Krit Kitisin

Progenitor/stem cells give rise to liver cancer due to aberrant TGF-β and IL-6 signaling

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-β-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-β 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-β 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-β pathway.

Tgf-Beta signaling in development.

The transforming growth factor–β (TGF-β) superfamily comprises nearly 30 growth and differentiation factors that include TGF-βs, activins, inhibins, and bone morphogenetic proteins (BMPs). Multiple members of the TGF-β superfamily serve key roles in stem cell fate commitment. The various members of the family can exhibit disparate roles in regulating the biology of embryonic stem (ES) cells and tumor suppression. For example, TGF-β inhibits proliferation of multipotent hematopoietic progenitors, promotes lineage commitment of neural precursors, and suppresses epithelial tumors. BMPs block neural differentiation of mouse and human ES cells, contribute to self-renewal of mouse ES cells, and also suppress tumorigenesis. ES cells and tumors may be exposed to multiple TGF-β members, and it is likely that the combination of growth factors and cross-talk among the intracellular signaling pathways is what precisely defines stem cell fate commitment. This Connections Map Pathway in the Database of Cell Signaling integrates signaling not only from TGF-β and BMP but also from the ligands nodal and activin, and describes the role of the signaling pathways activated by these ligands in mammalian development. Much of the evidence for the connections shown comes from studies on mouse and human ES cells or mouse knockouts. This pathway is important for understanding not only stem cell biology, but also the molecular effectors of TGF-β and BMP signaling that may contribute to cancer suppression or progression and thus are potential targets for therapeutic intervention.

Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation

Transforming growth factor-β (TGF-β) signaling members, TGF-β receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf+/− mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-β signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

Role of Transforming Growth Factor β Signaling and Expansion of Progenitor Cells in Regenerating Liver

Adult hepatic progenitor cells are activated during regeneration when hepatocytes and bile duct epithelium are damaged or unable to proliferate. On the basis of its role as a tumor suppressor and in the potential malignant transformation of stem cells in hepatocellular carcinoma, we investigated the role of key transforming growth factor beta (TGF‐β) signaling components, including the Smad3 adaptor protein β2‐Spectrin (β2SP), in liver regeneration. We demonstrate a streaming hepatocyte‐specific dedifferentiation process in regenerating adult human liver less than 6 weeks following living donor transplantation. We then demonstrate a spatial and temporal expansion of TGF‐β signaling components, especially β2SP, from the periportal to the pericentral zone as regeneration nears termination via immunohistochemical analysis. This expansion is associated with an expanded remaining pool of octamer 3/4 (Oct3/4)‐positive progenitor cells localized to the portal tract in adult human liver from more than 6 weeks posttransplant. Furthermore, disruption of TGF‐β signaling as in the β2SP (β2SP+/−) knockout mouse demonstrated a striking 2 to 4‐fold (P < 0.05) expanded population of Oct3/4‐positive cells with activated Wnt signaling occupying an alpha‐fetoprotein (AFP)+/cytokeratin‐19 (CK‐19)‐positive progenitor cell niche following two‐thirds partial hepatectomy. Conclusion: TGF‐β signaling, particularly β2SP, plays a critical role in hepatocyte proliferation and transitional phenotype and its loss is associated with activation of hepatic progenitor cells secondary to delayed mitogenesis and activated Wnt signaling. (HEPATOLOGY 2010.)

Hepatocellular Cancer Arises from Loss of Transforming Growth Factor Beta Signaling Adaptor Protein Embryonic Liver Fodrin Through Abnormal Angiogenesis

We have previously demonstrated that 40%‐70% of elf+/− mice spontaneously develop hepatocellular cancer (HCC) within 15 months, revealing the importance of the transforming growth factor‐beta (TGF‐β) signaling pathway in suppressing tumorigenesis in the liver. The current study was carried out to investigate mechanisms by which embryonic liver fodrin (ELF), a crucial Smad3/4 adaptor, suppresses liver tumor formation. Histological analysis of hyperplastic liver tissues from elf+/− mice revealed abundant newly formed vascular structures, suggesting aberrant angiogenesis with loss of ELF function. In addition, elf+/− mice displayed an expansion of endothelial progenitor cells. Ectopic ELF expression in fetal bovine heart endothelial (FBHE) cells resulted in cell cycle arrest and apoptosis. Further analysis of developing yolk sacs of elf−/− mice revealed a failure of normal vasculature and significantly decreased endothelial cell differentiation with embryonic lethality. Immunohistochemical analysis of hepatocellular cancer (HCC) from the elf+/− mice revealed an abnormal angiogenic profile, suggesting the role of ELF as an angiogenic regulator in suppressing HCC. Lastly, acute small interfering RNA (siRNA) inhibition of ELF raised retinoblastoma protein (pRb) levels nearly fourfold in HepG2 cells (a hepatocellular carcinoma cell line) as well as in cow pulmonary artery endothelial (CPAE) cells, respectively. Conclusion: Taken together these results, ELF, a TGF‐β adaptor and signaling molecule, functions as a critical adaptor protein in TGF‐β modulation of angiogenesis as well as cell cycle progression. Loss of ELF in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers. Our studies propose that ELF is potentially a powerful target for mimetics enhancing the TGF‐β pathway tumor suppression of HCC. (HEPATOLOGY 2008.)

Hepatocellular stem cells

The liver has enormous regenerative capacity. Restitution of the liver in response to different injuries involves proliferation of cells at different levels of liver lineage. Mature hepatocytes, which are normally dormant, could undergo rapid replication with a near infinite capacity to proliferate. When the replication of mature hepatocytes is inhibited, a reserve compartment of bipotential hepatic progenitor/stem cells is activated. The degree of activation appears to correlate with the degree of inflammation and stage of chronic liver disease. Deregulation of key regulatory signaling pathways such as transforming growth factor-beta, Wnt, hepatocyte growth factor, insulin-like growth factor, transforming growth factor-alpha and epidermal growth factor in this progenitor/stem cell population could give rise to HCC. Further understanding of these key signaling pathways and the molecular and genetic alterations associated with HCC could provide major advances in new therapeutic and diagnostic modalities.