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. 2023 Feb 13:2023:8334881.
doi: 10.1155/2023/8334881. eCollection 2023.

Interleukin-6 and Hypoxia Synergistically Promote EMT-Mediated Invasion in Epithelial Ovarian Cancer via the IL-6/STAT3/HIF-1 α Feedback Loop

Tongshuo Zhang  1   2   3 , Jing Yang  2 , Yang Sun  4 , Jiangnan Song  2   5 , Dandan Gao  1   2 , Suhui Huang  2   6 , Aibo Pang  5 , Jianhui Zhang  7 , Junhong Wang  1 , Yue Wang  1   2 , Yanqiu Li  8
Affiliations

Interleukin-6 and Hypoxia Synergistically Promote EMT-Mediated Invasion in Epithelial Ovarian Cancer via the IL-6/STAT3/HIF-1 α Feedback Loop

Tongshuo Zhang et al. Anal Cell Pathol (Amst). .

Abstract

Extensive peritoneal spread and capacity for distant metastasis account for the majority of mortality from epithelial ovarian cancer (EOC). Accumulating evidence shows that interleukin-6 (IL-6) promotes tumor invasion and migration in EOC, although the molecular mechanisms remain to be fully elucidated. Meanwhile, the hypoxic microenvironment has been recognized to cause metastasis by triggering epithelial-mesenchymal transition (EMT) in several types of cancers. Here, we studied the synergy between IL-6 and hypoxia in inducing EMT in two EOC cell lines, A2780 cells and SKOV3 cells. Exogenous recombination of IL-6 and autocrine production of IL-6 regulated by plasmids both induced EMT phenotype in EOC cells characterized by downregulated E-cadherin as well as upregulated expression of vimentin and EMT-related transcription factors. The combined effects of IL-6 and hypoxia were more significant than those of either one treatment on EMT. Suppression of hypoxia-inducible factor-1α (HIF-1α) before IL-6 treatment inhibited the EMT phenotype and invasion ability of EOC cells, indicating that HIF-1α occupies a key position in the regulatory pathway of EMT associated with IL-6. EMT score was found positively correlated with mRNA levels of IL-6, signal transducer and activator of transcription 3 (STAT3), and HIF-1α, respectively, in 489 ovarian samples from The Cancer Genome Atlas dataset. Next, blockade of the abovementioned molecules by chemical inhibitors reversed the alteration in the protein levels of EMT markers induced by either exogenous or endogenous IL-6. These findings indicate a positive feedback loop between IL-6 and HIF-1α, and induce and maintain EMT phenotype through STAT3 signaling, which might provide a novel rationale for prognostic prediction and therapeutic targets in EOC.

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Conflict of interest statement

The author(s) declare(s) that they have no conflicts of interest.

Figures

Figure 1
Figure 1
IL-6 promotes the EMT phenotype in EOC cell lines. A2780 cells were treated with exogenous IL-6 (50 ng/ml) for 24 hours, and SKOV3 cells were treated with exogenous IL-6 (10 ng/ml) for 48 hours under normoxic conditions. The expression and localization of E-cadherin and vimentin were then detected by immunofluorescence. The nuclei were stained with DAPI (blue) as an internal reference. E-cadherin and vimentin proteins were visualized by Alexa 488 fluorescent labeling (green) and 549 fluorescent labeling (red), respectively. Scale bars: 100 μm.
Figure 2
Figure 2
Hypoxia-induced HIF-1α regulates the levels of EMT markers and invasion in EOC cell lines. (a) Relative protein levels of EMT markers in A2780 and SKOV3 after YC-1 treatment under normoxic (21% O2) or hypoxic (1% O2 or CoCl2) conditions, respectively. (b) The invasion ability of A2780 and SKOV3 treated as described for (a) was detected by Matrigel invasion assay. Scale bars: 100 μm. In each experiment, the numbers of cells that penetrated the membrane were counted in five microscopic fields per filter. The invasion cell counts are presented as the mean ± SD of three independent experiments. ∗P < 0.05 compared with the control group, △P < 0.05 compared with the 1% O2 group, and ▲P < 0.05 comparedwith the CoCl2 group by one-way ANOVA with LSD multiple comparison test. (c) Protein levels of EMT markers in transfected A2780 cells overexpressing HIF-1α and transfected SKOV3 cells with repression of HIF-1α under normoxic conditions. (d) The invasion ability of transfected A2780 cells overexpressing HIF-1α and transfected SKOV3 cells with repression of HIF-1α was detected by Matrigel invasion assay. Scale bars: 100 μm.
Figure 3
Figure 3
Exogenous and endogenous IL-6 combined with hypoxia regulates the protein expression of EMT markers in EOC cell lines. (a) A2780 and SKOV3 cells were treated with or without IL-6 as described in Figure 1 under normoxic (21% O2) or hypoxic (1% O2 or CoCl2) conditions, respectively. After treatment, the protein levels of EMT markers were analyzed by western blotting. (b) A2780 clones overexpressing IL-6 and SKOV3 clones with depletion of IL-6 were treated under normoxic (21% O2) or hypoxic (1% O2 or CoCl2) conditions as described for (a), respectively. After treatment, the protein levels of EMT markers were analyzed by western blotting. Representative images from three independent experiments are shown.
Figure 4
Figure 4
HIF-1α inhibits the induction effect of IL-6 on EMT and invasion. (a) Protein levels of EMT markers in transfected SKOV3 cells with repression of HIF-1α after treatment with exogenous IL-6 (10 ng/ml) for 48 hours under normoxic conditions. (b) The invasion ability of transfected SKOV3 cells with repression of HIF-1α was detected by Matrigel invasion assay after treatment as described for (a). Scale bars: 100 μm. In each experiment, the numbers of cells that penetrated the membrane were counted in five microscopic fields per filter. The invasion cell counts are presented as the mean ± SD of three independent experiments. ∗P < 0.05 compared with the SKOV3/scramble shRNA group and #P < 0.05 compared with the IL-6-treated SKOV3/scramble shRNA group by one-way ANOVA with LSD multiple comparison test.
Figure 5
Figure 5
The IL-6/STAT3/HIF-1α loop mediates IL-6-induction EMT in EOC. (a) Correlation between the generic EMT score and gene expression of each molecule in the signaling loop. Scatter plots showed EMT signature scores and normalized mRNA levels of 489 EOC samples based on TCGA dataset. Spearman's rank correlation analysis. (b) Blocking STAT3 and HIF-1α with corresponding inhibitors (AG490 inhibits STAT3 and YC-1 inhibits HIF-1α) reversed IL-6-induced EMT in A2780, SKOV3, and A2780 clones overexpressing IL-6. Protein levels displayed are representative of three independent experiments with similar results.
Figure 6
Figure 6
Schematic of the IL-6/STAT3/HIF-1α autocrine signaling loop that mediates the interaction between IL-6 and HIF-1α in the acquisition of EMT and invasion under hypoxic conditions. Solid lines indicate a direct action and dotted lines show an indirect action. IL-6 in the tumor microenvironment binds to its receptor and thereupon triggers the phosphorylation and nuclear localization of STAT3 by Janus kinase 2. Activated STAT3 dimers bound to specific sites in target gene promoters inducing transcription of HIF-1α. In turn, HIF-1α facilitates IL-6 production indirectly through proinflammatory mediators, such as NF-κB, COX-2, or TLR4. More IL-6 is secreted out of EOC cells, further reinforcing this loop. Among these components of the loop, STAT3 and HIF-1α have been shown to regulate the expression of EMT markers as well as EMT-related transcription factors, leading to the invasion and metastasis of EOC cells.
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