The Regulation and Function of the Amino Acid Transporters LAT1, ASCT2, xCT in Urological Cancers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors attempted to give an overview of the most recent research concerning the regulation and function of the Amino Acid Transporters LAT1, ASCT2, xCT in urological cancers

While the paper addresses an interesting issue, it is not publishable in its current form.

The description of data and the studies presented lack important details and in general, the text is too poor in content and analysis.

 

 

 

Comments on the Quality of English Language

Moderate editing of English language is required

Author Response

comment1：The Authors attempted to give an overview of the most recent research concerning the regulation and function of the Amino Acid Transporters LAT1, ASCT2, xCT in urological cancers

While the paper addresses an interesting issue, it is not publishable in its current form.

The description of data and the studies presented lack important details and in general, the text is too poor in content and analysis.

 

response１: Thank Reviewer1 for giving us the opportunity to strengthen our manuscript with valuable comments and queries.

We have enhanced the details of the references cited in the paper, with a particular focus on elaborating the regulatory mechanisms involved in the cited studies. We have strengthened the sections on ASCT2 and xCT, adding new references and analyses of their results, which illustrate the close connections and potential mutual regulatory relationships among the three amino acid transporters (Line 185-205). Additionally, we have expanded our discussion on the relationship between ASCT2, xCT, and urological tumors (Chapter 3.1.2-3.1.3; 3.2.2-3.2.3; 3.3.2-3.3.3; ), as well as their inhibitors(Chapter 4.2-4.3; ), by including relevant literature and detailed descriptions of the studies. We have also added our perspectives on future research directions for targeting or inhibiting amino acid transporters (Chapter 5).

We have redrawn the figure and table, removing any obscure or confusing markings to make them more concise and clear (Figure 1). We have also adjusted the content of the inhibitors table to include not only urological tumors but also other significant and valuable tumor-related information that we consider important in the paper (Table 1).

We have corrected the spelling and grammatical errors in the paper, as well as some errors in the order of the headings.

Reviewer 2 Report

Comments and Suggestions for Authors

The review regulation and function of the Amino Acid Transporters (LAT1, ASCT2, xCT) in urological cancer submitted to journal receptors compiles the importance of amino acid transporters in urological cancer and focus on the review on expression regulation and clinical implications followed by suggestions on therapeutical targeting. The review paper is written elegantly in the first half; however, the later part appears as a quick summary and often appears incomplete.

I have noted some of the points which can improve the manuscript:

1.        In line 35, glucose is wrongly indicated as an amino acid. It must be corrected.

2.        Bladder and renal cancers are not covered in detail, and it needs more description of these malignancies is needed.

3.        There is brief information on the expression and regulation of LAT1. However, it needs to be included for ASCT2 and xCT and expanded for LAT1. It must be added to understand how malignant conditions change the regulation for their benefit.

4.        I am almost sure that more than what is described in section 4.3 is available. This section must be updated.

5.        In many places, an abbreviated form needs to be described. Any short form must be expanded on the first appearance in the text. Eg: LDH and NLR in line 184.

6.        There is a mention of Arsenic in line 188. However, the context needs to be clarified. It only says that arsenic might increase ASCT2 and Beta-catenin; however, how it could be linked to some of the malignancies mentioned here is unclear.

7.        Line 233 mentions a BCH’s chemical name. Please check this. It says 2-amino-2-ring. It should be 2-amino-bicyclo-. It needs to be corrected.

8.        My biggest concern with this review paper is that it is a partial compilation of the literature. More literature is available on the subject and focus of the review and must be added to make it more informative and useful.

 

Comments on the Quality of English Language

Minor editing is required and at some places choice of word is not appropriate.

Author Response

The review regulation and function of the Amino Acid Transporters (LAT1, ASCT2, xCT) in urological cancer submitted to journal receptors compiles the importance of amino acid transporters in urological cancer and focus on the review on expression regulation and clinical implications followed by suggestions on therapeutical targeting. The review paper is written elegantly in the first half; however, the later part appears as a quick summary and often appears incomplete.

