RAC1 Activation as a Potential Therapeutic Option in Metastatic Cutaneous Melanoma

Round 1

Reviewer 1 Report

In this review, the authors discuss the role of RAC1 in metastatic melanoma.

Whilst metastatic melanoma is indeed a topic worthy of review, there are already other recent reviews on the role of RAC1 in melanoma (for example: "Rac1, A Potential Target for Tumor Therapy" in Front. Oncol., May 2021 and "RAC1 as a Therapeutic Target in Malignant Melanoma" in Trends Cancer., June 2020) which are more comprehensive and better written than this review. Thus, I do not believe that this review will be much cited.

I have strong concerns as to why this this review is not suitable for publication.

(1) In this review, melanoma is treated as a single entity, however, cutaneous melanoma, mucosal melanoma and acral melanoma have distinct underlying genetics and signalling pathways, which are not considered.

(2) The manuscript has not been well written. 

(a) It is littered with typographical and grammatical errors. In several places, the sentences do not make sense. I have given some examples below, but there are numerous more: 

• Citation of reference [1] is missing. The first reference citations (line 26) are “[2-5]”.
• Line 26: “a n” needs to be changed to “an”.
• Line 27: “GDF” should be “GDP”.
• Line 30: remove the word “including”.
• Line 40: the comma should be removed.
• Line 42: “[7] [8]” should be “[7-8]”.
• Line 44: a comma is needed after “NRAS Q61”.
• Line 44: “RAC wit a requency” should be “RAC1 with a frequency”.
• Line 46: change “P29S” to “RAC1P29S”
• Line 56: “It has een” should be “It has been”.
• Lines 54-57 of the Introduction do not make sense (“Moreover, malignant melanocytes have elevated RAC activity that extended into the epidermis, deeper into the dermis, and was maintained during metastasis”). Similarly, the following lines (57-60) also do not make sense as there are gene names (some given in full, some only in abbreviated form) mixed in with biological processes (“T-cell invasion”).
• Line 73: “can shuttled from de cytoplasm” should be “can be shuttled from the cytoplasm.
• Line 91: “There is a growing number of evidence indicating that” should be “There is a growing body of evidence indicating that”.
• Line 94, 110 and many other places: you cannot start a sentence with “And”.
• Line 100: “Overexpression of RAC1 in melanoma cells increased ROS that inhibited PP2A preventing thereby dephosphorylation of Bcl-2”. This sentence does not make sense.
• Line 125: “its” should be “their”.
• Line 143: “parts” should be “organs”.
• Line 147: “RAC1 is in encharged of….” should be “RAC1 is responsible for…..”.
• Line 150: a comma is needed between “that when activated” and “RAC1 suppressed”.
• Line 151: “An important RAC1 effector is WAVE2, member…” should read “An important RAC1 effector is WAVE2, a member….”.
• Line 154: “MLC” is not defined. Please write in full.
• Line 155: “importance of WAVE2 as RAC1 effector” should be “importance of WAVE2 as a RAC1 effector”.
• Line 157: “invasivenees” should be “invasiveness”.
• Line 161: change to “Another pathway regulated by RAC1 that plays an important role in melanoma is PI3K-AKT, which has also been described to play a role in epithelial-mesenchymal transcription and migration [37] [38].”

(b) The authors are inconsistent with nomenclature, as the manuscript flicks between using “RAC”, “RAC1” and “RAC-1”.

(c) There are numerous places where statements are made but there is no reference cited to back up the statement.

(d) Section 5, subheading "Blocking GEF/RAC1 Interactions": Some paragraphs are only 1 sentence long! This section is not well written and is merely a listing of every paper related to the topic. No assimilation of the findings into a comprehensive report.

(3) It is worth noting that this review cites only 1 reference from 2021 (which in itself is a review!) and only 6 references from 2020. If there has been no more new information in this field, then it begs the questions of why this review is needed?

Author Response

(1) In this review, melanoma is treated as a single entity, however, cutaneous melanoma,  mucosal melanoma and acral melanoma have distinct underlying genetics and signalling pathways, which are not considered.  
          
