Inokuchi S, Aoyama T, Miura K, Osterreicher CH, Kodama Y, Miyai K, et al. Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis. Proc Natl Acad Sci U S A 2010;107:844-849. (Reprinted with permission.)
TGF-β-activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1ΔHEP) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.
Bettermann K, Vucur M, Haybaeck J, Koppe C, Janssen J, Heymann F, et al. TAK1 suppresses a NEMO-dependent but NF-kappaB-independent pathway to liver cancer. Cancer Cell 2010;17:481-496. (Reprinted with permission.)
The MAP3-kinase TGF-β-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-κB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-κB-independent functions of the IκB-kinase (IKK)-subunit NF-κB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-κB-independent function of NEMO in parenchymal liver cells.
Hepatocellular carcinoma (HCC) is one of the most common cancers and accounts for 600,000 deaths annually in the world.1 In the United States, the mortality due to HCC has doubled in the last 25 years. The increased frequency of HCC is due mainly to viral infections, but also emerging diseases such as nonalcoholic steatohepatitis.2 The impact of HCC on global health is further determined by its poor prognosis. The current 5-year survival rate of individuals with HCC is only 8.9%, making it the second most lethal malignancy.1
Understanding the molecular mechanisms of HCC development is expected to yield much-needed new agents for its prevention or eradication. Previous research suggests that HCC derives from dysplastic hepatocytes, which in turn are the product of chronic liver injury, inflammation, and fibrosis.1 Now Inokuchi et al.3 and Bettermann et al.4 have identified transforming growth factor (TGF) β-activated kinase 1 (TAK1) as an essential inhibitor of this fatal series of events in hepatocytes.
TAK1 belongs to the mitogen-activated protein kinase kinase kinase (MAP3K) family and acts as a mediator of stress, apoptosis, and inflammatory signals in the liver. Upon ligand activation, Toll-like receptor/interleukin-1 receptor (TLR/IL1R) and tumor necrosis factor receptor (TNFR) recruit and phosphorylate TAK1 through TNFR-associated factors (TRAFs). Phosphorylated TAK1 activates IκB kinase (IKK) and MAP kinase kinase 4/7 (MKK4/7), leading to the activation of nuclear factor-κ B (NF-κB) and c-Jun N-terminal kinase (JNK), respectively. NF-κB and JNK are important for mounting an immune response and causing tissue inflammation. Moreover, the current belief is that NF-κB protects hepatocytes from death, whereas JNK promotes apoptosis. Consequently, NF-κB and JNK act as regulators of injury, death, proliferation, and dysplastic transformation of hepatocytes,5, 6 making any molecule that activates these pathways, such as TAK1, a potential therapeutic target.
A role for TAK1 in the liver was first reported in 2001 in two studies from the Brenner group describing how TAK1 activated JNK and maintained hepatocyte quiescence7 and controlled the proliferation of stellate cells.8 One year later, Liedtke et al.9 elucidated TAK1's role in hepatocyte apoptosis by showing increased apoptosis after inhibition of the TAK1/JNK pathway. Because of the lack of mice with inactivation of Map3k7, the gene encoding TAK1, these studies relied on adenoviruses to express dominant-negative sequences in mice. Despite these intriguing findings, TAK1 was one of the least studied MAP3Ks in the last decade. However, the generation of mice carrying floxed Map3k7 alleles restored interest in this regulator.10 For example, Tang et al. crossed these mice with mice transgenic for Mx1-Cre, an interferon-inducible Cre recombinase. Upon injection of the interferon inducer polyinosinic:polycytidylic acid, Cre-mediated recombination resulted in TAK1 deficiency mainly in hematopoietic cells, but also in hepatocytes.11 Surprisingly, this caused cholestasis, massive hepatocyte apoptosis, and destruction of the normal liver architecture followed by death from liver failure 8-10 days after polyinosinic:polycytidylic acid injection. These results provided a starting point for two studies published this year that examined the function of TAK1 specifically in the liver. The first study by Inokuchi et al. appeared in Proceedings of the National Academy of Sciences of the U.S.A.3 and was quickly followed by the study from Bettermann et al. published in Cancer Cell.4
Both studies focused on the role of TAK1 in HCC development. Inokuchi et al. crossed mice carrying floxed Map3k7 alleles with mice expressing Cre from an albumin enhancer/promoter construct (Table 1).12 The resulting hepatocyte-specific TAK1 deficiency led to the spontaneous emergence of liver tumors as early as 4 months after birth, with 80% of the mice harboring large tumors at 9 months of age. Fetal liver genes, such as α-fetoprotein and the maternally imprinted noncoding transcript H19, were reactivated in the tumors, suggesting that they were HCCs. Bettermann et al. used a Cre-transgenic mouse with additional α-fetoprotein enhancer elements,13 leading to hepatocyte dysplasia and high penetrance of liver tumors that, similar to the study by Inokuchi et al., appeared as early as 16 weeks of age (Table 1). Histological and molecular analyses identified these tumors as HCCs that exhibited a remarkably coherent chromosomal aberration pattern.
