SIAH2–WNK1 Signaling Drives Glycolytic Metabolism and Therapeutic Resistance in Colorectal Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors explored SIAH2–WNK1 axis with chemoresistance and translational relevance, but there are some comments that needs to be discussed as follows:

• All rescue experiments rely on shRNA knock-down, adding experiments using inducible systems will strengthen this study, to remove the possibility of off-target effects.
• Glycolysis flux data was completely dependent on extracellular acidification. No stable-isotope tracer (e.g. 13C-glucose) or Seahorse ATP-rate assays are performed, so the contribution of mitochondrial respiration versus glycolysis is not quantified.
• Key glycolytic enzymes (PFKP, LDHA, GAPDH) are primarily assessed at the mRNA level, and additional protein or activity-based assays are important to support the metabolic conclusions. Similarly, CSC properties are evaluated by spheroid assays, these models may not fully capture long-term self-renewal or in vivo stemness,
• Statistics in Fig. 2 and Fig. 4 are shown only as t-tests; multiple comparisons are made without FDR correction.
• The patient cohort is relatively small and includes older samples with limited follow-up and clinical outcome, which should be recognized as a limitation when interpreting clinical correlations. Can the authors give some details on follow-up?
• Western blots panels are not presented in the figure. Can it be provided, not just quantification.
• Some methodological details for the qRT-PCR assays (such as primer information and validation) are not described.
• Raw data and code for bioinformatics analyses have not been deposited, or accession link has not been provided.

Author Response

Author's Reply to the Review Report

Title: SIAH2–WNK1 Signaling Drives Glycolytic Metabolism and Therapeutic Resistance in Colorectal Cancer

Reviewer #01

R1.1: All rescue experiments rely on shRNA knockdown; adding experiments using inducible systems will strengthen this study to remove the possibility of off-target effects.

A1.1: We appreciate this important methodological suggestion. To minimize potential off-target effects, we employed independent shRNA clones targeting SIAH2 and observed highly consistent phenotypic outcomes across proliferation, cell-cycle regulation, metabolic profiling, and stemness-associated assays. In addition, rescue experiments using a pcDNA-SIAH2 overexpression construct restored proliferative capacity and cell-cycle progression, supporting the specificity of SIAH2 depletion.

Although inducible knockdown systems were not implemented in this study, the strong concordance between independent shRNA clones and rescue experiments provides compelling evidence that the observed effects are attributable to SIAH2 suppression. This limitation and the rationale are now explicitly acknowledged in the revised Discussion. Kindly refer to the newly added western blot and qRT-PCR analysis for knockdown and overexpression results and its connected processes, in the main text result and supplementary information and here also.

R1.2: Glycolysis flux data was completely dependent on extracellular acidification. No stable-isotope tracer (eg 13C-glucose) or Seahorse ATP-rate assays are performed, so the contribution of mitochondrial respiration versus glycolysis is not quantified.

A1.2: In response to this comment, we have substantially strengthened the metabolic characterization by performing comprehensive Seahorse extracellular flux analyses, simultaneously measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Glycolytic capacity, glycolytic reserve, basal respiration, ATP-linked respiration, and maximal respiratory capacity were quantified using sequential injections of glucose, oligomycin, 2-deoxyglucose, FCCP, and rotenone/antimycin A. Additional result of this has been added to the main text at section 2.6 kindly refer to it. These analyses demonstrate that SIAH2 depletion disrupts both glycolytic output and metabolic flexibility, affecting the balance between glycolysis and mitochondrial respiration. While stable-isotope tracer or ATP-rate assays were not performed, the combined ECAR/OCR profiling provides direct functional evidence of altered metabolic contributions, which is now clearly described in the Results and Methods sections.

R1.3: Key glycolytic enzymes (PFKP, LDHA, GAPDH) are primarily assessed at the mRNA level, and additional protein or activity-based assays are important to support the metabolic conclusions.

A1.3: We agree with this comment and have strengthened the manuscript accordingly. Protein-level validation of key glycolytic enzymes (PFKP, LDHA, and GAPDH) is now provided by Western blot analysis, with representative blot images included in the revised figures. In addition, integrated transcriptomic–proteomic analyses using publicly available datasets demonstrate concordant regulation of glycolysis-associated enzymes.

Furthermore, transcriptomic analyses from TCGA-COAD and GSE17538 consistently support the association between SIAH2 expression and glycolytic gene programs, reinforcing the metabolic conclusions.

R1.4: Similarly, CSC properties are evaluated by spheroid assays, these models may not fully capture long-term self-renewal or in vivo stemness.

A1.4: We agree with this limitation. While three-dimensional spheroid formation, CAF-induced stemness assays, and chemotherapy-resistance experiments provide strong functional evidence of CSC-like behavior, we acknowledge that long-term self-renewal and in vivo tumor-initiating capacity were not directly assessed. This limitation and the need for future in vivo validation studies are now explicitly discussed in the revised Discussion. In this revised text we have addressed and added the tumor sphere assay image results.

R1.5: Statistics in Fig. 2 and Fig. 4 are shown only as t-tests; multiple comparisons are made without FDR correction.

