Auranofin Suppresses Cancer Cell Invasion by Inhibiting Heparanase-1 Expression via the aPKC–NF-κB Pathway

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewing report

This manuscript presents a well-motivated and technically rigorous investigation into the regulation of heparanase-1 (HPSE1) gene expression in human cancer cell lines, with auranofin (AUF) as a repurposed FDA-approved anti-rheumatic drug identified as a novel suppressor of HPSE1 transcription via the atypical protein kinase C (aPKC)–NF-κB signaling axis. The study combines high-throughput screening, gene knockdown experiments, invasion assays, and an in vivo xenograft model in a coherent experimental arc. The central mechanistic claim that aPKC inhibition by AUF reduces HPSE1 expression and consequently limits cancer cell invasion is generally well-supported by the data presented.

I want to be upfront: this is a paper worth publishing. The question it addresses is important. HPSE1 has been a tantalizing therapeutic target for decades, and the field's persistent failure to translate HPSE1 inhibitors into clinical use is precisely the problem the authors are trying to solve. Targeting the upstream regulators of HPSE1 expression rather than its enzymatic activity is a smart approach and the authors make a convincing case for it. The identification of AUF as a potential agent with this mechanism is genuinely interesting.

Comments:

• How many mice per group? Without this, the tumor volume and HPSE1 expression data cannot be properly evaluated.
• The immunohistochemistry data from Figure S1C/D is described qualitatively but statistical analysis of staining intensity is not mentioned. How was HPSE1 expression quantified in tissue sections?
• In Figures 2B, 2F, 2G, and 3C, western blots are shown but the corresponding quantification is not provided alongside the blots. Meanwhile, Figures 2C, 2D, and 2E include quantified mRNA data with statistical annotations. This inconsistency is distracting. For the protein-level data in Figures 2F, 2G, and 3C in particular where the authors are making claims about HPSE1 suppression by specific siRNAs densitometric quantification with statistical testing should be included. “Experiments were repeated three times and reproducible results were obtained” is not a substitute for quantification and statistics.
• The manuscript does not include any power analysis or explanation of how sample sizes were determined, either for the cell line experiments (replicates and biological repeats) or for the animal study. This should be addressed. For in vitro experiments, it’s important to clearly distinguish biological replicates from technical replicates throughout. In Section 4.13, only the statistical test used (one-way ANOVA with Dunnett’s) is mentioned no information about what constitutes n, how many independent experiments were run, or whether assumptions of normality were verified.
• The antibody used for immunohistochemistry (INS-26-2-0000-12, Insight Biopharmaceuticals) is not the same as the antibody used for western blotting (ab254254, Abcam).
• Provide densitometric quantification with statistics for all western blots where mechanistic claims rest on protein-level differences (Figures 2B, 2F, 2G, 3C).
• Report sample size (n per group) and statistical analysis for all xenograft data in Figure S1, including tumor volume and HPSE1 IHC quantification.
• Expand the Discussion paragraph on disaccharide composition to better contextualize what similar sulfation patterns in high-HS cells implies mechanistically.
• Verify and correct figure scale bar consistency in Figure 3A.

My recommendation is Minor Revision. The core science is solid, the experimental framework is well-conceived, and the biological question is important. The concerns I’ve raised the western blot quantification and in vivo statistics need to be brought up to standard. But none of these issues require fundamentally new experiments to resolve.

Author Response

Reviewer 1

This manuscript presents a well-motivated and technically rigorous investigation into the regulation of heparanase-1 (HPSE1) gene expression in human cancer cell lines, with auranofin (AUF) as a repurposed FDA-approved anti-rheumatic drug identified as a novel suppressor of HPSE1 transcription via the atypical protein kinase C (aPKC)–NF-κB signaling axis. The study combines high-throughput screening, gene knockdown experiments, invasion assays, and an in vivo xenograft model in a coherent experimental arc. The central mechanistic claim that aPKC inhibition by AUF reduces HPSE1 expression and consequently limits cancer cell invasion is generally well-supported by the data presented.

I want to be upfront: this is a paper worth publishing. The question it addresses is important. HPSE1 has been a tantalizing therapeutic target for decades, and the field's persistent failure to translate HPSE1 inhibitors into clinical use is precisely the problem the authors are trying to solve. Targeting the upstream regulators of HPSE1 expression rather than its enzymatic activity is a smart approach and the authors make a convincing case for it. The identification of AUF as a potential agent with this mechanism is genuinely interesting.

Comments:

Comment1: How many mice per group? Without this, the tumor volume and HPSE1 expression data cannot be properly evaluated. The immunohistochemistry data from Figure S1C/D is described qualitatively but statistical analysis of staining intensity is not mentioned. How was HPSE1 expression quantified in tissue sections?

