The inflammatory cytokine IL-6 induces FRA1 deacetylation promoting colorectal cancer stem-like properties

Colorectal cancer (CRC) has long been known for its tight association with chronic inflammation, thought to play a key role in tumor onset and malignant progression through the modulation of cancer stemness. However, the underlying molecular and cellular mechanisms are still largely elusive. Here we show that the IL-6/STAT3 inflammatory signaling axis induces the deacetylation of FRA1 at the Lys-116 residue located within its DNA-binding domain. The HDAC6 deacetylase underlies this key modification leading to the increase of FRA1 transcriptional activity, the subsequent transactivation of NANOG expression, and the acquisition of stem-like cellular features. As validated in a large (n = 123) CRC cohort, IL-6 secretion was invariably accompanied by increased FRA1 deacetylation at K116 and an overall increase in its protein levels, coincident with malignant progression and poor prognosis. Of note, combined treatment with the conventional cytotoxic drug 5-FU together with Tubastatin A, a HDAC6-specific inhibitor, resulted in a significant in vivo synergistic inhibitory effect on tumor growth through suppression of CRC stemness. Our results reveal a novel transcriptional and posttranslational regulatory cross-talk between inflammation and stemness signaling pathways that underlie self-renewal and maintenance of CRC stem cells and promote their malignant behavior. Combinatorial treatment aimed at the core regulatory mechanisms downstream of IL-6 may offer a novel promising approach for CRC treatment.


Sphere Formation Assay
Single cells were seeded at 1000 cells per well in 24-well ultra-low-attachment plates (3473; Corning Life Sciences, Lowell, MA, USA). Cells were grown in sphere medium consisting of DMEM/F12 (Invitrogen) supplemented with B27 serum-free supplement (1:50; Invitrogen), 20ng/ml epidermal growth factor and 10ng/ml basic fibroblast growth factor (PEPROTECH) at 37°C in 5% CO2。Supplemented medium was added to each well every two days to sustain supplement supply. After 1-2 weeks the developed spheres (>100 μm) were counted and analysed using ImageJ software, with their image captured under the light microscope.

Invasion and migration assays
The invasion assay was performed as described [3]. The migration assay was done with 24-well transwell (Corning). 4*10 5 DLD1 and HT-29 cells were suspended in 300μl serum-free RPMI 1640 and loaded onto the upper compartment whereas complete medium with or without 50ng/ml IL-6 was applied to the lower compartment. After incubation for 72h, the cells that had migrated to the lower surface of the membrane were fixed with 2% PFA and stained with crystal violet. The air-dried membrane was viewed under a microscope and four random 3 fields were selected for cell counting.

Chemo-resistance assay
Chemo-resistance of cells to 5-FU, Cisplatin and Tubastatin A were carried out using Annexin V/ PI staining assay. Cells were stained in 1x Annexin V binding buffer, FITC-conjugated Annexin V and PI solution as provided by the Annexin V/ PI Staining Kit (MultiSciences Biotech) according to manufacturer's instructions. The analysis was determined by a Cytomic flow cytometer 500 (BECKMAN COULTER).

Flow cytometry
For flow cytometry, the antibodies used included FITC-conjugated CD44 (eBioscience) and PE-conjugated CD133 (eBioscience). Rat IgG2b Iso Control FITC and Mouse IgG1 K Iso Control PE (eBioscience) were used to serve as controls. Cells were incubated with the antibodies in PBS containing 2% FBS. The samples were analyzed or sorted on the Cytomic flow cytometer 500 (BECKMAN COULTER) and BD FACS S ORP ARIA II (BD Biosciences), respectively. The data were analyzed by CXP Software and BD FACS S ORP ARIA II Software.
Sorted cells were seeded in 6-well plates with 1*10 5 cells per well. Plates were maintained at 37°C in a humidified incubator with 5% CO2 treated with or without IL-6.

Immunohistochemistry and Immunofluorescence
A total of 123 human CRC samples were collected at the Second Affiliated Hospital of Zhejiang University School of Medicine after informed consent had been given by all patients. The immunohistochemistry and the scoring for each slide were performed as described [3].
For staining human CRC tissues, the tissues were cut into frozen sections and fixed in ice-cold 4% paraformaldehyde (PFA). After dehydration in 30% sucrose, tissues were embedded with Optimum Cutting Temperature compound (O.C.T. Compound) into small silicone tubes. Frozen tissue was sectioned into 10 µm thick sections with a cryostat. Sections were blocked in PBT (0.3% Triton in 1X PBS) + 5% NGS (normal goat serum) + 0.1% NaN3 and then stained with primary antibodies. Following incubation, the sections were washed and stained with fluorochrome-conjugated secondary antibodies and counterstained with DAPI. Fluorescence signal was then visualized using OLYMPUS IX83-FV3000-OSR (Olympus). The primary antibodies used included anti-FRA1 (Santa Cruz), anti-NANOG (Abcam), anti-STAT3-pY705 (CST). The second antibodies used included Alexa Fluor 488 Goat Anti-Mouse IgG, Alexa Fluor 555 Goat Anti-Rabbit IgG and Alexa Fluor 647 Goat Anti-Chicken IgG (Life Technologies).

