Cancer-associated fibroblasts promote the migration and invasion of gastric cancer cells via activating IL-17a/JAK2/STAT3 signaling

Abstract

Background: Cancer-associated fibroblasts (CAFs), as the activated stroma cells, contribute to tumor progression via the release of cytokines, growth factors, and hormones. However, neither the factors produced by CAFs nor the molecular mechanisms were illuminated very well in gastric cancer (GC).

Methods: Immunohistochemical staining of alpha-smooth muscle actin (α-SMA) was applied to examine the number of CAFs in GC samples from 227 patients. ELISA and qRT-PCR were performed to detect the expression of interleukin 17a (IL-17a). The migration and invasion of GC cells were determined by the Transwell assay. The expressions of JAK2, STAT3, MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by western blotting. IL-17a was blocked with a polyclonal antibody, and JAK2/STAT3 signaling pathway was blocked by a specific inhibitor AG490.

Results: High CAFs in GC tissues were positively correlated with advanced TNM stage and perineural invasion. Furthermore, GC patients with high CAFs in tumor tissues had an obvious worse disease-free survival (DFS) and disease-special survival (DSS). Multivariate analysis showed that high CAFs in GC tissues were an independent risk factor for DFS and DSS. CAFs expressed IL-17a significantly after GC cell co-culture. CAFs markedly enhanced the migration and invasion abilities of AGS and SGC-7901 cells. Moreover, CAFs co-culture resulted in increased levels of MMP2/9, reduced expressions of TIMP1/2, and activation of the JAK2/STAT3 signaling pathway in the GC cells. IL-17a neutralizing antibody or JAK2 inhibitor AG490 can significantly inhibit the effects of CAFs on the migration, invasion, MMP2/9, TIMP1/2, and JAK2/STAT3 pathways of GC cells.

Conclusions: CAFs correlated with unfavorable clinical features and poor prognosis of GC patients. CAFs secreted IL-17a, which promoted the migration and invasion of GC cells through activating JAK2/STAT3 signaling. These results may identify IL-17a as a promising prognostic marker and therapeutic target of GC.

Keywords: Gastric cancer (GC); IL-17a; cancer-associated fibroblasts (CAFs); prognosis; tumor metastasis.

Figure 1

The detection of CAFs in GC tissues. CAFs were detected in GC tissues using immunohistochemical staining of α-SMA. (A) Representative IHC staining showed GC tissues with low CAFs; (B) representative IHC staining showed GC tissues with high CAFs. Scale bar: 50 µm. CAFs, cancer-associated fibroblasts; GC, gastric cancer.

Figure 2

The prognostic significance of CAFs in GC. GC patients with high CAFs in tumor tissues had more reduced disease-free survival (DFS) and disease-specific survival (DSS). CAFs, cancer-associated fibroblasts; GC, gastric cancer.

Figure 3

IL-17a is secreted by CAFs. (A) Immunofluorescence staining of IL-17a and α-SMA in GC tissue; (B) ELISA analysis was performed to determine the levels of IL-17a in the cell supernatant; (C) qRT-PCR was conducted to analyze the levels of IL-17a mRNA in CAFs or GC cells. *, P<0.05; **, P<0.001. CAFs, cancer-associated fibroblasts; GC, gastric cancer.

Figure 4

CAFs promote the migration and invasion of GC cells via IL-17a. (A) Cells that migrated to the bottom of the membranes were stained with 0.1% crystal violet for 30 min. The stained cells were observed and counted under a 100× microscope. CAFs promote the migration of AGS and SGC-7901 cells, which was then repressed by IL-17a neutralizing antibodies; (B) the IL-17a neutralizing antibody significantly abolished CAFs-induced GC cell invasion; (C) CAFs increased the level of MMP2/9 and decreased the expression of TIMP1/2 in GC cells, while the IL-17a neutralizing antibody blocked these effects. *, P<0.05; **, P<0.001. CAFs, cancer-associated fibroblasts; GC, gastric cancer.

Figure 5

CAFs-secreted IL-17a activates the JAK2/STAT3 pathway in GC cells. (A) CAFs enhanced the levels of p-JAK2 and p-STAT3 in AGS and SGC-7901 cells, which was then reduced by IL-17a neutralizing antibodies; (B) AG490 blocked CAFs-induced activation of the JAK2/STAT3 pathway in GC cells. *, P<0.05. CAFs, cancer-associated fibroblasts; GC, gastric cancer.

Figure 6

CAFs facilitate GC progression by activating the JAK2/STAT3 pathway. Inactivation of the JAK2/STAT3 pathway by AG490 abrogated the effects of CAFs on (A) cells that migrated to the bottom of the membranes were stained with 0.1% crystal violet for 30 min. The stained cells were observed and counted under a 100× microscope. Cell migration, (B) invasion, (C) MMP2/9, and TIMP1/2 expression in GC cells. *, P<0.05; **, P<0.001. CAFs, cancer-associated fibroblasts; GC, gastric cancer.