Response 1: For the xenograft study, n = 3 mice were included in each treatment and control group. For HPSE1 expression analysis, a stitched image was obtained from each animal. A random field (~67.2 μm² per field) was selected from each of the three sections of the stitched image obtained from each animal. HPSE1 expression was quantified by fluorescence intensity and area using ImageJ software. Differences between groups were assessed using one-way ANOVA. The quantification method and statistical results have been described in the revised manuscript.

Comment 2: In Figures 2B, 2F, 2G, and 3C, western blots are shown but the corresponding quantification is not provided alongside the blots. Meanwhile, Figures 2C, 2D, and 2E include quantified mRNA data with statistical annotations. This inconsistency is distracting. For the protein-level data in Figures 2F, 2G, and 3C in particular where the authors are making claims about HPSE1 suppression by specific siRNAs densitometric quantification with statistical testing should be included. “Experiments were repeated three times and reproducible results were obtained” is not a substitute for quantification and statistics.

Response 2: The statement “Experiments were repeated three times and reproducible results were obtained” has been removed.

Densitometric quantification of HPSE1 and proHPSE1 protein levels in cells treated with AUF or siRNAs was performed using data from three independent experiments, followed by statistical analysis. The corresponding quantitative data and statistical annotations have been added to Figs. 1D, 2B, 3C, 3D, and Fig. S1D.

Comment 3: The manuscript does not include any power analysis or explanation of how sample sizes were determined, either for the cell line experiments (replicates and biological repeats) or for the animal study. This should be addressed. For in vitro experiments, it’s important to clearly distinguish biological replicates from technical replicates throughout. In Section 4.13, only the statistical test used (one-way ANOVA with Dunnett’s) is mentioned no information about what constitutes n, how many independent experiments were run, or whether assumptions of normality were verified.

Response 3: All cell culture experiments were independently repeated three times (n = 3 independent biological experiments). No technical replicates were included in the statistical analyses. For the xenograft study, n = 3 mice were included in each treatment and control group. Sample sizes were selected based on the results published previously[1]; no formal power analysis was performed. Data distribution was assumed to be normal, and statistical analyses were conducted using parametric tests. These clarifications have been described into the “Materials and Methods” section (western blot; L437-438, Invasion assay; L515-517, Tumor-bearing model mice; L523-526 and L539-540) of the revised manuscript.

Comment 4: The antibody used for immunohistochemistryis not the same as the antibody used for western blotting (ab254254, Abcam).

Response 4: The anti-HPSE1 antibody used for western blotting (ab254254, Abcam) did not provide clear and specific immunohistochemical staining. Therefore, a different anti-HPSE1 antibody (INS-26-2-0000-12, Insight Biopharmaceuticals), which was validated for immunohistochemistry, was used for the immunostaining experiments.

Comment 5: Provide densitometric quantification with statistics for all western blots where mechanistic claims rest on protein-level differences (Figures 2B, 2F, 2G, 3C).

Response 5: Densitometric quantification of HPSE1 and proHPSE1 protein levels in cells treated with AUF or siRNAs was performed using data from three independent experiments, followed by statistical analysis. The corresponding quantitative data and statistical annotations have been added to Figs. 1D, 2B, 3C, 3D, and Fig. S1D.

Comment 6: Report sample size (n per group) and statistical analysis for all xenograft data in Figure S1, including tumor volume and HPSE1 IHC quantification.

Response 6: As mentioned above, n = 3 mice were included in each treatment and control group. Quantification of HPSE1 protein levels was performed using data from three independent experiments, followed by statistical analysis. Please heck in Figure S1.

Comment 7: Expand the Discussion paragraph on disaccharide composition to better contextualize what similar sulfation patterns in high-HS cells implies mechanistically.

Response 7: Thank you for this valuable suggestion. We carefully considered the potential mechanistic implications of the similar HS sulfation patterns observed among high-HS-producing cells. However, in our dataset, similar sulfation profiles were also observed in HT-29 cells, which are non-invasive. Therefore, we believe that the current data do not support a direct association between HS sulfation pattern and invasive capacity. While differences in the expression of cell-surface proteoglycans such as syndecans and glypicans may contribute to the functional consequences of HS production, these parameters were not examined in the present study. Because any mechanistic interpretation would be highly speculative without additional experimental evidence, we have chosen not to substantially expand this discussion and instead have retained the current text while acknowledging this limitation.

Comment 8: Verify and correct figure scale bar consistency in Figure 3A.

Response 8: The scale bars were reviewed throughout the manuscript and revised where necessary (Figs. 4A, 4D, 4E, 5A, 6A, and 6C).

