The Gene Master Regulators (GMR) Approach Provides Legitimate Targets for Personalized, Time-Sensitive Cancer Gene Therapy
Round 1
Reviewer 1 Report
The paper by Iacobos et al entitled "The Gene Master Regulators (GMR) approach provides the most legitimate targets for personalized, 3 time-sensitive cancer gene therapy" describes master regulator(s) gene expression (Gene Master Regulators) for personalized therapeutic targeting in cancers. Methods have been proposed to identify such GMRs human kidney, thyroid and prostate cancer biopsies/surgically excised tumors as well as using certain cancer cell lines. Identification of aberrantly expressed tumor specific genes or pathways for therapeutically targeting is the goal of cancer field and the software methodology described in this study though different, the approach is not entirely novel.
Specific comments
1. It is not clear in the cancer and normal tissues selected for analysis in this study were comprehensively analyzed for known cancer specific mutations. If they have been analyzed, the contributions of such mutations to overall changes gene expressions in cancers should be discussed.
2. The reason for choice of cancer samples or cell lines was not described.
3. It is not clear whether any of the treatment was undertaken by patients and its influence on GMRs identified in this manuscript.
4. Ploidy of the tumor and normal samples used in this study is not described. It should be evaluated and its influence on gene expression should be discussed.
5. It is not clear whether changes in transcript expression of GMRs is correlated with changes in protein expression.
Author Response
Reviewer 1
Comments and suggestions:
The paper by Iacobos et al entitled "The Gene Master Regulators (GMR) approach provides the most legitimate targets for personalized, 3 time-sensitive cancer gene therapy" describes master regulator(s) gene expression (Gene Master Regulators) for personalized therapeutic targeting in cancers. Methods have been proposed to identify such GMRs human kidney, thyroid and prostate cancer biopsies/surgically excised tumors as well as using certain cancer cell lines. Identification of aberrantly expressed tumor specific genes or pathways for therapeutically targeting is the goal of cancer field and the software methodology described in this study though different, the approach is not entirely novel.
Thank you very much for reviewing our manuscript. However, our approach is a significant departure from the “the goal of cancer field: identification of aberrantly expressed tumor specific genes” that may have caused cancerization. Instead, we are looking for the genes whose targeting would selectively kill the cancer cells from the tissue. Such genes have the expression so strongly correlated with that of many other genes that their forced alteration triggers unbearable perturbations of major functional pathways. In tens of previous papers on a large variety of cells and tissues from humans and animal (mouse, rat, rabbit, dog, chicken embryo) models of human diseases we proved that major functional pathways remodel in disease, with certain genes being promoted to command positions. We know that a gene is critical for a particular phenotype when its expression is strongly controlled by the homeostatic mechanisms, making it resistant to the small environmental fluctuations and thus very stably expressed across biological replicas. The biological replicas (e.g. the quarters of a quadrisected cancer nucleus) can be considered as being the same biological system but subjected to slightly different local conditions. The GMR is the top of the gene chain of command. We identify the GMRs by asking not the literature but the cancer nodules themselves what genes are critical for their very existence.
Specific comments:
1. It is not clear in the cancer and normal tissues selected for analysis in this study were comprehensively analyzed for known cancer specific mutations. If they have been analyzed, the contributions of such mutations to overall changes gene expressions in cancers should be discussed.
We have profiled the entire transcriptomes of both cancer and normal tissues from the selected samples with the main purpose to identify what genes command each phenotype. Although we identified also the regulated genes (and can provide supplementary tables with them for all analyzed tumors), our interest was not in what gene alteration might have caused cancerization but what genes should be targeted to selectively kill the cancer cells with minimal impact on the normal cells.
2. The reason for choice of cancer samples or cell lines was not described.
The purpose was to test our approach in as many as possible cancer samples and cells for the limited resources at our disposal. As a proof of concept, at this stage of the research, the types of cancer are less important. However, collaborative experiments with medical centers are under way to test our approach on several other cancers.
3. It is not clear whether any of the treatment was undertaken by patients and its influence on GMRs identified in this manuscript.
No treatment was undertaken by patients. It is a long way to get an FDA approved treatment. For now, we try to show the research community that there are other potential avenues in cancer gene therapy that deserve to be explored.
4. Ploidy of the tumor and normal samples used in this study is not described. It should be evaluated and its influence on gene expression should be discussed.
Even we have done our best to select as histo-pathologically homogeneous as possible regions to profile, we totally agree that our samples are still heterogeneous and cells of other than the dominant phenotype (normal or cancer tissue) affected the results. Experiments are under way with a Sony SH8005 sorter to separate the tumor cells into distinct phenotypes and then profile separately their transcriptome. However, our manuscript does not intend to provide definite results for kidney, papillary and anaplastic thyroid, androgen-sensitive and androgen-insensitive prostate or blood cancers, but to show a way to identify potential targets for gene therapy of any cancer type.
5. It is not clear whether changes in transcript expression of GMRs is correlated with changes in protein expression.
If the GMR transcript is coding, then its forced alteration affects directly the encoded protein. However, coding or non-coding, through the strong expression coordination, manipulation of the GMR affects the levels of numerous proteins involved in major functional pathways.