|Inokuchi et al.3||Bettermann et al.4|
|Floxed Map3k7 exon 2 (adenosine triphosphate–binding site of the kinase domain of TAK1)10|
|Cre transgene driven by mouse albumin promoter and albumin enhancer sequences12||Cre transgene driven by mouse albumin promoter and albumin and α-fetoprotein enhancer sequences13|
|Lack of NF-κB activation after stimulation with TNFα in vitro or lipopolysaccharide in vivo|
|Strong basal JNK activation in whole liver*||Strong JNK activation in whole liver after stimulation with lipopolysaccharide|
|Impaired JNK activation after stimulation with TNFα in primary hepatocytes|
|Spontaneous liver injury*|
|Loss of normal liver architecture|
|Biliary epithelial cell loss, bile duct paucity, and cholestasis†|
|Compensatory hepatocyte proliferation|
|Nodular hepatocyte dysplasia†|
|80% HCC at 9 months of age, beginning at 4 months of age||88% HCC between 16 and 33 weeks of age†|
|No lethality at 12 months||100% lethality at 40 weeks†|
Inokuchi et al. and Bettermann et al. identified hepatocyte injury and liver inflammation as the probable cause of spontaneous HCC formation in TAK1-deficient mice. Injury and inflammation led to hepatocyte apoptosis, which in turn caused compensatory proliferation of the surviving hepatocytes. This phenotype resembles previous findings made by Bradham et al. after expressing a dominant-negative TAK1 in the liver.7 Because accelerated hepatocyte turnover in the context of chronic liver injury or inflammation is believed to represent the mechanism by which HCC develops in human liver diseases, TAK1-deficient mice can be considered a truthful human hepatocarcinogenesis model. In support of this assessment, both groups observed progressive liver fibrosis, another hallmark of human liver cancer formation.
A striking difference between the two TAK1-deficient mouse models was the progressive loss of biliary epithelial cells and bile ducts found by Bettermann et al., causing marked cholestasis and death of their mice by 40 weeks of age. Similarly, cholestasis was previously observed in mice with floxed Map3k7 alleles transgenic for Mx1-Cre.11 In the Cre-transgenic mice used by Bettermann et al., Cre expression is known to be initiated in fetal liver progenitors before differentiation into hepatocytes or biliary epithelial cells.13 Thus, deficiency of TAK1 can be expected to affect both adult hepatocytes and biliary epithelial cells in this model. Similarly, the broad expression pattern of the Mx1-Cre transgene likely affords disruption of floxed Map3k7 in both parenchymal liver cell types. Importantly, these findings suggest that biliary epithelial cells are as sensitive to TAK1 deficiency as are hepatocytes.
To gain further insight into the molecular mechanisms revolving around TAK1's function in hepatocytes, the researchers generated mice that were additionally deficient for genes acting upstream or downstream of TAK1. By crossing their mice with mice ubiquitously lacking TNFR1, Inokuchi et al. showed that hepatocyte injury, apoptosis, and fibrosis in mice with TAK1-deficient hepatocytes are triggered by TNFα signaling. By deleting the regulatory IKK-subunit NEMO (NF-κB essential modulator) in TAK1-deficient hepatocytes and biliary epithelial cells in vivo, Bettermann et al. discovered that cholestasis and hepatocyte dysplasia and necrosis, but not hepatocyte injury, apoptosis, and compensatory proliferation, occur only in the presence of NEMO. Remarkably, the altered phenotype observed in response to additional loss of NEMO prevented early-onset HCC and death in these mice. Because NF-κB signaling was clearly blocked in TAK1-deficient mice, the results suggest that TAK1 suppresses a previously unrecognized NF-κB–independent, procarcinogenic effect of NEMO.
Another finding by Bettermann et al., namely the strong activation of JNK in livers of mice with TAK1-deficient hepatocytes and biliary epithelial cells after lipopolysaccharide injection, appears contradictory to previous reports of TAK1-dependent JNK activation7. Here, the study by Inokuchi et al. offers an explanation: Although JNK was activated in livers of mice with hepatocyte-specific deficiency of TAK1, stimulating hepatocytes isolated from these mice with TNFα in vitro had no effect on JNK. Moreover, Kupffer cell depletion blunted JNK activation in vivo, suggesting that nonparenchymal liver cells were likely responsible for JNK activation in whole liver samples.
The studies by Inokuchi et al. and Bettermann et al. identify TAK1 as an essential inhibitor of hepatocarcinogenesis. In its absence, the fatal interplay between chronic liver injury and inflammation, hepatocyte death and regeneration is unleashed and takes its course. The findings significantly improve our understanding of how inflammatory and stress-related signaling pathways affect liver cancer formation and suggest new therapeutic targets.