Unrestrained activation of the signal transducer and activator of transcription (Stat) signaling cascade frequently occurs in a wide variety of tumor types.1-3 In this pathway, binding of extracellular ligands such as cytokines, hormones, and growth factors to their specific receptors leads to the activation of Janus tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2).2, 3 These tyrosine kinases are able to phosphorylate a single tyrosine residue of each Stat protein. Similarly, nonreceptor tyrosine kinase such as Abelson murine leukemia viral oncogene homolog (c-ABL) and Schmidt-Ruppin A-2 viral oncogene homolog (c-SRC) can directly phosphorylate Stat proteins in the absence of ligand-induced receptor signaling.2, 3 Once phosphorylated, Stat proteins homodimerize or heterodimerize and translocate into the nucleus, where they activate a number of target genes involved in cell proliferation, survival, angiogenesis, invasion, and metastasis.2, 3 Nuclear localization of Stat proteins also results in the transcriptional activation of three families of inhibitory proteins, namely, the protein inhibitors of activated Stats (PIAS), the SH2-containing phosphatases (SHP), and the suppressors of cytokine signaling (SOCS).2-4 Induction of PIAS, SHP, and SOCS proteins following Stat activation represents an efficient negative feedback loop mechanism limiting the magnitude of Stat effects on target cells.2-4

In the liver, the best-characterized member of the Stat pathway is Stat3. Under physiological conditions, Stat3 is required for liver regeneration due to its ability to stimulate hepatic cell proliferation and survival.5 In hepatocellular carcinoma (HCC), presumably due to the loss of PIAS, SHP, and SOCS genes via promoter hypermethylation and/or the elevated synthesis of cytokines and growth factors that are able to activate the Stat cascade, Stat3 activity is almost ubiquitously elevated and unconstrained.6-8 Of note, high levels of Stat3 were detected in a vast HCC collection regardless of the liver tumor etiology, indicating that uncontrolled activation of Stat3 is a universal event in hepatocarcinogenesis.6 Also, it has been found that Stat3 levels are directly correlated with HCC biological aggressiveness and inversely with survival of patients with HCC, pointing to an important role of Stat3 in liver cancer prognosis.6, 7 Subsequent in vitro and in vivo studies have shown that suppression of Stat3 enhances the chemosensitivity of HCC cells and suppresses the growth and metastatic properties of human HCC in xenograft models.9, 10 Also, hepatocyte-specific Stat3-deficient mice were found to exhibit more than a six-fold reduction in HCC load compared to Stat3 wild-type mice when subjected to diethylnitrosamine treatment.8 In the latter model, disruption of the inhibitor of nuclear factor kappa-B kinase subunit beta–nuclear factor kappa-B (NF-κB) axis was the causative event leading to aberrant Stat3 activation, suggesting that the NF-κB pathway is a major negative regulator of Stat3 in HCC.8 Furthermore, carcinogen-induced HCC development in mice was significantly enhanced by heterozygous deletion of the Stat3 inhibitor SOCS1.11 Similarly, genetic knockout of the SOCS3 gene in the mouse liver resulted in prolonged Stat3 phosphorylation and increased expression of Stat3 target genes, ultimately leading to rapid HCC development when SOCS3 knockout mice were subjected to chemically induced hepatocarcinogenesis.12, 13 Altogether, these data establish Stat3 as a bona fide oncogene in the liver and suggest that Stat3 might be a critical target for innovative therapeutical approaches in HCC.

In this issue of HEPATOLOGY, the report by Schneller et al. provides new, significant experimental data on the role of Stat3 in liver carcinogenesis.14 In particular, the authors investigate the function of Stat3 in HCC progression in the presence of other molecular alterations, namely the activation of the rat sarcoma viral oncogene homolog (Ras) oncogenic cascade and the loss of expression of the p14ARF/p19ARF tumor suppressor gene. Unrestricted activation of the Ras pathway via suppression of Ras cellular inhibitors and epigenetic silencing of p14ARF (the human homolog of mouse p19ARF) are two recurrent molecular events in human hepatocarcinogenesis.6, 15 In their present investigation, Schneller et al. used an established mouse tumor transplantation model lacking p19ARF and transformed by oncogenic v-Ha-Ras that was retrovirally infected with various Stat3 gene variants. Interestingly, the results show that constitutive active Stat3 (referred to as Ca-Stat3) exerts a tumor-suppressive role in Ras-transformed p19ARF−/− hepatocytes, whereas the expression of Stat3 lacking phosphorylation at the Tyr-705 and Ser-727 residues (referred to as U-Stat3) enhances liver tumor formation.14 Similarly, Ras-transformed hepatocytes lacking both Stat3 and p19ARF displayed an increase in tumor growth compared to those expressing Stat3, implying an unexpected tumor suppressor activity of Stat3 in cells lacking p19ARF.14 Of note, endogenous expression of p19ARF led to either augmented or reduced HCC progression after expression of Ca-Stat3 or U-Stat3, respectively, in Ras-transformed hepatocytes. Furthermore, the analysis of diethylnitrosamine-induced liver tumors showed a remarkable up-regulation of p19ARF in Stat3 wild-type mice, whereas a significant reduction of p19ARF levels characterized HCC in Stat3-deleted mice following the same carcinogenesis protocol.14