 

A：Thank Reviewer2 for giving us the opportunity to strengthen our manuscript with valuable comments and queries.

 

I have noted some of the points which can improve the manuscript:

comment1：In line 35, glucose is wrongly indicated as an amino acid. It must be corrected.

response1：Yes, we apologize for this typographical error. We are sorry, and we have removed the incorrect mention of glucose.

 

comment2：Bladder and renal cancers are not covered in detail, and it needs more description of these malignancies is needed.

response2: We have strengthened the sections of the paper related to bladder cancer and kidney cancer, with particular emphasis on ASCT2 and xCT. We have added new references and provided additional descriptions and analyses of their results and mechanisms (Chapter 3.2-3.3). Furthermore, we have expanded our discussion on the relationship between ASCT2, xCT, and urological tumors, as well as their inhibitors, by including additional literature and detailed descriptions of the studies (Chapter 4).

 

comment3：There is brief information on the expression and regulation of LAT1. However, it needs to be included for ASCT2 and xCT and expanded for LAT1. It must be added to understand how malignant conditions change the regulation for their benefit.

response3: We have enhanced the details of the references cited in the paper, with a particular focus on elaborating the regulatory mechanisms involved in the cited studies. We have strengthened the sections on ASCT2 and xCT, adding new references and analyses of their results, which illustrate the close connections and potential mutual regulatory relationships among the three amino acid transporters (Line 185-205).

 

comment4：I am almost sure that more than what is described in section 4.3 is available. This section must be updated.

response4: we have expanded our discussion on the relationship between ASCT2, xCT, and their inhibitors, by including additional literature and detailed descriptions of the studies (Chapter 4.2-4.3).

 

comment5：In many places, an abbreviated form needs to be described. Any short form must be expanded on the first appearance in the text. Eg: LDH and NLR in line 184.

 response5：Yes, we have provided the full expansion of each abbreviation where it first appears.

 

commet6: There is a mention of Arsenic in line 188. However, the context needs to be clarified. It only says that arsenic might increase ASCT2 and Beta-catenin; however, how it could be linked to some of the malignancies mentioned here is unclear.

response6: Yes, we apologize for not clearly explaining the mechanisms involved. We have added the following paragraph to clarify that the pathogenic mechanism of arsenic remains unclear, but its role in promoting the progression of malignancies may be related to the upregulation of ASCT2 and its effects on beta-catenin and ROS.

“Arsenic exposure increases the risk of bladder cancer in humans, but the mechanisms behind it are unclear. Up-regulation of ASCT2 promotes the uptake of glutamine by cancer cells, which is a key factor in cancer cell proliferation and survival. The increase in ASCT2 further activates the β-catenin signaling pathway, which plays a key role in cell proliferation, self-renewal, and maintenance of tumor stem cell properties. By maintaining a balance of GSH/ROS, ASCT2 supports low levels of ROS, which is necessary for the stability and activity of beta-catenin. Thus, ASCT2 promotes cell proliferation and self-renewal by activating beta-catenin, crucial for maintaining GSH/ROS homeostasis. ASCT2 emerges as a potential therapeutic target for arsenic-induced urothelial cell proliferation and self-renewal[90,91].” (Line 401-411)

 

comment7: Line 233 mentions a BCH’s chemical name. Please check this. It says 2-amino-2-ring. It should be 2-amino-bicyclo-. It needs to be corrected.

response7：Yes, we apologize for this typographical error. We are sorry, and we have corrected the full name of BCH.

 

comment8: My biggest concern with this review paper is that it is a partial compilation of the literature. More literature is available on the subject and focus of the review and must be added to make it more informative and useful.

response8: We have refined the references in the paper, focusing on the regulatory mechanisms discussed in the studies. The sections on ASCT2 and xCT have been expanded with new references and analyses to highlight their interconnections and regulatory relationships among the three amino acid transporters (Lines 185-205). We also enhanced the discussion on ASCT2, xCT, and urological tumors (Chapters 3.1.2-3.1.3; 3.2.2-3.2.3; 3.3.2-3.3.3) and their inhibitors (Chapter 4.2-4.3), incorporating additional relevant literature and detailed study descriptions. Perspectives on future research directions for targeting or inhibiting amino acid transporters have been added (Chapter 5).

