Unveiling the Molecular Mechanism of Trastuzumab Resistance in SKBR3 and BT474 Cell Lines for HER2 Positive Breast Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

the manuscript describes very nicely the negative effects of transtuzumab resistance. It would be nice if the authors indicate quantitative data on the amount of resistant patients (I have heard is in the order of 50%) but it would be nice to have the data.

the authors carry out an experiment to identify the genes and miRNAs implicated in the resistance. The way is done is very nice, they use two different cell lines and maintain them with different concentrations of the antibodies for a long time, extracting RNAs at different times and analysing the RNAs and miRNAs the show significative differences, and after identifying them try to explain the implications the disregulaton of these genes. These explanations give us a clear idea of possible ways of targeting the resistance.

The work has been carrefully done, it is well explained and represents a nice piece of work highly interesting to people working in mammary tumors, but also in people working in immunotherapy of cancer in general since it points how to make these analyses whenever there is a resistance effect, which happens with a higher frequency tan expected.

Author Response

Comment :  Indicate quantitative data on the amount of resistant patients (I have heard is in the order of 50%) but it would be nice to have the data.

 

Author’s answer: We agree with the reviewer's advice. 

However, the goals and objective of the presented study have been confined to addressing a) in-vitro resistance development, b) determining the main biological and molecular processes affected using signalling pathways and protein networks study, and c) determining main molecular and biological mechanisms using integrative analysis of obtained high-throughput transcriptomic and microRNA expression data,  during drug resistance development. Therefore, quantitative analyses of gene expression at the protein level are left for future research.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a focused study on the molecular mechanisms behind trastuzumab resistance in breast cancer cell lines, offering insights that could potentially guide future therapeutic strategies. While the research approach is methodologically sound, certain aspects could be refined to enhance the study's robustness and the reliability of its conclusions.

Specific Comments:

·         The use of only two replicates at each time point limits the power to detect true differences and makes estimates of variability less robust. This could lead to unreliable identification of differentially expressed genes (DEGs). It is recommended to increase the number of replicates to improve the study's accuracy and reliability.

·         Given the small number of replicates, the application of additional cutoff parameters like Fold Change is advisable. This approach helps mitigate technical noise in genes detected at very low levels, which are more prone to technical variation. Incorporating such parameters would strengthen the analysis and the interpretation of the microarray data.

·         The absence of detailed data QC results in the manuscript is a notable omission, especially considering the limited number of replicates. Demonstrating that the data quality is comparable across conditions and that data between replicates are consistent is crucial. The inclusion of QC results in the supplemental materials would greatly enhance the credibility of the findings, particularly for results presented in figure 6, which could otherwise be misleading.

·         Investigating the expression of identified DEGs in patient samples and/or databases and their association with patient outcomes would significantly enrich the study. Such analysis could provide much-needed validation of the study's findings and their relevance to clinical practice, potentially highlighting new avenues for therapeutic intervention.

·         The authors should mention the limitation of this cell line-based assay. Additional factors, such as the immune system in vivo are not included in this experimental model.

Conclusion:

The study contributes valuable insights into the mechanisms of trastuzumab resistance. However, addressing the aforementioned concerns regarding sample size, data analysis robustness, quality control, and external validation would substantially reinforce the findings and their applicability to overcoming trastuzumab resistance in HER2 positive breast cancer.

 

 

Comments on the Quality of English Language

N/A

Author Response

Please see the attached.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript delivered an investigation into the resistance mechanisms against trastuzumab in HER2-positive breast cancer, highlighting the pivotal roles of specific genes and miRNAs through the use of SKBR3 and BT474 cell lines. This research meticulously outlines the development of trastuzumab-resistant cell lines and employs comparative analyses with control cell lines to identify molecular regulators contributing to resistance. The discovery of key genes and miRNAs underpinning trastuzumab resistance in HER2-positive breast cancer cell lines is a significant achievement of this study.

The structure of the study is commendably clear, guiding the reader from the significance of the research through to the experimental design, findings, and conclusions. The methodology is robust, employing a variety of techniques to reinforce the credibility of the results.

However, some areas for improvement are noted:

Figure 1: Clarify the number of biological replicates included in the figure within the legend.

Figure 2: Expand the legend to describe the time point symbols ("control", "T2", "T4", "T5", "T7") and their selection rationale. Clarify why control is associated with sensitive cell lines, while other symbols represent resistant cell lines, including an explanation for the observed proliferation rates.