We thank the reviewer for the comment. In this review we have considered cutaneous melanoma. We know that melanoma is not a single entity. The different types of melanoma, cutaneous melanoma,  mucosal melanoma and acral melanoma have distinct underlying genetics and signaling pathways. In this review we focus in the role of RAC1 activation in metastasis of cutaneous melanoma, thus we have included the “cutaneous melanoma“ concept in the tittle and other sections.

  ( 2)The manuscript has not been well written.
We thank the reviewer for the comment. We have corrected all the mistakes and improved the manuscript writing.

(a)    It is littered with typographical and grammatical errors. In several places, the    sentences do
not make sense. I have given some examples below, but there are numerous more: We thank the reviewer for these suggestions:

•    Citation of reference [1] is missing. The first reference citations (line 26) are “[2-5]”. Corrected
•    Line 26: “a n” needs to be changed to “an”. Corrected
•    Line 27: “GDF” should be “GDP”. Corrected
•    Line 30: remove the word “including”. Corrected
•    Line 40: the comma should be removed. Corrected
•    Line 42: “[7] [8]” should be “[7-8]”. Corrected
•    Line 44: a comma is needed after “NRAS Q61”. Corrected
•    Line 44: “RAC wit a requency” should be “RAC1 with a frequency”. Corrected
•    Line 46: change “P29S” to “RAC1P29S”. Corrected
•    Line 56: “It has een” should be “It has been”. Corrected
•    Lines 54-57 of the Introduction do not make sense (“Moreover, malignant melanocytes have elevated RAC activity that extended into the epidermis, deeper into the dermis, and was maintained during metastasis”). Similarly, the following lines (57-60) also do not make sense as there are gene names (some given in full, some only in abbreviated form) mixed in with biological processes (“T-cell invasion”). Corrected
•    Line 73: “can shuttled from de cytoplasm” should be “can be shuttled from the cytoplasm. Corrected
•    Line 91: “There is a growing number of evidence indicating that” should be “There is a growing body of evidence indicating that”. Corrected
•    Line 94, 110 and many other places: you cannot start a sentence with “And”.
•    Line 100: “Overexpression of RAC1 in melanoma cells increased ROS that inhibited PP2A preventing thereby dephosphorylation of Bcl-2”. This sentence does not make sense. Corrected
•    Line 125: “its” should be “their”. Corrected
•    Line 143: “parts” should be “organs”. Corrected
•    Line 147: “RAC1 is in encharged of….” should be “RAC1 is responsible for…..”. Corrected
•    Line 150: a comma is needed between “that when activated” and “RAC1 suppressed”. Corrected
•    Line 151: “An important RAC1 effector is WAVE2, member…” should read “An important RAC1 effector is WAVE2, a member….”. Corrected
•    Line 154: “MLC” is not defined. Please write in full. Corrected
•    Line 155: “importance of WAVE2 as RAC1 effector” should be “importance of WAVE2 as a RAC1 effector”. Corrected
•    Line 157: “invasivenees” should be “invasiveness”. Corrected
•    Line 161: change to “Another pathway regulated by RAC1 that plays an important role in melanoma is PI3K-AKT, which has also been described to play a role in epithelial-mesenchymal transcription and migration [37] [38].” Corrected

(b) The authors are inconsistent with nomenclature, as the manuscript flicks between using “RAC”, “RAC1” and “RAC-1”. Corrected

(c) There are numerous places where statements are made, but there is no reference cited to back up the statement. Corrected

(d) Section 5, subheading "Blocking GEF/RAC1 Interactions": Some paragraphs are only 1 sentence long! This section is not well written and is merely a listing of every paper related to the topic. No assimilation of the findings into a comprehensive report.
 We thank the reviewer for this comment. This section describes  GEF-RAC1 interaction as an interesting therapeutic option. We have included a table (Table 1) showing RAC inhibitors that block GEF/RAC1 interactions.

(3) It is worth noting that this review cites only 1 reference from 2021 (which in itself is a review!) and only 6 references from 2020. If there has been no more new information in this field, then it begs the questions of why this review is needed?
We thank the reviewer for this comment.  However, we think that our review  is a very detailed work, referring to several RAC mutations, signaling pathways, in vivo  xenografts, transcriptional programming and  therapy resistance. So, we think this review is needed.

