Review Reports - PrP<sup>C</sup>-Neutralizing Antibody Confers an Additive Benefit in Combination with 5-Fluorouracil in KRAS-Mutant Colorectal Cancer Models, Associated with Reduced RAS-GTP and AKT/ERK Phosphorylation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the role of extracellular cellular prion protein (PrPC) in KRAS-mutant colorectal cancer (CRC) and proposes that PrPC neutralization enhances 5-fluorouracil (5-FU) efficacy by attenuating RAS-AKT signaling, proliferation, and tumor-associated angiogenesis. The study combines in vitro analyses across a CRC cell-line panel with an in vivo KRAS G13D HCT-8 xenograft model, providing a coherent preclinical narrative.

Overall, the work is well organized, technically sound, and clinically relevant, particularly in the context of therapeutic limitations in KRAS-mutant CRC. However, several key mechanistic and quantitative issues need to be addressed before the conclusions can be fully supported. In its current form, the manuscript would benefit from major revision.

Major Comments

Although the manuscript repeatedly refers to modulation of the “RAS-AKT pathway,” the experimental evidence is limited to changes in AKT phosphorylation. No direct assessment of upstream RAS activity or parallel MAPK signaling is provided.
The manuscript concludes that anti-PrPC enhances 5-FU efficacy; however, it remains unclear whether this interaction is synergistic or merely additive.
The biological effects of the anti-PrPC antibody are interpreted as PrPC-dependent, but no genetic validation (e.g., PRNP knockdown or knockout) is provided.
While PrPC-RPSA interaction is convincingly shown by co-immunoprecipitation, the manuscript does not demonstrate that RPSA functionally mediates PrPC-dependent signaling.
The xenograft experiments are promising but would benefit from more rigorous reporting.
Use a single term throughout (e.g., “anti-PrPC antibody”) instead of alternating between “PrP antibody,” “PrPC antibody,” etc.
A brief table summarizing KRAS/NRAS/BRAF status of all cell lines would improve clarity.
Input levels of RPSA should be clearly shown to support interpretation of enhanced PrPC-RPSA association.
Since 200 ng/mL is relatively low, a short titration curve in the Supplementary Data would be helpful.

Author Response

In accordance with the constructive comments provided by the reviewers, we have carefully revised the manuscript throughout, and we believe that the clarity of the text and the presentation of the data have been improved. Detailed, point-by-point responses to each comment are provided in the accompanying “Response to Reviewers” document, in which each comment is cited verbatim and followed by our specific reply and a description of the corresponding changes made to the manuscript. All changes have been indicated in the revised manuscript in red font.

We are deeply grateful to the reviewers for their valuable and insightful comments, which have been very helpful in improving the quality of the manuscript. We hope that the revised version will be considered suitable for publication in your journal.

Thank you very much for your time and consideration.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors examined the effects of a PrPC-neutralizing antibody on both KRAS–wild-type and KRAS-mutant colorectal cancer cells by performing MTT assays, the cell cycle analysis and IFC staining. Anti-PrPC treatment suppressed cell proliferation in vitro and tumor growth in vivo, and also showed a synergistic effect in combination with 5-FU. Although the phenotypes are simple and clear, the mechanisms are yet to be determined. Anti-PrPC changed the levels of p-AKT, CD31, PCNA and α-SMA does not mean anti-PrPC inhibited colorectal cancer by attenuating RAS–AKT signaling. The correlation between its anti-tumor effect and KRAS mutation also needs further confirmation. Current form of manuscript is not suitable for publication of IJMS.

Author Response

Thank you very much for your time and consideration.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I recommend acceptance of the manuscript after the authors’ careful and thorough point-by-point revision.

Author Response

Reviewer 1

Comments and Suggestions for Authors

I recommend acceptance of the manuscript after the authors’ careful and thorough point-by-point revision.

: We sincerely thank the reviewer for the positive assessment and for recommending acceptance of our manuscript. We appreciate the recognition of our careful and thorough point-by-point revision. All reviewer comments have been fully addressed, and the manuscript has been revised accordingly, with changes incorporated in the revised text and figures as appropriate.

Reviewer 2 Report

Comments and Suggestions for Authors

The revison raised a question that why and how PrPᶜ antibody reduced RAS activity in colorectal cancer cells? From the data of the MS, I doubt whether upregulation of PrPᶜ in colorectal cancer has a relationship with KRAS-mutantion. In figure 2A and figure 3B, I do not see a significant change of PrPᶜ or RAS-GTP in expression, which may mean PrPᶜ promotes progression of colorectal cancer due to regulation of those reported signal pathways such as MAPK/ERK and PI3K/AKT, no mather what the RAS status is. As shown in Figures 3-5, RAS activation in KRAS-mutant cancer cells induces phosphoralation of both AKT and ERK, which could be attenuated by PrPᶜ antibody treatment. This expains why the combination of anti-PrPᶜ and 5-FU showed a synergistic effect in suppression of KRAS-mutant CRC. Since all the data were derived from cancer cell lines in vitro, it will strengthen the conclusion if the authors make a staining analysis of both RAS-GTP and PrPᶜ in RAS WT and mutant CRC clinical samples, which will tell a relationshio between RAS activation and PrPᶜ expression in patients.

