Hepatocellular carcinoma: thyroid hormone promotes tumorigenicity through inducing cancer stem-like cell self-renewal

Cancer stem-like cells (CSCs) play a key role in maintaining the aggressiveness of hepatocellular carcinoma (HCC), but the cell-biological regulation of CSCs is unclear. In the study, we report that thyroid hormone (TH) promotes cell self-renewal in HCC cells. TH also increases the percentage of CD90 + HCC cells and promotes drug resistance of HCC cells. By analyzing primary human HCC samples, we found that TRα transcript level is significantly elevated in primary liver cancer and portal vein metastatic tumor, compared to that of adjacent normal liver tissue. Knocking down TRα not only inhibits HCC self-renewal in vitro but also suppresses HCC tumor growth in vivo. Interestingly, treatment of TH leads to activation of NF-κB, which is required for the function of TH on inducing HCC cell self-renewal. We also found TRα and p65 cooperatively drive the expression of BMI1 by co-binding to the promoter region of BMI1 gene. In summary, our study uncovers a novel function of TH signaling in regulating the CSCs of HCC, and these findings might be useful for developing novel therapies by targeting TH function in HCC cells.


Extraction of nuclear protein
Nuclear protein was extracted and purified using the NE-PER kit from ThermoFisher (#78833), following the product's manual. The only modification was to extent the nuclear lysis step from forty minutes to one hour and a half on ice, with 10 times of 10-second vortex.

Immunoprecipitation
Immunoprecipitation was conducted as previously described 14 . Briefly, whole cell lysates were prepared from 5 x 10 7 CSQT-2 cells treated by T4 for 96 hrs using RIPA lysis buffer with protease inhibitor cocktail. Nuclear fraction of protein from CSQT-2 Cell lysates was harvested. The lysates were pre-cleared by incubating with protein A-Sepharose for 1 h at 4°C and centrifugation. The supernatant was immunoprecipitated with 1 μg rabbit IgG or anti-p65 antibody overnight at 4°C. Immune complexes were collected by incubation with protein A-Sepharose for 4 hrs at 4°C and washed for 5 times at 4°C with lysis buffer. The immune complexes adsorbed to the beads were centrifuged and the supernatant was removed. 50 μL of 1x loading buffer was added to the samples and boiled at 95°C for 5 minutes. Proteins were resolved by SDS-PAGE and immunoblotted by antibodies indicated in figures.

Western-blot
Cultured cells were washed twice with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer for 15 min on ice. Cell lysates were clarified by centrifugation at 10 000 g for 15 min, and protein concentration was determined by the Bradford Reagent. Lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins were then transferred to Immobilon membrane (Millipore, Bedford, MA, USA) and immunoblotted with indicated antibodies. All immunoblots were visualized by enhanced chemiluminescence. (Pierce, Rockford,

IL, USA)
Quantitative Real-Time RT-PCR Total RNA from cells was purified with TRIzol (Invitrogen). cDNA was prepared from 1 µg RNA.
Reaction mixtures (15 µl) contained 2.0 µl of cDNA, 7.5 µl of SYBR green master mix (Applied Biosystems) and appropriate primers. Product was monitored by SYBR green fluorescence. Control reactions lacking RT yielded little to no signal. Relative expression levels were determined from a delta-delta CT method and were normalized to 18S rRNA expression. Primer sequences are available upon request.
In vitro colony formation assay 1000 liver cancer cells were seeded in 3.5 cm dish with 2 ml of culture media, and cultured for up to 5 days. Cell-colony forming was measured by crystal violet staining at day 5 15 . The data was analyzed by the ImageJ software.
Chromatin immunoprecipitation 5x107 CSQT2 cells treated with or without T4 were used per IP. Protein-DNA complexes were crosslinked by incubating the cells with Formaldehyde at 1% final concentration. After sonication (12 × 5 sec), a small fraction of the chromatin was uncross-linked by heating the mixture to 67°C for 4 h, and the average size of the DNA fragments (300-400 bp) was determined by gel electrophoresis to verify effective sonication. The rest of the cross-linked chromatin was immunoprecipitated using anti-p65 and anti-TRα specific antibodies overnight at 4°C. The crosslinked protein was next uncoupled from DNA by heating. DNA was purified using Qiaquick gel extraction columns (Qiagen) and resuspended in 50 μL of H2O. Two microliters of immunoprecipitated sample or 2 μL of diluted input DNA were amplified in a 20-μL reaction volume (final) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 200 μM each dNTP, 0.8 U of HotStart Taq DNA polymerase (Takara), 100 nM FITC, 1 U of SYBR Green, and the sequence-specific primer pair (5 pmol) with temperature cycles of denaturation for 10 sec at 95°C, annealing for 10 sec at 58°C, and extension for 30 sec at 72°C. The primer sequences used for this assay were hBMI1-F 5' -GAGGTAAGCGCCGAACCAAGG -3'; hBMI1-R 5'-GACACTCGCATCCTGGTAACTGG-3'