Reviewer 2 Report

I really enjoyed reviewing ''RAC1 activation as a potential therapeutic option in metastatic melanoma''. RAC1 is a member of the RHO family of small guanosine phosphatases with great potential in cancer migration, invasion, Angiogenesis and metastasis.

It is a very detailed work, referring to several mutations, signalling pathways, in vivo  xenografts, transcriptional programming, therapy resistance.

What I would like to see as a detailed introductory part is the effect of ECM molecules and signalling cascades in melanoma metastasis. Start by describing melanoma stages, existing melanoma therapeutic strategies. You can be inspired by the publications by Stephane Brezillon, where his laboratory is devoted in melanoma metastasis, invadopodia formation, signalling cascades in metastatic melanoma, e.t.c. .

Minor details: where is ref 1 cited in the manuscript ?

line 56 & line 73: check grammar  errors

in vivo, in vitro    in italics

lines 161- 163 : expand or delete this paragraph

graph 1 is very nicely detailed with reference to tumor growth & survival, migration & invasion, angiogenesis, therapy resistance  

graph 2 could have improved analysis 

Author Response

We would like to express our sincere appreciation for the reviewers’ constructive comments concerning our review titled “RAC1 activation as a potential therapeutic option in metastatic cutaneous melanoma”(manuscript ID:  biomolecules-1385426).

Thanks to their invaluable and professional feedbacks, we believe that the quality of our review has now been significantly improved. We have made extensive modifications to our review according to the reviewers’ comments to finally make our results more comprehensive.

I really enjoyed reviewing ''RAC1 activation as a potential therapeutic option in metastatic melanoma''. RAC1 is a member of the RHO family of small guanosine phosphatases with great potential in cancer migration, invasion, Angiogenesis and metastasis. It is a very detailed work, referring to several mutations, signalling pathways, in vivo xenografts, transcriptional programming, therapy resistance.

We  really thank the reviewer for this comment.

What I would like to see as a detailed introductory part is the effect of ECM molecules and signalling cascades in melanoma metastasis. Start by describing melanoma stages, existing melanoma therapeutic strategies. You can be inspired by the publications by Stephane Brezillon, where his laboratory is devoted in melanoma metastasis, invadopodia formation, signalling cascades in metastatic melanoma, e.t.c. ..

We thank the reviewer for the suggestion. We have included the effect of RAC1 activation regulating ECM in melanoma metastasis.

Minor details: where is ref 1 cited in the manuscript ? Thank you for the correction. It was a mistake, we have  corrected it.

line 56 & line 73: check grammar  errors. We have  corrected it.

in vivo, in vitro    in italics We have corrected it.

lines 161- 163 : expand or delete this paragraph. We have expanded this paragraph including new data about the role of RAC1 signaling regulating ECM.

graph 1 is very nicely detailed with reference to tumor growth & survival, migration & invasion, angiogenesis, therapy resistance  We thank the reviewer for this comment.

graph 2 could have improved analysis We thank the reviewer for this comment.

Reviewer 3 Report

The article “RAC1 activation as a potential therapeutic option in metastatic melanoma” review a growing body of literature on this RHO-family member of small guanosine triphosphatases in malignant melanoma

While the manuscript is generally well written and informative, it may benefit from a more accurate revision of the language in few points; for example:

• Page 4, lines 147-148: “RAC1 is in encharged of ….”
• Page 4, lines 161-163: “epithelial-mesenchymal transcription….”
• Page 5, line 186
• Page 7, lines 285-286:

And others throughout the manuscript.

I believe that the article may also benefit from the inclusion of a table summarizing the compounds developed targeting RAC1 with a description of the mechanism of action and reference to the original paper.

Author Response

We would like to express our sincere appreciation for the reviewers’ constructive comments concerning our review titled “RAC1 activation as a potential therapeutic option in metastatic cutaneous melanoma”(manuscript ID:  biomolecules-1385426). 

Thanks to their invaluable and professional feedbacks, we believe that the quality of our review has now been significantly improved. We have made extensive modifications to our review according to the reviewers’ comments to finally make our results more comprehensive.

Reviewer 3#

The article “RAC1 activation as a potential therapeutic option in metastatic melanoma” review a growing body of literature on this RHO-family member of small guanosine triphosphatases in malignant melanoma.

