Liver Stem Cells and TGF-β in Hepatic Carcinogenesis

Abstract

Hepatocellular carcinoma is the fifth most common malignancy worldwide. Recent trends indicate a rising incidence in the United States, with a 5-year survival rate of less than 5%. New therapeutics targeting advanced-stage hepatocellular carcinoma, such as sorafenib, have marginally improved the median overall survival by 3 months. There is an urgent need for new targeted agents that are associated with minimal local and systemic toxicities. Up to 40% of hepatocellular carcinomas are clonal, potentially arising from stem cells and increased activation of multiple pathways including IL-6/STAT3, WNT, CDK4, and hedgehog, as well as loss of response to the transforming growth factor-beta (TGF-β) signaling pathway. Our hypothesis has been that these “cancer stem cells” or cancer sustaining cells may prove to be strong genetic and therapeutic targets. Modulating stem cell renewal factors such as STAT3, NANOG, and OCT4 may reduce hepatocellular carcinoma formation. These points are discussed in detail in this review.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and third most frequent cause of cancer deaths worldwide, with 600,000 new cases diagnosed each year.¹ The prognosis of HCC patients remains extremely poor, with a 5-year survival rate of less than 5%. Furthermore, the incidence of this tumor is rising in the United States.^2,3 Currently, the only curative therapeutic option for HCC is surgical intervention, including hepatic resection and liver transplantation.^3,4 However, 70% of HCC cases are ineligible for potentially curative surgical therapy because the disease has reached an advanced stage at the time of diagnosis. Current chemotherapy is ineffective for the most part and patients often have significant liver dysfunction; median survival is approximately 6 to 16 months.^5,6 The development of HCC is a multi-step process often beginning with cirrhosis and progressing to adenoma and dysplastic nodule formation.⁷

One potential mechanism of HCC resistance to chemotherapy may lie in the plasticity of the cell of origin, which is often a dysfunctional progenitor or stem cell. As many as 40% of HCCs are clonal and thus are considered to originate from progenitor/stem cells (Figure 1).^8–11 In addition, several signaling pathways, such as STAT3 (signal transducer and activator of transcription 3), NOTCH, hedgehog, and transforming growth factor-beta (TGF-β) are commonly deregulated in HCC (Figure 1).¹² We and others have identified TGF-β pathway inactivation in HCCs that have a stem cell phenotype.^12–15

Figure 1:

Schematic diagram of TGF-β/ELF and IL-6/STAT3/ITIH4 signaling in hepatic stem cell renewal and differentiation. Abbreviations: ELF = embryonic liver fodrin; ES = embryonic stem cells; IL-6 = interleukin-6; ITIH4 = inter-α-trypsin inhibitor-4; OCT4 = ornithine carbamoyltransferase-4; STAT3 = signal transducer and activator of transcription 3; TBRII (or TGFBR2) = TGF-β receptor II; TGF-β = transforming growth factor-beta.

THE TGF-β SIGNALING PATHWAY

Several signaling networks coordinate the development and differentiation of embryonic stem cells (ES) and somatic stem cells into functional neuronal, hematopoietic, mesenchymal and epithelial lineages. Among these, the signaling mechanisms activated by TGF-β family proteins have emerged as important players in the selfrenewal and maintenance of stem cells in their undifferentiated state, the selection of a differentiation lineage, and the progression of differentiation along individual lineage. ¹⁶ The TGF-β signaling pathway is activated upon ligand binding to type I and II transmembrane receptor serine-threonine kinases; TGF-β receptor I (TGFBR1) and TGF-β receptor II (TGFBR2), respectively. Activated TGFBR1 subsequently phosphorylates SMAD (mothers against decapentaplegic homolog) transcription factors, such as receptor regulated SMAD3, resulting in heterodimerization with the common mediator SMAD4, SMAD3/SMAD4 nuclear translocation, and activation (or repression) of TGF-β target genes.^17–18 Type II β-spectrin adaptor proteins such as embryonic liver fodrin (ELF) are crucial for SMAD regulation and cell specificity. ELF associates with SMAD3, presenting it to the cytoplasmic domain of the TGF-β receptor complex. Dysfunction of TGF-β pathway members, such as TGFBR2, SMAD3, SMAD4, and ELF, may lead to progenitor/stem cell deregulation, and possibly, cancer formation. ELF, a stem cell adaptor protein, has been found to play a pivotal role in TGF-β signaling.¹⁶ This β-spectrin is a major dynamic scaffolding molecule involved in generating functionally distinct membrane protein domains, conferring cell polarity and regulating endocytic traffic.¹⁹ Our previous analysis revealed that mice with complete loss of ELF (elf −/−) displayed the similar phenotypes to SMAD2^+/−/SMAD3^+/− mutant mice with midgestational death, hypoplastic livers, as well as gastrointestinal, neural and heart defects.^20,21 Elf^+/− heterozygotes develop liver fibrosis and dysplasia,²² and up to 40% of these mice spontaneously develop hepatocellular cancers with markedly increased expression of several oncogenes.¹²