Figures and tables have been redrawn for clarity, removing any confusing markings (Figure 1). The inhibitors table has been updated to include information on both urological tumors and other significant tumors (Table 1).

Reviewer 3 Report

Comments and Suggestions for Authors

The review article titled "The Regulation and Function of the Amino Acid Transporters (LAT1, ASCT2, xCT) in Urological Cancers" provides a comprehensive overview of the role of these amino acid transporters in urological cancers. However, other amino acid transporters that might be also relevant in urological cancer like SLC6A14 (ATB0,+) and SLC38A2 (SNAT2) should be considered. 

The abstract effectively summarizes the key points of the review

The introduction provides a good background and rationale for the review. However, the figure is really confusing and lacks a proper description. In a review article, images should be easy to consult and well connected to the main text, but everywhere is cited (lines 84, 91 and 109) I can’t see what is written in the text clearly represented in the figure. I suggest to draw it differently and maybe split it into 2 figures to make it more understandable and to have the possibility to add features representative of each transporter. The figure legend should also explain why some arrows present different thickness or are dashed or have different colours.

The paragraph 4.2 is divided into smaller paragraphs representing just a list of papers and adding almost nothing with respect to the following table. This paragraph deserves a better analysis of the pathways where ASCT2 is involved and what represents its inhibition with different compounds.

paragraph 4.3 also deserves a better discussion, even if is not directly linked to urological cancers but the paragraph should explain the entire state of art of the transporter and what is its function in cancers and how its inhibition may affect tumor viability

 

Author Response

comment1:The review article titled "The Regulation and Function of the Amino Acid Transporters (LAT1, ASCT2, xCT) in Urological Cancers" provides a comprehensive overview of the role of these amino acid transporters in urological cancers. However, other amino acid transporters that might be also relevant in urological cancer like SLC6A14 (ATB0,+) and SLC38A2 (SNAT2) should be considered. 

The abstract effectively summarizes the key points of the review

The introduction provides a good background and rationale for the review.

response1: Thank Reviewer3 for giving us the opportunity to strengthen our manuscript with valuable comments and queries. We apologize for not covering SLC6A14 (ATB0,+) and SLC38A2 (SNAT2), which we agree are also important amino acid transporters. This is a limitation of our review and will be discussed in future studies.

 

comment2: However, the figure is really confusing and lacks a proper description. In a review article, images should be easy to consult and well connected to the main text, but everywhere is cited (lines 84, 91 and 109) I can’t see what is written in the text clearly represented in the figure. I suggest to draw it differently and maybe split it into 2 figures to make it more understandable and to have the possibility to add features representative of each transporter. The figure legend should also explain why some arrows present different thickness or are dashed or have different colours.

response2: We have redrawn the figure and table, removing any obscure or confusing markings to make them more concise and clear (Figure 1). We have also adjusted the content of the inhibitors table to include not only urological tumors but also other significant and valuable tumor-related information that we consider important in the paper (Table 1).

 

comment3: The paragraph 4.2 is divided into smaller paragraphs representing just a list of papers and adding almost nothing with respect to the following table. This paragraph deserves a better analysis of the pathways where ASCT2 is involved and what represents its inhibition with different compounds. paragraph 4.3 also deserves a better discussion, even if is not directly linked to urological cancers but the paragraph should explain the entire state of art of the transporter and what is its function in cancers and how its inhibition may affect tumor viability

 

response3: We have enhanced the details of the references cited in the paper, with a particular focus on elaborating the regulatory mechanisms involved in the cited studies. We have strengthened the sections on ASCT2 and xCT, adding new references and analyses of their results, which illustrate the close connections and potential mutual regulatory relationships among the three amino acid transporters (Line 148-168). Additionally, we have expanded our discussion on the relationship between ASCT2, xCT, and their inhibitors(Chapter 4.2-4.3), by including relevant literature and detailed descriptions of the studies. We have also added our perspectives on future research directions for targeting or inhibiting amino acid transporters (Chapter 5).

