Mint3 as a Molecular Target Activated in the Early Stage of Hepatocarcinogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have previously described the role of Mint3 in various other tumor diseases. Here, they present evidence for the first time that Mint3 is involved in early tumor formation of HCC. Since angiogenesis has become a major backbone of HCC therapy, further understanding in the pathophysiology is important to develop further treatment options. Overall, the paper is well written and presents interesting new data. 

To further strengthen this paper, some points should be improved:

1. A clinical translational part showing the expression of Mint3 in primary human tumor samples should be added. This would also allow to connect Mint3 to different tumor stages (as discussed for bladder cancer in the paper) for HCC and maybe even link to MASH or fibrosis patterns. Was a TCGA (or similar) analysis performed for HCC before? This could be interesting as the paper esp looks into early stages which are well represented in TCGA.

2. The difference in Mint3 staining intensity is somewhat difficult to see. The supplementary figures do not provide enough overview and Fig 1 does not show a strong difference between the two areas. Stainings should also be quantified (H score etc).

3. AFP was the only parameter linked to Mint3 expression. Is there a correlation between AFP levels and Mint3 expression? This would also require additional Mint3 scoring but could then also be linked to stage and size of HCC.

4. Blots in Fig 2A should be quantified. It is somewhat intriguing that also the weak knockdown of Mint3 by shMint3#1 leads to the same suppression of target genes. Please comment and correct the statement in the text (l. 104). No statistical difference is given for the two knockdown constructs. Please calculate here, too.  How could an error bar be calculated for the tumor size of shMint3#1 with an n = 1?

5. For the chemical carcinogenesis, why was a different group size used (Fig 3A)? Results are difficult to interpret and the % numbers given may be misleading. Was a sample size calulcation done for this experiment?

Author Response

Comments 1: [A clinical translational part showing the expression of Mint3 in primary human tumor samples should be added. This would also allow to connect Mint3 to different tumor stages (as discussed for bladder cancer in the paper) for HCC and maybe even link to MASH or fibrosis patterns. Was a TCGA (or similar) analysis performed for HCC before? This could be interesting as the paper esp looks into early stages which are well represented in TCGA.]

Response 1: [We thank the reviewer for this important comment. We presented the staining of Mint3 in both human-derived tumors and mouse-derived tumors. The staining status was described and added in the supplemental information (Supplementary Figure 5). As the reviewer pointed out, we are also very interested in the expression of Mint3 in MASH or fibrosis models, which we would like to evaluate in the future.

We thank the reviewer for this valuable comment. Accordingly, we evaluated the expression status of Mint3 at different BCLC stages in the Chinese Liver Cancer Atlas by using cBioPortal (https://www.cbioportal.org/). Mint3 expression tends to be higher at lower BCLC stages, which supports our results. We added these data as the supplemental information (Supplementary Figure 4) and we also explain the data in detail in the Discussion section. We would like to thank the reviewer again for the useful comments.]

 

Comments 2: [The difference in Mint3 staining intensity is somewhat difficult to see. The supplementary figures do not provide enough overview and Fig 1 does not show a strong difference between the two areas. Stainings should also be quantified (H score etc).]

Response 2: [We greatly appreciate the reviewer’s suggestion. Accordingly, we have quantified the staining of Mint3 using ImageJ [1]. In Figure 1, the staining of Mint3, VEGFA, and HK2 in well-differentiated HCC was evaluated and compared to moderately-differentiated HCC, indicated as a ratio. The ratio of staining intensity of well- to that of moderately- was 1:1.14 (Mint3), 1:1.25 (VEGFA), 1:1.11 (HK2), respectively, which was slightly higher in well-differentiated HCC. We added these data and revised the manuscript accordingly. We also performed some modification of the photographs presented in Figure 1 (C-F) for readers to make them comparable to MRI images.

We thank the reviewer for this valuable comment. Mint3 expression status in surgically resected HCC was also quantified using ImageJ [1]. Mint3 -positive or -negative was defined as membranous and/or cytoplasmic staining detected in ≦10% of the tumor as score 0, 10%< ～≦50% as score 1, and >50% as score 2, with score 0 being negative and score 1 and 2 being positive. We added representative photomicrographs of score 0,1,2 in the supplemental information (Supplementary Figure 5).]

 

Comments 3: [AFP was the only parameter linked to Mint3 expression. Is there a correlation between AFP levels and Mint3 expression? This would also require additional Mint3 scoring but could then also be linked to stage and size of HCC.]

Response 3: [We apologize the reviewer that the data was not easy to recognize. Indeed, we investigated the expression of Mint3 in surgically resected 100 HCC tissues and evaluated the expression patterns of Mint3 as described above (Table 1). Mint3 expression was associated with low serum AFP levels (p = 0.029). We also presented the data that higher Mint3 expression was associated with lower BCLC stages, as indicated in the response to comment 1. We further evaluated the association between Mint3 expression and tumor size, and found no correlation. We revised the Results section accordingly.]

 

Comments 4: [Blots in Fig 2A should be quantified. It is somewhat intriguing that also the weak knockdown of Mint3 by shMint3#1 leads to the same suppression of target genes. Please comment and correct the statement in the text (l. 104). No statistical difference is given for the two knockdown constructs. Please calculate here, too.  How could an error bar be calculated for the tumor size of shMint3#1 with an n = 1?]