Figure 3: Consider relocating this figure to the supplementary information section since it does not directly contribute to the core conclusions, or alternatively, present a single representative graph in the main text and relegate the rest.

Figure 4: Specify the number of biological replicates in the legend for clarity.

Figures 5 & 6: Suggest focusing on comparing different time points to "T0" rather than conducting pairwise comparisons across all time points. Presenting the data as line graphs could better illustrate the treatment's time-course effects.

Tables 8-12: Enhance readability and informativeness by incorporating graphical representations of the GO and KEGG analysis results.

Line 97: Provide a concise rationale for the selection of the treatment doses (5 µg/mL and 10 µg/mL), including references to existing literature.

Line 104: Highlight the initial observation that the high proliferation rates of the trastuzumab-resistant cell lines indicate successful establishment, despite differences in sensitivity to trastuzumab.

Line 280: The selection of 25 genes and miRNAs for interaction investigation appears somewhat arbitrary. A broader gene pool should be analyzed to achieve more universal results.

By the way, to enhance the reliability of gene expression findings, it is advisable to conduct quantitative analyses of gene expression at the protein level. This step will serve as a validation for the microarray data, ensuring the results' accuracy and comprehensiveness.

Overall, the manuscript is well-crafted, presenting significant contributions to our understanding of trastuzumab resistance in HER2-positive breast cancer. These recommendations aim to enhance the clarity, impact, and scientific rigor of the findings.

Author Response

Please see the attached.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

1. Applying established statistical model to a data set does not necessarily mean the output is always accurate and reliable. This is why it should be cautious when data with only one replicate is fit into a statistical model that was designed to make use of multiple replicates. The result could be still be informative to some extent but should be treated with caution.

2. fitting a linear model using the LIMMA package does not directly address the concern I mentioned in comment 2. Actually, fold change filter is a common practice that is recommended by the authors of LIMMA/edgeR.

3. thanks the authors for including the QC files. However, the most critical one: reproducibility between replicates. This is critical as there is only one replicate per sample. Low reproducibility will results in questionable differentially expressed gene list. Applying statistical methods is not sufficient to remove all technical artifacts.

4&5 without any evidence of validation from the functional perspective, it undermines the significance and impact of such analysis 

Comments on the Quality of English Language

N/A

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript presented a comprehensive investigation into the molecular mechanism of the resistance to trastuzumab in HER2-positive breast cancer cells with integrated transcriptomic and microRNA expression data analysis. Given the high prevalence of HER2-positive breast cancer worldwide and the associated mortality despite the advances in targeted therapies, the work exhibited great significance in breast cancer therapy.

The authors employed a robust two-fold experimental approach, utilizing well-characterized breast cancer cell lines, SKBR3 and BT474, to explore the development of trastuzumab resistance. The methodology involving the optimization of trastuzumab doses based on cell proliferation rates, followed by a longitudinal study of gene and miRNA expression, is well-designed and appropriate for the objectives of the study. The identification of differentially expressed genes and miRNAs, such as BIRC5, E2F1, TFRC, USP1, and several hsa-miRs, provided valuable insights into the complex network of molecular interactions leading to drug resistance.

However, the paper would benefit from a clearer articulation of its novelty and significance in the broader context of HER2-positive breast cancer research. While the identification of molecular players in trastuzumab resistance is undoubtedly valuable, a more detailed discussion on how these findings advance the current understanding or open new therapeutic avenues would enhance the manuscript's impact.

Additionally, while the experimental design is sound, the manuscript could be improved by providing more details on the validation of the identified targets. For instance, functional assays to confirm the role of the highlighted genes and miRNAs in mediating trastuzumab resistance would strengthen the conclusions. Furthermore, considering the complex nature of cancer and drug resistance, integrating the findings with existing knowledge on cancer signaling pathways and resistance mechanisms could offer a more comprehensive view.

In conclusion, this manuscript contributed important findings to the field of breast cancer research, particularly in understanding trastuzumab resistance. With some revisions to deepen the analysis and contextualize the results within the existing literature, this paper has the potential to be a valuable resource for researchers and clinicians alike. Incorporating suggestions for future research directions and potential clinical implications of the findings could also enhance the manuscript's relevance and applicability.

Author Response

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Author Response File: Author Response.pdf