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Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Farnebo L, Shahangian A, Lee Y, Shin JH, Scheeren FA, Sunwoo JB - Oncotarget (2015)

Bottom Line: Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation.TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice.In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA, USA.

ABSTRACT
Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

No MeSH data available.


Related in: MedlinePlus

Zymosan activates MAPK/ERK and NF-κB signaling pathways in HNSCC, which can be blocked by anti-TLR2 antibodyA. UM-SCC-6 cell line was treated with 10 μg/ml zymosan or 10 ng/ml recombinant TNFα for the indicated times and analyzed for expression of phospho-ERK and phospho-NF-κB p65 by Western blots. Total ERK was used as the loading control. B. UPCI:SCC103 cells were treated with media alone, 10 μg/ml zymosan, 5 μg/ml mIgG1 antibody and 10 μg/ml zymosan, or 5 μg/ml α-TLR2 antibody and 10 μg/ml zymosan. Cells were collected after 60 min and analyzed for expression of phospho-NF-κB p65 by Western blots.
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Figure 3: Zymosan activates MAPK/ERK and NF-κB signaling pathways in HNSCC, which can be blocked by anti-TLR2 antibodyA. UM-SCC-6 cell line was treated with 10 μg/ml zymosan or 10 ng/ml recombinant TNFα for the indicated times and analyzed for expression of phospho-ERK and phospho-NF-κB p65 by Western blots. Total ERK was used as the loading control. B. UPCI:SCC103 cells were treated with media alone, 10 μg/ml zymosan, 5 μg/ml mIgG1 antibody and 10 μg/ml zymosan, or 5 μg/ml α-TLR2 antibody and 10 μg/ml zymosan. Cells were collected after 60 min and analyzed for expression of phospho-NF-κB p65 by Western blots.

Mentions: Because we observed significant inhibition of zymosan-induced tumor growth with an α-TLR2 mAb, we assessed if zymosan could induce the activation of signaling pathways downstream of TLR2. Although activation of NF-κB and MAPK pathways by TLR2 is well described in immune cells, little is known about TLR2 signaling in HNSCC. To investigate this, HNSCC cells were incubated in low-serum conditions with zymosan, and phosphorylation of the p65 subunit of NF-κB and ERK was assessed by Western immunoblot over a time course. Exposure of the cells to zymosan resulted in increased phosphorylation of p65 and ERK at 30 minutes, with levels returning to baseline by 90 minutes after stimulation. Interestingly, the addition of zymosan was able to increase phosphorylation of p65 and ERK above the constitutive baseline (Fig. 3A), consistent with the effects of zymosan on tumor growth in the organoid assays (Fig. 1C and 2D). Notably, incubation of the cells with α-TLR2 mAb was able to inhibit the phosphorylation of p65 (Fig. 3B), confirming that zymosan is acting through TLR2. Together, these data indicate that TLR2 signaling is intact in HNSCC cells and that activation of TLR2 results in the enhanced activation of the NF-κB and MAPK pathways, both of which have previously been shown to be associated with a survival and growth advantage in this malignancy [41, 42].


Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Farnebo L, Shahangian A, Lee Y, Shin JH, Scheeren FA, Sunwoo JB - Oncotarget (2015)

Zymosan activates MAPK/ERK and NF-κB signaling pathways in HNSCC, which can be blocked by anti-TLR2 antibodyA. UM-SCC-6 cell line was treated with 10 μg/ml zymosan or 10 ng/ml recombinant TNFα for the indicated times and analyzed for expression of phospho-ERK and phospho-NF-κB p65 by Western blots. Total ERK was used as the loading control. B. UPCI:SCC103 cells were treated with media alone, 10 μg/ml zymosan, 5 μg/ml mIgG1 antibody and 10 μg/ml zymosan, or 5 μg/ml α-TLR2 antibody and 10 μg/ml zymosan. Cells were collected after 60 min and analyzed for expression of phospho-NF-κB p65 by Western blots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496405&req=5

Figure 3: Zymosan activates MAPK/ERK and NF-κB signaling pathways in HNSCC, which can be blocked by anti-TLR2 antibodyA. UM-SCC-6 cell line was treated with 10 μg/ml zymosan or 10 ng/ml recombinant TNFα for the indicated times and analyzed for expression of phospho-ERK and phospho-NF-κB p65 by Western blots. Total ERK was used as the loading control. B. UPCI:SCC103 cells were treated with media alone, 10 μg/ml zymosan, 5 μg/ml mIgG1 antibody and 10 μg/ml zymosan, or 5 μg/ml α-TLR2 antibody and 10 μg/ml zymosan. Cells were collected after 60 min and analyzed for expression of phospho-NF-κB p65 by Western blots.
Mentions: Because we observed significant inhibition of zymosan-induced tumor growth with an α-TLR2 mAb, we assessed if zymosan could induce the activation of signaling pathways downstream of TLR2. Although activation of NF-κB and MAPK pathways by TLR2 is well described in immune cells, little is known about TLR2 signaling in HNSCC. To investigate this, HNSCC cells were incubated in low-serum conditions with zymosan, and phosphorylation of the p65 subunit of NF-κB and ERK was assessed by Western immunoblot over a time course. Exposure of the cells to zymosan resulted in increased phosphorylation of p65 and ERK at 30 minutes, with levels returning to baseline by 90 minutes after stimulation. Interestingly, the addition of zymosan was able to increase phosphorylation of p65 and ERK above the constitutive baseline (Fig. 3A), consistent with the effects of zymosan on tumor growth in the organoid assays (Fig. 1C and 2D). Notably, incubation of the cells with α-TLR2 mAb was able to inhibit the phosphorylation of p65 (Fig. 3B), confirming that zymosan is acting through TLR2. Together, these data indicate that TLR2 signaling is intact in HNSCC cells and that activation of TLR2 results in the enhanced activation of the NF-κB and MAPK pathways, both of which have previously been shown to be associated with a survival and growth advantage in this malignancy [41, 42].

Bottom Line: Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation.TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice.In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA, USA.

ABSTRACT
Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

No MeSH data available.


Related in: MedlinePlus