The human colorectal cancer cell lines HCT-116, HCT-8 and HT-29 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were maintained in RPMI-1640 medium (Corning Incorporated, Corning, NY, USA), supplemented with 10% fetal bovine serum (FBS; RMBIO, Missoula, MT, USA), antibiotics and glutamine at 37°C in 5% CO₂. LPS and baicalein were obtained from Sigma-Aldrich (St. Louis, MO, USA). SB216763 was purchased from Selleckchem (Houston, TX, USA).

Cells were washed in phosphate-buffered saline (PBS) and lysed in NP-40 buffer (Elpis Biotech, Inc., Daejeon, Korea) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein phosphorylation states were preserved through the addition of phosphatase inhibitors (Cocktail II; Sigma-Aldrich) to the NP-40 buffer. Protein concentrations were quantified using a BCA assay kit (Pierce, Rockford, IL, USA). Proteins (10 μg/sample) were separated by 10–15% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% skim milk prior to western blot analysis. Chemiluminescence was detected using an ECL kit (Advansta, Inc., Menlo Park, CA, USA) and the multiple Gel DOC system (Fujifilm, Tokyo, Japan). Primary antibodies against the following proteins were used: E-cadherin, N-cadherin, Snail, vimentin, TCF8/Zeb1, β-actin, MyD88, LKB1, SirT1, AceCS1, ACSL1, FASN, phospho-ACLY (Ser⁴⁵⁵), ACLY, phospho-ACC (Ser⁷⁹), ACC, phospho-AMPK (Thr¹⁷²), AMPK, phospho-GSK3β (Ser⁹), GSK3β, phospho-Lyn (Tyr⁵⁰⁷), Lyn, phospho-PI3K (Tyr⁴²⁸), PI3K, phospho-Akt (Ser⁴⁷³), Akt, phospho-Src (Tyr⁴¹⁶) and Src (Cell Signaling Technology, Beverly, MA, USA); TAp63 (BioLegend, Inc., San Diego, CA); α-SMA (Bioss, Inc., Woburn, MA, USA); FLAG (Sigma-Aldrich); CD74, CD44, phospho-PTEN (Ser³⁸⁰/Thr^382/383), and PTEN (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Experimentally verified human CD44-small interfering RNA (siRNA) duplex, CD74-siRNA duplex, and negative control-siRNA were obtained from Bioneer (Daejeon, Korea). Cells were seeded at a concentration of 1×10⁵/well in a 6-well plate and grown overnight prior to transfection with 200 nM siRNA using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were used for further experiments 48 h after transfection.

Transient transfection of cultured cells was performed using Lipofectamine 2000 according to the manufacturer's instructions. Cells were plated at a density of 2×10⁵/well in a 6-well culture plate and transfected the next day. Typically, 10 ng of construct DNA was transfected with 9 μl Lipofectamine. Cultured cells were transiently transfected with either a TAp63 expression vector of TAp63 cDNA cloned into pcDNA3.1 (Addgene, Cambridge, MA, USA) or empty vector pcDNA3.1 (Invitrogen). Cells were transfected for 48 h and analyzed by western blotting.

PGE2 and MIF concentrations in the culture supernatants were quantified using a single cytokine ELISA kit from Abcam (Cambridge, UK). VEGF and IL-8 were quantified by a single cytokine ELISA kit from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's protocol. The data are expressed as the average of the number of biological replicates ± standard deviation (SD).

Cell invasion was determined using the CultreCoat 96-well Medium BME Cell Invasion Assay kit (R&D Systems) according to the manufacturer's protocol. Cells (2.5×10⁴) in serum-free RPMI-1640 containing 0.1% FBS were seeded into the upper chamber and the lower compartment was filled with RPMI-1640 containing 10% FBS as a chemoattractant. After incubation for 24 h, non-invading cells on the upper membrane surface were removed by wiping with a cotton swab. Invaded cells were stained with calcein-AM and quantified using a microplate reader.

