PONE-D-23-42914PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancerPLOS ONE

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Additional Editor Comments (if provided):

While the study investigated resistance mechanisms to MET tyrosine kinase inhibitors in MET-amplified lung cancer cell lines and addressed an important clinical challenge, it presents several significant limitations that undermine its scientific significance and clinical relevance. The reviewers have raised several concerns that need to be addressed before the manuscript could be accepted for publication.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study investigating resistance mechanisms to MET tyrosine kinase inhibitors in MET-amplified lung cancer cell lines, while addressing an important clinical challenge, presents several significant limitations that undermine its scientific significance and clinical relevance:

1. The study focuses on only two lung cancer cell lines, EBC-1 and H1993. This narrow scope may not adequately represent the genetic and phenotypic heterogeneity observed in non-small cell lung cancer (NSCLC) patients. Broader inclusion of cell lines could provide a more comprehensive understanding of resistance mechanisms.

2. The absence of in vivo studies, such as patient-derived xenograft models, limits the validation of the proposed resistance mechanisms and their therapeutic implications. In vivo studies are crucial to confirm the relevance of in vitro findings in a more complex biological context.

3. While the study identifies SERPINE1 overexpression and epithelial-to-mesenchymal transition in EBC-1 cells and MAP kinase pathway activation in H1993 cells as resistance mechanisms, it may overlook other potentially significant pathways. A more comprehensive approach could provide a fuller picture of resistance dynamics.

4. The study does not include clinical data or samples to correlate the in vitro findings with patient outcomes. Clinical validation is essential to confirm the relevance of the identified resistance mechanisms in actual patient populations.

5. Although the study suggests combination therapy with a PAI-1 inhibitor and MEK inhibitors as potential strategies, these suggestions are not experimentally validated within the study. More experimental testing of combination therapies is necessary to assess their efficacy and safety.

6. The study does not explore the development of biomarkers for predicting or monitoring resistance to MET-TKIs, which could be crucial for personalized therapy in NSCLC.

In summary, while the study contributes to understanding resistance mechanisms to MET-TKIs in MET-amplified lung carcinoma, its limited scope, reliance on in vitro models, lack of in vivo validation, and absence of clinical correlation significantly restrict its impact on the field. Further research involving a broader range of cell lines, in vivo studies, and clinical validation is required to enhance the translational potential of these findings.

Reviewer #2: 1. Title: "PAI-1 as resistance..." - Can you precise it as "acquired resistance" or do you have any signals that it also can be intrinsic? Furthermore, the title address only one resistance mechanism while the whole article dedicates also two other, EMT driven and via ERK upregulation. Please consider re-phrasing the title.

2. line 44: add "often" to "mutually exclusive"

3. Line 77: "second site mutation" should be changed to "off-target resistance"

4. Line 85: you have used cell lines both from squamous and adenocarcinoma. Please, explain the reason and address the issue of possible different resistance mechanism depending of the primary phenotype. Is the type of phenotype predisposing to a certain resistance. What was the level of MET amplification, as it a priori defines response to Crizotinib.

5. Line 148. please provide reference for the human genome (hg38)

6. Discussion: The capital should be enriched by more discussion about validation of PAI-overexpression and clinical implementation. What is the next steps for employing your finding into the clinic? Proposal of clinical trials?

Line 513: Neither Tiplaxatin or Trametinib are chemotherapeutic agents.

To provide more focused and comprehensive insight in your findings, a table collecting possible resistance mechanism, their features and suggestion for overcoming resistance will be an advantage for readers.

Reviewer #3: Major concern

1. Since all the cell viability assays are performed with six replicates, data for cell viability shown in Fig 1A need to be expressed with a standard deviation.

2. Signal transduction responses, shown in fig 1C, between two resistant-induced cell lines are different. The authors need to discuss the fundamental differences between these two induction processes and how they reflect on the clinical outcomes of patients.

