Janus-faced bacterial regulation of epithelial cell death and survival: Association with colon carcinogenesis

Abstract

Gut microbiota and lipopolysaccharide (LPS) signaling have been associated with colon cancer development. Our recent findings demonstrated that LPS receptor subunits expressed on colonocytes have antagonistic roles in cell death and tumorigenesis: epithelial toll-like receptor 4 (TLR4) confers resistance to the apoptosis induced by its co-receptor CD14 and contributes to epithelial transition to cancer.

Colon carcinoma is characterized by unlimited cell proliferation and resistance to cell death in broad contrast to the dynamic yet balanced turnover of epithelial cells in the normal intestine, which is maintained by cell proliferation in the crypts and cell death at villi surfaces. Gut microbiota and receptors of the innate immune system are involved in both myeloid and epithelial signaling and have been found to be associated with colon cancer formation. The bacterial lipopolysaccharide (LPS) receptor consisting of 3 subunits—CD14/toll-like receptor 4 (TLR4)/lymphocyte antigen 96 (MD2)—is a well-known proinflammatory receptor complex that is involved in septic injury. Upregulation of LPS receptors was reported in the intestinal mucosa of patients with inflammatory bowel disease and colorectal cancers.¹ Our recent study in Cell Death and Differentiation (2015) demonstrated that enteric bacteria regulate epithelial apoptosis and colon carcinogenesis in part through functional antagonism of the LPS receptor subunits CD14 and TLR4, which are expressed on colonocytes.² We showed that epithelial CD14-mediated lipid signaling triggered caspase-dependent cell apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development (Fig. 1).² Our study provided evidence of a regulatory mechanism derived from bacterial LPS signaling for physiological apoptosis that eliminates premalignant epithelial cells in the gut. Dysregulation of the expression pattern of LPS receptor subunits on epithelial cells holds the key to the opposing life-and-death responses associated with tumorigenesis.

Figure 1.

Bacterial lipopolysaccharide (LPS) receptors have antagonistic roles in epithelial apoptosis and tumorigenesis. (A) Physiologically, proliferating stem cells in crypts differentiate into epithelial cells that migrate upward to the colon surface where apoptosis occurs. Apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells (arrowheads) are found on the luminal surface, whereas proliferating ki67-immunoreactive cells (yellow fluorescence) are present at the crypt bottom. A balance between death and proliferation in epithelium is crucial for maintaining gut homeostasis. Colonic carcinoma is characterized by reduced cell death and heightened cell proliferation, as shown by decreased numbers of TUNEL-positive cells and increased ki67 staining, respectively. (B) Normal colonocytes express CD14 constitutively with negligible to low levels of toll-like receptor 4 (TLR4), whereas tumorous tissues display enhanced levels of CD14 and TLR4. Bacterial LPS binds to apical CD14 to activate cell apoptosis via lipid signaling by ceramide synthesis and activation of protein kinase C, zeta (PKCζ). Upregulated TLR4 expression provides antiapoptotic signals that oppose its co-receptor CD14 and cause cellular hyperproliferation, leading to disturbance of gut homeostasis and predisposition to cancer development.

The LPS receptor, originally identified and constitutively expressed in monocytic cell lineages, is responsible for transmitting signals via myeloid differentiation primary response 88 (MyD88), mitogen-activated protein kinases, and nuclear factor κB for the production of proinflammatory cytokines. Despite being the first identified subunit in the receptor, CD14, which lacks a cytoplasmic tail, has been considered merely a component for LPS binding that is needed for scavenging or transfer to the transmembrane signaling moiety TLR4. Evidence for recruitment of TLR4 to lipid raft domains to form complexes with CD14 following LPS/CD14-dependent activation of lipid messengers was later found in monocytic cells.³ This indicates an autonomous, individual signaling pathway mediated by CD14 alone, as well as spatial disparity of the 2 subunits on the surface of myeloid cells in the resting state.

