Association of MACC1 expression with lymphatic metastasis in colorectal cancer: A nested case-control study

Zheying Zhang; Huijie Jia; Yuhang Wang; Baoshun Du; Jiateng Zhong

doi:10.1371/journal.pone.0255489

Peer Review History

Original SubmissionDecember 31, 2020
16 Apr 2021 Decision Letter - Ajay Goel, Editor PONE-D-20-41076 Association of MACC1 Expression with Lymphatic Metastasis in Colorectal Cancer: a nested case-control study PLOS ONE Dear Dr. Zhong, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 31 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This study by Zhang et al. investigated an association of MACC1 with lymphatic metastasis in a nested case control study. Public datasets were employed for bioinformatic analysis and after assessing for MACC1 mRNA levels in different age groups, the authors concluded that an association only applied to a specific age group (64-75 years old). While the study is of interest, due to the lack of clarity with figures presented, it is difficult to assess the validity of their findings. Comments: 1. Information presented is extensive bioinformatic analysis. Of key significance, missing in this study, is validation at the protein level comparing primary tumors from patients with and without lymphatic metastasis. 2. Authors reach the conclusion that there is a correlation between MACC1 and MMP2, MMP7, CD44, etc). However, according to the Pearson correlation shown of R < 0.5 (Figure 3), this is a very weak association and should be clearly stated in the manuscript. 3. Figure 1 is difficult to interpret. There is no legend to inform the reviewer what each of the lines represent. Figure 1C has a legend in the graph but is blurry and difficult to read. 4. Figure 2A is also blurry and difficult to read. 5. Figure 2C. Unclear what each bar graph represents. Further, what do values on the y-axis represent? Furthermore, does tumor data represent patients with lymphatic metastasis? 6. A more updated reference for #15 should be used. The one cited in manuscript is from 2012. 7. A description of MACC1 (domains, protein size and chromosomal location) do not belong in the discussion session. 8. A few typos and grammatical errors were found. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0255489.r001
Revision 1
24 May 2021 Author Response Response to Reviewer #1: Reviewer #1: This study by Zhang et al. investigated an association of MACC1 with lymphatic metastasis in a nested case control study. Public datasets were employed for bioinformatic analysis and after assessing for MACC1 mRNA levels in different age groups, the authors concluded that an association only applied to a specific age group (64-75 years old). While the study is of interest, due to the lack of clarity with figures presented, it is difficult to assess the validity of their findings. Response: Thank you for your review. We increased the clarity of the image, and marked the vertical and horizontal coordinates in detail, and explained them in detail in the Figure legends. The expression level of MACC1 protein (Figure 4) and mRNA (Figure 2C) in colorectal cancer tissues with and without lymph node metastasis were supplemented. Comments: [1] Information presented is extensive bioinformatic analysis. Of key significance, missing in this study, is validation at the protein level comparing primary tumors from patients with and without lymphatic metastasis. Response: Thanks for your kind suggestion. In the results and discussion section, we supplemented the expression level of MACC1 protein in colorectal cancer tissues with and without lymph node metastasis.Thanks! Supplementary contents in results： Expression level of MACC1 protein in colorectal cancer tissues with and without lymph node metastasis We examined the expression of MACC1 protein in normal control tissues, colorectal cancer tissues from patients with lymph node metastasis, non-metastatic colorectal cancer tissues, and metastatic lymph node samples in patients aged 64-75 years, and patients younger than 64 years and older than 75 years, respectively. It was found that MACC1 protein expression was higher in colorectal cancer tissues with lymph node metastasis than in colorectal cancer tissues without lymph node metastasis in 64-75 age group. It is rarely expressed in normal tissues. The expression was up-regulated in metastatic lymph nodes compared with normal tissues, but not significantly (Fig 4). Figure 4 Fig 4. MACC1 protein expression levels in colorectal cancer tissues with and without lymph node metastasis (A) Immunohistochemical method was used to detect the expression level of MACC1 protein in normal control tissues, colorectal cancer tissues from patients with lymph node metastasis, non-metastatic colorectal cancer tissues, and metastatic lymph node tissues in patients aged 64-75 years. (B) Immunohistochemical method was used to detect the expression level of MACC1 protein in normal control tissues, colorectal cancer tissues from patients with lymph node metastasis, non-metastatic colorectal cancer tissues, and metastatic lymph node tissues in patients younger than 64 years and older than 75 years. p < 0.05;p < 0.01; *p < 0.001. Supplementary contents in Discussion：We analyzed the expression level of MACC1 mRNA in CRC through online database. The mRNA level of MACC1 was increased in the cancer