A1.5: We have revised the statistical analyses to fully address this concern. Multi-group comparisons are now analyzed using one-way or two-way ANOVA, followed by Tukey’s post-hoc test where appropriate. For transcriptomic, correlation, and pathway-level analyses involving multiple testing, false discovery rate (FDR) correction using the Benjamini–Hochberg method has been applied. Adjusted q-values are now reported, and these changes are detailed in the revised Statistical Analysis section and figure legends.

R1.6: The patient cohort is relatively small. Can the authors give some details on follow up?

A1.6: We have updated the “Patients and Samples” section to include follow-up details. The median follow-up duration of the cohort was 16.9 months, with a range of 2.7 to 36.7 months. The limited cohort size and follow-up duration are also acknowledged as study limitations.

R1.7: Western blot panels are not presented in the figure. Can they be provided, not just quantifications?

A1.7: Representative Western blot images corresponding to all quantified data have now been included in the revised figures and Supplementary Information. Detailed antibody information expected molecular weights, dilution ratios, and loading controls are also provided to ensure transparency and reproducibility.

R1.8: Some methodological details for the qRT-PCR assays (such as primer information and validation) are not described.

A1.8: We have addressed this comment by adding a comprehensive information of primer sequences (forward and reverse) in the main text. Primer validation procedures, melt-curve analyses, normalization strategy (GAPDH), and data analysis using the 2⁻ΔΔCt method are now fully described in the Methods section.

R1.9: Raw data and code for bioinformatics analysis have not been deposited, or accession links have not been provided.

A1.9: We have clarified data availability and bioinformatics transparency in the revised manuscript. Public datasets analyzed in this study include TCGA-COAD (accessed via UALCAN), DepMap resources, and the GEO dataset GSE17538. Differential expressions, correlation, and pathway analyzed were conducted using established R-based pipelines and GSEA. Detailed methodological descriptions, for online access and database use has been reported in the main text.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to congratulate the authors on a manuscript of high scientific quality that stands out for both its methodological soundness and conceptual originality. The study rigorously and coherently integrates bioinformatic analysis, experimental validation, and functional models, allowing for an in-depth and coherent examination of the role of the SIAH2-WNK1 axis in tumour metabolism and therapeutic resistance in colorectal cancer. The breadth of the approaches employed, together with a clear and well-structured narrative, reinforces the robustness of the results and their biological relevance. Overall, this is a carefully designed and executed study that contributes significant advances to our understanding of tumour metabolism and provides a solid foundation for future research with potential translational impact.

Despite the above, I believe that some changes could be made to improve the manuscript.

MANUSCRIPT

• Review all the headings in which the acronyms appear in lowercase.

MATERIALS AND METHODS:

• Line 173: Glut1 and Glut4 must be appear in capital letters
• Figures 1A to 1E are too small to see the data. Consider including a single analysis that can be read and adding the rest to the supplementary material.

RESULTS:

• Line 268: “As illustrated in” could be deleted
• Lines 351-368: This is not results. You must summarize it

DISCUSSION:

• The discussion in general should be reconsidered since there are many paragraphs that give the same information as in the introduction.
• On the other hand, despite its originality, I believe the results should be discussed further. It's noteworthy that only four references are used throughout the entire discussion.
• It would be advisable to add a paragraph outlining the strengths and limitations of the study, as well as to continue this work in the future.

Author Response

Reviewer #02

R2.1: Review all the headings in which the acronyms appear in lowercase.

A2.1: All headings have been carefully revised to ensure correct capitalization of acronyms, including CRC, SIAH2, and WNK1.

R2.2: Line 173: Glut1 and Glut4 must appear in capital letters.

A2.2: This has been corrected to GLUT1 and GLUT4 throughout the manuscript now this belongs to line 84,85 specially. In addition, the manuscript has undergone professional English language editing provided by Taipei Medical University (TMU)–Wallace Academic Editing Service (internal) to improve clarity, grammar, and readability. The editing certificate has been included with the revised submission for reference. Kindly find the certificate attached.

R2.3: Figures 1A to 1E are too small to see the data. Consider including a single analysis that can be read.

A2.3: Figure 1 has been redesigned with improved resolution and layout, including clearer UMAP visualizations and feature plots, to enhance readability and data interpretation.

R2.4: Lines 351-368: This is not resulting. You must summarize it.

A2.4: This section has been condensed and rewritten to focus strictly on the key findings derived from the GSE17538 dataset, removing interpretive discussion from the Results section.

R2.5: The discussion in general should be reconsidered since there are many paragraphs that give the same information as in the introduction.

A2.5: The Discussion has been substantially revised to minimize redundancy and to emphasize mechanistic interpretation, CAF-mediated regulation, metabolic reprogramming, and therapeutic implications of the SIAH2/WNK1 axis.

R2.6: It would be advisable to add a paragraph outlining the strengths and limitations of the study.

A2.6: A dedicated paragraph discussing the strengths, limitations, and future directions of the study has been added, together with a schematic model summarizing the proposed mechanism.

We once again thank the reviewers for their valuable comments and constructive suggestions. We believe that the revised manuscript has been significantly strengthened and now meets the scientific and editorial standards of the International Journal of Molecular Sciences.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed the reviewer concerns, strengthened the experimental and analytical rigor of the study, and appropriately acknowledged remaining limitations.