3. The authors described that the SERPINE1 gene is six-fold higher in EBC-1 CRH cells. Please explain the disproportionate cell viability response in combination with MET-TKI and PAI-1 inhibitors (fig 1C).

4. There are no significant morphology differences between CRH and CRS in Fig 1B but substantial differences in the EMT-marker protein expression in Fig 3 and Fig 4. Please explain it.

5. It is not appropriate to conclude that EMT-induced antiapoptotic Bcl-2 expression in line 321-324 without any data support.

6. The authors need to conduct all the Western analyses of the EMT transition marker proteins with significant expression differences in gene analysis.

Reviewer #4: This manuscript reports the identification of mechanisms underlying crizotinib resistance in two MET-amplified lung cancer cell lines (EBC-1 and H1993). The authors demonstrated that multiple factors, such as PAI-1 overexpression and activation of EMT and MAPK pathways, were involved in the crizotinib resistance mechanism in these cell lines. The findings provide insights into potential therapeutic strategies for crizotinib-resistant MET-amplified lung cancer.

I would like to have some issues to be addressed by the authors.

1. One of the MET-amplified lung cancer cell lines used in this study was squamous cell carcinoma and the other was adenocarcinoma (page 5, line 85). In their introduction, the authors noted that MET amplification accounts for 1.7-2.5% of lung adenocarcinomas, but they did not mention MET amplification in squamous cell carcinoma. The authors should explain why they used squamous cell carcinoma cell lines for this study. Also, what is the percentage of MET amplification in lung squamous cell carcinoma?

2. The p-ERK activation has been observed in EBC-1 CRH, EBC-1 CRS, and H1993 CRH cell lines (page 11, line 218). Have you examined whether the MEK inhibitor combination is also effective in EBC-1 CRH and EBC-1 CRS?

3. As mentioned above, EBC-1 is a lung squamous cell carcinoma cell line, and it would be desirable to explain whether this finding that PAI-1 is involved in crizotinib resistance in EBC-1 CRH can be extrapolated to adenocarcinoma as well (page 13, line 24). (page 13, line 242).

4. Please describe how you titrated the concentration of tiplaxtinin (page 13, line 255).

5. Please describe which data were used for the analysis of survival data of NSCLC patients and the method of testing in the method section (page 14, line 268). In addition, since this study was focused on MET-amplified lung cancer patients, it may be better to consider conducting the survival analysis using only MET-amplified lung cancer patients.

6. What is the possible interpretation of the fact that only SNAI1 was not changed in CRS but up-regulated in CRH in the gene expression analysis of EMT markers in EBC-1 CRS cell lines (page 16, line 304)?

7. In the western blotting of EMT-related proteins (Fig. 3C), the authors stated that N-cadherin and vimentin are highly expressed in EBC-1 CRS, but this does not seem to be the case in the images (especially vimentin) (page 16, line 309). There does not seem to be a significant change compared to CRH.

8. H993 seems to be a misnomer for H1933 (page 19, line 354).

9. Cleaved PARP expression in H1993 CRH cell lines does not appear to be significantly different between crizotinib, trametinib, and combination treatment groups (page 19, line 355). Have you considered quantification or evaluation by other apoptosis markers?

10. What is the possible interpretation of the lower expression of SNAI1 and SNAI2 in H1993 CRH cell lines compared to parental cells (page 21, line 382)?

11. Regarding MET in lung cancer, treatment targeting MET exon 14 skipping mutation has been attracting much attention. How do the authors think the results of this study can be applied to MET ex14 lung cancers?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Jun Jen Liu

Reviewer #4: No

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Attachments

Attachment

Submitted filename: Major concern.docx

Attachment

Submitted filename: Yin_Crizotinib_manuscript_review.docx

PAI-1 mediates acquired resistance to MET-targeted therapy in non-small cell lung cancer

PONE-D-23-42914R1

Dear Dr. Suzawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Abeer El Wakil, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: The authors successfully addressed my comments and I would be satisfied with the authors' response.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #4: No

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