Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

Abstract

Toll-like receptors (TLRs) are innate immune receptors for sensing microbial molecules and damage-associated molecular patterns released from host cells. Double-stranded RNA and the synthetic analog polyinosinic:polycytidylic acid (poly(I:C)) bind and activate TLR3. This stimulation leads to recruitment of the adaptor molecule TRIF (Toll/IL-1 resistance (TIR) domain–containing adapter-inducing interferon β) and activation of the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), classically inducing IFNβ production. Here we report that, unlike non-metastatic intestinal epithelial cells (IECs), metastatic IECs express TLR3 and that TLR3 promotes invasiveness of these cells. In response to poly(I:C) addition, the metastatic IECs also induced the chemokine CXCL10 in a TLR3-, TRIF-, and IRF3-dependent manner but failed to produce IFNβ. This was in contrast to healthy and non-metastatic IECs, which did not respond to poly(I:C) stimulation. Endolysosomal acidification and the endosomal transporter protein UNC93B1 was required for poly(I:C)-induced CXCL10 production. However, TLR3-induced CXCL10 was triggered by immobilized poly(I:C), was only modestly affected by inhibition of endocytosis, and could be blocked with an anti-TLR3 antibody, indicating that TLR3 can still signal from the cell surface of these cells. Furthermore, plasma membrane fractions from metastatic IECs contained both full-length and cleaved TLR3, demonstrating surface expression of both forms of TLR3. Our results imply that metastatic IECs express surface TLR3, allowing it to sense extracellular stimuli that trigger chemokine responses and promote invasiveness in these cells. We conclude that altered TLR3 expression and localization may have implications for cancer progression.

Alternative MyD88 -Cyclin D1 signaling in breast cancer cells regulates TLR3 mediated cell proliferation

“…Induction of cell proliferation via surface localization of TLR3 has been shown by our research group in breast cancer cells (Bondhopadhyay et al, 2014) and by other groups in diverse types of cancers (Glavan and Pavelic 2014; Bugge et al, 2017). This mechanism is supposedly an alternative to the endosomal mediated action but the exact mechanism of alternating TLR3 signaling lacks clarity.…”

“…Stimulation of TLR3 with ligand poly (I:C) it gets translocated from ER into the endosomal compartment (Johnsen et al, 2006). Though, regulation of this translocation is reported to be controlled by UNC93B1 protein, its inhibition has only a partial effect on TLR3 mediated signaling (Bugge et al, 2017). Further, cell surface expression of TLR3 has been reported in a variety of cells such as pulmonary cells, hepatocytes, breast cancer, prostate cancer and epithelial adenocarcinoma (Salaun et al, 2006; Gambara et al, 2014; Helminen et al, 2016) indicate that it signaling occurs through the plasma membrane.…”

“…Dynasore, a dynamin inhibitor that inhibits endocytosis of the receptors, only partially affects the poly(I:C) mediated TLR3 signaling. This suggests that TLR3 signaling may occur independent to ligand-internalization (Bugge et al, 2017). Our result shows the increase in surface expression of TLR3 in breast MDA-MB-231 and T47D cells upon TLR3 ligand activation.…”

“…TLR3 synthetic ligands were used with conventional chemotherapies or radiotherapy in clinical trials for the treatment of cancer patients (Braunstein et al, 2018; Aranda et al, 2014; Smith et al, 2018). This reported tumor suppressive and apoptotic effect of TLR3 is achieved predominantly by induction of type I IFN and activation of effector cells, when TLR3 is located within the endosomal compartment (Bugge et al, 2017; Gambar et al, 2014; Braunstein et al, 2018). TLR3 synthetic ligand poly-ICLC with Sorafenib significantly reduces tumor growth, both in-vitro and in-vivo in hepatocellular carcinoma (Ho et al, 2015).…”

“…Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral origin, small interfering RNAs, and self-RNAs derived from damaged cells (Bugge et al, 2017; Kawasaki and Kawai 2014). TLR3 induces a potent antigen-specific CD8+ T-cell responses that directly induces effector CD8+ T-cell and natural killer (NK) cells for IFN-γ release (Conforti et al, 2010).…”