is recognized nowadays that LPS binding to Online (http://cancerres.aacrjournals.org/). CD14 leads to ligand transfer onto the TLR4/MD2 complex, Corresponding Author: Linda Chia-Hui Yu, National Taiwan University College which dimerizes to initiate downstream signaling (11, 25, 26). of Medicine, Suite 1020, #1 Jen-Ai Rd. Sec. I, Taipei 100, Taiwan. Phone: 886-02- However, eritoran (with only 4 acyl chains) does not induce 23123456-88237; Fax: 886-02-23964350; E-mail: [email protected] TLR4/MD2 complex dimerization or cytoplasmic signaling doi: 10.1158/0008-5472.CAN-16-0172 (27). Animal studies using eritoran have demonstrated an inhi- Ó2016 American Association for Cancer Research. bition of proinflammatory TLR4 signaling, improvement of 4684 Cancer Res; 76(16) August 15, 2016 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 21, 2016; DOI: 10.1158/0008-5472.CAN-16-0172 Tumor Progression by Dysregulated LPS Signaling intestinal microcirculation, and higher survival rates in endotoxe- tumor formation was clearly observed on day 63 following the mic rodents (22, 28) and in influenza- and syncytial virus– start of the AOM/DSS protocol (19), and eritoran was given on infected mice (29, 30). However, whether eritoran binds to CD14 days 63, 70, and 77. Based on pilot studies, the intracolonic (i.c.) and triggers CD14-mediated apoptosis in colon cancer cells dose of eritoran was 10 mg dissolved in 150 mL of PBS per bolus per remains unclear. mouse. For intragastric (i.g.) administration, 500 mg of eritoran The aim of our study was to investigate whether the adminis- was dissolved in 250 mL of PBS per bolus per mouse. For tration of eritoran inhibits colorectal carcinoma development in intravenous (i.v.) administration, mice were injected with 5, mouse models. In the current study, we explored the potential 10, or 20 mg/kg of eritoran dissolved in 250 mL of PBS per bolus therapeutic use of eritoran in tumor reduction and examined its per mouse (22, 28, 29). underlying anticancer mechanisms using primary mouse tumor spheroids and human adenocarcinoma cell lines. Measurement of cell apoptosis Intestinal sections were stained using a terminal deoxynucleo- Materials and Methods tidyl transferase dUTP-biotin nick end labeling (TUNEL) detec- Animals tion kit (Merck). The percentage of TUNEL-positive cells was 2 BALB/c mice 7 to 10 weeks of age were used in this study. calculated, normalized, and expressed per 1 mm from 10 tumor Experimental procedures were approved by the Laboratory Ani- sections from each group of mice. In addition, cancer cells were mal Care Committee of the National Taiwan University College of homogenized, and the supernatant was measured for DNA frag- Medicine. mentation by using a cell death detection ELISA kit (Roche; refs. 19, 20, 31, 32). Antibodies All antibodies were purchased from Cell Signaling Technology, Primary intestinal spheroid cultures except otherwise stated. Primary antibodies included mouse anti- Colonic tumors were cultured in vitro as spheroids following human PCNA (1:2,000), phospho (p)- and total (t)-IkB previously methods (33, 34) with modification. Briefly, intestinal (1:2,000), p- and t-ERK1/2 (1:4,000), p- and t-JNK (1:2,000), fragments with tumors were incubated in a chelation buffer for 60 p- and t-Akt (1:2,000), p-PKCz (Thr410; 1:2,000), p-PKCz minutes on ice to remove most of the normal epithelial cells. (Thr560; 1:5,000; Abcam Epitomics), t-PKCz (1:2,000), p-tyro- Tumor fractions were then incubated in a digestion buffer with sine (1:2,000; EMD Millipore), CD14 (1:50 for immunofluores- collagenase and dispase for 2 hours at 37C. After counting cence and 1:2,000 for Western blot; R&D Systems), TLR4 (1:100 isolated single tumor cells, approximately 10,000 cells were for immunofluorescence and 1:2,000 for Western blot; Santa Cruz plated in 300 mL of ice-cold Matrigel (BD Biosciences; 3:1 ratio Biotechnology), TLR2 (1:2,000; Santa Cruz Biotechnology), with crypt culture medium) per well in 24-well plates. After MyD88 (1:2,000), bromodeoxyuridine (BrdUrd; 1:200; Abcam), Matrigel polymerization, 300 mL of crypt culture medium was mouse IgG1 and IgG2a isotype controls (R&D Systems), rat IgG2a overlaid. The overlaying medium was refreshed every 2 to 3 days, isotype controls (Abcam), and b-actin (1:10,000; Sigma; refs. 19, and images were captured under a light microscope. Spheroids 31). The secondary antibodies used were goat anti-mouse IgG were collected by dissolving the Matrigel with a recovery solution conjugated to horseradish peroxidase (1:2,000), and to Alexa 594 (BD Biosciences). or 488 (1:1,000; ThermoFisher). Spheroids grown for 5 days were challenged by adding LPS (from E. coli O26:B6; Sigma) or eritoran to the overlaying medi- Models of chemically induced colorectal carcinoma um, and the effects this had on cell proliferation and cell death Mouse models of colitis-associated colorectal carcinoma were measured. Spheroid area was determined using imaging were prepared according to previously established methods software (AxioVision Rel. 4.8). Alternatively, time lapse imaging (19). Briefly, mice were injected i.p. with azoxymethane (AOM; for spheroid growth was performed using a real-time cultured cell 10 mg/kg body weight; Sigma) at the beginning of the exper- monitoring system (ASTEC Co.). iment (day 0). After 7 days, 2% dextran sodium sulfate (DSS; MP Biomedicals) was administered via the drinking water for 4 Cell lines days, and this was followed by 3 days of regular water.