While the manuscript is generally well written and informative, it may benefit from a more accurate revision of the language in few points; for example:

We thank the reviewer for these corrections

• Page 4, lines 147-148: “RAC1 is in encharged of ….” Corrected
• Page 4, lines 161-163: “epithelial-mesenchymal transcription….” Corrected
• Page 5, line 186 Corrected
• Page 7, lines 285-286: Corrected

And others throughout the manuscript. We thank the reviewer for this comment. We have corrected the mistakes.

I believe that the article may also benefit from the inclusion of a table summarizing the compounds developed targeting RAC1 with a description of the mechanism of action and reference to the original paper. We thank the reviewer for this suggestion.  We have included a table summarizing the compounds developed targeting RAC1 with a description of the mechanism of action and reference to the original paper (Table1).

Round 2

Reviewer 1 Report

Overall the manuscript has been improved, however, there are still some remaining issues:

• Figure 1 still says “RAC” throughout instead of “RAC1”.

• These sentences do not make sense (i.e., I do not understand the point that is being made, so cannot correct them):

Lines 282 – 284

Lines 339 – 340

Lines 353 – 356

Lines 430 – 431

Lines 455 – 457

• The following corrections (“>>>”) need to be made:

Line 15: melanoma >>> cutaneous melanoma

Line 25: cancers >>> cancer

Line 27: define EMT (as this is the first time you use this abbreviation)

Line 69: RAS induced >>> RAS-induced

Line 94, 214, 219 and 484: wt >>> wildtype

Line 97: de >>> the

Line 107: anti-metastasis >>> anti-metastatic

Line 108, 110 and 116: melanoma >>> cutaneous melanoma

Line 120: italicize gene names

Line 123: RAC-1 >>> RAC1

Line 125: renders >>> confers

Line 135: remove “known”

Line 145: RAC1 >>> RAC1 (as it’s a gene name)

Line 146: BRAF >>> BRAF (as it’s a gene name)

Line 206: shRNA >>> knockdown

Line 198: use the abbreviation ‘EMT’

Line 200: promoting >>> and promotes

Line 210: et al –> et al.

Line 211: RAC1 wt >>> wildtype RAC1

Line 212: had >>> showed

Line 215: remove the full stop in the middle of the line

Line 222: is the word “prevent” missing? i.e., “Merlin, a tumor suppressor known to downregulate cyclin D1 and prevent cell cycle progression”

Line 223: both, >>> , both

Line 227 pro metastatic >>> pro-metastatic

Line 233: invasion >>> undergoing invasion

Line 245: pointed out >>> highlighted

Line 245: in >>> during

Line 277: [[53] . >>> [53].

Line 279: major vessels >>>major vessel

Line 280: probably >>> likely

Line 285: Matrix Metalloproteinases >>> matrix metalloproteases

Line 286:remodel >>> remodel the

Line 286: remove “Extracellular Matrix” as ECM has already been defined previously

Line 286: ECM >>> ECM,

Line 287: allow >>> allows

Line 287: cells >>> cell

Line 290: “comprising”….did you mean “involving”?

Line 292: “downstream VEGF”…..did you mean “downstream of VEGF”

Line 298: caused >>> causes

Line 302: did you mean to include “ROS”?

Line 305: VEGFR mediating >>> VEGFR, mediating

Line 306: if you are referring to the IQGAP1 gene then it needs it be italicized (and in lower case if it’s a mouse gene)

Line 306: VEGFR2-RAC1 >>> the VEGFR2-RAC1

Line 309: RAC1-PAK1 >>> the RAC1-PAK1

Line 312: conquers >>> overcomes

Line 316: be used >>> prove useful

Line 320: melanoma >>> cutaneous melanoma

Line 348: remove “and”

Line 350: remove the comma

Line 365: interfere >>> interferes

Line 366: PATs >>> PAT

Line 367: its >>> their

Line 367: shown >>> been shown

Line 367: decreasing >>> decrease

Line 369: palmitoylation >>> palmitoylation,

Line 370: when you say “its inhibition”…..did you mean ‘RAC1 inhibition’? This is not clear

Line 374: change to: “allow attachment of downstream effectors”

Line 374: remove sentence starting “Since RAC1 has high….”. The following sentence should be: “EHT-1864 is an inhibitor of the Rac family GTPases and blocks activation by direct binding to all RAC isoforms [reference]. Critically, it has been shown to block RAC1-mediated transformation [64], although it has also shown off-target effects [reference]”.

