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Dear Authors,
After recommendation of our referees and checking the rebuttal letters thoroughly, I am pleased to inform you that the manuscript is accepted for publication in PeerJ in its current form. Please note that this is only an editorial acceptance and you need to complete prepublication tasks, so I advise you to be available for few days to avoid any delays in publication.
All the best for future submissions.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
Title & Abstract
The authors have revised the title as per suggestion. It clearly shows that the manuscript focuses on PLK1 as a marker to predict outcomes and as a target for treatment. No further changes are suggested.
The abstract mentions PLK1's unclear role in LUAD, briefly explains the methods using bioinformatics from TCGA/GEO/GTEx, functional analyses, and lab tests and the results (overexpression, links to prognosis, effects on cell cycle and immune system, and inhibition effects). The conclusion is in line with the findings.
Introduction
The introduction gives a clear background for the research. It discusses the impact of LUAD and highlights treatment issues, such as resistance to certain drugs. It describes PLK1's roles in cell division and other functions like EMT and immune changes and also identifies knowledge gaps in LUAD. The authors have incorporated the suggestions and removed the lines 65-71 which were redundant. They have then improved the flow of the information by introducing drugs like onvansertib to bridge preclinical findings to therapeutic potential, and address PLK1's role in radio resistance. No further changes are suggested.
Figures & Tables
The tables and figures are clear, legible and free from unnecessary modification.
Material and Methods
The methods are clear, the revised manuscript includes the critical details that were suggested. The manuscript now mentions STR profiling for A549 cell line. For Western Blotting, the clone numbers for the Cyclin B1 and CDK1 antibodies have now been included. GSK461364’s IC50 for PLK1 has also been added. For flow cytometry data, gating strategy has now been added. No further changes are suggested.
Results
The results show some new findings by combining PLK1's ability to predict outcomes with practical testing (such as GSK461364 causing G2/M arrest and Cyclin B1/CDK1 build-up). This goes beyond earlier studies that only used bioinformatics. The authors have changed the word “upregulation” to “accumulation” in reference to Cyclin B1 and CDK1 as was suggested. No further changes are necessary.
Discussion
The discussion matches the results well, and the revised version now highlights the model of mitotic catastrophe which improves the understanding. The authors have also rewritten the limitations section, acknowledging the problems with the use of KRAS-mutant line. The revised version needs no further corrections.
Conclusion
The conclusions align well with the findings and the revised version is concise as well as precise. No further changes are suggested.
GOOD
GOOD
GOOD
GOOD
Dear Authors,
Our referees pointed out some important issues to be addressed before going for further processing. Please do the needful and resubmit asap.
All the best
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
Title & Abstract
Title: The title clearly shows that the manuscript focuses on two things about PLK1. First, it looks at PLK1 as a marker to predict outcomes, using data analysis, survival links, and prediction models. Second, it sees PLK1 as a target for treatment, shown by lab tests that stop cell growth, cause cell death, and slow down cell division. To enhance focus, consider revising the title slightly to "PLK1 Overexpression as a Dual-Role Biomarker and Therapeutic Vulnerability in Pulmonary Adenocarcinoma" for smoother phrasing.
Abstract:
The abstract gives a clear summary. It mentions PLK1's unclear role in LUAD, briefly explains the methods using bioinformatics from TCGA/GEO/GTEx, functional analyses, and lab tests, and the results (overexpression, links to prognosis, effects on cell cycle and immune system, and inhibition effects). The conclusion is in line with the findings. No changes are suggested.
Introduction
The introduction gives a clear background for the research. It discusses the impact of LUAD, including death rates from studies by Molina et al ( 2008 and Bray et al. (2024. It highlights treatment issues, such as resistance to certain drugs. It describes PLK1's roles in cell division and other functions like EMT and immune changes. It identifies knowledge gaps in LUAD, such as PLK1's interaction with the tumor environment and prediction models, and cites important studies like Seki et al., 2008; Wu et al., 2016; Kong et al., 2022. This introduction is easy for non-experts to understand and presents new ideas for specialists.
Lines 65-71, including “Despite these……clinical validation,” appear redundant and can be shortened. They can be summarized in a single sentence since the latter part of the introduction speaks about the significance of research on PLK1.
The introduction could also incorporate more on recent PLK1 inhibitors' clinical trials in lung cancer (e.g., onvansertib or volasertib) to bridge preclinical findings to therapeutic potential, and address PLK1's role in radioresistance.
Figures & Tables
The tables and figures are clear, legible, and free from unnecessary modification.
Material and Methods
The methods are mostly clear but lack critical details necessary for replication, particularly in the experimental sections.
• The manuscript must state that the A549 cell line was recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination. This is a standard requirement for publication.
• For Western Blotting, the catalog numbers are provided, but the clone numbers for the Cyclin B1 and CDK1 antibodies must also be included.
