LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

Abstract

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide

“…In vitro studies have shown a direct effect of LPS via TLR4/MD2 signaling on the stimulation of intestinal epithelial cell proliferation and a resistance to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (16)(17)(18). Our recent study demonstrated a functional antagonism between CD14 and TLR4 in the regulation of epithelial apoptosis and intestinal carcinogenesis (19). TLR4 counteracted CD14-dependent epithelial apoptosis and promoted survival in developing cancer cells (19)(20)(21).…”

“…Primary antibodies included mouse antihuman PCNA (1:2,000), phospho (p)-and total (t)-IkB (1:2,000), p-and t-ERK1/2 (1:4,000), p-and t-JNK (1:2,000), p-and t-Akt (1:2,000), p-PKCz (Thr410; 1:2,000), p-PKCz (Thr560; 1:5,000; Abcam Epitomics), t-PKCz (1:2,000), p-tyrosine (1:2,000; EMD Millipore), CD14 (1:50 for immunofluorescence and 1:2,000 for Western blot; R&D Systems), TLR4 (1:100 for immunofluorescence and 1:2,000 for Western blot; Santa Cruz Biotechnology), TLR2 (1:2,000; Santa Cruz Biotechnology), MyD88 (1:2,000), bromodeoxyuridine (BrdUrd; 1:200; Abcam), mouse IgG1 and IgG2a isotype controls (R&D Systems), rat IgG2a isotype controls (Abcam), and b-actin (1:10,000; Sigma; refs. 19,31). The secondary antibodies used were goat anti-mouse IgG conjugated to horseradish peroxidase (1:2,000), and to Alexa 594 or 488 (1:1,000; ThermoFisher).…”

“…Mouse models of colitis-associated colorectal carcinoma were prepared according to previously established methods (19). Briefly, mice were injected i.p.…”

“…Our recent study demonstrated a functional antagonism between CD14 and TLR4 in the regulation of epithelial apoptosis and intestinal carcinogenesis (19). TLR4 counteracted CD14-dependent epithelial apoptosis and promoted survival in developing cancer cells (19)(20)(21).…”

Functional Optical Imaging of Primary Human Tumor Organoids: Development of a Personalized Drug Screen

“…Optical imaging technologies used to study organoids include bright-field microscopy, light microscopy, phase microscopy, fluorescence microscopy, confocal microscopy, time-lapse microscopy, multiphoton fluorescence imaging, fluorescence lifetime imaging, and optical coherence tomography (OCT) (8,12,(18)(19)(20)29,30). In wide-field or whole-field microscopy, an entire sample or field of view is illuminated by a light source, and images are captured by a camera.…”