STAT3 is Activated by CTGF-mediated Tumor-stroma Cross Talk to Promote HCC Progression

Abstract

Background & aims: Signal transducer and activator of transcription 3 (STAT3) is known as a pro-oncogenic transcription factor. Regarding liver carcinogenesis, however, it remains controversial whether activated STAT3 is pro- or anti-tumorigenic. This study aimed to clarify the significance and mechanism of STAT3 activation in hepatocellular carcinoma (HCC).

Methods: Hepatocyte-specific Kras-mutant mice (Alb-Cre Kras^LSL-G12D/+; Kras^G12D mice) were used as a liver cancer model. Cell lines of hepatoma and stromal cells including stellate cells, macrophages, T cells, and endothelial cells were used for culture. Surgically resected 12 HCCs were used for human analysis.

Results: Tumors in Kras^G12D mice showed up-regulation of phosphorylated STAT3 (p-STAT3), together with interleukin (IL)-6 family cytokines, STAT3 target genes, and connective tissue growth factor (CTGF). Hepatocyte-specific STAT3 knockout (Alb-Cre Kras^LSL-G12D/+ STAT3^fl/fl) downregulated p-STAT3 and CTGF and suppressed tumor progression. In coculture with stromal cells, proliferation, and expression of p-STAT3 and CTGF, were enhanced in hepatoma cells via gp130/STAT3 signaling. Meanwhile, hepatoma cells produced CTGF to stimulate integrin/nuclear factor kappa B signaling and up-regulate IL-6 family cytokines from stromal cells, which could in turn activate gp130/STAT3 signaling in hepatoma cells. In Kras^G12D mice, hepatocyte-specific CTGF knockout (Alb-Cre Kras^LSL-G12D/+ CTGF^fl/fl) downregulated p-STAT3, CTGF, and IL-6 family cytokines, and suppressed tumor progression. In human HCC, single cell RNA sequence showed CTGF and IL-6 family cytokine expression in tumor cells and stromal cells, respectively. CTGF expression was positively correlated with that of IL-6 family cytokines and STAT3 target genes in The Cancer Genome Atlas.

Conclusions: STAT3 is activated by CTGF-mediated tumor-stroma crosstalk to promote HCC progression. STAT3-CTGF positive feedback loop could be a therapeutic target.

Keywords: IL-6 Family Cytokines; Intercellular Interactions; Liver Cancer; Tumor Microenvironment.

Graphical abstract

Figure 1

Hepatocyte-specific Kras-mutant mice developed liver tumors with STAT3 activation and upregulation of CTGF and IL-6 family cytokine expression in hepatoma cells and stromal cells, respectively. Kras^G12D mice and their littermate Alb-Cre transgenic control mice were sacrificed at 7 to 12 months of age and their phenotypes were compared (A-G). A, Representative images of the livers at 9 months of age. B, HE staining of a liver tumor (left; ×40, right; ×100). C, Immunohistochemistry results showing α-SMA, CD3, F4/80, and PECAM-1 in liver tumors in Kras^G12D mice (400×). D, Western blot showing liver tissue proteins. E, Immunohistochemistry results showing CTGF in the liver of Kras^G12D mice (200×). F, mRNA expression of STAT3 target genes, IL-6 family cytokines, ctgf, and tgf-b1 in liver tissues (n = 7-12). G, Correlation of mRNA expression between CTGF and STAT3-target molecules and IL-6 family cytokines (n = 20). Liver tumors were collected from a 12-month-old mouse harboring the Kras^G12D/+ allele and subjected to single-cell RNA sequencing (scRNA-seq) (H-I). In total, 1303 cells from 2 tumors were analyzed. H, UMAP plots of IL-6 family cytokines. I, Heat map of IL-6 family cytokines, STAT3-target genes, tgf-b1, and ctgf according to the cell types. For immunohistochemistry, a representative image of each molecule from 6 samples was presented. NT, Nontumor tissues; T, tumor tissues. ∗P < .05.

Figure 2

Hepatocyte-specific STAT3 knockout suppressed tumor progression and CTGF expression in liver tumors of Kras^G12Dmice. Kras^G12D mice and STAT3-floxed mice were mated to generate hepatocyte-specific STAT3-knockout Kras^G12D mice. After mating STAT3^fl/+ Kras^+/+ Alb-Cre mice and STAT3^fl/+ Kras^LSL-G12D/+ mice, the offspring: (1) Kras^wild STAT3^+/+ (STAT3^+/+ Kras^+/+Alb-Cre); (2) Kras^wild STAT3^-/- (STAT3^fl/fl Kras^+/+Alb-Cre); (3) Kras^G12D STAT3^+/+ (STAT3^+/+ Kras^LSL-G12D/+ Alb-Cre); (4) Kras^G12D STAT3^+/- (STAT3^fl/+ Kras^LSL-G12D/+ Alb-Cre); and (5) Kras^G12D STAT3^-/- (STAT3^fl/fl Kras^LSL-G12D/+ Alb-Cre) littermates were sacrificed at 8 months of age. A, Representative images of the livers. B, Macroscopic liver tumorigenesis rate, liver/body weight ratio, tumor number, and maximum tumor diameter (n = 8-12 per group). C, Representative images of HE staining of liver tumors. D, Representative images of immunohistochemical staining for PCNA (400×) and the number of PCNA-positive cells in liver tumors per each high-power field (HPF) (n = 10 per group). E, Western blot showing liver tumor proteins. F, Gene expression of STAT3-target genes, IL-6 family cytokines, and CTGF in liver tumors (n = 5-9). ∗P < .05.