• GSK461364 is stated to be selective, but a sentence on its known half-maximal inhibitory concentration for PLK1 versus other kinases would increase the confidence in the specificity of the observed effects.
• For flow cytometry data (Apoptosis and Cell Cycle), a brief description of the gating strategy used to identify the cell populations should be included in the methods, or a supplementary figure showing representative gates should be provided.
• There is no mention of PLK1 knockdown confirmation (e.g., via qPCR/Western for inhibitor specificity).
Results
The results show some new findings by combining PLK1's ability to predict outcomes with practical testing (such as GSK461364 causing G2/M arrest and Cyclin B1/CDK1 build-up). This goes beyond earlier studies that only used bioinformatics. Only a minor revision is suggested.
Lines 285-286 mention that “Western blot analysis demonstrated concomitant upregulation of Cyclin B1 and CDK1 (Fig. 7E)…….” The term “upregulated” typically implies transcriptional activation, but here it reflects protein accumulation due to post-translational stabilization. While technically correct, it could mislead without context (e.g., suggesting PLK1 inhibition drives proliferation, contrary to Figs. 7A-C).
Discussion
The discussion matches the results well, but it needs a closer look at the main finding and a better understanding of the study's limitations.
Lines 321-325: The discussion of the Cyclin B1/CDK1 upregulation is good, but needs to be more prominent and detailed. This is a central finding. Expand on the model of "mitotic catastrophe": PLK1 inhibition prevents the activation of the APC/C complex, which is required for the degradation of Cyclin B1. This leads to an accumulation of Cyclin B1 and CDK1, but the kinase is improperly regulated/activated, leading to a futile cycle that triggers cell death arrest in G2/M. This paradox is a known effect of PLK1 inhibition and should be highlighted as a key mechanism of action.
Lines 358-363 (Limitations): The point about A549 cells (a KRAS-mutant line) not representing all LUAD subtypes is crucial. The discussion should briefly speculate on how PLK1 dependency might differ across major LUAD genomic subtypes (e.g., EGFR-mutant, KRAS-mutant, etc.), as this has direct clinical relevance for patient stratification.
Conclusion
The conclusions align well with the findings but are slightly generic. Make the conclusion more precise. Rather than saying "elevated PLK1 levels were linked to disease progression," specify "We demonstrate that PLK1 overexpression serves as a significant independent prognostic indicator in LUAD, correlated with an immunosuppressive TME and driven by cell cycle dysregulation." Clearly articulate the dual role: "This study confirms PLK1's dual function as both a strong biomarker and a potential therapeutic target in LUAD."
1. The manuscript makes compelling claims about PLK1's role in modulating an immunosuppressive tumor microenvironment (TME), based on bioinformatic deconvolution of bulk transcriptomic data. While this analysis is informative and generates an interesting hypothesis, it remains correlational. The study lacks direct experimental evidence to prove that PLK1 causally regulates the infiltration or function of immune cells like Tregs and CD8⁺ T cells in LUAD. The authors should revise the manuscript to more cautiously interpret these findings.
2. In the Discussion section, line 331, the text refers to (Fig. 7) when discussing immune cell infiltration. The correct citation should be to Figure 6, which contains the relevant data.
3. The sentence on line 339 is a near-verbatim repeat of the preceding sentence on line 338.
4. The "Materials & Methods" section should explicitly state the final concentration of GSK461364 used for each of the downstream assays (clonogenic survival, apoptosis, cell cycle, and Western blotting). Although 40 nM is mentioned for the CCK-8 assay and can be inferred for the others, stating it clearly for each method is crucial for reproducibility.
5. Figure 1 contains Chinese characters. Please correct them immediately and pay close attention to all details to ensure that all details in the manuscript are accurate.
6. To enhance the robustness and generalizability of the findings, it is recommended that the study include validation of PLK1's function using additional datasets
7. The description of the methodology in the manuscript is somewhat lacking in detail. It would be helpful if the authors could provide more specific information regarding the experimental procedures. Furthermore, citing previously published studies that have used similar experimental approaches could enhance the reliability and validity of the methods presented, as it would provide additional support for the chosen techniques.
8. In the discussion section, it is crucial to address the complex interactions within the tumor microenvironment (TME) when applying immunotherapy. The TME plays a significant role in modulating immune responses and can impact the effectiveness of treatments. The authors could strengthen this section by discussing how various components of the TME, such as immune cells, cytokines, and tumor-associated factors, influence the efficacy of immunotherapy. Including relevant and recent studies on the TME’s role in immunotherapy would add credibility and depth to the discussion. They could offer valuable insights into these complex interactions and provide a more comprehensive understanding of how cytokines and the TME influence the therapeutic outcomes in gastric cancer. Incorporating these references would help clarify the dynamic relationship between immune cells, tumor cells, and the broader TME, which is essential for predicting treatment response.
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