CANCER STEM CELLS

Although the existence of cancer stem cells was first proposed more than 40 years ago,^23,24 only in the past decade have these cells been identified in hematologic malignancies, and more recently, in solid tumors including breast, prostate, brain, and colon cancer.²⁵ Identifying the difference between cancer stem cells and normal stem cells is crucial not only for understanding tumor biology but also for the development of specific therapies to target these cells in patients.¹⁶ However, the origin of cancer stem cells and mechanisms by which they arise remain elusive. For tumors comprised of a subpopulation of cancer stem cells, there are at least two proposed mechanisms as to how these cancer stem cells could have arisen: oncogenic mutations that inactivate the constraints on normal stem cell expansion; or, in a more differentiated cell, oncogenic mutations could generate continual proliferation of cells in cell cycle that no longer enter a post-mitotic differentiated state, thereby creating a pool of self-renewing cells in which further mutations can accumulate. The flexibility of such cells is reflected by recent studies where pluripotent stem cells could be induced from embryonic or adult fibroblasts by introducing four factors: OCT 3/4 (ornithine carbamoyltransferase 3/4), SOX2 (SRY-box containing gene 2), c-MYC and KLF4 (Krupple-like factor 4) under embryonic stem cell culture conditions.²⁶ Potentially, biologically significant pathways that regulate these stem/progenitor cells in cancer tissues could be identified through dual roles in embryonic stem cell development and tumor activation or suppression.¹⁶

STEM CELL MARKERS AND THE LOSS OF TGF-β SIGNALING

Through gene knockout experiments and observation with embryonic stem cells, we have identified putative liver progenitor or stem cells from human living-donor liver transplant specimens. These normal human progenitor/stem cells expressed the stem cell markers STAT3, OCT4, and NANOG, as well as the TGF-β signaling proteins, TGFBR2 and ELF (Figure 1).¹²

We also demonstrated that, in contrast, in human HCC tissues, STAT3/OCT4-positive cells are prominently negative for TGFBR2 and ELF (Figure 1). Therefore, STAT3/OCT4-positive HCC cells, which have dysfunctional TGF-β signaling, are likely cancer progenitor cells that have the potential to give rise to HCCs, similar to the HCCs that develop in the elf-mutant mice with loss of response to TGF-β.¹² Utilizing mouse genetics, we were able to modify STAT3 signaling in the elf-mutant mice and markedly reduce HCC formation.

TGF-β-family signaling is most prominent at the interface between development and cancer in gut epithelial cells. TGF-β-family proteins have emerged as dual regulators of the maturation of cells in each of the lineages mentioned above and as suppressors of carcinogenesis.²⁷ When TGF-β signaling is interrupted, the imbalance can result in an undifferentiated phenotype and cancer may ensue.²⁸

Hypothesis

Our observations demonstrating the presence of the TGF-β signaling components TGFBR2 and ELF in human hepatic stem cells led us to explore the impact of these components on liver development and tumorigenesis. We hypothesized that the interruption of the TGF-β pathway resulted in HCC through disruption of a normal pattern of cellular differentiation by hepatic progenitor/stem cells.

We began by examining human HCC tissue specimens from 10 individuals, and observed a small, strongly positive cluster of three to four OCT4+ cells that were negative for TBFBR2 and ELF in 9 of the 10 HCC tissues. Cells with this phenotype were never observed during our surveys of either normal liver or biopsies from regenerating organs. We speculate that these STAT3+/OCT4+ positive human HCC cells that have lost TGF-β signaling proteins have the potential to give rise to HCCs.

