Review Reports - EMT-Inducing Transcription Factors, Drivers of Melanoma Phenotype Switching, and Resistance to Treatment

Round 1

Reviewer 1 Report

This manuscript is a timely review on the control of melanoma phenotype switching and resistance to therapy by a group of transcription factors that controls epithelial-to-mesenchymal transition (EMT-TFs). This review first summarizes the role of EMT-TFs as regulators of melanoblast migration and patterning which regulate melanocyte homeostasis. Then this manuscript highlights how the plasticity of cancer cells underlies their capacity to adapt to the selective pressures they encounter during tumor development. The authors described EMT-TFs as major regulators of phenotype switching between differentiated/proliferative and neural crest stem cell-like/invasive states in melanoma. Various mechanisms of how the EMT-TFs create cancer cell plasticity and fuel both tumor initiation, metastasis and resistance to treatment are summarized. Moreover, the author discussed the therapeutic potential of targeting cell EMT-TFs /plasticity to prevent resistance to treatment in melanoma.

The subject is important, interesting and well presented by experts in the field. This topic is of interest to the readership of Cancers. Considering all the above achievements this review has reached, this article is suitable for publication with minor modifications. The manuscript could be improved by developping the regulation of EMT-TFs by microRNAs and the relationship between EMT-TFs and the metabolic plasticity of melanoma.

Author Response

We thank this reviewer for his/her helpful comments.

We agree that the role of microRNAs in the regulation of EMT-TFs needed to be mentionned. This is now done p7:

“A double-negative feedback loop involving miR-200 family members and ZEB transcription factors is known to regulate carcinoma cell plasticity [45]. However, miR-200 expression levels are low in melanoma and regulation of ZEB1/2 was shown to be largely miR-200 independent.” This reversible switch in EMT-TF expression is rather controlled downstream of ERK by a member of the AP-1 complex, FRA1 (FOS related 1), a master transcription factor of the gene regulatory network of the invasive phenotype in melanoma cells [30].

The relationship between EMT-TFs and metabolic plasticity has now been addressed in the « targeting of cell plasticity » section and promising approaches are now highlighted in Table1.

p17:

« Since metabolic plasticity is intimately linked to EMT, promising pharmacological inhibitors of metabolic pathways are currently investigated for their capacity to target EMT [95,96]. »

This is also further highlighted in the conclusion.

"Finally, future investigations will decipher the molecular crosstalk of EMT-TFs with epigenetic and metabolic regulators. This should lead to the development of novel therapeutic strategies aiming at targeting cell plasticity, which may be tested in combination with targeted or immunotherapies."

Reviewer 2 Report

The review treats the proposed topic in a complete and exhaustive way, providing a clear and complete overview of the EMT-inducing transcription factors. The text is well written although at times it is not very flowing.

Following my suggestions.
- pay more attention to punctuation. e.g. abstract line 11 "transcription factors [,] extensively described for their role in epithelial-mesenchymal 12 transition (EMT-TFs) in epithelial cells [,] also display essential functions in the melanocyte lineage"; abstract line 15 "as major regulators of phenotype [,] switching" etc

Line 62. I think it would be better to briefly explain the difference between invasive and proliferative state

Line 147. I would modify the phrase "from nevi to melanoma" as the transformation from nevus to melanoma is a rare event (30%). It is possible to say "from melanocytes to melanoma".

- Line 327. what is meant by "tumor resilience"?

- Line 364. "Nearly 40% of sustained responses were observed with anti-PD1 (Nivolumab, Pembrolizumab) [75]". Reference 75 refers to nivolumab + ipilimumab combination, while the text refers to the use of anti-PD-1 in monotherapy. I believe it is correct to give both information, since in some countries the combination ipilimumab + nivolumab is not yet approved. It would therefore be possible to leave the reference (75) and briefly inserting the results achieved with the immune combination into the text; at the same time, i suggest to add a second reference to what has already been written (eg Immune-checkpoint inhibitors for the treatment of metastatic melanoma: a model of cancer immunotherapy. Semin Cancer Biol. 2019 Dec; 59: 290-297. Doi: 10.1016 / j.semcancer.2019.08.001. Epub 2019 Aug 17).

Author Response

Following my suggestions.
- pay more attention to punctuation. e.g. abstract line 11 "transcription factors [,] extensively described for their role in epithelial-mesenchymal 12 transition (EMT-TFs) in epithelial cells [,] also display essential functions in the melanocyte lineage"; abstract line 15 "as major regulators of phenotype [,] switching" etc

We thank this reviewer for his/her useful comments and have carefully checked punctuation throughout the manuscript.

Line 62. I think it would be better to briefly explain the difference between invasive and proliferative state

This is done p9:

« The model based on MITF-mediated phenotype switching describes the reversible transitions of melanoma cells [27]. Gene expression analyses of melanoma short-term cultures enabled their classification into two states, proliferative or invasive [53]. Xenograft of either proliferative or invasive cells resulted in tumors displaying a similar level of intra-tumor heterogeneity with MITFhigh and MITFlow cells [28], thus demonstrating that melanoma cells are able to shift in a reversible way between these two states [29]. »

Line 147. I would modify the phrase "from nevi to melanoma" as the transformation from nevus to melanoma is a rare event (30%). It is possible to say "from melanocytes to melanoma".

We agree with this comment and this modification has been done.

- Line 327. what is meant by "tumor resilience"?

The term resilience was replaced by resistance.

- Line 364. "Nearly 40% of sustained responses were observed with anti-PD1 (Nivolumab, Pembrolizumab) [75]". Reference 75 refers to nivolumab + ipilimumab combination, while the text refers to the use of anti-PD-1 in monotherapy. I believe it is correct to give both information, since in some countries the combination ipilimumab + nivolumab is not yet approved. It would therefore be possible to leave the reference (75) and briefly inserting the results achieved with the immune combination into the text; at the same time, i suggest to add a second reference to what has already been written (eg Immune-checkpoint inhibitors for the treatment of metastatic melanoma: a model of cancer immunotherapy. Semin Cancer Biol. 2019 Dec; 59: 290-297. Doi: 10.1016 / j.semcancer.2019.08.001. Epub 2019 Aug 17).

A sentence and the proposed reference (n°76) were added p15:

« In addition to MAPK targeted therapies, another major breakthrough in the treatment of metastatic melanoma was achieved through immunotherapies targeting negative regulatory checkpoints in immune cells, CTLA4 and PD1 [76]. Nearly 40% of sustained responses were observed with anti-PD1 (Nivolumab, Pembrolizumab), and combination with anti-CTLA4 may further increase this efficacy [77]. However, despite impressive clinical responses around 60% of patients still present primary or acquired resistance to these therapies. »

Reviewer 3 Report

The article is intersting and well written. I would only recommend to the authors to provide a table with a list of the trials that are testing the hypothesis of interfering with EMT-inducing transciption factors to treat cancer in order to provide the readers with a stronger evidence on the feasibility of this approach.

Author Response

As recommended by this reviewer, a table has now been included.

Table1: Short list of the main therapeutic strategies under evaluation to target EMT-dependent cell plasticity in cancers.