STAT3 Induces Immunosuppression by Upregulating PD-1/PD-L1 in HNSCC - PubMed (original) (raw)

STAT3 Induces Immunosuppression by Upregulating PD-1/PD-L1 in HNSCC

L L Bu et al. J Dent Res. 2017 Aug.

Abstract

Head and neck cancer is one of the most prevalent cancers around the world. Head and neck squamous cell carcinoma (HNSCC) accounts for nearly 90% of head and neck cancer. In recent years, significant advances have been made in immunotherapy for HNSCC. Although some clinical trials targeting immune checkpoints have shown success, the molecular mechanism for regulation of programmed death 1 (PD-1) and its ligand (PD-L1) is partially understood. In an effort to explore the effect of activation of signal transducers and activators of transcriptions (STAT3) on PD-1/PD-L1, the expression and correlation between phosphorylation of STAT3 and PD-1/PD-L1 were determined with immunostaining of human and mouse HNSCC tissue sections. PD-1/PD-L1 overexpression was found to be significantly associated with p-STAT3 in human and mouse HNSCC. Targeting STAT3 by a small molecule effectively inhibited the expression of PD-L1 in the CAL27 cell line. Furthermore, we found that blockade of STAT3 signaling downregulated PD-1/PD-L1 in a Tgfbr1/Pten 2cKO HNSCC mouse model. These findings suggest that STAT3 signaling plays an important role in PD-1/PD-L1 regulation and the antitumor immune response of HNSCC.

Keywords: S3I-201; cytokine; immune checkpoint; immune escape; immunotherapy; mice model.

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Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.

Figure 1.

p-STAT3 and PD-1/PD-L1 expression levels in human HNSCC. (A) Representative immunostaining pictures of p-STAT3 (substance), PD-L1, p-STAT3 (mesenchyme), and PD-1 in human HNSCC. Scale bar = 50 µm. (B) Linear regression analysis of p-STAT3 (substance) and PD-L1 histoscore of immunostaining in a human HNSCC tissue microarray; P < 0.001, r = 0.4495. (C) linear regression analysis of p-STAT3 (mesenchyme) and PD-1 histoscore of immunostaining in a human HNSCC tissue microarray; P < 0.001, r = 0.5051. HNSCC, head and neck squamous cell carcinoma; PD-1, programmed death 1; PD-L1, programmed death 1 ligand; STAT, signal transducers and activators of transcription; p-STAT3, phosphorylated STAT3.

Figure 2.

Figure 2.

The expression level of PD-L1 was measured by Western blotting in a head and neck squamous cell carcinoma cell line (Cal 27 and FaDu), with and without S3I-201. β-Actin was used as the internal protein loading control. PD-L1, programmed death 1 ligand; STAT, signal transducers and activators of transcription; p-STAT3, phosphorylated STAT3.

Figure 3.

Figure 3.

p-STAT3 and PD-1/PD-L1 levels in a Tgfbr1/Pten 2cKO mice model. (A) Representative immunostaining pictures of p-STAT3, PD-L1, and PD-1 in_Tgfbr1/Pten_ 2cKO HNSCC mice. Scale bars = 50 µm. (B) Linear regression analysis of p-STAT3 (substance) and PD-L1 histoscore of immunostaining in Tgfbr1/Pten 2cKO HNSCC mice; P < 0.001, _r_= 0.8041. (C) Linear regression analysis of p-STAT3 (mesenchyme) and PD-1 histoscore of immunostaining in Tgfbr1/Pten 2cKO HNSCC mice; P < 0.001, _r_= 0.7226. HE, hematoxylin and eosin; HNSCC, head and neck squamous cell carcinoma; PD-1, programmed death 1; PD-L1, programmed death 1 ligand; STAT, signal transducers and activators of transcription; p-STAT3, phosphorylated STAT3.

Figure 4.

Figure 4.

Inhibition of phosphorylation of STAT3 reduced tumor growth in a Tgfbr1/Pten 2cKO mice model. (A) Schematic picture shows the tamoxifen gavage and drug delivery strategy of S3I-201 in Tgfbr1/Pten 2cKO mice. (B) Representative pictures of mice with head and neck tumors after treatment with S3I-201 or PBS on days 18 and 38. (C) Total volume of tumor was measured in the S3I-201 and control groups once a week. Data are presented as mean ± SEM (unpaired Student’s t test;**P < 0.01,***P < 0.001). (D) Relative growth of body (g) was measured in the S3I-201 and control groups once a week. Data are presented as mean ± SEM (unpaired Student’s t test; NS, not significant). (E) Representative spleen pictures of Tgfbr1/Pten 2cKO mice after treatment with S3I-201 or PBS. (F) Spleen index shows that S3I-201 significantly inhibited compensatory growth of spleen in Tgfbr1/Pten 2cKO mice (1-way analysis of variance with post hoc Tukey test; ***P < 0.001). PBS, phosphate-buffered saline; STAT, signal transducers and activators of transcription.

Figure 5.

Figure 5.

S3I-201 treatment downregulates the PD-1/PD-L1 signaling axis in Tgfbr1/Pten 2cKO mice. (A) Representative flow cytometry showed decreased staining population of PD-1 in the S3I-201 treatment group and a quantitative PD-1+cell population in spleen, LN, blood, and tumor in the S3I-201 treatment and control groups (1-way analysis of variance with post hoc Tukey test;*P < 0.05). (B) The intratumor levels of STAT3, p-STAT3, PD-1, and PD-L1 were measured by Western blotting in the S3I-201 treatment and control groups. β-Actin was used as the internal protein loading control. (C) Representative immunostaining pictures of p-STAT3, PD-L1, and PD-1 in tumor derive from Tgfbr1/Pten 2cKO mice in the S3I-201 treatment and control groups. Scale bar = 100 µm. (D) The expression level of INF-γ was accessed by immunohistochemistry in Tgfbr1/Pten 2cKO mice after treatment with S3I-201 or PBS. Scale bar = 100 µm. LN, lymph node; PBS, phosphate-buffered saline; PD-1, programmed death 1; PD-L1, programmed death 1 ligand; STAT, signal transducers and activators of transcription; p-STAT3, phosphorylated STAT3.

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