To obtain a molecular signature of hepatic cancer that arises when TGF-β signaling is inactivated and to define the intracellular pathways that are engaged, we performed a series of microarray and proteomic analyses in elf^+/−, elf^+/−/itih4^−/−, itih4^−/− tissues. Significantly increased expression of the IL-6 (interleukin-6)/STAT3, WNT (wingless-type MMTV integration site family), and CDK4 (cyclin-dependent kinase 4) signaling pathways were observed. The previously described association of increased IL-6 signaling activity in hepatic tumorigenesis led us to focus our attention on the IL-6 pathway.^29,30

We hypothesized that the increased activity of the IL-6 pathway, occurring in hepatic progenitor/stem cells lacking competent TGF-β circuitry, directly resulted in disturbed growth and differentiation of the liver stem/progenitor cells. To test the hypothesis that the increased IL-6 pathway activity was a critical step in – and not a consequence of – tumorigenesis, we attempted to construct a mouse defective in IL-6 signaling on a heterozygous elf background. However, STAT3-null mice are embryonic lethal and the IL-6–null mice were similarly too fragile to intercross to obtain a homozygous IL-6 null on a heterozygous elf background.^31,32

We have engineered a mouse in which the gene for IL-6–regulated protein, ITIH4 (inter-α-trypsin inhibitor-4), has been deleted. ³³ ITIH4 is a member of a liver-restricted serine protease inhibitor family, expressed in hepatoblasts, and is a biomarker of foregut cancers of uncertain function.^34–36 Mice homozygous for the ITIH4 mutation (itih4^−/−) are normal and fertile, suggesting that the ITIH4 mutation does not show dominant effects. Surprisingly, IL-6/STAT3 signaling is one of the most significantly suppressed pathways we detected in the itih4^−/− liver tissues. Furthermore, hepatocytes remained well differentiated in the itih4^−/− mice. We suspect that ITIH4, an acute phase protein, might be associated in a positive feedback loop with IL-6.^37–39

IL-6 ACTIVITY IS INCREASED IN HCC IN THE ABSENCE OF TGF-β SIGNALING

To explore the role of IL-6 activation in HCC associated with ELF deficiency, we generated mouse intercrosses between elf^+/− mice and itih4^−/− mice. Only 1 of 25 (4%) elf^+/−/itih4^−/− mice developed HCC, compared with 10 of 25 (40%) elf^+/− mice. The tumor that developed in the elf^+/−/itih4^−/− mouse was 0.4 cm³ in size, which was smaller than those in the elf^−/− mice (ie, 3 to 4 cm³). Microarray profiles of itih4^−/− and elf^+/−/itih4^−/− liver tissues indicated a significant suppression of IL-6 signaling. Similarly, immunoblot and immunohistochemical analyses showed that expression of IL-6/STAT3 was decreased in the itih4^−/− and elf^+/−/itih4^−/− liver tissues, whereas IL-6 was activated in elf^+/− mice. STAT3 phosphorylation was also dramatically decreased in the itih4^−/− and elf^+/−/itih4^−/− liver tissues. The disruption of IL-6/STAT3 signaling in elf^+/−/itih4^−/− mutant liver tissues was similar to that seen in the itih4^−/− liver tissues.

Furthermore, in human HCC tissues, we demonstrated markedly elevated expression of STAT3, p-STAT3, and ITIH4 by immunohistochemistry.⁸ In addition, markedly increased STAT3 and p-STAT3 expression was observed in SNU-398 cells derived from a human HCC cell line that does not express TGFBR2 and ELF.

Deregulation of TGF-β signaling potentially contributes to impaired differentiation and allows for the development of cancers, linking the differentiation of stem cells with suppression of carcinogenesis. Further, the development of HCC appears to require IL-6 in addition to the loss of TGF-β signaling. Additionally, increased ITIH4, an IL-6 target, appears to be a critical mediator of hepatocarcinogenesis. This demonstrates an important functional role of the serine protease inhibitor ITIH4 in hepatocellular transformation, previously identified as an IL-6–regulated biomarker for cancers of the foregut. Added support for this concept comes from current successful anti-cancer strategies aimed at blocking IL-6 signaling. Modulation of IL-6/STAT3 signaling may potentially manipulate stemness and induce cell differentiation rather than cell death, thereby reducing HCC formation.

CONCLUSION

Targeting of signaling pathways critical for the proliferation and survival of cancer stem cells may represent a powerful therapeutic strategy. We have previously reported that mice spontaneously develop HCC when TGF-β signaling is disrupted.¹² Several TGF-β signaling components are established tumor suppressors with the ability to constrain cell growth and inhibit cancer development at its early stages. Inactivation of at least one of these components, such as TGFBR2, SMAD2, or the common mediator SMAD4, occurs in almost all gastrointestinal tumors.^27,40,41 Given the important role of TGF-β signaling in liver development as well as in suppression of hepatocarcinogenesis, searching for signaling pathways that interact with TGF-β signaling may reveal mechanisms of cancer stem cell self-renewal, differentiation, and apoptosis. Modulation of the IL-6/STAT3 pathway, a major pathway in stem cell renewal, is a potentially attractive target for reducing HCC formation and offers a new avenue for the development of novel therapies with minimal systemic toxicities.