Figure 3

Hepatocyte-specific STAT3 knockout suppressed tumor progression and CTGF expression in model mice with DEN-induced liver carcinogenesis. To generate hepatocyte-specific STAT3-knockout mice, STAT3^fl/+ Alb-Cre mice were mated with STAT3^fl/+ mice. The offspring littermates: (1) STAT3^+/+ (STAT3^+/+ Alb-Cre); (2) STAT3^+/- (STAT3^fl/+ Alb-Cre); and (3) STAT3^-/- (STAT3^fl/fl Alb-Cre), were administered 20 mg/kg DEN at 2 weeks of age and sacrificed at 9 months of age. A, Macroscopic appearance of the liver. B, Macroscopic tumorigenesis rate, tumor number and diameter, and liver/body weight ratio (n = 11-17 per group). C, HE-stained liver tumors (200×). D, Immunohistochemical staining for α-SMA, CD3, F4/80, and PECAM-1 in the liver of STAT3^+/+ mice (400×). E, Representative image of immunohistochemical staining for PCNA (400×) and the number of PCNA-positive cells in liver tumors per high-power field (HPF) (n = 10 per group). F, Western blot showing liver tissue proteins. G, Immunohistochemical staining for CTGF in the liver of STAT3^+/+ mice. H, mRNA expression levels in the liver tissues (n = 6-12 per group). ∗P < .05.

Figure 4

Stromal cells increased p-STAT3 and CTGF expression and cell growth in hepatoma cells. HepG2 or Alex cells were transfected with STAT3 siRNA or control siRNA (A-B). WST-8 assay (A) and CTGF mRNA expression (B) of HepG2 or Alex cells after siRNA transfection. Total RNA was collected 48 hours after siRNA transfection. HepG2 or Alex cells were incubated in monoculture or cocultured with LX-2, THP-1, MTA, and TMNK-1 cells, using Transwell insert system (C-G). THP-1 cells were pretreated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce macrophage differentiation before coculturing. Total proteins and RNA were collected after 24 hours of incubation (C, E, F). C, Western blot showing protein expression of HepG2 or Alex cells. D, WST-8 assay of HepG2 or Alex cells in coculture at the indicated time points. HepG2 or Alex cells were transfected with STAT3 siRNA, gp130 siRNA, or control siRNA 48 hours before coculturing (E-G). E, Western blot showing protein expression and qPCR showing CTGF mRNA expression in HepG2 or Alex cells transfected with STAT3 siRNA or control siRNA. F, Western blot and CTGF mRNA expression in HepG2 or Alex cells transfected with gp130 siRNA or control siRNA. G, WST-8 assay of HepG2 or Alex cells transfected with STAT3 siRNA, gp130 siRNA, or control siRNA 48 hours after incubation (n = 3-4). ∗^{, †, ‡, §}P < .05 vs control.

Figure 5

IL-6 family cytokines increased p-STAT3 and CTGF expression in hepatoma cells and enhanced cell proliferation. Concentrations of IL-6, LIF, and OSM in the culture supernatant (A). Culture supernatant was collected 24 hours after the initiation of HepG2, Alex, LX-2, THP-1, MTA, and TMNK-1 cell monoculture. IL-6, LIF, or OSM concentration was measured by enzyme-linked immunosorbent assay. THP-1 cells were pretreated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce macrophage differentiation, and the culture medium was replaced with PMA-free medium 24 hours before supernatant collection. HepG2 cells and Alex cells were treated with recombinant proteins of IL-6 (20 ng/mL), LIF (1:1000), or OSM (20 ng/mL) (B-D). Cells were serum-starved for 16 hours before the addition of recombinant proteins. B, Protein expression 1 hour after recombinant protein treatment. C, mRNA expression of CTGF 3 hours after recombinant protein treatment. D, WST-8 assay at the indicated time points after treatment with recombinant proteins. HepG2 cells and Alex cells were transfected with gp130 siRNA, STAT3 siRNA, or control siRNA 48 hours before the addition of recombinant proteins (E-F). Recombinant proteins were added 16 hours after serum starvation. E, mRNA expression of CTGF 3 hours after treatment with recombinant proteins. F, WST-8 assay at the indicated time points after recombinant protein treatment (n = 3-4). ∗P < .05.

