Aberrant Expression of ZIP and ZnT Zinc Transporters in UROtsa Cells Transformed to Malignant Cells by Cadmium

Round 1

Reviewer 1 Report

It has been a pleasure to review this work, even I have some comments, the results seems to show interesting results about the realationship between cancer and zinc homeostasis.

Minor points:

Introduction:

1. What´s the interest of study Zn/Cd homeostasis in cancer? Why Cadmium (Cd) can alter Zinc (Zn) homeostasis (mimetic action of Cd). You may need to relate Zn and Cd and explain why it is importan in cancer.
2. Can you indicate if there are differences in the cadmium blood concentration between smokers and no smokers?
3. It will be very useful that you explain the function and location of each ZnT and ZIP proteins, in the presence as absence of zinc, how the mechanisms woks, for example in a figure. It will help to the reader to follow the explanations and the importance of the variations in the exression of its genes. Also the explanations of the disscusion will be better understood.

Rest of the text:

4. The tables will be easier to read if its each one completed on one page, maybe due to the review format it has been cut.
5. Show the data that you talk about. For example, in line 113, you talk about the ZIP7 data expression, but you do not show them in none of the figures or graphics.
6. Unify writing, if number of clones are used, then do not use percentages. In my opinion, in the point number 2.2 of the results, as the number of clones are small, talk about number of clones is more representative than percentages.
7. Can you explain why did you choose the clone number six for the "versus expression analysis" with the clone 1?
8. Don´t you think that introducing the parental expression level in the graphs of figure 1 will show better the differences in each transformed clone? I think it will bring a very visual approach to the reader.
9. In the discussion, the first sentence must be referred to the table1.
10. Why do you use 3 µM of Cd to transform the cells? Is it the maximum concentration the cells supports? And why 1µM for the maintenance? When you have the clones, did you use Cd to re-grow it, did they conserved it miss zinc homeostasis desregulation in the absence of Cd?
11. It will be interesting that you can better explain the paragraph between lines 179 and 184, as you do in the next paragraph?

Mayor points:

Results:

12. You explain how you transformed UROtsa cells, but you do not explain nor in the Results or in the Materials and Methods sections the culture conditions you have used to grow the UROtsa cells (parental / cadmium-transformed) to obtain the RNA for the expression studies. Which was the concentration of Cd and Zinc in the employed medium? Is the RNA obtained from cultures supplemented with 3 µM of Cd? Depending on the Zn and Cd concentration the expression will change and to know the real their real concetration and the balance between these tow metals will be important to interpret and understand the results.
13. It will be interesting to include in this study if the UROtsa transformed cells present changes in the expression, in the zinc homeostasis transcription factors (ZNF658, MTF1...), the metallothioneins or in some hotspot genes that encode proteins involved in the cell cycle, for example: CDC45, TOR... It will indicate if the changes in zinc homeostasis can be blamed to changes in the expression of the regulons or it affects to genes of the hot spots cell cycle, or help to better understand the results: Why is the expression in the Cd1 clon different to the others, may be due to the toxicity of Cd affectes other mechanisms that in the others?
14. Do you have the tumors that you had obtained from the heterotransplants you realized with the UROtsa Cd-transformed cells? I think that will be very interesting to know if there are changes in the expression of all the genes, or in the most important in this cell, in vivo and compare the results that you have obtained with the in vitro
15. I can´t understand why and how do you "envisioned" (line 169), that ZIP14 and ZnT1 can "influence uptake, accumulation levels and resultant toxicity of cadmium" when they are having relatively high expression levels, can you explain it clearly?
16. In lines 172 and 173, you say that UROtsa cells with high levels of ZnT1 acquired tolerance to cadmium toxicity. To suggest this, did you had done assays to measure the tolerance of these cells to Cd toxicity?  
17. Review the first part of the discussion (line 166 to 192), you have to better explain it and indicates how your results supports your conclusions.

Author Response

Reviewer 1

It has been a pleasure to review this work, even I have some comments, the results seems to show interesting results about the relationship between cancer and zinc homeostasis.

We thank the Reviewer for advice, constructive and insightful comments.  Accordingly, we have revised our paper extensively, and its title has been changed to read, “Aberrant Expression of ZIP and ZnT Zinc Transporters in UROtsa Cells Transformed to Malignant Cells by Cadmium”. Part of abstract has been rewritten.  We provide below point-by-point response to the issues raised.

Minor points:

Introduction:

Point 1:  What´s the interest of study Zn/Cd homeostasis in cancer? Why Cadmium (Cd) can alter Zinc (Zn) homeostasis (mimetic action of Cd). You may need to relate Zn and Cd and explain why it is important in cancer.