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have worked hard to improve the manuscript, which is now more complete and detailed.

Nevertheless, some improvements are needed:

- in the title, please remove the parentheses

- line 35: add the abbreviation of leucine  (Leu) as you did for glutamine (Gln) and use it through the whole text

- line 45: "Cancer cells accomplish this primarily....." please,Rewrite the sentence replacing "this" with the concept it refers to

-line 65: please specify the 4 subtypes names of LAT transportes

- line 72: plase replace "The study" with "the study of the author did the study

- fig 1 replace "cyto membrane" with "plasma membrane"

-line 136: Glutathione is not an aminoacid. Please correct the sentence

- line 164: please rewrite the sentence because it is not clear

- line 193 : please, replace the preposition " with" used in each title of the section 3  with " in" 

- line 202: please, add the abbreviation CRPC, used then at line 224

- line 245: please  specify the abbreviation used for ADT

 - line 308 and 338: please use this format: [¹⁸ F] and [¹⁴C]

- line 562: please replace "Francesca"  ( the first name) with "Oppalesano" ( the last name" as you can see in  ref 121

- line 648: please replace P53 with p53

 

Comments on the Quality of English Language

Minor editing of English language is required.

Author Response

Reviewer1

The authors have worked hard to improve the manuscript, which is now more complete and detailed.

A: Thank Reviewer 1, for providing detailed feedback that has helped us improve our manuscript.

 

Nevertheless, some improvements are needed:

- in the title, please remove the parentheses

A：Yes, we removed the parentheses in the title.

 

- line 35: add the abbreviation of leucine (Leu) as you did for glutamine (Gln) and use it through the whole text

A: Yes, we have added the abbreviation for leucine (Leu) and replaced the part of the full article that refers to leucine. (line 34)

 

- line 45: "Cancer cells accomplish this primarily....." please,Rewrite the sentence replacing "this" with the concept it refers to

A: Yes, we rewrote the sentence to make it easier to understand.

‘Cancer cells accomplish the reprogramming of their metabolic pathways primarily by upregulating various transporters that mediate glucose and amino acid uptake.’ (line 48-49)

 

-line 65: please specify the 4 subtypes names of LAT transporters’

A: Yes, we added the names of the 4 subtypes of LAT (LAT1, LAT2, LAT3, LAT4). (line 67)

 

- line 72: plase replace "The study" with "the study of the author did the study

A: Yes, we rewrote the sentence to make it easier to understand.

‘The study by the author underscores the necessity of 4F2hc for LAT1 transportation to the plasma membrane, with LAT1 influencing the transport properties of heterodimers.’ (line 73-75)

 

- fig 1 replace "cyto membrane" with "plasma membrane"

A: Yes, we replaced the "cyto membrane" in Figure 1 with "plasma membrane".

 

-line 136: Glutathione is not an aminoacid. Please correct the sentence

A: We are sorry for the clerical error. We've replaced amino acids with substances.

‘The amino acid transporter xCT is mainly responsible for transporting two substances, cysteine and glutathione.’ (line 140-141)

 

- line 164: please rewrite the sentence because it is not clear

A: Yes, we rewrote the sentence to make it easier to understand.

‘Since cysteine is essential for the synthesis of glutathione (GSH), intracellular cysteine is mainly obtained through xCT-mediated cystine uptake. When cystine is depleted or xCT is inhibited either genetically or pharmacologically, it induces strong ferroptosis in many cancer cells. On the other hand, the upregulation of xCT in cancer cells boosts GSH pro-duction, providing resistance to ferroptosis[59,62,63].’ (line 156-161)

 

- line 193 : please, replace the preposition " with" used in each title of the section 3  with " in" 

A: Yes, we changed the "with" to "in" in all the titles of Section 3.

 

- line 202: please, add the abbreviation CRPC, used then at line 224

A: Originally, we added the full name of CRPC (castration-resistant prostate cancer) where CRPC first appeared in lines 170-171. We also added the full name of CRPC where you mentioned (line213-214).

 

- line 245: please specify the abbreviation used for ADT

A: Originally, we added the full name of ADT (androgen deprivation therapy) where ADT first appeared in lines 231. We also added the full name of ADT where you mentioned (line233-234).