Response 4: [We greatly appreciate the reviewer’s suggestion. Blots quantification was performed using ImageJ [1] with the following protocol; Kenji Ohgane, Hiromasa Yoshioka 2019. Quantification of Gel Bands by an Image J Macro, Band/Peak Quantification Tool. protocols.io, https://dx.doi.org/10.17504/protocols.io.7vghn3w. We defined the ratio of Mint3 to β-actin in quantified the gel band signals as relative expression. Relative expression of control was 0.96, shMint3#1 was 0.80, and shMint3#2 was 0.38 and shMint3#2 was most strongly suppressed. There was a trend that HIF-1 target genes VEGFA and HK2 were more suppressed in shMint3#2 compared with shMint3#1. The similar tendency was observed in the experiments of spheroid formation capacity, although no statistically significant difference was observed. We revised the manuscript accordingly.

We completely agree with the reviewer’s comment and the error bar in Figure 2 have been removed.]

 

Comments 5: [For the chemical carcinogenesis, why was a different group size used (Fig 3A)? Results are difficult to interpret and the % numbers given may be misleading. Was a sample size calulcation done for this experiment?]

Response 5: [We thank the reviewer for this important comment. As for the sample size of the DEN model, initially, we started with an equal number of WT and Mint3 KO mice (n=7), however the HCC incidence in Mint3 KO mice were significantly lower and no tumor development was detected. To further confirm the experimental accuracy, we doubled the number of Mint3 KO mice to that of the WT mice. We believe this experiment allowed us to prove the effect of Mint3 absence on inhibition of HCC development with enough statistical power. We revised the Materials and Methods section accordingly.

We agree with the reviewer that the % number in Figure 3 and Figure 4 may be misleading, and revised the figure accordingly.]

 

＊According to the reviewer’s Comments and Suggestions, we have corrected minor details in Figures and Supplementary figures.

References

1. Abramoff, M.D., Magelhaes, P.J., Ram, S.J. Image Processing with ImageJ. Biophotonics International 2004, 11, 36-42.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In the present study, Nishitani et al. showed the expression and function in Mint3 in hepatocellular carcinoma. They found that the down-regulation of Mint3 inhibits development and growth of HCC. Although the manuscript was well presented, the overall data were very preliminary and no mechanism studies were performed.

1. Mint3 was elevated in well-differentiated HCC, but was correlated with low AFP and well/moderate differentiation. The author should expain more on these correlations.

2. Only Mint3 KO mice was used in the experiments. The overexpression of Mint3 (such as injection of Mint3) to HCC mouse model should be performed to investigate the Mint3 upregulation to HCC development.

3. The signaling of Mint3 should be analyzed.

Author Response

Comments 1: [Mint3 was elevated in well-differentiated HCC, but was correlated with low AFP and well/moderate differentiation. The author should expain more on these correlations.]

Response 1: [We apologize the reviewer that the data was not easy to recognize. We investigated the expression of Mint3 in surgically resected 100 HCC tissues and evaluated the expression patterns of Mint3 (Table 1). Mint3 expression was associated with low serum AFP levels (p = 0.029). We considered that because AFP is typically expressed in moderately to poorly differentiated HCC, the inverse correlation between Mint3 expression and serum AFP levels could be attributed to the role of Mint3 in promotion and progression in well-differentiated HCC. We evaluated the expression status of Mint3 at different BCLC stages in the Chinese Liver Cancer Atlas by using cBioPortal (https://www.cbioportal.org/). Mint3 expression tends to be higher at lower BCLC stages. which supports our results. We added the supplemental information (Supplementary Figure 4) and the description for this has been added to the Results and Discussion.]

 

Comments 2: [Only Mint3 KO mice was used in the experiments. The overexpression of Mint3 (such as injection of Mint3) to HCC mouse model should be performed to investigate the Mint3 upregulation to HCC development.]

Response 2: [We greatly appreciate the reviewer’s suggestion. Indeed, our experiments performed in this manuscript used only Mint3 KO mice, and the overexpression model was not used. Overexpression experiment may allow us to further evaluate the involvement of Mint3 in HCC. However, unfortunately, there is no report to evaluate the effect of Mint3 overexpression in HCC cell lines in vitro and in mouse liver in vivo. There are reports on the expression status of Mint3 in various types of cell lines [1], but Mint3 was basically detected in various cell lines, and different cell lines might have more fundamental genetic differences such as p53/HIF status in addition to the Mint3 status. Clearly, further studies are needed to determine whether these cell lines can be used to create a Mint3 overexpression model. We understand that your suggestion is invaluable to clarify the role of Mint3 in HCC development, but transgenic mice approach is not always successful and will take a considerable long time to get the results. We would like to evaluate the effect of Mint3 overexpression in the tumor as future challenges. We added the description in the Discussion section as a limitation and revised the manuscript.]

 

Comments 3: [The signaling of Mint3 should be analyzed.]

Response 3: [We apologize the reviewer that the signaling pathways mediated by Mint3 was not well described. Indeed, we previously reported that Mint3 activates HIF-1 target genes through the interaction with FIH-1 [2, 3]. We therefore evaluated the expression of HIF-1 targets such as VEGFA, HK2 in HCC. We clarified the points and revised the manuscript.]

 

＊According to the reviewer’s Comments and Suggestions, we have corrected minor details in Figures and Supplementary figures.

References

1. Sakamoto T, Niiya D, Seiki M. Targeting the Warburg effect that arises in tumor cells expressing membrane type-1 matrix metalloproteinase. J Biol Chem 2011, 286, 14691-14704.
2. Sakamoto T, Seiki M. Mint3 enhances the activity of hypoxia-inducible factor-1 (HIF-1) in　macrophages by suppressing the activity of factor inhibiting HIF-1. J Biol Chem 2009, 284, 30350-303509.
3. Tanaka N, Sakamoto T. Mint3 as a Potential Target for Cooling Down HIF-1alpha-Mediated Inflammation and Cancer Aggressiveness. Biomedicines 2023, 11.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All comments are nicely answered and the manuscript was significantly improved now. There are no further questions.