My recommendation is Minor Revision. The core science is solid, the experimental framework is well-conceived, and the biological question is important. The concerns I’ve raised the western blot quantification and in vivo statistics need to be brought up to standard. But none of these issues require fundamentally new experiments to resolve.

 

Reference

1. Hatem E, Azzi S, El Banna N, He T, Heneman-Masurel A, Vernis L, et al. Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer. J Natl Cancer Inst. 2019;111(6):597-608. doi: 10.1093/jnci/djy149. PubMed PMID: 30779852.

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, Komeno et al. investigated the effects of auranofin on cancer cell invasion by suppressing heparanase-1 expression through the aPKC–NF-κB signaling pathway. Overall, this study is interesting; however, some issues should be addressed before the manuscript can be considered for publication in the International Journal of Molecular Sciences.
1.    I recommend that the authors use a live-cell imaging system, such as IncuCyte, to monitor and quantify cancer cell invasion in real time.
2.    The Introduction should include a more comprehensive description of auranofin, including its pharmacological properties, current clinical applications, and previously reported anticancer activities.
3.    The Western blot results should be quantified and presented as graphs showing the relative protein expression levels. Statistical analysis should also be included.
4.    The invasion assay results should be quantified and presented graphically.
5.    In the Materials and Methods section, the authors state that cancer cell lines were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin G, and 50 U/mL streptomycin. However, in Figures 3A, 4A, 5A, and 5C, it appears that FBS was omitted from the experimental conditions. The authors should clarify the culture conditions used in these experiments and explain any deviations from the standard culture protocol.
6.    Given the relevance of the Warburg effect, the authors should clearly specify the culture medium composition, including glucose concentration, used throughout the study. 

Author Response

In this study, Komeno et al. investigated the effects of auranofin on cancer cell invasion by suppressing heparanase-1 expression through the aPKC–NF-κB signaling pathway. Overall, this study is interesting; however, some issues should be addressed before the manuscript can be considered for publication in the International Journal of Molecular Sciences.

Comment 1: I recommend that the authors use a live-cell imaging system, such as IncuCyte, to monitor and quantify cancer cell invasion in real time.

Response 1: We appreciate the reviewer's suggestion. Unfortunately, a live-cell imaging system such as IncuCyte is not available in our laboratory/institution, and therefore we were unable to perform these experiments in the present study. We acknowledge the value of this approach and consider it an important direction for future work.

Comment 2: The Introduction should include a more comprehensive description of auranofin, including its pharmacological properties, current clinical applications, and previously reported anticancer activities.

Response 2: The sentence was revised as follows.

Here, we found that auranofin (AUF), a thioredoxin reductase inhibitor currently under clinical investigation in combination with chemotherapy, immuno-therapy, and targeted therapies[20], markedly suppressed HPSE1 expression and exhibited significant anti-tumor activity. Additionally, the atypical protein kinase C (aPKC)–NF-κB signaling axis plays a critical role in regulating HPSE1 expression in epithelial cancer cell lines.

Comment 3: The Western blot results should be quantified and presented as graphs showing the relative protein expression levels. Statistical analysis should also be included.

Response 3: Densitometric quantification of HPSE1 and proHPSE1 protein levels in cells treated with AUF or siRNAs was performed using data from three independent experiments, followed by statistical analysis. The corresponding quantitative data and statistical annotations have been added to Figs. 1D, 2B, 3C, 3D, and Fig. S1D.

Comment 4: The invasion assay results should be quantified and presented graphically.

Response 4: Relative invasion activity was quantified using stitched pictures from three independent experiments, followed by statistical analysis. The corresponding quantitative data and statistical annotations have been added to Figs. 4D, 4E.

Comment 5: In the Materials and Methods section, the authors state that cancer cell lines were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin G, and 50 U/mL streptomycin. However, in Figures 3A, 4A, 5A, and 5C, it appears that FBS was omitted from the experimental conditions. The authors should clarify the culture conditions used in these experiments and explain any deviations from the standard culture protocol.

Response 5: Thank you for pointing it out. Serum-free conditions were used in the upper chamber to eliminate the chemotactic effects of serum components, which could interfere with cell migration and invasion. Sentence was revised as follows.

Cells were seeded in 200 µL of serum free DMEM onto the Chemotaxel upper layer.

Comment 6: Given the relevance of the Warburg effect, the authors should clearly specify the culture medium composition, including glucose concentration, used throughout the study.

Response 6: The composition of Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/L glucose; Nacalai Tesque Inc., Kyoto, Japan) is available on the supplier’s website.

https://www.e-nacalai.jp/ec2/EC-srchdetl.cfm?Dum=1&syohin=0848945&syubetsu=3

Therefore, the glucose concentration has been added in the revised manuscript.