Figure 6

Inhibition of gp130/STAT3 signaling abolished the accelerated growth of HepG2 cells injected with LX-2 cells and downregulated CTGF expression in xenograft tumors. Protein and mRNA expression and proliferation in HepG2 cells stably expressing STAT3, gp130, or control shRNA in vitro (A). RNA and proteins were extracted 24 hours after plating. WST-8 assay was performed to evaluate cell proliferation at indicated time points. Xenograft models of HepG2 cells stably expressing STAT3, gp130, or control shRNA with or without LX-2 cells (B-C). HepG2 cells stably expressing gp130 shRNA (B), STAT3 shRNA (C), or control shRNA were subcutaneously injected alone or with the same number of LX-2 cells into the left and right flank portions of NOG mice, respectively. Representative images of xenograft tumors, tumor volume measured sequentially, mRNA expression, and immunohistochemistry for p-STAT3 (scale bar: 50 μm) in xenograft tumors are presented (n = 4-5). ∗P < .05 vs control.

Figure 7

Knockdown of CTGF in HepG2 cells decreased IL-6 family cytokine expression in stromal cells, downregulated p-STAT3 expression in HepG2 cells and reduced the proliferation of HepG2 cells. Western blotting of HepG2 or Alex cells treated with recombinant CTGF protein under monoculture (A). Protein lysate was collected from cells 0, 0.5, 1, and 3 hours after the addition of 5 nM recombinant CTGF protein. HepG2 cells were incubated under monoculture or coculture with stromal cell lines (B-E). HepG2 cells were transfected with CTGF siRNA or control siRNA 48 hours before coculturing. Total proteins and RNA were collected after 24 hours of coculture. THP-1 cells had been pretreated with 100 ng/m phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce macrophage differentiation before coculture. B, Western blot showing HepG2 cell proteins in monoculture or cocultured with LX-2, THP-1, MTA, and TMNK-1 cells. C, WST-8 assay of HepG2 cells performed 48 hours after initiation of monoculture or coculture (n = 3). ∗P < .05. D, mRNA expression of IL-6 family cytokines in stromal cell lines in monoculture or cocultured with HepG2 cells transfected with CTGF siRNA or control siRNA (n = 4). E, IL-6 concentration in supernatant measured via enzyme-linked immunosorbent assay (n = 4). ∗P < .05 vs monoculture; ∗∗P < .05 vs coculture with HepG2 control si.

Figure 8

Recombinant CTGF treatment upregulated IL-6 family cytokine expression in stromal cell lines via integrin/NF-kB signaling. LX-2, THP-1, MTA, and TMNK-1 cells were serum-starved for 16 hours and then treated with 5 nM recombinant CTGF protein. THP-1 cells had been pretreated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) to induce macrophage differentiation for 48 hours before starvation. Total protein was collected at the indicated time points after the addition of recombinant CTGF protein. Total RNA was extracted 6 hours after incubation with recombinant CTGF protein. Protein and gene expression after recombinant CTGF treatment (A-C). Cells were treated with recombinant CTGF alone (A) or pretreated with 1 mM RGDS peptide (B) or 50 ng/mL SN-50 (C) for 2 hours before the addition of recombinant CTGF protein. n = 4 (A, B) or n = 3 (C). ∗P < .05.

Figure 9

Hepatocyte-specific knockout of CTGF downregulated IL-6 family cytokine expression in liver tumors of Kras^G12Dmice and suppressed STAT3 activation and tumor progression. By mating Kras^G12D mice with CTGF-floxed mice, we generated hepatocyte-specific CTGF-knockout Kras^G12D mice. After crossing CTGF^fl/+ Kras^+/+ Alb-Cre mice and CTGF^fl/+ Kras^LSL-G12D/+ mice, the following littermates were obtained: (1) Kras^G12D CTGF^+/+ (CTGF^+/+ Kras^LSL-G12D/+ Alb-Cre); (2) Kras^G12D CTGF^+/- (CTGF^fl/+ Kras^LSL-G12D/+ Alb-Cre); and (3) Kras^G12D CTGF^-/- (CTGF^fl/fl Kras^LSL-G12D/+ Alb-Cre). The phenotypes were evaluated at 8 months of age. A, Representative images of 8-month-old livers. B, Western blot showing liver tumor proteins. C, mRNA expression of CTGF, STAT3 target genes, and IL-6 family cytokines in liver tumors (n = 7-13). ∗P < .05.

Figure 10

CTGF expression was positively correlated with that of STAT3 target genes and IL-6 family cytokines which were mainly expressed in stromal cells in human HCC. Immunohistochemical staining for CTGF, aSMA, CD3, CD68, and PECAM-1 in human HCC (200×) A,. A representative image of each molecule from 6 samples is presented. Samples taken from surgically resected HCC tissues were subjected to scRNA-seq (n = 12) (B-C). B, UMAP showing CTGF and IL-6 family cytokines. Correlation of mRNA expression between CTGF and STAT3-target genes or IL-6 family cytokines in 373 HCC samples obtained from the The Cancer Genome Atlas database (C). Correlation coefficients presented in red were statistically significant. ∗P < .05.