The results obtained in the mouse models were successfully recapitulated by Schneller et al. in human HCC cells. Indeed, silencing of p14ARF in the Hep3B cell line via short hairpin RNAs was associated with reduced tyrosine phosphorylated levels of Stat3 during tumor growth in order to circumvent the tumor-suppressive function of Stat3.14 Subsequent experiments aimed at inhibiting the Janus tyrosine kinases revealed that Jak caused tyrosine phosphorylation and activation of Stat3 independently of p14ARF levels, implying that p14ARF modulates the oncogenic function of tyrosine-phosphorylated Stat3 downstream of Jak.14

In summary, this interesting study implies the existence of pro- and antioncogenic roles played by Stat3 in Ras-induced liver cancer that directly depend on p19ARF/p14ARF expression (Fig. 1). In accordance with the results obtained by Schneller et al., it has been previously shown that the HepG2 and PLC/PRF/5 HCC cell lines, which are p14ARF-negative, are partly resistant to treatment with the Stat3 inhibitor NSC 74859. On the other hand, Huh-7 and SNU-398 cells, which express p14ARF, showed a remarkable decline in cell proliferation after the same therapeutic approach.10 Therefore, the present data also envisage a predictive value of Stat3 and p14ARF status in the treatment of human HCC with Stat3 inhibitors.

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Figure 1. Schematic representation of the hypothetical interplays between Stat3 and p19ARF (and its human homolog, p14ARF) in hepatocarcinogenesis induced by activated Ras. (A) In the presence of p19ARF (p19ARF+/+), overexpression of constitutive active Stat3 (Ca-Stat3) promotes liver tumor development, whereas both genetic disruption of Stat3 (Stat3−/−) or overexpression of unphosphorylated Stat3 (U-Stat3) drive liver tumor suppression. In this genetic background, p19ARF sequesters an unknown factor termed ARF-X. (B) In the absence of p19ARF (p19ARF−/−), U-Stat3 functionally interacts with ARF-X to trigger an oncogenic program. Similarly, genetic inactivation of Stat3 induces hepatocarcinogenesis in the p19ARF−/− background. In striking contrast, overexpression of Ca-Stat3 induces tumor suppression, presumably through the activation of an alternative group of Stat3-specific target genes with antioncogenic activity.

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The apparently paradoxical tumor-suppressive role of Stat3 in liver cancer is in accordance with cumulative findings in other tumor types. In glioblastoma, deficiency of the phosphatase and tensin homolog tumor suppressor (PTEN) led to astrocyte malignant transformation upon Stat3 inhibition, arguing for an antioncogenic function of Stat3.16 Similarly, overexpression of Ca-Stat3 was able to consistently block c-myc–induced transformation of p53−/− mouse fibroblasts.17 A dual role of Stat3 was also described in a well-characterized mouse model of intestinal cancer. In ApcMin/+ (adenatomous polyposis coli) mice, Stat3 promoted early adenoma formation, whereas Stat3 deficiency triggered rapid tumor progression and invasion.18 The latter observation suggests that, similar to other genes, Stat3 might play diverse and seemingly paradoxical roles in cancer, reflecting specific requirements during different stages of tumorigenesis. According to this hypothesis, inactivation of the E-cadherin (CDH1) tumor suppressor gene was predominantly detected in HCC with no vascular invasion, whereas increased CDH1 expression was a predominant feature of liver tumors with marked vascular invasion and adverse prognosis.19 Similarly, although lysyl oxidase (LOX) methylation and down-regulation has been found in various tumor types, LOX up-regulation is a hallmark of highly aggressive tumors, due to its ability to confer migration and invasive advantages to cancer cells during hypoxia-induced metastasis.20

Although the study by Schneller et al. has significantly improved our knowledge on the role, or roles, of Stat3 in liver cancer, many questions on this topic remain unanswered. Indeed, although it is clear that Stat3 activation possesses oncogenic and antioncogenic properties in the liver, the genes that cooperate with p14ARF/p19ARF to modulate Stat3 functions remain poorly delineated. In their work, Schneller et al. hypothesize the presence of a putative transcription factor, designated as ARF-X, which might mediate the p14ARF/p19ARF control over Stat3 transcriptional activity.14 Additional experiments are required to address this issue. Because a negative modulation by the NF-κB over Stat3 in experimental hepatocarcinogenesis has been demonstrated,8 it would be significant to determine whether NF-κB contributes to Stat3 regulation by p14ARF/p19ARF. Furthermore, the genes that modulate the function of Stat3 in the liver independent of p14ARF/p19ARF deserve additional investigation. In this regard, preliminary data by Schneller et al. suggest that the tumor-suppressive role of Stat3 in the liver is independent of the activity of both p16INK4A and p53 tumor suppressors.14 Also, it is worth mentioning that HCC cells with a disrupted transforming growth factor beta pathway have been found to be peculiarly sensitive to treatments with Stat3 inhibitors, implying the existence of a cross-talk between these two signaling cascades.10 Finally, it will be pivotal to determine the cellular (proliferation, apoptosis, senescence) events as well as the molecular mechanisms whereby the Stat3 function is modulated by its interactors in liver cancer.

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Since this Editorial was originally submitted, a recent study from Wang et al.21 has shown that hepatocyte-specific Stat3 knockout mice are resistant to DEN-induced hepatic carcinogenesis but more susceptible to CCl4-induced liver fibrosis and liver tumor development than wild-type mice, suggesting that Stat3 could either promote or inhibit liver tumorigenesis in different models. These observations further substantiate the notion of the paradoxical oncogenic and tumor suppressive role of Stat3 in liver cancer.


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