Fig 2: The TGF-ß/SCF signaling loop neutralizes TGF-ß tumor suppressor function and drives tumor progression in vitro. (A) TGF-ß/SCF positive feedback loop. TGF-ß–activated SMAD2 transcriptionally induces SCF expression via direct binding to the SCF promoter. SCF expression and secretion result in autocrine and paracrine stimulation of the c-KIT receptor, followed by activation of JAK1/STAT3 signaling. pTyr705STAT3 translocates to the nucleus where it binds directly to the TGF-ß ligand gene, positively regulating its expression. Following activation of secreted TGF-ß precursor, it can activate the TGF-ß receptor. In addition, TGF-ß/c-KIT drive tumor progression via STAT3 through neutralization of TGF-ß antiproliferative functions and induction of EMT, tumor cell migration, and invasion. Disruption of the TGF-ß/c-KIT signaling loop on the level of the SCF/STAT3 axis restores TGF-ß tumor suppressor function by inhibition of EMT, tumor cell migration, and invasion, and restoration of its cell cycle inhibitory functions. (B) Migration assay for TGF-ß1–induced tumor cell migration in parental liver tumor cells in comparison to their SMAD2-, SCF-, and STAT3-knockdown clones. (C) Invasion assay for TGF-ß1–induced tumor cell invasion in parental liver tumor cells, and their SMAD2-, SCF-, and STAT3-knockdown clones. (D) MTT cell viability assay of parental HCC cells versus their SMAD2-, SCF- and STAT3-knockdown clones.

Fig 3: c-KIT signaling is the mediator of TGF-ß autoregulation resulting in a positive feedback loop. (A) qRT-PCR for TGF-ß1 ligand transcription in parental and SCF-knockdown liver tumor cells stimulated with TGF-ß1; results were normalized for GAPDH. (B) qRT-PCR for TGF-ß1 ligand transcription in SCF-stimulated parental and STAT3-knockdown HCC cells; results were normalized for GAPDH. (C) ChIP analysis of TGF-ß1–treated and untreated liver tumor cells immunoprecipitated with an anti-STAT3 antibody followed by PCR using TGF-ß specific primers for STB-1 and STB-2; STB-2 also serves as a positive control for STB-1. (D) TGF-ß1–induced (10 ng/ml, 24 hours) luciferase activity using luciferase expression plasmids with STAT3 bindings site–containing TGFB gene fragments (Suppl. Figure 6B) transfected in liver tumor cells. Construct (1) contains STB-1 and STB-2, construct (2) contains STB-2 only, and construct (3) contains STB-1 only. (E) ChIP analysis for TGF-ß1–induced STAT3 binding to the STB-2 binding site in parental versus SCF-knockdown liver tumor cells.

Fig 4: SCF is overexpressed in advanced HCC and is correlated with pSMAD2 and decreased survival. (A) Representative sections of stage I to IV human HCC tissue immunohistochemically stained for SCF. (B) Allred scoring system–based quantification of SCF-positive tumors of HCC patients (n = 116); patient’s characteristics are summarized in Suppl. Table 2. (C) Stage-dependent quantification of intratumoral pSer465/467Smad2/SCF co-positivity in human HCC tumors (n = 116); immunohistochemical positivity defined as > 30% of tumor cells per tumor being positive for pSer465/467Smad2 and SCF. (D) SCF transcription in tumor versus matching nonmalignant hepatic tissue in an independent set of HCC patients (n = 36). Data were normalized for GAPDH and are shown as fold increase in SCF mRNA in tumor versus matching nonmalignant hepatic tissue; the blue line represents the median. SCF transcription was significantly higher in advanced- versus early-stage HCC (P = .029). (E) Correlation analysis for intratumoral PAI-1 and SCF mRNA in human HCC tumors. The red line represents the trend line based upon linear regression analysis. Intratumoral PAI-1 and SCF mRNA was significantly (P = .0006) correlated in advanced-stage HCC with a correlation coefficient R = 0.84 (95% CI 0.49-0.96). (F) Representative sections of human stages I to IV HCC tumors evaluated by co-immunofluorescence for SCF and pSer465/467SMAD2 co-localization. (G) Quantification of human HCC tumors (n = 116) analyzed for intracellular SCF/pSer465/467SMAD2 co-positivity. Shown is the percentage of SCF/pSer465/467SMAD2 co-positive tumor cells in pSer465/467SMAD2-positive tumors (median number of cells of five high-power fields per sample).