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, 105 (7), 2445-50

Progenitor/stem Cells Give Rise to Liver Cancer Due to Aberrant TGF-beta and IL-6 Signaling

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Progenitor/stem Cells Give Rise to Liver Cancer Due to Aberrant TGF-beta and IL-6 Signaling

Yi Tang et al. Proc Natl Acad Sci U S A.

Abstract

Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ "cancer stem cells," such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000-50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-beta-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF. elf(+/-) mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-beta signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC in elf(+/-) mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-beta signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-beta pathway.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of liver progenitor/stem cells in posttransplant human liver tissues. Immunohistochemical labeling of posttransplant human liver tissue taken from living donor liver transplant 4 weeks after transplantation. The tissue is labeled for the presence of ELF (A, arrows) and Oct4 (B, arrows). Sections are taken consecutively to enable identical localization. (C–N) Confocal images of human liver at 3 months after living-related liver transplantation. (C–E) The tissue is labeled with stem cell proteins Stat3 and Oct4 and prodifferentiation TGF-β signaling component ELF. (F) These proteins coexpress in a small cluster of two to four cells. (G) DAPI represents nuclear labeling. (H) Differential interference chromatography (DIC) represents a transmission image of this cluster of cells. (I–K) Regenerative liver tissue from another liver transplant is labeled with p-histone H3 (Ser10), Oct4, and ELF. (L) These proteins coexpress in this cluster of progenitor-like cells. (M and N) DAPI represents nuclear labeling (M), and DIC represents transmission images (N). Arrows point to the nuclei of the progenitor-like cells. (Scale bars for all figures are in micrometers.)
Fig. 2.
Fig. 2.
Identification of liver progenitor/stem cells in posttransplant human liver and HCC tissues. Immunohistochemical labeling of posttransplant human liver tissues taken from living donor liver transplant recipient 4 weeks after transplantation. (A–D) The tissue is labeled for the presence of ELF (A and C) and Oct4 (B and D). Sections are taken consecutively to enable identical localization. (E–J) Equivalent areas are marked by red dotted lines, and green arrows point to the positive labeling. Immunohistochemical labeling of normal human liver (E and H) and HCC tissues (F, G, I, and J). The loss of ELF is evident when comparing the immunohistochemical labeling of normal (E and H) and HCC samples (F and I). Strikingly, there are small pockets of three to four Oct4 positively stained cells present in the midst of transformed hepatocellular cells (G and J, arrows). These Oct4-positive cells are stained negatively for ELF (I, area marked by blue dotted line). (K–M) Confocal images of human HCC labeled to highlight prodifferentiation TGF-β signaling component ELF (K) and progenitor cell proteins Stat3 and Oct4 (L and M). (N, white arrow) The overlay image demonstrates a cell that labeled positively for Stat3 and Oct4 but lacks nuclear expression of ELF. (O) DIC represents transmission image of this cluster of cells. (P) DAPI represents nuclear labeling. The white arrow points to the nucleus of the HCC progenitor/stem cell lacking ELF. PT represents portal tract.
Fig. 3.
Fig. 3.
Decreased incidence of hepatocellular cancer is observed by genetic modulation of IL-6-stat-3 signaling. (A and B) Heatmap microarray assay illustrating gene expression in mouse liver or HCC tissues. Targeted disruption of the ITIH-4 gene and generation of itih4−/− mice. Exp1: elf+/− liver tissue vs. wild-type liver tissue; Exp2: itih4−/− liver tissue vs. wild-type liver tissue; Exp3: elf+/−/itih4−/− liver tissue vs. wild-type liver tissue. The signal gradients are located below each image. (C) The targeting vector for itih4 gene; the targeting strategy deletes a 1.8-kb SmaI-ClaI fragment that contains second and third exons of the itih4 gene. (D) Southern blot analysis shows ES cells heterozygous (i151, i155, and i160) with correct homologous recombination events within the itih4 locus. Genomic DNA from these clones was digested with EcoRV, followed by Southern blot using a 1.8-kb fragment 3′ to the targeting vector. (E) Immunoblot analysis using the antibody specific for ITIH4 shows loss of ITIH4 expression in itih4−/− and elf+/−/itih4−/− liver tissue lysates compared with the wild-type and elf+/− samples. (F) Kaplan–Meier tumor-free survival curves of wild-type (control), elf+/−, itih4−/−, and elf+/−/itih4−/− (experimental) animals.
Fig. 4.
Fig. 4.
Analysis of gene expression in mouse liver tissues and human HCC tissues and cell lines. (A, C, and D) Immunohistochemical labeling demonstrates low/absent expression of phosphorylated Stat3 in normal (wild-type) mouse liver (A), ITIH4−/− liver (C), and Elf+/−/ITIH4−/− liver (D). (B) In contrast, Elf+/− HCC liver tissue shows increased expression of P-Stat3. (E and F) Immunohistochemical detection shows increased expression of Stat3 in human HCC tissues (F, arrows) compared with normal liver tissues (E). (H and G) Phosphorylated-Stat3 is also increased in human HCC tissues (H, arrows) compared with normal liver tissues (G). (Scale bar is in micrometers.)

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