Hypermethylated in cancer 1(HIC1) suppresses non-small cell lung cancer progression by targeting interleukin-6/Stat3 pathway

Abstract

Non-small cell lung cancer (NSCLC), which accounts for more than 80% of lung cancers, is a leading cause of cancer mortality worldwide. However, the mechanism underlying its progression remains unclear. Here we found that HIC1 promoter was heavily methylated in NSCLC cell lines and tissues contributing to its low expression compared to normal controls. Restoring HIC1 expression inhibited migration, invasion and promoted inducible apoptosis of NSCLC cells. Notably, HIC1 is a tumor suppressor through inhibiting the transcription of IL-6 by sequence-specific binding on its promoter. Restoring IL-6 expression could partially rescue these phenotypes induced by HIC1 in vitro and in vivo. Mechanistic analyses show that autocrine secretion of IL-6 induced by loss of HIC1 activated STAT3 through IL-6/JAK pathway and was associated with NSCLC progression. The HIC1/IL-6 axis may serve as a prognostic biomarker and provide an attractive therapeutic target for NSCLC.

Keywords: HIC1/IL-6 axis; NSCLC; hypermethylation.

Figure 1. Methylation of HIC1 promoter impairs its expression in NSCLC

A. Genomic DNAs from NSCLC cell lines were treated with sodium bisulfate, the PCR products amplificated with HIC1 MSP primers were confirmed by agarose gel electrophoresis. M: methylation; U: unmethylation. B. and C. Genomic DNAs from NSCLC cell lines and tissues were treated with sodium bisulfate, PCR products amplificated with HIC1 BSP primers were sequenced and the percentage of methylation was calculated. D. and E. Quantitative real-time RCR analysis of HIC1 gene in NSCLC cell lines and tissues.

Figure 2. HIC1 inhibits invasion, migration and promotes apoptosis of NSCLC cells

A. and B. The invasive properties of A549 and H292 cells expressing control vector or HIC1 were detected through extracellular matrices in porous culture chambers, the invasive cells were stained by crystal violet and then photographed by fluorescence inversion microscope system. C. and D. The migration abilities of A549 and H292 expressing control vector or HIC1 were detected by scratch wound healing assay. E. and F. A549 and H292 cells expressing control vector or HIC1 were treated with 0.1 μmol/L of staurosporine for 12 h, and apoptosis was performed by tunel assay using the In Situ Cell Death Detection Kit, TMR red (Roche). G. HIC1 knockdown by shHIC1-1 and shHIC1-2 in A549 cells promoted migration, H. inhibited apoptosis. The experiments were performed 3 times; representative images were shown (× 100). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. IL-6 is a direct target gene of HIC1

A. The map of IL-6 gene promoter. HIRE, HIC1 responsive element. B. The truncated IL-6 promoter was constructed to the pGL3-basic vector. The length of different promoter constructs used in reporter assays was shown. C. IL-6 promoter activity was detected in A549 and 293T cells co-transfected with full length construct (−996/+100) and PC3.1 or HIC1 expression vectors by luciferase reporter assay. Basic, control for promoter construct; PC3.1, control for HIC1 expression vector. D. The activities of IL-6 promoter were assayed in A549 and 293T cells co-transfected with truncated IL-6 promoter constructs and PC3.1 or HIC1 expression vector. NS, not significant. E. Nucleotide sequence of −210/+100 construct showed two potential HIC1 binding sites (TGCC). TGCC in the two sites was replaced by CATT and marked as M1 and M2. The arrows indicate the primers which are used to amplify the IL-6 promoter fragment in the ChIP experiment presented in Figure 3G. F. Luciferase reporter assays showed that ΔM1 and ΔM2 mutated construct significantly decreased the repression ability of HIC1. G. ChIP analysis of HIC1 binding to the IL-6 promoter region in A549 and H292 cells. Three independent experiments were performed. Data are represented as mean ± SD. **p < 0.01, ***p < 0.001.

Figure 4. HIC1 inactivates the activity of STAT3 through targeting IL-6/JAK pathway

A549 cells with HIC1 knockdown were treated with multiple signaling pathways inhibitors for 6 hours. The activities of these pathways were detected by Western blot, A. 5μM TKI reduced the levels of p-EGFR^Y1086, but had no effect on p-STAT3^Y705. B. 1μM Saracatinib reduced the levels of p-SRC^Y416, but had no effect on p-STAT3^Y705. C. and D. 1 nM Ruxolitinib and 2 ng/ml IL-6 Neu Ab reduced the levels of both p-JAK2^Y1007/1008 and p-STAT3^Y705. E. and F. Quantitative real-time PCR assayed the expression of IL-6 family factors such as IL-11, CNTF, LIF, IL-6 and the receptors IL-6R and gp130 in A549 ^HIC1 and H292^HIC1 cells and their controls. Three independent experiments were performed. Data are represented as mean ± SD.*p < 0.05.

Figure 5. IL-6 partially rescues HIC1-induced phenotypes of NSCLC cells

A. and B. Exogenous IL-6(40 ng/ml) stimulation in A549 cells could partially rescued the reducing invasion and migration caused by HIC1 re-expression. C. ELISAs determined the expression of IL-6 in A549^GFP, A549^HIC1and A549^HIC1+IL-6 cells. D. Re-expression of IL-6 in A549^HIC1 cells partially rescued HIC1-induced enhancement of apoptosis. Representative images were shown (× 100). E. p-STAT3^Y705, MMP2, Bcl-2 and Survivin levels were detected in A549^GFP, A549^HIC1 and A549^HIC1+IL-6 cells by Western blot. Three independent experiments were performed. Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Figure 6. Effect of restoring HIC1 expression on tumor metastasis in vivo

A. ELISA assays detected the level of IL-6 secretion in luciferase-tagged A549^GFP, A549^HIC1 and A549^HIC1+IL-6 cells. B. Restoring HIC1 expression in A549 cells significantly reduced lung metastasis compared with the control group (p < 0.05) in transplantable nude balb/c mouse models, but re-expression of IL-6 in A549^HIC1 cells(noted as A549^HIC1+IL-6) partially rescued HIC1-induced reduction of metastasis compared with the A549^HIC1 cells (p < 0.05). On the left, representative images of BLI animals at the eighth week were shown. The up right statistical graph showed the fluorescence signal intensity collected from the metastasis loci of each mouse. Each of group n = 6, the p value was calculated by Mann Whitney U test. C. HE staining of tumor tissue and immunohistochemical evaluation of p-STAT3^Y705, MMP2 and Bcl-2 in lung micrometastases grown in nude balb/c mice. Representative microscopic images of HE staining (×50, scale bar, 100μm) and tumor tissues stained with an anti-human p-STAT3^Y705 antibody (1:50), MMP2 antibody (1:100) and Bcl-2 antibody (1:100) (× 200, scale bar, 50μm) were shown.

Figure 7. Schematic model for the function of HIC1/IL-6 axis in NSCLC progression

HIC1 expression is impaired in NSCLC due to the hypermethylation modification, leading to the higher secretion of IL-6. Autocrine of IL-6 can activate JAK/STAT3 pathway and the expression of downstream targets, such as MMP2, Bcl-2 and Survivin, therefore promoting NSCLC progression.