Line 378: remove sentence starting “Unluckily, EHT1864 has shown….”

Line 383: depicts >>> represents

Line 402: Change the sentence starting “Then, these molecules…” to read “Another inhibitor that was identified, known as ‘Compound 4’, impeded RAC1 binding to TIAM1, TRIO and VAV2 [87]”

Line 408: cells >>> cell

Line 423: this summary sentence can be removed as it doesn’t contribute anything.

Line 426: Regardless attempts >>> Regardless of attempts

Line 429: druggable RAC1 target PAKs proteins that take part in >>> druggable RAC1 effectors are the PAKs, which take part in….

Line 443: SRF/MRTF inhibitors (CCG-1423 and CCG-203971) usage >>> use of SRF/MRTF inhibitors (CCG-1423 and CCG-203971)

Line 445: overcome >>> overcame

Line 447: progressioN >>> progression

Line 449: could be used in RAC1 mutant tumours treatment >>> could be used in the treatment of RAC1 mutant tumors

Line 451: Regarding PI3K network >>> Regarding the PI3K network

Line 452: cells >>> cell

Line 453: RAC1 and BRAF --- if these are genes (not proteins) they should be italicized

Line 469: RAC1P29S mutant >>> mutant RAC1P29S

Line 470: dabrafenib conferring >>> dabrafenib, thus conferring

Line 472: did you mean “RAC1P29S triggers the PAK, AKT….”

Line 475: inhibitors >>> inhibitor

Line 475: las >>> last

Line 476: and used >>> and been used

Line 480: melanoma [106]. Ha Linh Vu and collaborators, found….. >>> melanoma [106], Vu et al. [14] found…..

Line 487: remove the sentence starting “Further research on aberrant” as it doesn't make sense. Then the following sentence (“Therefore it may be valuable….”) can come straight after the sentence (“These observations suggest that….”). This flows better.

Line 514: RAC1 mutant >>> mutant RAC1

Author Response

We would like to express our sincere appreciation for reviewer 1’ constructive comments concerning our review titled “RAC1 activation as a potential therapeutic option in metastatic cutaneous melanoma”(manuscript ID: biomolecules-1385426). 

Thanks to their invaluable and professional feedbacks, we believe that the quality of our review has now significantly improved.

 We have made modifications to our review according to reviewer 1’s comments to finally make our results more comprehensive. 

Overall the manuscript has been improved, however, there are still some remaining issues:

• Figure 1 still says “RAC” throughout instead of “RAC1”.

We  really thank the reviewer for this comment. We corrected it

• These sentences do not make sense (i.e., I do not understand the point that is being made, so cannot correct them):

 We  really thank the reviewer for this comment. We corrected them.

Lines 282 – 284

Lines 339 – 340

Lines 353 – 356

Lines 430 – 431

Lines 455 – 457 

• The following corrections (“>>>”) need to be made:

We thank the reviewer for this comment. Thanks to her/his exhaustive corrections. We corrected all of them.  We believe that the quality of our review has now significantly improved.

Line 15: melanoma >>> cutaneous melanoma

Line 25: cancers >>> cancer

Line 27: define EMT (as this is the first time you use this abbreviation)

Line 69: RAS induced >>> RAS-induced

Line 94, 214, 219 and 484: wt >>> wildtype

Line 97: de >>> the

Line 107: anti-metastasis >>> anti-metastatic

Line 108, 110 and 116: melanoma >>> cutaneous melanoma

Line 120: italicize gene names

Line 123: RAC-1 >>> RAC1

Line 125: renders >>> confers

Line 135: remove “known”

Line 145: RAC1 >>> RAC1 (as it’s a gene name)

Line 146: BRAF >>> BRAF (as it’s a gene name)

Line 206: shRNA >>> knockdown

Line 198: use the abbreviation ‘EMT’

Line 200: promoting >>> and promotes

Line 210: et al –> et al.