Response: We have rewritten the Introduction. In a revised version, we have stated our working hypothesis concerning how Cd perturbs zinc homeostasis (lines 62-69).  Clinical features of bladder cancer have also been provided (lines 39-47).  Additional studies implicating role of zinc dysregulation and zinc transporters in cancers are included and discussed (ref.39-41, 37).

Point 2: Can you indicate if there are differences in the cadmium blood concentration between smokers and non-smokers?

Response: We have inserted a statement on blood Cd levels in cigarettes smokers compared with non-smokers in the introduction (lines 32-34).

Point 3: It will be very useful that you explain the function and location of each ZnT and ZIP proteins, in the presence or absence of zinc, how the mechanisms works, for example in a figure. It will help to the reader to follow the explanations and the importance of the variations in the expression of its genes. Also the explanations of the discussion will be better understood.

Response: We agree that a Figure it would be most useful to readers.  However, because current knowledge on ZIP and ZnT is still fragmentary, we have not attempted to create such a figure. We have included two additional references that give excellent figures showing subcellular compartmentalization of zinc and function of zinc transporters.  

21. Nishito Y, Kambe T. Zinc transporter 1 (ZNT1) expression on the cell surface is elaborately controlled by cellular zinc levels. J. Biol. Chem. 2019, 294, 15686-15697.

22. Kambe, T.; Taylor, K.M.; Fu, D. Zinc transporters and their functional integration in mammalian cells. J. Biol. Chem. 2021, 296, 100320.

Rest of the text:

Point 4: The tables will be easier to read if each one completed on one page, maybe due to the review format it has been cut.

Response: Table 1 and Table 2 each now appears a whole table on a page.

Point 5: Show the data that you talk about. For example, in line 113, you talk about the ZIP7 data expression, but you do not show them in none of the figures or graphics.

Response: In the original manuscript, ZIP7 expression data were provided in Table 2 together with information on doubling time, tumor phenotypes and invasiveness potential.  In a revised version, we have rearranged data presentation in Table 2, and we have described expression of each zinc transporter in details (lines 109-114, 123-144). 

Point 6: Unify writing, if number of clones are used, then do not use percentages. In my opinion, in the point number 2.2 of the results, as the number of clones are small, talk about number of clones is more representative than percentages.

Response:  As advised, we have now uniformly stated number of clones throughout the text instead of percentages.

Point 7: Can you explain why did you choose the clone number six for the "versus expression analysis" with the clone 1?

Response: Reasons for our selection of UCdT6 to compare with UTCd1 are clarified in the Discussion (lines 243-251).

 Point 8: Don´t you think that introducing the parental expression level in the graphs of figure 1 will show better the differences in each transformed clone? I think it will bring a very visual approach to the reader.

Response: We believe that a table format is suitable for quantitative data (exact copy number of transcripts) because they form database for comparing expression of every zinc transporter in other cell types.  In a revised version, we have explained expression ratio values in the legends to Figures 1 and 2 (lines 184-185, lines 199-200).    .

Point 9: In the discussion, the first sentence must be referred to the table1.

Response: We have inserted “table 1” at the end of the statement and have reorganized the text such that the first paragraph is pertaining to parent UROtsa cells.

Point 10: Why do you use 3 µM of Cd to transform the cells? Is it the maximum concentration the cells supports? And why 1µM for the maintenance? When you have the clones, did you use Cd to re-grow it, did they conserved it miss zinc homeostasis desregulation in the absence of Cd?

Response: We have used 1µM Cd concentration throughout to induce UROtsa cells to transform to cancer cells.  We have not used 3 µM Cd because it would have killed more than 50% of cells.  At 1 µM Cd, cell viability is reduced by by 10-15 % in 24 h and continuing presence of 1µM Cd over a long period (10 weeks) would allow surviving cells to propagate. Cells were exposed to 1µM Cd in culture medium only during cell transformation. Transfomed clones analyzed in the present study were not from sub-cloning, but they from the seven independent cell culture batches (UTCd1-7) that found to form tumors in nude mice as indicated in Table 2.

Point 11: It will be interesting that you can better explain the paragraph between lines 179 and 184, as you do in the next paragraph?

Response: We have rewritten the Discussion, and information pertaining to ZIP8 has been inserted in a referred paragraph (lines 227-238). We have added two papers (ref. 48, 49) that showed ZIP8 down regulation in Cd resistant cells via epigenetic mechanisms.

