Smad3 Phospho-Isoform Signaling in Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis with insulin resistance, oxidative stress, lipotoxicity, adipokine secretion by fat cells, endotoxins (lipopolysaccharides) released by gut microbiota, and endoplasmic reticulum stress. Together, these factors promote NAFLD progression from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and eventually end-stage liver diseases in a proportion of cases. Hepatic fibrosis and carcinogenesis often progress together, sharing inflammatory pathways. However, NASH can lead to hepatocarcinogenesis with minimal inflammation or fibrosis. In such instances, insulin resistance, oxidative stress, and lipotoxicity can directly lead to liver carcinogenesis through genetic and epigenetic alterations. Transforming growth factor (TGF)-β signaling is implicated in hepatic fibrogenesis and carcinogenesis. TGF-β type I receptor (TβRI) and activated-Ras/c-Jun-N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3 to create two phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TβRI/pSmad3C signaling terminates cell proliferation, while constitutive Ras activation and JNK-mediated pSmad3L promote hepatocyte proliferation and carcinogenesis. The pSmad3L signaling pathway also antagonizes cytostatic pSmad3C signaling. This review addresses TGF-β/Smad signaling in hepatic carcinogenesis complicating NASH. We also discuss Smad phospho-isoforms as biomarkers predicting HCC in NASH patients with or without cirrhosis

Smad Phospho-Isoforms for Hepatocellular Carcinoma Risk Assessment in Patients with Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) sometimes occurs in mildly fibrotic livers, while HCC incidence in NASH-related cirrhosis is lower than and less predictable than in hepatitis C virus (HCV)-related cirrhosis. Transforming growth factor (TGF)-β signaling in hepatocytic nuclei is implicated in fibrosis and carcinogenesis. TGF-βtype I receptor (TβRI) and c-Jun N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3, resulting in 2 distinct phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). In mature hepatocytes, oncogenic signaling via the JNK/pSmad3L pathway antagonizes signaling via the tumor-suppressive TβRI/pSmad3C pathway. We immunohistochemically examined domain-specific Smad3 phosphorylation in liver biopsy specimens from 30 NASH patients representing different fibrotic stages and 20 chronically infected hepatitis C patients as controls, correlating Smad3 phosphorylation with clinical course. HCC occurred during follow-up in 11 of 12 NASH patients with abundant pSmad3L and limited pSmad3C but in only 2 of 18 with limited pSmad3L. In contrast, HCC developed in 12 of 15 NASH patients with limited pSmad3C but only 1 of 15 with abundant pSmad3C. Two of fourteen NASH patients with mild fibrosis developed HCC, their hepatocytic nuclei showed abundant pSmad3L and limited pSmad3C. Five of sixteen patients with severe fibrosis did not develop HCC, their hepatocytic nuclei showed limited pSmad3L and abundant pSmad3C. Smad phospho-isoforms may represent important biomarkers predicting HCC in NASH and potential therapeutic targets for preventing NASH-related HCC

Phospho-Smad3 signaling is predictive biomarker for hepatocellular carcinoma risk assessment in primary biliary cholangitis patients

Introduction: Patients with primary biliary cholangitis (PBC) are at increased risk for development of hepatocellular carcinoma (HCC), particularly in the presence of comorbidities such as excessive alcohol consumption. Although liver fibrosis is an important risk factor for HCC development, earlier predictors of future HCC development in livers with little fibrosis are needed but not well defined. The transforming growth factor (TGF)-β/Smad signaling pathway participates importantly in hepatic carcinogenesis. Phosphorylated forms (phospho-isoforms) in Smad-related pathways can transmit opposing signals: cytostatic C-terminally-phosphorylated Smad3 (pSmad3C) and carcinogenic linker-phosphorylated Smad3 (pSmad3L) signals. Methods and results: To assess the balance between Smad signals as a biomarker of risk, we immunohistochemically compared Smad domain-specific Smad3 phosphorylation patterns among 52 PBC patients with various stages of fibrosis and 25 non-PBC patients with chronic hepatitis C virus infection. HCC developed in 7 of 11 PBC patients showing high pSmad3L immunoreactivity, but in only 2 of 41 PBC patients with low pSmad3L. In contrast, 9 of 20 PBC patients with minimal Smad3C phosphorylation developed HCC, while HCC did not occur during follow-up in 32 patients who retained hepatic tumor-suppressive pSmad3C. Further, PBC patients whose liver specimens showed high pSmad3L positivity were relatively likely to develop HCC even when little fibrosis was evident. Conclusion: In this study, Smad phospho-isoform status showed promise as a biomarker predicting likelihood of HCC occurrence in PBC. Eventually, therapies to shift favorably Smad phospho-isoforms might decrease likelihood of PBC-related HCC