Line 211: RAC1 wt >>> wildtype RAC1

Line 212: had >>> showed

Line 215: remove the full stop in the middle of the line

Line 222: is the word “prevent” missing? i.e., “Merlin, a tumor suppressor known to downregulate cyclin D1 and prevent cell cycle progression”

Line 223: both, >>> , both

Line 227 pro metastatic >>> pro-metastatic

Line 233: invasion >>> undergoing invasion

Line 245: pointed out >>> highlighted

Line 245: in >>> during

Line 277: [[53] . >>> [53].

Line 279: major vessels >>>major vessel

Line 280: probably >>> likely

Line 285: Matrix Metalloproteinases >>> matrix metalloproteases

Line 286:remodel >>> remodel the

Line 286: remove “Extracellular Matrix” as ECM has already been defined previously

Line 286: ECM >>> ECM,

Line 287: allow >>> allows

Line 287: cells >>> cell

Line 290: “comprising”….did you mean “involving”?

Line 292: “downstream VEGF”…..did you mean “downstream of VEGF”

Line 298: caused >>> causes

Line 302: did you mean to include “ROS”?

Line 305: VEGFR mediating >>> VEGFR, mediating

Line 306: if you are referring to the IQGAP1 gene then it needs it be italicized (and in lower case if it’s a mouse gene)

Line 306: VEGFR2-RAC1 >>> the VEGFR2-RAC1

Line 309: RAC1-PAK1 >>> the RAC1-PAK1

Line 312: conquers >>> overcomes

Line 316: be used >>> prove useful

Line 320: melanoma >>> cutaneous melanoma

Line 348: remove “and”

Line 350: remove the comma

Line 365: interfere >>> interferes

Line 366: PATs >>> PAT

Line 367: its >>> their

Line 367: shown >>> been shown

Line 367: decreasing >>> decrease

Line 369: palmitoylation >>> palmitoylation,

Line 370: when you say “its inhibition”…..did you mean ‘RAC1 inhibition’? This is not clear

Line 374: change to: “allow attachment of downstream effectors”

Line 374: remove sentence starting “Since RAC1 has high….”. The following sentence should be: “EHT-1864 is an inhibitor of the Rac family GTPases and blocks activation by direct binding to all RAC isoforms [reference]. Critically, it has been shown to block RAC1-mediated transformation [64], although it has also shown off-target effects [reference]”.

Line 378: remove sentence starting “Unluckily, EHT1864 has shown….”

Line 383: depicts >>> represents

Line 402: Change the sentence starting “Then, these molecules…” to read “Another inhibitor that was identified, known as ‘Compound 4’, impeded RAC1 binding to TIAM1, TRIO and VAV2 [87]”

Line 408: cells >>> cell

Line 423: this summary sentence can be removed as it doesn’t contribute anything.

Line 426: Regardless attempts >>> Regardless of attempts

Line 429: druggable RAC1 target PAKs proteins that take part in >>> druggable RAC1 effectors are the PAKs, which take part in….

Line 443: SRF/MRTF inhibitors (CCG-1423 and CCG-203971) usage >>> use of SRF/MRTF inhibitors (CCG-1423 and CCG-203971)

Line 445: overcome >>> overcame

Line 447: progressioN >>> progression

Line 449: could be used in RAC1 mutant tumours treatment >>> could be used in the treatment of RAC1 mutant tumors

Line 451: Regarding PI3K network >>> Regarding the PI3K network

Line 452: cells >>> cell

Line 453: RAC1 and BRAF --- if these are genes (not proteins) they should be italicized

Line 469: RAC1P29S mutant >>> mutant RAC1P29S

Line 470: dabrafenib conferring >>> dabrafenib, thus conferring

Line 472: did you mean “RAC1P29S triggers the PAK, AKT….”

Line 475: inhibitors >>> inhibitor

Line 475: las >>> last

Line 476: and used >>> and been used

Line 480: melanoma [106]. Ha Linh Vu and collaborators, found….. >>> melanoma [106], Vu et al. [14] found…..

Line 487: remove the sentence starting “Further research on aberrant” as it doesn't make sense. Then the following sentence (“Therefore it may be valuable….”) can come straight after the sentence (“These observations suggest that….”). This flows better.

Line 514: RAC1 mutant >>> mutant RAC1