Major points:

Results:

Point 12:  You explain how you transformed UROtsa cells, but you do not explain nor in the Results or in the Materials and Methods sections the culture conditions you have used to grow the UROtsa cells (parental / cadmium-transformed) to obtain the RNA for the expression studies. Which was the concentration of Cd and Zinc in the employed medium? Is the RNA obtained from cultures supplemented with 3 µM of Cd? Depending on the Zn and Cd concentration the expression will change and to know the real their real concetration and the balance between these two metals will be important to interpret and understand the results.

Response: We have revised the Materials and Method section to provide information concerning cells and culture maintenance (lines 264-270). We used the same culture (growth) medium (Dulbeco’s modified Eagle’s medium supplemented with 5% vol/vol fetal bovine serum) with no other additional supplements.  Cells were exposed to 1µM of Cd in culture medium during cell transformation only as clarified in response to point 10 above.

Point 13: It will be interesting to include in this study if the UROtsa transformed cells present changes in the expression, in the zinc homeostasis transcription factors (ZNF658, MTF1...), the metallothioneins or in some hotspot genes that encode proteins involved in the cell cycle, for example: CDC45, TOR... It will indicate if the changes in zinc homeostasis can be blamed to changes in the expression of the regulons or it affects to genes of the hot spots cell cycle, or help to better understand the results: Why is the expression in the Cd1 clone different to the others, may be due to the toxicity of Cd affects other mechanisms that in the others?

Response: We thank the reviewer for offering the suggestions that will distinguish the two possible mechanisms.  They all are important future undertakings to advance our knowledge.

Point 14: Do you have the tumors that you had obtained from the heterotransplants you realized with the UROtsa Cd-transformed cells? I think that will be very interesting to know if there are changes in the expression of all the genes, or in the most important in this cell, in vivo and compare the results that you have obtained with the in vitro.

Response: We have characterized the tumors generated from the seven cadmium-transformed clones used in the present study and results have been reported in two papers (ref. 36,37). These two papers have been included in our revised manuscript.

36. Hoggarth et al. The urothelial cell line UROtsa transformed by arsenite and cadmium display basal characteristics associated with muscle invasive urothelial cancers. PLoS One 2018, e0207877.
37. Ajjimaporn et al. ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer. Cancer Cell Int. 2012, 12, 16.

Point 15: I can´t understand why and how do you "envisioned" (line 169), that ZIP14 and ZnT1 can "influence uptake, accumulation levels and resultant toxicity of cadmium" when they are having relatively high expression levels, can you explain it clearly?

Response: The word “envisioned” has been removed and we have rewritten the referred statement sin the first paragraph of the Discussion (lines 202-207).

Point 16: In lines 172 and 173, you say that UROtsa cells with high levels of ZnT1 acquired tolerance to cadmium toxicity. To suggest this, did you had done assays to measure the tolerance of these cells to Cd toxicity?  

Response: We agree with the Reviewer that quantitation of intracellular Cd concentration, which is an integrative function of efflux and influx zinc transporters, would be required. However, we know of at least four reports in which Cd accumulation levels were measured and were related to resistance/sensitivity to Cd toxicity (ref. 34, 35, 48, 49).  In revision we have discussed these previous works in details (lines 227-242).  Of note, epigenetic silencing of the ZIP8 gene by DNA hypermethylation has been shown to be a plausible molecular mechanism by which the ZIP8 gene is down-regulated leading to a reduction in Cd accumulation and tolerance (ref. 48).

Point 17: Review the first part of the discussion (line 166 to 192), you have to better explain it and indicates how your results supports your conclusions.

Response: As advised, we have rephrased statements in conclusions (lines 330-332).

Author Response File: Author Response.pdf

Reviewer 2 Report

Transformation of UROtsa clones with cadmium stimulates an increase in ZIP8 expression and decrease in ZIP1, ZIP2, and ZIP3 expression, as measured by qPCR.  Aberrant expression of ZIP7, ZnT5, ZnT6, and ZnT7 were also observed. Cadmium-transformation of UROtsa clones leads to aberrant zinc homeostasis in malignant cells.

