Toshihito Seki

Validation of a New Prognostic Staging System for Hepatocellular Carcinoma: the JIS Score Compared With the CLIP Score

The Japan Integrated Staging score (JIS score), which combines the Child‐Turcotte‐Pugh classification and tumor‐node‐metastasis staging, has been proposed as a better prognostic staging system for hepatocellular carcinoma (HCC) than the Cancer of the Liver Italian Program (CLIP) scoring system. In this study, validation was performed among a larger patient population. A total of 4,525 consecutive patients with HCC who had been diagnosed at five institutions were included. Stratification ability, prognostic predictive power, and reproducibility were analyzed and compared with results from the CLIP scoring system. Only 45% (1,951 of 4,525) of all patients were categorized as early stage HCC according to JIS score (0 or 1), whereas 63% (2,878 of 4,525) of the patients were categorized as having a CLIP score of 0 or 1. Significant differences in survival curves were not observed among CLIP scores 3 to 6. In contrast, survival curves showed significant differences among all the JIS scores. The same JIS scoring subgroups showed a similar prognosis, and good internal reproducibility was observed in each of the institutions. Multivariate analysis of the prognosis in all 4,525 patients proved the JIS score to be the best prognostic factor. Furthermore, the Akaike information criteria proved that the JIS scoring system was statistically a better model for predicting outcome than the CLIP scoring system. In conclusion, the stratification ability and prognostic predictive power of the JIS score were much better than that of the CLIP score and were simple to obtain and remember. (HEPATOLOGY 2004; 40:1396–1405.)

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor β signaling, promoting cirrhosis and hepatocellular carcinoma†

Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF‐β) activates not only TGF‐β type I receptor (TβRI) but also c‐Jun N‐terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). Whereas the TβRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21WAF1 transcription, JNK/pSmad3L‐mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI‐1). We studied the domain‐specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV‐infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI‐1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21WAF1 decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro‐inflammatory cytokine interleukin‐1β stimulated the pSmad3L/PAI‐1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF‐β‐dependent tumor‐suppressive activity by the pSmad3C/p21WAF1 pathway. Conclusion: These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF‐β signaling from tumor‐suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC. (HEPATOLOGY 2007;46:48–57.)

TGF-β and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-β (TGF-β) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-β treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-β or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-β signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-β allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-β-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-β signals. Moreover, a combined treatment with HGF and TGF-β led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-β-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-β transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Transforming Growth Factor-β and Platelet-Derived Growth Factor Signal via c-Jun N-Terminal Kinase-Dependent Smad2/3 Phosphorylation in Rat Hepatic Stellate Cells after Acute Liver Injury

After liver injury, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-β-triggered cascade are not completely understood. TGF-β signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl4 administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of α-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-β and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-β and PDGF signals. Moreover, treatment of HSCs with both TGF-β and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-β and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Multicenter prospective analysis of newly diagnosed hepatocellular carcinoma with respect to the percentage of Lens culinaris agglutinin-reactive α-fetoprotein1

Background and Aim: The Lens culinaris agglutinin‐reactive fraction of α‐fetoprotein (AFP‐L3) has been reported to be a highly useful marker for hepatocellular carcinoma (HCC) compared with a conventional serum AFP concentration, which allows earlier detection of HCC compared with using other imaging modalities and predicting prognosis after therapy. A collaborative prospective study involving nine Japanese hospitals was conducted to analyze the relationships between the tumor characteristics of a HCC patient and the percentage of AFP‐L3/AFP total at the initial detection.

Combination therapy with transcatheter arterial chemoembolization and percutaneous microwave coagulation therapy for hepatocellular carcinoma.

A small number of microwave electrode insertions and microwave irradiations were used to obtain complete tumor necrosis in hepatocellular carcinomas (HCC) measuring > 2.0 cm but ≤ 3.0 cm in greatest dimension. The efficacy of combining transcatheter arterial chemoembolization (TACE) with subsequent percutaneous microwave coagulation therapy (PMCT) was assessed in this study.

Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-β signaling from tumor suppression to oncogenesis in early chronic hepatitis B†

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor β (TGF‐β) signaling involves both tumor suppression and oncogenesis. TGF‐β activates TGF‐β type I receptor (TβRI) and c‐Jun N‐terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3‐mediated signaling between tumor suppression and oncogenesis in HBx‐expressing hepatocytes indicated that TβRI‐dependent pSmad3C transmitted a tumor‐suppressive TGF‐β signal, while JNK‐dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L‐ and pSmad3C‐mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx‐activated JNK/pSmad3L/c‐Myc oncogenic pathway was enhanced, while the TβRI/pSmad3C/p21WAF1 tumor‐suppressive pathway was impaired as human and mouse HBx‐associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor‐suppressive pSmad3C developed HCC within 12 years. Conclusion: HBx shifts hepatocytic TGF‐β signaling from the tumor‐suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV‐infected liver specimens should prove clinically useful for predicting risk of HCC. (HEPATOLOGY 2009.)

Reversible Smad-Dependent Signaling between Tumor Suppression and Oncogenesis

Cancer cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor-beta (TGF-beta) together with stimulation of its oncogenic activity as in Ras-transformed cells; however, molecular mechanisms remain largely unknown. TGF-beta activates both its type I receptor (TbetaRI) and c-Jun NH2-terminal kinase (JNK), which phosphorylate Smad2 and Smad3 at the COOH-terminal (pSmad2/3C) and linker regions (pSmad2/3L). Here, we report that Ras transformation suppresses TbetaRI-mediated pSmad3C signaling, which involves growth inhibition by down-regulating c-Myc. Instead, hyperactive Ras constitutively stimulates JNK-mediated pSmad2/3L signaling, which fosters tumor invasion by up-regulating plasminogen activator inhibitor-1 and matrix metalloproteinase-1 (MMP-1), MMP-2, and MMP-9. Conversely, selective blockade of linker phosphorylation by a mutant Smad3 lacking JNK-dependent phosphorylation sites results in preserved tumor-suppressive function via pSmad3C in Ras-transformed cells while eliminating pSmad2/3L-mediated invasive capacity. Thus, specific inhibition of the JNK/pSmad2/3L pathway should suppress cancer progression by shifting Smad-dependent signaling from oncogenesis to tumor suppression.

Smad2 and Smad3 Phosphorylated at Both Linker and COOH-Terminal Regions Transmit Malignant TGF-β Signal in Later Stages of Human Colorectal Cancer

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

Down-regulation of TGF-β receptors in human colorectal cancer : implications for cancer development

Many colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-β (TGF-β). TGF-β also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-β and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-β1, type I and type II receptor mRNA and immunohistochemical staining of TGF-β1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-β receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-β receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-β1 mRNA and the protein were detected in all colorectal cancers. TGF-β receptor mRNAs and TGF-β1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-β stimulation. The escape of human colon cancer from TGF-β -mediated growth inhibition by down-regulation of TGF-β receptors as well as the effects of TGF-β on stroma formation and angiogenesis indicate a possible role for TGF-β in the progression of colon cancer in an intact host.