A novel small-molecule inhibitor of trefoil factor 3 (TFF3) potentiates MEK1/2 inhibition in lung adenocarcinoma

TFF3 has been identified as a novel biomarker to distinguish between lung adenocarcinoma (ADC) and lung squamous-cell carcinoma (SCC). Herein, we determined the oncogenic functions of TFF3 and demonstrated the potential of pharmacological inhibition of TFF3 in lung ADC using a novel small-molecule inhibitor of TFF3 dimerization (AMPC). Forced expression of TFF3 in lung ADC cells enhanced cell proliferation and survival, increased anchorage-independent growth, cancer stem cell behavior, growth in 3D Matrigel, and cell migration and invasion. In contrast, depleted expression of TFF3 suppressed these cellular functions. Mechanistically, TFF3 exerted its oncogenic function through upregulation of ARAF and hence enhanced downstream activation of MEK1/2 and ERK1/2. Pharmacological inhibition of TFF3 by AMPC, resulted in markedly decreased cell survival, proliferation, 3D growth and foci formation, and impaired tumor growth in a xenograft mouse model. Moreover, the combination of various MEK1/2 inhibitors with AMPC exhibited synergistic inhibitory effects on lung ADC cell growth. In conclusion, this study provides the first evidence that TFF3 is a potent promoter of lung ADC progression. Targeting TFF3 with a novel small-molecule inhibitor alone or in combination with conventional MEK1/2 inhibitors are potential strategies to improve the outcome of lung ADC.

Representative flow cytometry plots with percentages of ALDH-positive cells, corresponding to Figure   3I are shown. B. Expression levels of cancer stem cell-related genes in H1299 cells with forced expression of TFF3 or in H1299 cells treated with AMPC. Relative mRNA expression was detected by qPCR. The ratios are shown in the figure, which indicates increased or decreased gene expression in H1299-TFF3 cells relative to H1299-Vec cells, or AMPC (10µM for 24h) treated H1299 cells relative to DMSO treated H1299 cells. Data are expressed as mean ± SD. *, p < 0.05; **, p < 0.01. C. Western blot analysis of the expression of cancer stem cell-related proteins in DMSO or in H1299 cells treated with 10 µM AMPC for 24 h. β-ACTIN was used as input control. D. Western blot analysis of ARAF protein levels in cell lysates of H1299 and H1975 treated with DMSO or 10µM AMPC for 24h. β-ACTIN was used as input control. The genes involved in cell cycle control, apoptosis, adhesion and invasion, and signal transduction were examined by qPCR in H1299-Vec and H1299-TFF3 cells. The ratios indicate increased or decreased gene expression in H1299-TFF3 cells relative to H1299-Vec cells. The results are shown as mean and P value of triplicates in a representative experiment.

Total cell number counting
Cells were plated into 6-well cell culture plate at 2 ×10 4 cells/well with 10% FBS condition media or 1 ×10 5 cells/well with 2% FBS condition media. Plates were settled in 37°C in incubator with 5% CO2 and culture media was changed every two days. Cells were counted every 1-2 days during culture until the sixth day or eighth day. On indicated days, cells in each well were trypsinised with 0.5% trypsin and harvested by centrifuging at 200 × g for 5 minutes. Collected cells were resuspended and counted by haemocytometer.

Cell viability assay
Cells were plated into 96-well cell culture plate at 2×10 3 cells/well and cultured at 37°C in incubator.
During the drug treatments, drugs were dissolved in DMSO and added in the 2% FBS condition media while the control groups were added same amount of DMSO. AlamarBlue™ Cell Viability Reagent (Invitrogen) was using to measure cell viability. After the drug treatments for 48h or 72h, culture media was replaced with media containing 10% AlamarBlue reagent. Plates were incubated 4-6 hours and proceeded for plate reading using a fluorescence/ luminesence microplate reader (Tecan). Increased fluorescence was detected by fluorescence using an excitation between 530-560 and an emission at 590 nm.

Cell cycle analysis
Cultured cells after treatment were trypsinized and suspended in 10% FBS medium. After centrifuged for 5 mins, cells pellet was resuspended in 0.5 ml phosphate-bufferedsaline (PBS). Cells were fix by adding 2 ml cold 80% EtOH into cell suspension with vortexing the suspension to prevent clustering of cells during the fixation. Incubation could be 10-15 mins on ice or more at -20°C. Fixed cells were centrifuged and washed with 2 ml PBS. Finally, cell pellet was suspended in 500 µL staining solution (50 µg/ml RNaseA and 0.1% Triton X-100 in PBS) and incubated for 30-40 min at room temperature.
Propidium Iodide (PI) (10 µg/mL, from the stock) was added directly to the cell solution and vortex gently. Samples were incubated at least 5 min at room temperature before fluorescence-activated cell sorting (FACS) analysis. Results were analyzed by Modfit software and population of G1, S, and G2 phase was compared between samples.