Major Concerns

• The authors claim to have explored the “utility of zinc transporters as biomarkers for bladder cancer progression,” but this is not the case (line 67). No analysis of these transporters’ expression levels in bladder cancer patient tissues, nor their correlation to survival or disease stage, was performed in this paper.
• It is logical that exposure to cadmium dysregulates the expression of genes encoding metal transporters. However, the authors should directly link these transporters to the malignant phenotype of cells (vs just their cadmium exposure). For example, what effect does overexpression of ZIP8 (or any other) have on non-exposed, non-transformed UROtsa cells? Does silencing of ZIP8 reduce UTCd5 tumorigenicity? Mechanistic studies would strengthen the conclusion that “Aberrant expression of multiple zinc transporters may thus account for the pathogenesis of bladder cancer associated with cadmium exposure.”
• The expression levels of these genes are clearly fluctuating, but that is no guarantee that expression of the protein transporters have also changed. Modifications and decay of mRNA, as well as interference by noncoding RNA, may inhibit their translation. Protein expression levels – at the level of function – would be more compelling.
• Real-time PCR was performed in triplicate. However, were these experiments repeated multiple times to determine the mean? If not, they need to be repeated. Data from a single experiment do not meet the requirements for reproducibility.
• There are results reported that are not depicted in any figure. See example: Lines 116-120, describing changes in ZIP6, 10, and 14 and ZnT2. Why are these expression levels omitted from Figure 1?
• Line 127-130 – A claim is made that all several clones produced tumors in nude mice. The tumor sizes and pathology are not shown. Are the cell clones used to generate these tumors the same ones produced for the Somji et al (2010) paper that is referenced? If so, the generation of the clones themselves (not just the preparation of the tumors) in the Materials and Methods need to cite that article, and this should be explicitly stated - not presented as new work. If not, then the new tumors and their pathologies need to be shown.

Minor Concerns

• Some text in the results section reads like a figure legend. See example: Lines 93-97. This is awkward.
• What does the far-left asterisk in Figure 1C refer to? I assume ZnT6 expression, but it is floating in space.
• Line 35 should read “dissect the carcinogenic mechanism”
• Line 52 should read “characteristics resembling primary transitional”

Author Response

Reviewer 2

Transformation of UROtsa clones with cadmium stimulates an increase in ZIP8 expression and decrease in ZIP1, ZIP2, and ZIP3 expression, as measured by qPCR.  Aberrant expression of ZIP7, ZnT5, ZnT6, and ZnT7 were also observed. Cadmium-transformation of UROtsa clones leads to aberrant zinc homeostasis in malignant cells.

Response to top comments: We thank the Reviewer for evaluating the merit of our paper and for constructive comments. Accordingly, we have revised our paper extensively, and its title has been changed to read, “Aberrant Expression of ZIP and ZnT Zinc Transporters in UROtsa Cells Transformed to Malignant Cells by Cadmium”.  We provide below point-by-point response to issues raised.

Major Concerns

Point 1: The authors claim to have explored the “utility of zinc transporters as biomarkers for bladder cancer progression,” but this is not the case (line 67). No analysis of these transporters’ expression levels in bladder cancer patient tissues, nor their correlation to survival or disease stage, was performed in this paper.

Response:  The referred statement has been deleted and the issue is no longer pertaining to a revised manuscript. We have now clarified the purpose of the present work (78-88) and working hypothesis (lines 59-66). Clinical features of human bladder cancer have been provided (lines 37-44). Part of Abstract and Conclusion have been changed to be congruent with stated purposes and working hypothesis.

Point 2: It is logical that exposure to cadmium dysregulates the expression of genes encoding metal transporters. However, the authors should directly link these transporters to the malignant phenotype of cells (vs just their cadmium exposure). For example, what effect does overexpression of ZIP8 (or any other) have on non-exposed, non-transformed UROtsa cells? Does silencing of ZIP8 reduce UTCd5 tumorigenicity? Mechanistic studies would strengthen the conclusion that “Aberrant expression of multiple zinc transporters may thus account for the pathogenesis of bladder cancer associated with cadmium exposure.”

Response:  The referred statement in the conclusion has been deleted.  In a revised, we have clarified that our paper has its focus on dysregulation of zinc homeostasis in cancer cells. In an attempt to understand how Cd exposure increases risk of bladder cancer in humans (lines  76-86), we studied bladder cancer cells that have experimentally been produced in our laboratory.  We have provided, in the original manuscript, the protocol we used to induce UROtsa cells to transform to cancer cells. In revision, we have provided a summary of molecular and histological characterization done in our laboratory that has led to the present study (lines 79-83).

Point 3: The expression levels of these genes are clearly fluctuating, but that is no guarantee that expression of the protein transporters have also changed. Modifications and decay of mRNA, as well as interference by noncoding RNA, may inhibit their translation. Protein expression levels – at the level of function – would be more compelling.

Response:  We thank the Reviewer the suggestion concerning assessment of protein expression levels. The suggested lines of investigation are in progress in our laboratory and results will be reported in a future paper.  At least, Western blot analysis showing expression of ZIP8 at protein level has been reported (lines 207-208, ref. 37).

Point 4: Real-time PCR was performed in triplicate. However, were these experiments repeated multiple times to determine the mean? If not, they need to be repeated. Data from a single experiment do not meet the requirements for reproducibility.