tissues and was associated with lymph node metastasis, but there was no difference in the degree of lymph node metastasis. The protein expression of MACC1 was detected by immunohistochemistry. It was consistent with mRNA results. The expression of MACC1 was significantly upregulated in primary lesions in patients with lymph node metastasis compared with patients without lymph node metastasis in 64-75 age group. These results all indicate that MACC1 plays an important role in CRC lymph node metastasis and can be used as a marker to determine whether lymph node metastasis occurs in 64-75 age group. [2] Authors reach the conclusion that there is a correlation between MACC1 and MMP2, MMP7, CD44, etc. However, according to the Pearson correlation shown of R < 0.5 (Figure 3), this is a very weak association and should be clearly stated in the manuscript. Response: Thanks for your careful comments. We have made a modification in the results and discussion part of the manuscript. Modified contents in Results: “The results from GEPIA 2 showed that MACC1 had a relatively high correlation with CDH1 and MET, and a weak correlation with MMP9 and SNAI1. MACC1 is also highly correlated with proliferation-related genes MYC and CDK13 (Fig 3). ” Modified contents in Discussion: “We also analyzed the co-expression of MACC1 and tumor-related genes through GEPIA database. The results showed that MACC1 had a relatively high correlation with CDH1 and MET, and a weak correlation with MMP9 and SNAI1. MACC1 may play a metastasis role through CDH1 and MET genes, and MACC1 is also highly correlated with proliferation-related genes MYC and CDK13. This suggests that in addition to promoting metastasis, MACC1 may also be closely related to tumor proliferation.” Figure 3 Fig 3. Bioinformatics analysis of MACC1 co-expression genes. Pearson’s correlation analysis of MACC1 and related genes. We used the services provided by the website GEPIA (http://gepia.cancer-pku.cn/index.html) to examine the correlations between CDH1, MET, MMP9, SNAI1, MYC and CDK13. [3] Figure 1 is difficult to interpret. There is no legend to inform the reviewer what each of the lines represent. Figure 1C has a legend in the graph but is blurry and difficult to read. Response: Thank you for your question. We relabeled the coordinates in Figure 1 and explained them in detail in the Figure legends. Figure 1 Figure legend： Fig 1. Relationship between MACC1 expression level and lymphatic metastasis. (A) A linear relationship between MACC1 expression level and lymphatic metastasis was observed. The horizontal ordinate represents the expression level of MACC1, and the longitudinal coordinates represents the probability of lymph node metastasis. The blue circles represent 95% confidence intervals and the red dots represent the fitting curves. (B) A nonlinear relationship between MACC1 expression level and lymphatic metastasis was observed after adjusting all variables. The horizontal ordinate represents the expression level of MACC1, and the longitudinal coordinates represents the probability of lymph node metastasis. The blue circles represent 95% confidence intervals and the red dots represent the fitting curves. (C) A nonlinear relationship between MACC1 expression level and lymphatic metastasis was observed after adjusting all variables in middle age group. The horizontal ordinate represents the expression level of MACC1, and the longitudinal coordinates represents the probability of lymph node metastasis. Divide the age into three groups: the red dots represent the lower age group, the green circles represent the middle age group, and the blue triangle represents the higher age group. [4] Figure 2A is also blurry and difficult to read. Response: Thanks for reviewer’s kind suggestion. We add red and blue box notes to Figure 2A and annotated Figure 2A in detail in the Figure legends. Figure legend： Fig 2. Expression levels of MACC1 mRNA in CRC tissues. (A) Expression levels of MACC1 mRNA with CRC tissues and control tissues in Oncomine. Red box represents the number of microarray results that were up-regulated in colorectal cancer. Blue box represents the number of microarray results that were down-regulated in colorectal cancer.The graph on the right is the results from dataset. The results showed that MACC1 expression was upregulated 3.733-fold in CRC specimens compared with control colon tissue specimens. [5] Figure 2C. Unclear what each bar graph represents. Further, what do values on the y-axis represent? Furthermore, does tumor data represent patients with lymphatic metastasis? Response: Thank you for your question. We mark the ordinate and describe the picture in detail in the annotation. We removed the unimportant original Figure 2B and changed Figure 2C to Figure 2B, and increased the expression level of MACC1 in Colon adenocarcinoma (COAD) and Rectum adenocarcinoma (READ) (Fig 2B) and lymph node metastasis (Fig 2C). Thanks! Modified contents in results: We used GEPIA 2 (http://gepia2.cancer-pku.cn/#index) online databases in May 2021 to analyze the expression of MACC1 mRNA in Colon adenocarcinoma (COAD) and Rectum adenocarcinoma (READ) [20]. The results showed that MACC1 upregulated in CRC tissues compared with normal tissues (Fig 2B). We used UALCAN (http://ualcan.path.uab.edu/analysis.html) online databases in May 2021 to analyze