Cell apoptosis measurement
Apoptotic cell death was determined using Annexin-V AlexaFluor® 488 Propidium Iodide (PI) Dead Cell Apoptosis Kit (Life Technologies) following the recommended protocols. Cells were seeded in 6well plates at the density of 2×10 5 cells/well and incubated 8 hours for cells to attach to the plates.
Apoptosis was induced by serum-free starvation or drug treatment for 24 hours or 48 hours. Cells were harvested, washed with PBS, and suspended in 100 µL 1X Annexin-binding buffer. After that, 100 µg/mL working solution of PI and 5 µL of FITC Annexin V was added to each sample and incubated for 15 mins. For single stain control samples, only PI or Annexin V was added. Another 400 µL 1X Annexin-binding buffer was added into samples after the incubation. Samples were kept on ice and analyzed by flowcytometry as soon as possible. The population was separatde into three groups: live cells with low level of fluorescence, early apoptotic cells with green fluorescence, and late apoptotic cells with both red and green fluorescence.
Apoptotic cell death was also determined using Caspase-Glo caspase 3/7 kit (Promega Madison, WI) following the recommended protocols. Cells were plated at a density of 1 × 10 4 cells/ well in whitewalled 96-well plates. After serum-free starvation or drug treatment for inducing apoptosis, prepared Caspase-Glo 3/7 reagent was added into per well at the ratio of 1:1 with the culture media. Reagent and culture media were mixed at 300-500 rpm using a plate shaker for 30 seconds and followed by a 1-hour incubation at room temperature in dark. Finally, luminescence of each well was measured with the fluorescence/ luminesence microplate reader (Tecan, Zurich, Switzerland).

Anchorage-independent growth
Assays including colony formation and foci formation were performed to investigate anchorageindependent growth of ADC cells. For colony formation, each well of a 24 well plate was covered with a base agar layer of 0.5% agarose in serum free medium. Cells re-suspended in 0.35% agarose and culture medium was added to each well on the top of the base agarose. Normal culture media was added on the top of each well and refreshed every three days. After two weeks of culture, pictures of cell colonies were taken under microscope and colony numbers for each well were counted at 40 × magnification (Olympus, Tokyo, Japan).
For foci formation, 1×10 3 cells were seeded in 6-well plates and cultured for two weeks. Cold methanol was used for fixing the cell. After leaving the plates open to dry for two hours, 1% crystal violet in 20% EtOH was added into the well. Cell foci were stained in purple color with crystal violet. Pictures were taken for each well.

Three-dimensional culture assay
Cells were grown in three-dimensional culture on a layer of growth factor-reduced Matrigel (BD Biosciences) in 96 well plate. First, 40µL Matrigel was added to coat the surface of cell culture plate.
After that, 1×10 3 cells were cultured in 4% Matrigel/medium solution upon the Matrigel base for 9 days.
After 9 days, pictures of cells colonies were taken under microscope and stained with AlamarBlue to examine the cell viability. 10 µL of AlamarBlue was added to each well, and left for 4 hours in the CO2 incubator before spectrofluorometric reading were taken at 560nm/590nm. For the drug treatment experiments, cells were growth in 4% Matrigel for three days without drugs. At the fourth day, half of the 4% Matrigel/medium solution in the plates was removed with the same amount of solution with drugs or DMSO. Later, the medium solution was refreshed every two days until the tenth day.

Migration and invasion assay
In vitro cell migration and invasion assays were performed using 24-well cell invasion chamber (8 µm pore size) following the provided protocol (BD Biosciences). For invasion assessment, inserts were coated with 10% Matrigel before seeding the cells. Cells (2× 10 4 cells) were seeded with serum free medium in the upper chamber and complete medium were added in the bottom well. After 12h incubation for migration and 24h incubation for invasion, cells on the lower surface of the inserts were washed with PBS and fixed with 4% paraformaldehyde. After removing the non-migrating or noninvading cells on the upper surface of the inserts with cotton tipped swab, the cells on lower surface of 12 inserts were stained with Hoechst 33342 (Sigma). The migrated and invaded cells were observed and counted using fluorescence microscope.
For the wound healing assay, cells were cultured in a 6-well plate until 100% confluence. Three perpendicular equal straight artificial wounds were drawn using a pipette tip. Cells were maintained with 10%FBS culture medium and pictures were taken at 0, 12, and 24h after drawing the wounds.

Histological analysis
Morphologic analyses were carried out on 4% paraformaldehyde-fixed for 24 to 48 hours before being processed and embedded into paraffin wax. Deparaffinized, rehydrated sections were immunolabeled with anti-Ki67 antibody (Abcam ab-15580) and anti-Trefoil factor3 (TFF3) antibody (Abcam ab-108599) and stained with hematoxylin (Goodbio technology, Wuhan, China). The antigen antibody was visualized using 3,3-diaminobenzidine. Sections were then dehydrated, cleared with xylene, and mounted with resinous mounting medium. In every tumor section, the percentage of Ki67 positive cells in three different regions was counted. The percentage of positive TFF3 staining area was conducted with Image J software, and calculated by the following formula: The percentage of TFF3 = TFF3 staining positive area÷Region area×100%

TUNEL Staining Assay
Cell apoptosis was detected through Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL) assay using a TUNEL Apoptosis Detection Kit (Alexa Fluor 488) (YeSen Biotechnology, Shanghai, China) as per manufacturer's instruction. The relative fluorescence intensity was calculated as the ratio of the mean fluorescence intensity of the TUNEL staining to that of DAPI staining. Two fields per section of four independent sections in each group were evaluated.