Author Response

Reviewer 2

: Thank you for the reviewer’s comment. We note that our study did not include mass spectrometry–based analyses, and no MS data are presented in the manuscript. We address why/how the anti-PrPᶜ antibody reduces RAS activity in colorectal cancer (CRC) cells as follows.

First, Ras activity was measured using a GST–RAF1-RBD pull-down assay to isolate GTP-bound Ras (Ras-GTP). Ras-GTP in the pull-down fraction was detected by Western blot using an anti-Ras antibody, so this readout reflects the total active Ras pool without distinguishing K-, H-, or N-Ras and is not specific to mutant KRAS. Thus, the reduction in Ras-GTP after anti-PrPᶜ treatment indicates reduced signaling input at the cell surface that contributes to total Ras activation, rather than any change in KRAS genotype.

Second, our co-immunoprecipitation (Co-IP) analyses showed an association between PrPᶜ and the 37/67-kDa laminin receptor (RPSA). Following anti-PrPᶜ treatment, Ras-GTP decreased, and AKT and ERK phosphorylation (p-AKT and p-ERK) was reduced. We did not test whether RPSA is required for these changes. Accordingly, we revised the manuscript to avoid causal wording and describe RPSA as a candidate mediator. Future studies will test RPSA involvement using RPSA siRNA/shRNA knockdown and/or CRISPR/Cas9 knockout, with readouts including Ras-GTP and p-AKT/p-ERK.

Third, although the representative blots in Fig. 2A and Fig. 3B may appear modest, we quantified band intensities from independent biological replicates (n ≥ 3) using ImageJ densitometry. PrPᶜ was normalized to a loading control, and Ras-GTP was normalized to input (total Ras) before statistical testing. The quantified data showed statistically significant differences, and the quantification plots and statistical details are provided in the figure legends.

In addition, while the signaling readouts (Ras-GTP, p-AKT, p-ERK) were assessed primarily in vitro, we also evaluated in vivo efficacy in a xenograft model established using HCT-8 (KRAS G13D) mutant cells. In this model, the combination of anti-PrPᶜ antibody and 5-FU produced greater tumor growth inhibition than either monotherapy. In Fig. 8, PCNA immunofluorescence was reduced in tumors from the combination group, and vascular staining also showed reduced CD31 (microvessels) and reduced α-SMA (arteriolar-type vessels) signals in the combination group.

Taken together, we do not claim that KRAS mutations directly drive PrPᶜ upregulation. Instead, our data show that PrPᶜ neutralization is accompanied by reduced total Ras-GTP and reduced AKT/ERK phosphorylation.

We agree that patient-based validation would strengthen the clinical context. In practice, RAS activity is commonly assessed using lysate-based GST–Raf1-RBD pull-down or ELISA-type assays. Direct quantitative IHC for GTP-bound (active) RAS in FFPE tissues varies with fixation and antigen retrieval and requires careful reagent validation. For clinical evaluation, it is reasonable to combine PrPᶜ IHC with downstream markers (p-ERK/p-AKT) together with confirmed mutation status. At the revision stage, we do not have access to a well-annotated clinical cohort to add these experiments; patient-based validation will be pursued in a subsequent project.

References

Gauczynski S, Peyrin J-M, Haïk S, et al. The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. EMBO J. 2001;20(21):5863–5875.

de Rooij J, Bos JL. Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras. Oncogene. 1997;14(5):623–625.
van Triest M, de Rooij J, Bos JL. Measurement of GTP-bound Ras-like GTPases by activation-specific probes. Methods Enzymol. 2001;333:343–348.
Theiss AP, Chafin D, Bauer DR, Grogan TM, Baird GS. Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation. PLoS One. 2014;9(11):e113608.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

In my previous round of review comments, "MS" refers to the manuscript, not mass spectrometry.
The authors still need to address the question regarding the reduction of GTP-bound Ras following anti-PrPᶜ treatment. What is the proposed mechanism for this phenomenon?
The representative data in Fig. 2A and Fig. 3B do not match the quantified results. I recommend that the authors either provide a more representative Western blot result from replicates or present all replicate data along with a quantitative analysis.

Author Response

Re: Manuscript ID ijms-4081822

Dear Editor,

We hereby submit the third revised version of our manuscript entitled “PrPᶜ-neutralizing antibody confers an additive benefit to 5-fluorouracil activity in KRAS-mutant colorectal cancer by attenuating RAS–AKT signaling” (Manuscript ID: ijms-4081822) for consideration for publication in the International Journal of Molecular Sciences, Special Issue “KRAS-Associated Cancer Signaling”.

In this revision, we have addressed the remaining reviewer and editorial comments and updated the manuscript accordingly. The text and figures have been revised where appropriate, and the corresponding quantitative analyses and statistical results have been updated as needed. A detailed, point-by-point response is provided in the accompanying “Response to Reviewers” document, in which each comment is reproduced and followed by our response and a description of the revisions made. Changes in the revised manuscript are marked in red.

Thank you for your time and consideration.

Sincerely,

Sang Hun Lee, Ph.D.
Department of Biomedical Sciences, College of Medicine,
and Program in Biomedical Science & Engineering,
Inha University, Incheon 22332, Republic of Korea
E-mail: shlee2309@inha.ac.kr

Author Response File: Author Response.pdf