A tumor suppressor enhancing module orchestrated by GATA4 denotes a therapeutic opportunity for GATA4 deficient HCC patients

Rationale: Effective targeting therapies are limited in Hepatocellular carcinoma (HCC) clinic. Characterization of tumor suppressor genes (TSGs) and elucidation their signaling cascades could shed light on new strategies for developing targeting therapies for HCC. Methods: We checked genome-wide DNA copy number variation (CNV) of HCC samples, focusing on deleted genes for TSG candidates. Clinical data, in vitro and in vivo data were collected to validate the tumor suppressor functions. Results: Focal deletion of GATA4 gene locus was the most prominent feature across all liver cancer samples. Ectopic expression of GATA4 resulted in senescence of HCC cell lines. Mechanistically, GATA4 exerted tumor suppressive role by orchestrating the assembly of a tumor suppressor enhancing module: GATA4 directly bound and potently inhibited the mRNA transcription activity of β-catenin; meanwhile, β-catenin was recruited by GATA4 to promoter regions and facilitated transcription of GATA4 target genes, which were TSGs per se. Expression of GATA4 was effective to shrink GATA4-deficient HCC tumors in vivo. We also showed that β-catenin inhibitor was capable of shrinking GATA4-deficient tumors. Conclusions: Our study unveiled a previously unnoticed tumor suppressor enhancing module assembled by ectopically expressed GATA4 in HCC cells and denoted a therapeutic opportunity for GATA4 deficient HCC patients. Our study also presented an interesting case that an oncogenic transcription factor conditionally functioned as a tumor suppressor when recruited by a TSG transcription factor.

Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote SD. *P < 0.05; **P < 0.01; ***P < 0.001    Right: statistics of the positive percentage of senescence cells.

Silver staining and Mass spectrometry assays
Silver staining was performed as Silver staining Rapid silver staining kit's description.
Cutting the target strip or dividing the strip into several components (strip width is about 2mm) and placed them in 500 l EP tube. Then experiments were performed according to the protocol of In-gel digestion as described in In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nature Protocols volume 1, pages 2856-2860 (2006), product of In-gel digestion was taken into the Mass spectrometry assays. Mass spectrometry assays was performed on ABI 5600 Triple TOF.

Transfection and Top-Flash Assay
HEK293 cells were transfected by lipofectamine 3,000. Empty control plasmid was added to ensure that each transfection receives the same amount of total DNA. To normalize for transfection efficiency, 0.1 g of pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. After 24 hours transfection, ICG001 (1 µg/mL) and DOX (1 µg/ml) were added for 48 hours. Luciferase assays were performed using a dual-specific luciferase assay kit (PROMEGA).

Chromatin immunoprecipitation (ChIP) PCR
HepG2i cells were seeded in eight 150 mm plates, when the cells reached a confluence of 30%~40%, cells received DOX treatment. After 3~4 days of culture, the cells were crosslinked with 1% formaldehyde (final concentration) and sonicated using the following parameters: 5s on, 10s off, 15 cycles at the 25% set power (VCX500, SONICS, CT, USA). Immunoprecipitation was performed with 5 μg of GATA4 antibody or 5 μg of β-catenin antibody, and the immune complexes were absorbed with protein A/G beads. Finally, the eluted DNA was resolved in ddH2O and processed for further PCR.

Fluorescent confocal microscopy
HepG2 cells were transfected with the indicated plasmids by lipofectamine 3,000. At 24 hours after transfection, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were observed with a Zeiss LSM700 confocal microscope under a ×60 oil objective.

GST pull down
Transfect plasmid PEGX-catenin and PEGX-GATA4 into BL21 E.coli and GST pull down assay performed using GST Pull-down standard protocol of Cold Spring Harbor Laboratory Press.
Incubate cells at 37 °C overnight. Add thymidine to a final concentration of 2 mM.
Culture cells in a tissue culture incubator at 37 °C for 18 hours. Remove thymidine by washing cells through addition of 10 ml pre-warmed 1x PBS and discard PBS. Add 10 ml of pre-warmed fresh medium and incubate for 9 hours in a tissue culture incubator at 37 °C. Add second round of thymidine to a final concentration of 2 mM. Culture cells at the tissue culture incubator for another 18 hours at 37 °C. Release cells by washing with pre-warmed 1× PBS and incubating cells in pre-warmed fresh media.
Cells are collected at 12hours for analysis.

Histopathological findings
Xenograft tumor and liver tissue samples were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Samples were subsequently sectioned at 5 µm thickness and stained with hematoxylin and eosin (H&E) for histopathology.

IHC Staning
IHC staning utilized IHC Protocal-1 of Bond max (Leica) and Hot antigen repair applied HIER 25 min with ER2 Protocal of Bond max (Leica).

Statistical analysis
The data are presented as means ± SD of three independent determinations. Statistical analyses were carried out by Student's t-test. p<0.05 and p<0.01 were considered to be significant.