Toll-like receptor 2 activation contributes to oral squamous cell carcinoma development and miRNA-mediated drug resistance

Tue, 05/14/2019 - 08:56

Nuclear Factor kappa B (NFkB) signaling pathway poster, Novus Biologicals

By Jamshed Arslan, Pharm. D., PhD.

Squamous cell carcinoma is the most common cancer in the oral cavity.1 The tumor surface biofilms in oral cancers contain high levels of aerobic and anaerobic microorganisms.1,2 Periodontal pathogens can activate epithelial Toll-like receptors (TLRs) on tumor cells to promote oral squamous cell carcinoma (OSCC) progression. In this regard, TLR2 is known to recognize micro-organisms in the oral cavity by forming a heterodimer with TLR1 or TLR6, but the precise role of TLR2 in OSCC is unclear. TLR2-activating signals from bacteria can induce various noncoding RNAs called miRNAs, the role of which is yet to be understood in the context of OSCC. In other words, how bacterial pathogens in the oral cavity may contribute to TLR2-induced OSCC progression is unknown. To answer this question, researchers in Japan examined TLR2 and miRNA expression in human OSCC tissues and cell lines.2 They found that bacterial ligands can activate TLR2 expression, which contributes to OSCC progression and miR-146a-mediated resistance to cisplatin.

Activated TLR2 signaling in oral squamous cell carcinoma (OSCC)

To determine the role of TLR2 in OSCC, the researchers first determined expression of TLR2 and its co-receptors (TLR1 and TLR6) in OSCC tissues. The results of immunohistochemistry, immunocytochemistry and western blotting showed higher levels of TLR2, TLR1 and TLR6 in human OSCC tissues relative to adjacent noncancerous tissues. Human OSCC cell lines (HSC3, HSC3-M3) also showed elevated TLR2, TLR1 and TLR6 levels. To determine the functional role of these receptors, the team transfected OSCC cell lines with a luciferase reporter vector that contained NF-kB response element. The choice of NF-kB activation assay stemmed from the fact that bacterial lipoprotein-mediated TLR signaling leads to NF-kB activation. Researchers used synthetic bacterial lipopeptides Pam3CSK4 and FSL-1 as TLR-ligands for heterodimers TLR2/TLR1 and TLR2/TLR6, respectively. These ligands, dose-dependently, increased the luciferase activity, validating a functional role of activated TLR2 signaling in OSCC.

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TLR2 detection by western blot analysis of transfected cell lysate

TLR2 Antibody[NB100-56720] - Analysis using the Azide Free version of NB100-56720. Detection of TLR2 in transfected cell lysate using this antibody.

miR-146a-induced CARD10 downregulation in OSCC due to TLR2 signaling

To determine the mechanism behind the above mentioned TLR2 signaling in OSCC, the team performed miRNA microarray and found miR-146a-5p to be specifically upregulated by both ligands (Pam3CSK4 and FSL-1) in human OSCC cells. In silico analysis revealed six tumor-suppressive genes targeted by miR-146a. Among the six genes, the researchers singled out CARD10 because its mRNA was repressed in OSCC cells when the cells were treated with TLR2-ligand or transfected with miR-146a-5p.

This means that TLR2 activation induces miR-146a-5p expression and CARD10 downregulation in OSCC. The next question was to determine therapeutic consequence of TLR2 signaling in OSCC.

Caspase-3 detection by western blot analysis of HEK293 cell extract

Caspase-3 Antibody (CPP32 4-1-18) [NB500-210] - Detection of Caspase (19 and 35 kDa) from HEK293 cell extract using NB500-210. Lanes 1 and 2 contain inactive and active Caspase, respectively.

TLR2 induced drug resistance in OSCC

CARD10 regulates caspase-induced apoptosis. To determine the role of TLR2-induced silencing of CARD10, the OSCC cells were transfected with CARD10 siRNA and treated with cisplatin. As expected, the researchers found reduced cisplatin-induced cell death and decreased levels of cleaved-caspase3 protein in transfected cells relative to untreated OSCC cells. In other words, TLR2 signaling contributes to the resistance against cisplatin-induced apoptosis in OSCC by downregulating CARD10.

Significance for TLR2 targeting strategies in OSCC

This2 is the first study linking human OSCC progression with CARD10 silencing and the role of TLR2 signaling in this process. This research prompts identification and targeting of bacterial ligands that lead to OSCC progression through TLR2 activation.

Jamshed Arslan Jamshed Arslan, Pharm D., PhD.
Previously at the University of Alabama at Birmingham, School of Medicine.
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans and transgenic mice


  1. Gallimidi, Adi Binder, et al., "Periodontal Pathogens Porphyromonas gingivalis and Fusobacterium nucleatum Promote Tumor Progression in an Oral-Specific Chemical Carcinogenesis Model."Oncotarget, vol. 6, no. 26, 2015, pp. 22613–22623.
  2. Ikehata, Naoki, et al. "Toll-Like Receptor 2 Activation Implicated in Oral Squamous Cell Carcinoma Development." Biochemical and Biophysical Research Communications, vol. 495, no. 3, 2018, pp. 2227–2234.


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