For assessment of free fatty acids, colorectal cancer cells were harvested, homogenized with chloroform/Triton X-100 solution (1% Triton X-100 in pure chloroform), and centrifuged at top speed to collect the organic phase (lower phase). The supernatant was vacuum dried to remove the chloroform and the dried lipid samples were dissolved in fatty acid assay buffer. Concentrations of free fatty acids were determined using the Free Fatty Acid Quantification kit (Abcam) according to the manufacturer's instructions.

Data were expressed as the mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of the variance. A P-value <0.05 was considered statistically significant.

First, we investigated whether TLR4 stimulation affects the expression of TAp63-related signal molecules and the expression of lipogenic enzymes in CRC cells. LPS-treated colon cancer cells (HCT-116, HCT-8 and HT-29) showed activation of the PI3K/AKT signaling pathway, whereas phosphorylation of AMPK, LKB1 and SirT1, key metabolic regulators, was significantly reduced (Fig. 1A). Although the expression of TAp63 was downregulated, upregulation of CD74 and CD44 was observed in LPS-activated HCT-116, HCT-8 and HT-29 cells (Fig. 1A). Enzymes associated with lipogenesis in CRC were activated after engagement of LPS with TLR4. Cytoplasmic acetyl-CoA synthetase (AceCS1), ATP-citrate lyase (ACLY), phospho-ACLY (p-ACLY), long-chain acyl-CoA synthetase (ACSL1), ACC, phospho-ACC (p-ACC), and fatty acid synthase (FASN) were all upregulated. In addition, LPS activation induced phosphorylation of GSK-3β in colon cancer cells (Fig. 1B). Our results suggest that the TLR4-mediated signaling pathway is one of the key regulators of metastasis-related mechanisms in colon cancer cells.

To confirm the correlation of GSK-3β with lipogenic enzyme activity and invasion of LPS-activated colon cancer cells, the cells were pretreated with the GSK-3β inhibitor SB216763. Treatment with SB216763 blocked phosphorylation of GSK-3β and attenuated activation of lipogenic enzymes in LPS-treated colon cancer cells (Fig. 2A). The levels of intracellular long chain fatty acid increased >3-fold compared to non-treated control cells after stimulation with LPS. In addition, pretreatment with SB216763 significantly blocked fatty acid synthesis in LPS-activated colon cancer cells (Fig. 2B). Prostaglandin E₂ (PGE2) increases the phosphorylation of GSK-3β and consequently stimulates β-catenin/T cell factor (TCF)-mediated transcription for growth and invasion in colon cancer (29). We observed that stimulation with LPS increased the secretion of PGE2 in colon cancer cells; however, inhibition of GSK-3β reduced the LPS-activated PGE2 production (Fig. 2C). Treatment with SB216763 effectively blocked the expression of CD74 and CD44 in LPS-treated colon cancer cells (Fig. 2D). SB216763 also reduced secretion of MIF in LPS-treated colon cancer cells (Fig. 2E). In addition, suppression of GSK-3β recovered the expression of TAp63 in LPS-stimulated colon cancer cells (Fig. 2D). Our results suggest that GSK-3β not only plays a critical role in lipogenesis and cancer metabolism, but also regulates the level of TAp63 in LPS-stimulated colon cancer cells.

Next, we investigated the role and connection of TAp63 in GSK-3β-mediated lipogenesis and EMT processes in CRCs after LPS stimulation. After transfection with the TAp63-expressing plasmid, the levels of TAp63-related proteins did not significantly change compared to control cells or cells transfected with a control vector (Fig. 3A). TAp63 overexpression in LPS-activated HCT-116 cells not only blocked the attenuation of phosphorylated AMPK, LKB1 and SirT1, but also prevented the upregulation of phosphorylated GSK-3β and activation of lipogenic enzymes (Fig. 3B and C). Although, the upregulated CD74 and CD44 expression following LPS stimulation was maintained (Fig. 3B), the downregulation of lipogenic enzymes was accompanied by decreased intracellular fatty acid synthesis (Fig. 3D). Inhibition of GSK-3β by TAp63 transfection also reduced the production of PGE2 (Fig. 3D). TAp63-expressing HCT-116 cells activated by LPS showed concomitant reduction of the mesenchymal markers N-cadherin, snail, vimentin, α-SMA and TCF8 and decreased migratory activity (Fig. 3E and F). Our results suggest that TAp63 expression regulates GSK-3β-related intracellular lipogenesis and influences mesenchymal characteristics and invasion activity in LPS-activated CRCs.