Clinico-Pathological Importance of TGF-β/Phospho-Smad Signaling during Human Hepatic Fibrocarcinogenesis

Chronic viral hepatitis is a global public health problem, with approximately 570 million persons chronically infected. Hepatitis B and C viruses increase the risk of morbidity and mortality from liver cirrhosis, hepatocellular carcinoma (HCC), and extrahepatic complications that develop. Hepatitis virus infection induces transforming growth factor (TGF)-β, which influences microenvironments within the infected liver. TGF-β promotes liver fibrosis by up-regulating extracellular matrix production by hepatic stellate cells. TGF-β is also up-regulated in patients with HCC, in whom it contributes importantly to bringing about a favorable microenvironment for tumor growth. Thus, TGF-β is thought to be a major factor regulating liver fibrosis and carcinogenesis. Since TGF-β carries out regulatory signaling by influencing the phosphorylation of Smads, we have generated several kinds of phospho-specific antibodies to Smad2/3. Using these, we have identified three types of phospohorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C), linker phosphorylated Smad2/3 (pSmad2L and pSmad3L), and dually phosphorylated Smad3 (pSmad2L/C and pSmad3L/C). TGF-β-mediated pSmad2/3C signaling terminates cell proliferation; on the other hand, cytokine-induced pSmad3L signaling accelerates cell proliferation and promotes fibrogenesis. This review addresses TGF-β/Smad signal transduction in chronic liver injuries and carcinogenic processes. We also discuss the reversibility of Smad signaling after antiviral therapy

Narrowing the Patient–Physician Gap Based on Self-Reporting and Monthly Hepatologist Feedback for Patients With Alcohol-Related Liver Disease: Interventional Pilot Study Using a Journaling Smartphone App

BackgroundScreening and intervention for alcohol use disorders (AUDs) are recommended to improve the prognosis of patients with alcohol-related liver disease (ALD). Most patients’ smartphone app diaries record drinking behavior for self-monitoring. A smartphone app can be expected to also be helpful for physicians because it can provide rich patient information to hepatologists, leading to suitable feedback. We conducted this prospective pilot study to assess the use of a smartphone app as a journaling tool and as a self-report–based feedback source for patients with ALD. ObjectiveThe aims of this study were assessment of whether journaling (self-report) and self-report–based feedback can help patients maintain abstinence and improve liver function data. MethodsThis pilot study used a newly developed smartphone journaling app for patients, with input data that physicians can review. After patients with ALD were screened for harmful alcohol use, some were invited to use the smartphone journaling app for 8 weeks. Their self-reported alcohol intake, symptoms, and laboratory data were recorded at entry, week 4, and week 8. Biomarkers for alcohol use included gamma glutamyl transferase (GGT), percentage of carbohydrate-deficient transferrin to transferrin (%CDT), and GGT-CDT (GGT-CDT= 0.8 × ln[GGT] + 1.3 × ln[%CDT]). At each visit, their recorded data were reviewed by a hepatologist to evaluate changes in alcohol consumption and laboratory data. The relation between those outcomes and app usage was also investigated. ResultsOf 14 patients agreeing to participate, 10 completed an 8-week follow-up, with diary input rates between 44% and 100% of the expected days. Of the 14 patients, 2 withdrew from clinical follow-up, and 2 additional patients never used the smartphone journaling app. Using the physician’s view, a treating hepatologist gave feedback via comments to patients at each visit. Mean self-reported alcohol consumption dropped from baseline (100, SD 70 g) to week 4 (13, SD 25 g; P=.002) and remained lower at week 8 (13, SD 23 g; P=.007). During the study, 5 patients reported complete abstinence. No significant changes were found in mean GGT and mean %CDT alone, but the mean GGT-CDT combination dropped significantly from entry (5.2, SD 1.2) to the week 4 visit (4.8, SD 1.1; P=.02) and at week 8 (4.8, SD 1.0; P=.01). During the study period, decreases in mean total bilirubin (3.0, SD 2.4 mg/dL to 2.4, SD 1.9 mg/dL; P=.01) and increases in mean serum albumin (3.0, SD 0.9 g/dL to 3.3, SD 0.8 g/dL; P=.009) were recorded. ConclusionsThese pilot study findings revealed that a short-term intervention with a smartphone journaling app used by both patients and treatment-administering hepatologists was associated with reduced drinking and improved liver function. Trial RegistrationUMIN CTR UMIN000045285; http://tinyurl.com/yvvk38t