Th17 cell-derived IL-17A promoted tumor progression via STAT3/NF-κB/Notch1 signaling in non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases, which is the leading cause of cancer deaths worldwide. IL-17░A, the major effector cytokine derived from Th17 cells, is a key cytokine in tumor pathogenesis and modulates tumor progression. We aimed to identify whether IL-17░A derived from Th17 cells promotes the progression of NSCLC. Here we found that the level of Th17 cells was increased in NSCLC and IL-17░A was mainly produced by CD4⁺ cells (Th17 cells) in NSCLC. IL-17░A enhanced the migration, invasion and stemness of NSCLC via STAT3/NF-κB/Notch1 signaling. Blockade of this signaling inhibited the migration, invasion and stemness of NSCLC mediated by IL-17░A. Th17 cells in NSCLC were closely associated with poor prognosis of NSCLC patients. Our results indicated that Th17 cell-derived IL-17░A plays an important role in tumor progression of NSCLC via STAT3/NF-κB/Notch1 signaling. Therefore, therapeutic strategies against this pathway would be valuable to be developed for NSCLC treatment.

Keywords: Th17 cells; Tumor microenvironment; interleukin-17A (IL-17A); non-small cell lung cancer (NSCLC); tumor progression.

Figure 1.

The level of Th17 cells in NSCLC was increased. (A) CD3⁺CD4⁺IL-17░A⁺IFN-γ⁻Th17 cells from peripheral blood were analyzed by flow cytometry in NSCLC patients and healthy donors. (B) Comparison of Th17 cell phenotypes in peripheral blood from NSCLC patients and healthy donors represented as scatter. (C) Comparison of Th17 cell frequencies in NSCLC tissues and non-tumor tissues represented as scatter. (D) Comparison of Th17 cell frequencies in peripheral blood and tumor tissues from NSCLC patients represented as scatter. (E) Tumor tissues and non-tumor tissues from NSCLC patients were subjected to double immunofluorescence for CD4 (red), IL-17░A (green) and DAPI (blue). *, ** and *** indicate P < 0.05, P < 0.01 and P< 0.001, respectively. Scale bar represents 50 μm.

Figure 2.

IL-17░A promoted the migration and invasion of NSCLC cells in vitro. The migration activities of A549 (A) and H460 (B) cells, and the invasion activities of A549 (C) and H460 (D) cells before and after treatment of rhIL-17░A was analyzed using transwell assay. One representative analysis is shown. Data are presented as a histogram. (E) The expression of N-cadherin in A549 and H460 cells before and after treatment of rhIL-17░A was analyzed using western blotting. (F) The relative expression of N-cadherin in A549 and H460 cells before and after treatment with rhIL-17░A was analyzed using qPCR. (G) The expressions of IL-17░A and CD31 in tumor tissues from NSCLC patients were detected by immunofluorescence. * indicates P < 0.05. Scale bar represents 50 μm.

Figure 3.

The STAT3/NF-κB/Notch1 signaling was critical for IL-17░A-induced migration and invasion in NSCLC cells. (A) The activation of STAT3, NF-κB and Notch1in A549 and H460 cells treated with rhIL-17░A was analyzed using western blotting. (B) The migration activities of A549 and H460 cells treated with or without rhIL-17░A and STAT3, or NF-κB, or Notch1 inhibitor were assessed by transwell assay. One representative analysis is shown. The data from A549 (C) and H460 (D) cells are presented as histogram. (E) The invasion activities of A549 and H460 cells treated with or without rhIL-17░A and these molecular inhibitors were assessed by transwell assay. One representative analysis is shown. The data from A549 (F) and H460 (G) cells are presented as a histogram. (H) The expression of N-cadherin in A549 and H460 cells treated with or without rhIL-17░A and STAT3, or NF-κB, or Notch1 inhibitor was analyzed using western blotting. * indicates P < 0.05. Scale bar represents 50 μm.

Figure 4.

IL-17░A promoted the CSC-like properties of NSCLC cells. A549 (A) and H460 (B) cells were cultured with rhIL-17░A for 7 days, and then collected for sphere assay. One representative photomicrograph is shown. Data are presented as a histogram. (C) The expression of Oct4 in A549 and H460 cells before and after treatment of rhIL-17░A was analyzed using western blotting. The apoptosis of A549 (D) and H460 (E) cells treated with or without docetaxel and rhIL-17░A was analyzed by flow cytometry. IL-17░A expression in IL-17░A-expressing and control vectors-tranfected H460 cells was analyzed by western blotting (F) and qPCR (G). (H) IL-17░A promotes NSCLC tumor growth in vivo. IL-17░A expressing A549 cells were injected subcutaneously into nude mice (n = 6 mice/group). Tumor volumes were measured at 21 days after cell implantation. (I) Lump images of xenograft tumors that were formed in nude mice at 21 days after cell implantation. (J) The result of tumor weights at 21 days after cell implantation was shown as histogram. (K) Tumor growth curve from 7 to 21 days after cell implantation was shown as statistical graph. * indicates P < 0.05. Scale bar represents 100 μm.

Figure 5.

Blockade of STAT3/NF-κB/Notch1 signaling inhibited NSCLC stemness promoted by IL-17░A. (A) After the treatment with STAT3░or NF-κB or Notch1 inhibitor, the sphere forming of A549 and H460 cells induce by rhIL-17░A was blocked. One representative photomicrograph is shown. The results from A549 (B) and H460 (C) cells were shown as histogram. (D) The expression of Oct4 in A549 and H460 cells treated with or without rhIL-17░A and these molecular inhibitors were assessed by western blotting. The apoptosis of A549 (E) and H460 (F) cells treated with or without rhIL-17░A and these molecular inhibitors were assessed by transwell assay. * indicates P < 0.05. Scale bar represents 100 μm.

Figure 6.

The level of Th17 cells was associated with poor survival in NSCLC patients. The relationship between Th17 frequency and TNM stage (A), tumor grade (B) and lymph node metastasis (C) of NSCLC patients, respectively. The changes of Th17 cell frequency in peripheral blood from NSCLC patients before and after chemotherapy with the status of PD (D), PR (E) and SD (F). (G) IL-17░A expression in tumor tissues of NSCLC was analyzed by immunohistochemistry. (H) Kaplan-Meier survival curves for NSCLC patients with lower and higher IL-17░A expression (immunohistochemistry analysis). *, ** and ns indicate P < 0.05, P < 0.05 and non-significance, respectively. Scale bar represents 50 μm.