Quantitative real-time PCR analysis
Total RNA was isolated from cells using RNAiso Plus (TaKaRa, Kyoto, Japan). Reverse transcription was performed with the PrimeScript RT reagent Kit (TaKaRa). Quantitative real-time PCR (qRT-PCR) was achieved using the CFX96 Real-Time System (BIO-RAD).
Target gene expression was normalized to GAPDH levels in respective samples as an internal control, and the results are representative of at least three independent experiments.

Enzyme-linked immunosorbent assay (ELISA)
For the determination of IL-6 concentration in the culture medium of DLD1 and HT-29 CRC cell lines, human IL-6 ELISA kit (430501; Biolegend) was used according to manufacturer's instruction.

Cell Viability Assay
The Viability of cells was carried out using CELL-Counting Kit-8 (CCK8) (DOJINDO) according to the manufacturer's instruction.
The biotinylated DNA probes were pre-incubated with M-280 streptavidin Dynabeads (Invitrogen) in binding and washing buffer (5mM Tris-HCl pH7.5, 500nM EDTA, 1M NaCl) according to the manufacturer's instructions. The beads were then added to the nuclear extracts and shaken for 4h at 4 C. After four washes with wash buffer (25mM HEPES pH7.5, 20% glycerol, 0.4% TritonX-100, 0.5mM EDTA, 150mM NaCl), protein loading buffer was added to the precipitates, boiled for 5min, and separated on 10% SDS-PAGE. Proteins pulled down by the DNA probes were analyzed using western blot with Ku80 as a loading control.

Metastases formation in nude mice
Luciferase-labeled DLD1 cells (3*10 5 /0.2 ml) were injected into the lateral tail vein of BALB/C nude mice. In weekly intervals, anesthetized mice were injected i.p. with D-luciferin (150 mg/kg) and imaged 10 min after injection using the IVIS Illumina System (Caliper Life Sciences). The acquisition time was 2 min. Eleven weeks after tail vein injection, mice were sacrificed and examined for lung metastases using H&E staining.

Subcutaneous tumor growth in nude mice
For subcutaneous (s.c.) injections, indicated cells were resuspended with 100 μl of 1×PBS and injected into each flank of BALB/C nude mice (male, 5 weeks of age). The mice were sacrificed three weeks later. Tumor tissues were harvested and tumor weight was measured.

Homology modeling and residue mutation
The

System preparation and MD simulations
The three prepared structures were used as the initial structure for the conventional MD simulations. The topology and coordinate files of each system were generated by tleap in AMBER16. The ff14SB and OL15 force field was used [43]. Each system was solvated into a cubic TIP3P water box with 12 Å away from the surface of the complex. Then, an appropriate number of Na + ions were added to neutralize each system. The minimization, heating and Finally, 100 ns MD simulations were conducted in the NPT ensemble. The temperature was controlled by the Langevin temperature equilibration scheme with a collision frequency of 2.0 ps −1 . The particle mesh Ewald (PME) algorithm was used to handle the long-range electrostatic interactions under periodic boundary condition and a cutoff of 8 Å was used for the real-space interactions [44]. The SHAKE algorithm was used to constrain all covalent bonds involving hydrogen atoms and the time step was set to 2 fs.

Trajectory analysis and MM/GBSA calculation
Trajectory analysis was carried out with cpptraj module in Amber16. In order to monitor the stability of each system, the root mean square deviation (RMSD) of twenty residues in DNA combine region of FRA1 (Arg107 to Arg126) was calculated with their Cα atoms. (f) Western blot analysis of DLD1 cells cultured in the presence/absence of IL-6 (50 ng/ml), TCZ (5μg/ml) and siSTAT3. Protein levels of STAT3-pY705, STAT3, FRA1, and α-tubulin were examined.
(g) Sphere formation assays were performed with DLD1 cells cultured in the presence/absence of IL-6 (50 ng/ml), TCZ (5μg/ml) and siSTAT3. Results are shown as representative images and as histograms of the sphere numbers in triplicate (mean ± SD).
(h) DLD1 and HT-29 cells were cultured in the presence of IL-6 for 7 days and the CD44 + /CD133 + subpopulation was assessed by FACS.
(k) CD44 + /CD133 + and CD44 -/CD133 -DLD1 cells were sorted by FACS and cultured in the presence/absence of IL-6 for the indicated times. These cultures were monitored at the indicated time points by FACS analysis. Representative FACS plots are depicted.
The percentage of apoptotic (Annexin V + ) cells indicate that shNANOG HT-29 cells were characterized by increased sensitivity when compared to the shScramble cells. IL-6 enhances chemo-resistance in shNC DLD1 cells, an effect that is abrogated by the NANOG knockdown.