Reviewer 2
Comments and suggestions:
The article "The Gene Master Regulators (GMR) approach provides the most legitimate targets for personalized, time-sensitve cáncer gene therapy” suggest the use of GMR approach for a cancer personalized treatments. Results are very interesting and well organized but there are some experiments that could increase the interest of this article:
Thank you so much for reviewing our work, appreciative remark and interesting suggestions.
Specific comments:
1.- I have some concerns about the low sample size. Authors use different tumor and cell models but the number of tumors analyzed for each tumor type is really low.
The purpose of our paper was to introduce and validate the GMR paradigm on all tumor samples and cancer cell lines we had access so far as the basis for a really personalized cancer gene therapy. Owing to the astronomic number of possibilities, each cancer patient has his/her unique gene hierarchy and therefore the GMRs should be identified for each patient separately. Larger number of analyzed tumors (that we’ll accomplish in time) may (or may not) identify common GMRs but will increase the confidence in the conclusions of this study that: 1) cancer nuclei and surrounding normal tissue have different GMRs and 2) that manipulation of the GMR expression has catastrophic consequences only on the (cancer or normal) cell phenotype it commands.
2.- How authors explain that control regions showed different GCH scores?
GCH analysis is highly sensitive and reveals that, although similar, humans are not identical. Nor are the gene hierarchies, even in the normal regions or in cancer nodules with the same Gleason score (as in Table 3).
3.- In my opinion authors did not show any conclusive evidence about the clinical utility of GMR theory. There are any relation between chemotherapy treatment in different cell lines and top gene expression in each model? Authors should incubate cells with different drugs in order to analyze if GMR approach could improve drug treatment selection.
This is a very interesting experiment to be performed in future. However, what we propose here is how to select the targets for cancer gene (not drug) therapy. With regard to the clinical utility, we’ve shown that manipulating the expression of a gene has transcriptomic consequence proportional to its GCH score. Therefore, targeting the cancer nodule GMR should have the largest destructive effect. As limited as it is for now as number of cases studied, our work may open novel research avenues.
In conclusion, from my point of view this article is interesting but these points must be clarified for publication in Genes.
Thank you so much for the positive conclusion and hope that our responses provided acceptable clarifications.
Reviewer 2 Report
The article "The Gene Master Regulators (GMR) approach provides the most legitimate targets for personalized, time-sensitve cáncer gene therapy” suggest the use of GMR approach for a cancer personalized treatments. Results are very interesting and well organized but there are some experiments that could increase the interest of this article:
1.- I have some concerns about the low sample size. Auhors use different tumour and cell models but the number of tumours analized for each tumour type is really low.
2.- How authors explain that control regions showed different GCH scores?
3.- In my opinion authors did not show any conclusive evidence about the clinical utility of GMR theory. There are any relation between chemotherapy treatment in different cell lines and top gene expression in each model? Authors should incubate cells with different drugs in order to analyze if GMR approach could improve drug treatment selection.
In conclusion, from my point of view this article is interesting but these points must be clarified for publication in Genes.
Author Response
Reviewer 2
Comments and suggestions:
The article "The Gene Master Regulators (GMR) approach provides the most legitimate targets for personalized, time-sensitve cáncer gene therapy” suggest the use of GMR approach for a cancer personalized treatments. Results are very interesting and well organized but there are some experiments that could increase the interest of this article:
Thank you so much for reviewing our work, appreciative remark and interesting suggestions.
Specific comments:
1.- I have some concerns about the low sample size. Authors use different tumor and cell models but the number of tumors analyzed for each tumor type is really low.
The purpose of our paper was to introduce and validate the GMR paradigm on all tumor samples and cancer cell lines we had access so far as the basis for a really personalized cancer gene therapy. Owing to the astronomic number of possibilities, each cancer patient has his/her unique gene hierarchy and therefore the GMRs should be identified for each patient separately. Larger number of analyzed tumors (that we’ll accomplish in time) may (or may not) identify common GMRs but will increase the confidence in the conclusions of this study that: 1) cancer nuclei and surrounding normal tissue have different GMRs and 2) that manipulation of the GMR expression has catastrophic consequences only on the (cancer or normal) cell phenotype it commands.
2.- How authors explain that control regions showed different GCH scores?
GCH analysis is highly sensitive and reveals that, although similar, humans are not identical. Nor are the gene hierarchies, even in the normal regions or in cancer nodules with the same Gleason score (as in Table 3).
3.- In my opinion authors did not show any conclusive evidence about the clinical utility of GMR theory. There are any relation between chemotherapy treatment in different cell lines and top gene expression in each model? Authors should incubate cells with different drugs in order to analyze if GMR approach could improve drug treatment selection.
This is a very interesting experiment to be performed in future. However, what we propose here is how to select the targets for cancer gene (not drug) therapy. With regard to the clinical utility, we’ve shown that manipulating the expression of a gene has transcriptomic consequence proportional to its GCH score. Therefore, targeting the cancer nodule GMR should have the largest destructive effect. As limited as it is for now as number of cases studied, our work may open novel research avenues.
In conclusion, from my point of view this article is interesting but these points must be clarified for publication in Genes.
Thank you so much for the positive conclusion and hope that our responses provided acceptable clarifications.