relationship between MACC1 expression and lymph node metastasis [21]. The results showed that MACC1 upregulated in CRC tissues with lymph node metastasis compared with non-lymph node metastatic (Fig 2C). Figure legend： Fig 2. Expression levels of MACC1 mRNA in CRC tissues. (B) Expression levels of MACC1 mRNA in CRC tissues and normal tissues from GEPIA 2 database. Red represents the expression in tumor tissue and gray represents the expression in normal tissue. p < 0.05. (C) Expression levels of MACC1 mRNA with lymphatic metastasis CRC tissues and non-lymphatic metastasis control tissues in UALCAN database. The abscissa represents the number of tumor samples with different grade of lymph node metastasis. **p < 0.001. Figure 2 [6] A more updated reference for #15 should be used. The one cited in manuscript is from 2012. Response: Thanks for reviewer’s kind suggestion. We have updated the reference up to date. Modified reference: 24. Bray F, Ferlay J, Soerjomataram I, Siegel R L, Torre L A,Jemal A, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394-424. 10.3322/caac.21492 Modified contents in discussion: The latest global cancer statistics show that the incidence rate of colorectal cancer ranks the third, after lung cancer and breast cancer, accounting for about 10.2%. The case fatality rate was the second highest at 9.2%, second only to lung cancer [24]. [7] A description of MACC1 (domains, protein size and chromosomal location) do not belong in the discussion session. Response: Thanks for reviewer’s careful comments. We have put the description of MACC1 in the introduction part. [8] A few typos and grammatical errors were found. Response: We are very sorry for our writing. We have corrected spelling mistakes and asked two English teachers to help modify the manuscript. Thank you! Attachments Attachment Submitted filename: Response to Reviewers.doc* https://doi.org/10.1371/journal.pone.0255489.r002
23 Jun 2021 Decision Letter - Ajay Goel, Editor PONE-D-20-41076R1 Association of MACC1 Expression with Lymphatic Metastasis in Colorectal Cancer: a nested case-control study PLOS ONE Dear Dr. Zhong, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Your manuscript was re-reviewed by one of the original reviewers. While the article is of interest, one of the reviewers have requested a few minor revisions. Please submit your revised manuscript in next 2-4 weeks. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Additional information and editing of the content provided in figure legend 1 is strongly recommended. There are two blue lines and information for only one is given and this needs clarification. Next, redundancy in the description on what the x and y coordinates are as it is given for each graph. State it at the very end and just once. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0255489.r003
Revision 2
25 Jun 2021 Author Response Response to Reviewer #1: Comments: [1] Additional information and editing of the content provided in figure legend 1 is strongly recommended. There are two blue lines and information for only one is given and this needs clarification. Next, redundancy in the description on what the x and y coordinates are as it is given for each graph. State it at the very end and just once. Response: Thanks for your kind suggestion. We have modified the figure legend for Figure 1. The explanation of the two blue lines is added, and the redundant part is removed. Thanks! Modified contents in Figure legend 1： Fig 1. Relationship between MACC1 expression level and lymphatic metastasis. (A) A linear relationship between MACC1 expression level and lymphatic metastasis was observed. The blue circle lines on both sides represent 95% confidence interval and the red dot line represents the fitting curves. (B) A nonlinear relationship between MACC1 expression level and lymphatic metastasis was observed after adjusting all variables. The blue circle lines on both sides represent 95% confidence interval and the red dot line represents the fitting curves. (C) A nonlinear relationship between MACC1 expression level and lymphatic metastasis was observed after adjusting all variables in middle age group. Divide the age into three groups: the red dot line represents the lower age group, the green circle line represents the middle age group, and the blue triangle line represents the higher age group. In this figure, the horizontal ordinate represents the expression level of MACC1, and the longitudinal coordinate represents the probability of lymph node metastasis. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0255489.r004
19 Jul 2021 Decision Letter - Ajay Goel, Editor Association of MACC1 Expression with Lymphatic Metastasis in Colorectal Cancer: a nested case-control study PONE-D-20-41076R2 Dear Dr. Zhong, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ajay Goel Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No https://doi.org/10.1371/journal.pone.0255489.r005
Formally Accepted
26 Jul 2021 Acceptance Letter - Ajay Goel, Editor PONE-D-20-41076R2 Association of MACC1 Expression with Lymphatic Metastasis in Colorectal Cancer: a nested case-control study Dear Dr. Zhong: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ajay Goel Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0255489.r006

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