GATA4 downregulation enhances CCL20-mediated immunosuppression in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is a deadly cancer with a high global mortality rate, and the downregulation of GATA binding protein 4 (GATA4) has been implicated in HCC progression. In this study, we investigated the role of GATA4 in shaping the immune landscape of HCC.

Methods: HCC tumor samples were classified into "low" or "normal/high" based on GATA4 RNA expression relative to adjacent non-tumor liver tissues. The immune landscapes of GATA4-low and GATA4-normal/high tumors were analyzed using cytometry by time-of-flight, bulk/spatial transcriptomic analyses and validated by multiplex immunofluorescence.

Results: GATA4-low tumors displayed enrichment in exhausted programmed cell death protein 1+ T cells, immunosuppressive regulatory T cells, myeloid-derived suppressor cells, and macrophages, highlighting the impact of GATA4 downregulation on immunosuppression. Spatial and bulk transcriptomic analyses revealed a negative correlation between GATA4 and C-C Motif Chemokine Ligand 20 (CCL20) expression in HCC. Overexpressing GATA4 confirmed CCL20 as a downstream target, contributing to an immunosuppressive tumor microenvironment, as evidenced by increased regulatory T cells and myeloid-derived suppressor cells in CCL20-high tumors. Lastly, the reduced expression of GATA4 and higher expression of CCL20 were associated with poorer overall survival in patients with HCC, implicating their roles in tumor progression.

Conclusions: Our study reveals that GATA4 downregulation contributes to an immunosuppressive microenvironment, driven by CCL20-mediated enrichment of regulatory T cells and myeloid-derived suppressor cells in HCC. These findings underscore the critical role of GATA4 reduction in promoting immunosuppression and HCC progression.

Graphical abstract

FIGURE 1

Comparison of differentially expressed genes between GATA4-norm/high and GATA4-low tumors. (A) Two to 5 tumor sectors (T) and 1 adjacent nontumor sector (N) sample were collected from each of the 37 patients with HCC and analyzed by RNA sequencing (seq) and CyTOF for their transcriptomic and immunomic profiles. (B) Volcano plot highlighting the significant genes upregulated in GATA4-norm/high and GATA4-low from bulk RNA sequencing. (C) Functional pathway analysis of the differentially expressed genes using the Database for Annotation, Visualization, and Integrated Discovery. Abbreviations: BHMT, betaine-homocysteine S-methyltransferase; CNTN1, contactin 1; COMP, cartilage oligomeric matrix protein; CRYAA, crystallin alpha A; CTLA4, cytotoxic T lymphocyte-associated protein 4; CyTOF, cytometry by time-of-flight; FC, fold change; FNDC5, fibronectin type III domain containing 5; FXYD3, FXYD domain-containing ion transport regulator 3; GATA4, GATA binding protein 4; HAO2, hydroxyacid oxidase 2; HAVCR2, hepatitis A virus cellular receptor 2; IGFALS, insulin-like growth factor binding protein acid labile subunit; MUC13, mucin 13; PD-1, programmed cell death protein 1; TCP10L, t-complex 10 like; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TPM, transcripts per million.

FIGURE 2

Immune profiling of the lymphoid subsets from GATA4-norm/high and GATA4-low tumors. (A) Median expression of selective immune markers in CD4 T cells, CD8 T cells, NKT cells, and NK cells clusters (C). (B) t-distributed stochastic neighbor embedding plots of the 13 significantly enriched lymphoid clusters (C) in GATA4-norm/high or GATA4-low tumor sectors. (C–E) Proportions of enriched immune clusters and validation of immune subsets by manual gating using markers as stated on y-axis for (C) CD4 T cells, (D) CD8 T cells and (E) NK/NKT cell subsets. Abbreviations: CCR, C-C Motif Chemokine Receptor; FoxP3, Forkhead box P3; GATA4, GATA binding protein 4; NK, natural killer cells; NKT, natural killer T cells; Tbet, T-box transcription factor TBX21; TCM, central memory T cell; Tregs, regulatory T cells; T_RM, resident memory T cells.