 

 - line 308 and 338: please use this format: [¹⁸ F] and [¹⁴C]

A: Yes, we have changed the numbers [¹⁸F] and [¹⁴C] in the whole paper to superscript.

 

- line 562: please replace "Francesca"  ( the first name) with "Oppalesano" ( the last name" as you can see in  ref 121

A: Yes, we replaced the author's first name with the last name. Sorry for the error.

 

- line 648: please replace P53 with p53

A: Yes, we changed all uppercase P53 to lowercase p53.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript, “The Regulation and Function of the Amino Acid Transporters 2 (LAT1, ASCT2, xCT) in Urological Cancers,” is significantly improved. I must say that the authors have done tremendous work to improve the manuscript. I have no major concerns regarding the content. However, I recommend a careful language review as there are a few errors, such as in line 34, where “cancer cell synthesis” should be “cancer cell proliferation.” Additionally, I suggest including a discussion on the drug JHU083. This glutamine antagonist can suppress tumor growth by impacting glutamine metabolism and mTOR signaling. It would be relevant to add a few reports on JHU083 in bladder cancer to the section on ASCT2. These are my only remaining minor comments.

Comments on the Quality of English Language

Minor improvement is needed.

Author Response

Reviewer2

The manuscript, “The Regulation and Function of the Amino Acid Transporters 2 (LAT1, ASCT2, xCT) in Urological Cancers,” is significantly improved. I must say that the authors have done tremendous work to improve the manuscript. I have no major concerns regarding the content. However, I recommend a careful language review as there are a few errors, such as in line 34, where “cancer cell synthesis” should be “cancer cell proliferation.” Additionally, I suggest including a discussion on the drug JHU083. This glutamine antagonist can suppress tumor growth by impacting glutamine metabolism and mTOR signaling. It would be relevant to add a few reports on JHU083 in bladder cancer to the section on ASCT2. These are my only remaining minor comments.

A: We would like to extend our sincere thanks to Reviewer 2 for the detailed feedback, which has greatly contributed to the improvement of our manuscript. In response to your suggestions, we have replaced "cancer cell synthesis" with "cancer cell proliferation" in line 33. Additionally, we have carefully proofread and corrected the grammar and spelling as requested. We have also incorporated the information about JHU083 in relation to ASCT2 and bladder cancer, and included the details of JHU083 and reference 94 in the final table as indicated.

“Studies have also shown that in prostate and bladder cancers, the increased expres-sion of ASCT2 is associated with an increase in tumor-associated macrophages (TAMs) with immunosuppressive properties[94]. JHU083 is a prodrug of a glutamine antagonist, which inhibits tumor growth by blocking glutamine metabolism. In prostate and bladder cancer models, JHU083 significantly inhibits tumor growth and alters immune cells within the tumor microenvironment, particularly TAMs. JHU083 suppresses glutamine utilization, promoting a shift in TAMs from the immunosuppressive M2 type to the in-flammatory M1 type. This shift increases TAMs' phagocytic activity and reduces their pro-angiogenic capabilities, disrupting tumor cell metabolic pathways, including glycoly-sis, the TCA cycle, and purine metabolism. This metabolic disruption in tumor cells may ultimately lead to apoptosis. In prostate and bladder cancer models, JHU083 as a mono-therapy can significantly inhibit tumor growth, and this effect is not entirely dependent on T cells. As a novel glutamine antagonist, JHU083 offers a potential therapeutic strategy for tumors rich in immunosuppressive TAMs, especially for tumor types that respond poorly to immune checkpoint blockade therapy[94].” (line346-360)

Reviewer 3 Report

Comments and Suggestions for Authors the author's reply and corrections satisfy my concerns, making the paper suitable for publication.

Author Response

Reviewer3

the author's reply and corrections satisfy my concerns, making the paper suitable for publication.

A: Thank Reviewer 3, for providing detailed feedback that has helped us improve our manuscript.

In addition to the above revisions, we also corrected some formatting issues, such as unnecessary spaces.