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

Round 1

Reviewer 1 Report

The manuscript reports interesting results on the relationship between the differential phosphorylation of Smad3 protein and the progression of NASH and HCV to hepatocellular carcinoma. Smad3 mediates signalling pathways in the nucleus which can enhance either tumorigenesis or tumour suppression, depending on the input signal.

Comments

While the trends observed in differential phosphorylation are reasonably clear (Figs 2 to 5) the number of patient liver biopsy samples is relatively low (30 NASH, 20 HCV). The observed trends are reasonably descriptive. It would seem best therefore to be more circumspect about the main conclusion and to write in Abstract and Discussion and other relevant parts that Smad isoforms “MAY” represent biomarkers predicting HCC in NASH.

I think it will take some time for the informed reader who is not an expert/ immediately familiar in this field, to understand the rationale and results and main conclusions of this manuscript. For example, the first paragraph of the Discussion is a reasonably lengthy summary of the results as they stand, which is good, but it is really difficult to see the main conclusions. The Introduction section might be more clear if it included a precise statement of the aims of the study. The context would be so much easier to see if there was included a Scheme/Figure of the main pathways involving Smad3, analogous to that made by this group in one of their previous papers, Yoshida et al 2018, Fig. 1. The last paragraph of the Introduction seems somewhat imprecise and unclear. It needs appropriate references and a clear distinction between what is already published, what is planned for present study, and what has been found in present study.

The idea proposed by the authors (Smad phosphorylation as a predictor---repressor, oncogenesis) seems valuable and interesting, but it seems buried in a lot of words which are difficult to understand.

Results first paragraph. Part of this does not really make sense eg “…distribution of Smad phos… in NASH…” Can the authors make a really clear simple understandable statement of what the experiments have involved?

Last paragraph of Results and Fig. 6. This paragraph and the Scheme Fig. 6 seem to me to need some sorting out and precision. There needs to be a clear statement/ distinction between the results obtained in present study, previous results of others, and the inclusion of references to previous results. The Scheme (Fig 6) does not really make sense or does not seem to mean anything over and above what is stated in the text. Suggest omit Scheme or clarify in a much better way than present.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors describe their study of the role of transforming growth factor (TGF)-β signaling in hepatocytic nuclei which is playing a significant role in fibrosis and carcinogenesis. Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) can occur in mildly fibrotic livers, while HCC incidence in NASH-related cirrhosis is less frequent and is less predictable than in hepatitis C virus (HCV)-related cirrhosis. Transforming growth factor (TGF)-β signaling in hepatocytic nuclei is of clinical significance in fibrosis and carcinogenesis. It is known that the TGF-β type 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). The authors demonstrate by the use od phosphospecific Sma3 antibodies that in mature hepatocytes, oncogenic signaling is transduced via the JNK/pSmad3L pathway antagonizes signaling via the tumor-suppressive TβRI/pSmad3C pathway. The study provide convincing evidence for the conclusions drawn from data presented in this manuscript.  Immunohistochemical investigations of 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, demonstrated that Smad3 phosphorylation (in linker region by JNK versus C-terminal phosphorylation) correlated with clinical course.

Author Response

Dear, reviewer 2.

Thank you very much for reviewing our paper.