FIGURE 3

Immune profiling of the myeloid subsets from GATA4-norm/high and GATA4-low tumors. (A) Median expression of immune markers in MDSCs, macrophages, nonclassical monocytes, and cDC2 clusters (C). (B) t-distributed stochastic neighbor embedding plots of the enriched myeloid clusters (C) in GATA4-norm/high and GATA4-low. (C) Proportions of enriched immune clusters in GATA4-low tumors and further validation by manual gating using markers as stated on y-axis for MDSCs and immunosuppressive macrophages. (D) Proportions of enriched immune clusters in GATA4-norm/high tumors and further validation by manual gating using markers as stated on y-axis for ncMos, proinflammatory macrophages, and cDC2. Abbreviations: cDC2, conventional dendritic cells; GATA4, GATA binding protein 4; MDSCs, myeloid-derived suppressor cell; PMN, polymorphonuclear; TIE2, tunica intima endothelial kinase 2; VISTA, V-domain Ig suppressor of T-cell activation.

FIGURE 4

Spatial correlation of GATA4 and CCL20 in HCC tumors. (A) H&E staining of HCC1 and HCC2 tissues. (B) Clustering and uniform manifold approximation and projections of HCC1 and HCC2 tissues according to spatial RNA expression. (C, D) Images showing the RNA expression of (C) GATA4 and (D) CCL20 in the tumor (T) and surrounding non-tumor (NT) tissue (n=2). Abbreviations: CCL20, C-C Motif Chemokine Ligand 20; GATA4, GATA binding protein 4; H&E, hematoxylin and eosin; N, Non-tumor; T, Tumor.

FIGURE 5

Identification of CCL20 as a downstream target of GATA4. (A) Correlation between GATA4 fold change and CCL20 expression according to bulk RNA sequencing. (B) Quantitative RT-PCR showing mRNA overexpression of GATA4 on doxycycline (Dox) treatment in PLC/PRF/5 and Huh-7 cell lines containing lentiviral system with Dox-inducible GATA4 gene. (C) Venn diagram representing genes downregulated more than 2-fold (adjusted p-value<0.05) in both Dox-treated PLC/PRF/5 and Huh-7 Tet GATA4 cells, overlapped with genes highly expressed in GATA4-Low and not GATA4-Norm/High tumors from our previous HCC cohort study (PLANet) data set (37). These 5 overlapping genes included CCL20. (D) mRNA levels of CCL20 were reduced on Dox-induced GATA4 overexpression in PLC/PRF/5 and Huh-7 cell lines, measured by quantitative RT-PCR. Abbreviations: CCL20, C-C Motif Chemokine Ligand 20; CXCL10, C-X-C Motif Chemokine Ligand 10; GATA4, GATA binding protein 4; TPM, transcripts per million.

FIGURE 6

Comparison of Treg and MDSC populations between CCL20-high and CCL20-low tumors. Spearman correlation between frequencies of total (A) Treg and (B) MDSC from CyTOF data, with CCL20 gene expression (TPM) from bulk RNA-seq data of matched tumor tissue. (C) mIF staining was performed on tissue microarray sections from an independent cohort of 49 patients with HCC. (D–F) Representative images showing protein expression of (D) CCL20 (yellow), (E) Tregs (FoxP3=green; CD4=red) as denoted by white arrows and (F) MDSCs (CD11b=green; CD33=red, co-stained with DAPI=blue), all based on their respective median densities. Scale bars: 150 μm. (G) Box plot showing the area of CCL20 expression mm² per core in tumors from CCL20-low and CCL20-high groups. (H) Box plot showing densities of Tregs or MDSCs, quantified as count per 1 mm² core in CCL20-low versus CCL20-high tumors. Abbreviations: CCL20, C-C Motif Chemokine Ligand 20; CyTOF, cytometry by time-of-flight; FoxP3, Forkhead box P3; MDSCs, myeloid-derived suppressor cell; mIF, multiplexed immunofluorescence; TPM, transcripts per million; Tregs, regulatory T cells.

FIGURE 7

Clinical implications of GATA4 and CCL20 status in patients with HCC. (A, B) Kaplan-Meier analysis of overall survival profiles of the Cancer Genome Atlas liver cancer cohort (n=364) segregated by high (red) or low (black) expression of (A) GATA4 (cutoff value=946, range of expression: 68–6067) and (B) CCL20 (cutoff value=286, range of expression: 0–28000). (C–D) Kaplan-Meier analysis of overall survival profiles of the Cancer Genome Atlas liver cancer cohort segregated by high (red) or low (black) expression of CCL20 in (C) GATA4-low (cutoff value=351, range of expression: 0–28,000) and (D) GATA4-high tumors (cutoff value=2066, range of expression: 0–23,583). Abbreviations: CCL20, C-C Motif Chemokine Ligand 20; GATA4, GATA binding protein 4.