Response: Measurement was done twice.  The results were in good agreement with microarray data obtained by the Affymetrix 133 Plus 2.0 chip showing that Cd may have caused up-regulation of at least 285 genes and down-regulation of at least 215 genes to induce cancer transformation (ref 38).

Point 5: There are results reported that are not depicted in any figure. See example: Lines 116-120, describing changes in ZIP6, 10, and 14 and ZnT2. Why are these expression levels omitted from Figure 1?

Response: : In the original manuscript, data showing changes in expression levels of ZIP6, 10, and 14 and ZnT2 were provided in Table 2 together with information on doubling time, tumor phenotypes and invasiveness potential.  In a revised version, we have rearranged data presentation in Table 2, and we have described expression of every zinc transporter in details (lines 109-114, 123-144). 

Point 6: Line 127-130 – A claim is made that all several clones produced tumors in nude mice. The tumor sizes and pathology are not shown. Are the cell clones used to generate these tumors the same ones produced for the Somji et al (2010) paper that is referenced? If so, the generation of the clones themselves (not just the preparation of the tumors) in the Materials and Methods need to cite that article, and this should be explicitly stated - not presented as new work. If not, then the new tumors and their pathologies need to be shown.

Response: Tumors generated from transformed UROtsa cells have been characterized and compared with clinical bladder cancer specimens and these have been reported in two papers (ref. 36, 37). We have indicated data pertaining to tumors formed the seven UROtsa clones analyzed in the present are published data (lines 121-122).

Minor Concerns

Point 7: Some text in the results section reads like a figure legend. See example: Lines 93-97. This is awkward.

Response: We have rewritten the referred part of Results Section.

Point 8: What does the far-left asterisk in Figure 1C refer to? I assume ZnT6 expression, but it is floating in space.

Response: We thank the Reviewers for pointing out that we have missed labelling Figures. In revision, we have added a text box to each Figure to explain “*”.  The “*” in question indicates a significant difference in ZnT6 expression in UTCd1 and UTCd6.  In addition, we have explained the meaning of expression ratio values in the legends to Figures 1 and 2 (lines 184-185, lines 200-202).   

Point 9: Line 35 should read “dissect the carcinogenic mechanism”

Response:  A correction has been undertaken.

Point 10: Line 52 should read “characteristics resembling primary transitional”

Response: A correction has been undertaken

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The paper is now easier to read and understand. In my oppinion there is only 1 point that you may need to improve.

In the discusion, first you say  in lines 117 to 119 a very logical affirmation but I´ll like you to better explain this paragraph: the up-regulation of ZIP8 reflects a compensatory response to a contraction of labile zinc pool because of the elevation to the ZIP1, 2 and 3 expresion (lines 223 to 227).

Author Response

Reviewer 1

The paper is now easier to read and understand. In my opinion there is only 1 point that you may need to improve.

In the discussion, first you say in lines 217 to 219 a very logical affirmation but I´ll like you to better explain this paragraph: the up-regulation of ZIP8 reflects a compensatory response to a contraction of labile zinc pool because of the elevation to the ZIP1, 2 and 3 expression (lines 223 to 227).

Response: We thank the Reviewer for giving us another opportunity to improve our manuscript.  As advised, we have inserted additional explanations in the Discussion to which the Reviewer has referred. The explanations are quoted below. 

"The increased ZIP8 expression seen in six transformed UROtsa clones in the present study may reflect, in part, a compensatory response to a contraction of labile zinc pool because expression levels of zinc uptake zinc transporters, namely ZIP1, ZIP2 and ZIP3 were simultaneously elevated (Fig. 1B) and/or induction of MT by Cd [15] as stated in our hypothesis. It may also reflect that expression of ZIP8 and ZnT1 genes are co-regulated. We have observed an increased expression of ZIP8 in all six clones showing low expression levels of ZnT1 (Fig. 1A). Conversely, ZIP8 expression was diminished in the transformed UROtsa clone in which ZnT1 expression was elevated (Fig. 1A, Table 2). It is conceivable that up-regulation of Zn influx transporters, notably ZIP8, simultaneously with down-regulation of the efflux transporter ZnT1 would be required to prevent excess Zn accumulation and toxicity from overdose. Hence, the maintenance of Zn homeostasis is an integrative function of influx and efflux transporters." 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have adequately addressed the issues with the original manuscript. I thank them for their hard work. 

Author Response

Reviewer 2

The authors have adequately addressed the issues with the original manuscript. I thank them for their hard work.

Response:  Once again, we sincerely thank the Reviewer for the guidance to improve our paper to an acceptable level.