The intestine, a unique organ with a high load of lumen-dwelling commensals, is considered immune-tolerant as a result of the absence or suppression of particular receptors or signaling molecules. Normal human colonocytes constitutively express CD14 in the presence of negligible to low levels of TLR4,^1,4 whereas the high expression of TLR4 in malignant tissues has been associated with cell hyperproliferation via cyclooxygenase 2- and epidermal growth factor receptor (EGFR)-dependent pathways.¹ Our report argues against the basal sequestration concept and supports the theory that LPS receptor subunits actively participate in the life-and-death decisions of gut epithelial cells.

Mounting evidence indicates alternative mechanisms for innate immune signaling in epithelial-derived tumorigenesis involving control of the balance between cell proliferation and apoptosis instead of the regulation of inflammation. Increased cell apoptosis associated with decreased tumor burden was documented in our study, as well as in other studies using genetically deficient mice lacking TLR4, MyD88, or epithelial-specific IκB kinase β (IKKβ).^1,5,6 In contrast, mice lacking nucleotide-binding oligomerization domain-like receptors (NLRs, such as NLR family, CARD domain containing 4 [NLRC4]) or caspase-1 (a component of the inflammasome complex) are more susceptible to cancer formation, as manifested by increased epithelial cell proliferation and reduced apoptosis in advanced tumors.⁷ While an inverse correlation between cell death and colon carcinogenesis was clearly shown in these models of aberrant innate signaling, the activation of surface TLRs and cytosolic NLRs following bacterial recognition appears to lead to opposite consequences in cancer development. How crosstalk among innate immune receptors might shape the outcome of host-microbial interactions in tumor progression warrants further investigation. Such research may provide novel perspectives regarding the evolution of innate immunity for Janus-faced bacterial regulation in gut homeostasis and carcinogenesis.

Cancer biology research has largely focused on mutations in oncogenes and tumor suppressor genes for the promotion of unlimited proliferation. Clinically, genotoxic drugs (e.g., 5-fluorouracil and oxaliplatin) are widely applied based on their effectiveness in killing tumor cells. To many oncologists, studies of antiapoptotic signaling are designed to decipher the cause of drug resistance, and the notion of death resistance as an underlying carcinogenic mechanism remains a theoretical hypothesis. Our finding indicates that gut epithelial cells undergo CD14-dependent apoptosis, whereas the ability to resist cell death through upregulation of TLR4 expression partly contributes to cancer transition.² A direct relationship between resistance to cell death and cancer development was shown in mice deficient in proapoptotic molecules (e.g., BCL2-antagonist/killer [Bak] and Fas),^8,9 which provide strong evidence that resistance to cell death increases susceptibility to tumor development.

Elucidating the mechanisms of cell death-associated gut epithelial homeostasis has been challenging because the relevant systemic- or tissue-specific knockout mice die perinatally, as reported for caspase-3 knockout mice, Fas-associated death domain (FADD)-deficient mice, and receptor-interacting protein kinase 1 (RIPK1) complete or epithelial-specific knockout mice.¹⁰ Recent findings have added further complexity to our understanding of cell death in gut homeostasis and tumorigenesis. Other mechanisms, such as necroptosis (programmed necrosis) may also be involved in the physiological turnover of epithelial cells and annihilation of premalignant cells. Studies using epithelial-specific double knockouts of Ripk1/Fadd genes demonstrated that RIPK1 is involved in epithelial homeostasis by inhibiting apoptosis and necroptosis through a kinase-independent scaffolding function.¹⁰ Investigating the interaction between innate signaling and necroptosis could be another approach to decoding the mechanisms of survival and malignant transformation of intestinal epithelial cells.

Last, we also demonstrated that intracolonic administration of a synthetic analog of LPS lipid A moiety that inhibits TLR4 signaling increased tumor cell apoptosis and decreased the tumor burden in mice, suggesting that manipulation of innate signaling in the epithelium has therapeutic potential.² By understanding how intestinal epithelium maintains homeostasis under physiological conditions, novel insights into the mechanisms contributing to the epithelial transition to carcinogenesis may evolve into therapeutic strategies for colon cancer.

Funding

This study was supported by funding from the Ministry of Science and Technology (MOST 102-2628-B-002-009-MY3) and National Taiwan University (NTU-CDP-104R7798).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.