We found that treatment with SB216763 recovered the expression of TAp63 in LPS-activated colon cancer cells, whereas overexpression of TAp63 also reduced the levels of phosphorylated GSK-3β. We estimated that another molecule plays an important role in regulating TAp63 expression and GSK-3β-mediated lipogenesis. From previous results, targeted inhibition of GSK-3β using SB216763 suppressed the expression of CD74 and CD44, but reversed the level of TAp63. Moreover, TAp63 overexpression and reduction of lipogenesis had no effect on the expression of CD74 and CD44 in LPS-treated colon cancer cells. Based on these results, we next evaluated whether LPS-induced expression of CD74 and CD44 affects the expression of TAp63 and phosphorylated GSK-3β, the activation of lipogenic enzymes, fatty acid synthesis and MIF secretion. Although TLR4-stimulation of CD44 or CD74-knockdown HCT-116 cells induced PI3K/AKT activation, gene silencing of CD44 or CD74 with siRNA blocked the downregulation of TAp63, phosphorylated AMPK, LKB1 and SirT1 in LPS-treated HCT-116 cells (Fig. 4A and B). Expression of FASN and the production of long chain free fatty acids in LPS-activated HCT-116 cells were decreased after knockdown of CD44 or CD74 with siRNA (Fig. 4A–C). Secretion of PGE2 and MIF was also attenuated in CD44 or CD74 knockdown HCT-116 cells after stimulation with LPS (Fig. 4D and E). Expression of mesenchymal markers and invasion activity were significantly suppressed when induction of CD44 or CD74 expression was inhibited in LPS-stimulated HCT-116 cells (Fig. 5A and B). The production of EMT-related cytokines, including VEGF and IL-8, after LPS stimulation was also suppressed in HCT-116 cells transfected with CD44 or CD74 siRNA (Fig. 5C). Our results suggest that the CD74/CD44 expression of TLR4-stimulated colon cancer cells plays a critical role in the suppression of TAp63 as well as lipogenesis-related EMT processes.

Baicalein, a natural plant extract, suppresses EMT of breast epithelial cells and the tumorigenic activity of breast cancer cells (30). We previously reported that treatment with baicalein upregulates TAp63 expression, resulting in apoptosis of B-cell lymphoma cell lines (31). Treatment with baicalein also decreased the lipid level in the livers of mice fed a high-fat diet through PI3K/AKT-mediated AMPK activation (32). Based on these results, we investigated whether baicalein prevents the lipogenesis-related EMT processes through the regulation of TAp63-dependent GSK-3β expression. Pre-exposure of LPS-activated HCT-116 cells to baicalein showed reduced phosphorylation of Syk/Src tyrosine kinase, resulting in blockage of PI3K/AKT activation (Fig. 6A). Treatment with baicalein not only prevented the downregulation of TAp63 and phosphorylated AMPK, LKB1, and SirT1, but also suppressed the expression of CD44, CD74, and phosphorylated GSK-3β in LPS-treated HCT-116 cells (Fig. 6B). The production of long chain fatty acid and PGE2 in TLR4-activated HCT-116 cells was efficiently blocked after treatment with baicalein (Fig. 6C and D). Expression of mesenchymal markers (Fig. 6E), production of MIF (Fig. 6F), and migratory activity (Fig. 6G) of LPS-stimulated HCT-116 cells were also inhibited by baicalein. Our results suggest that baicalein controls the EMT processes of LPS-stimulated CRCs through the regulation of TAp63/GSK-3β-mediated lipogenesis.