PONE-D-21-26390

GPC1 promotes the growth and migration of colorectal cancer cells through regulating the TGF-β1/SMAD2 signaling pathway

PLOS ONE

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #2: Yes

Reviewer #3: Yes

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: There are several questions needed to be discussed.

1. The manuscript has to be polished by professional language polishing companies because there are a lot of typographical or grammatical errors.

2. TGF-β signaling is initiated through binding of the TGF-β ligand to the receptor, which in turn phosphorylates the downstream target SMAD2/3. In this study, authors reported that GPC1 knockdown decreased the expression of TGF-β and p-SMAD2. However, why total SMAD2 expression also be affected?

3. In figure 2k, the GAPDH expression obviously differs in CRC cells and NCM460. therefore, please provide more accurate figures.

4. Please discuss how GPC1 influences the expression of TGF-β? It functions as a transcript factor?

Reviewer #2: PONE-D-21-26390.

GPC1 promotes the growth and migration of colorectal cancer cells through regulating the TGF-β1/SMAD2 signalling pathway

The authors investigate the potential role of GPC-1 in colorectal cancer using in silico and in vitro techniques. While the role of GPC-1 has been well described in other cancers such as pancreatic and prostate, its role in colorectal cancer is poorly understood.

While there has been some published data on GPC-1 mRNA expression in colorectal vs normal tissues, and in GPC-1 exosomes in colorectal cancer, there has been limited investigation of the mechanisms by which GPC-1 may play a role in the biology of CRC.

The authors demonstrate that knockdown of GPC-1 in colon cancer cell lines reduces proliferation and migration, consistent with what has been observed in other cancer types. They have identified the TGF-b1/SMAD2 signalling axis as a potential mediator of GPC-1 function in CRC.

A major challenge in determining the expression of GPC-1 in different cancers is that expression of the mRNA should be correlated to the expression of the GPC-1 protein using immunohistochemistry. If tissue availability is an issue for this, the authors should comment on the likelihood that the relative expression identified by mRNA is reflected in the protein expression in tumours.

Similarly, the detection of GPC-1 via western blot in this paper is problematic. GPC-1 is known to be heparan sulphated. While the core protein of ~55kDa can be detected without lysate treatment, use of Heparanase can result in much higher levels of GPC-1 detectable via western blot. The western blots shown in the figures and in the supplementary material do not have a positive control (e.g. recombinant GPC-1) to ensure that the band detected is actually GPC-1 (it is not clear what the molecular weight of the reactive bands are).

Specific comments:

Line 77 – reference 9 (Quach) does not show the expression in endometrial, lung or other tumours. Suggest referring to individual papers for each indication or a review article.

Line 177 – Western blot. GPC-1 is generally highly glycosylated – in particular with heparan sulfate chains. Was Heparanase treatment of samples attempted? If so was there any additional GPC-1 that became visible after treatment?

Line 186 – all glypicans seem to be differentially expressed at a statistically significant level between normal and tumour tissue. It appears GPC-3 and GPC6 are decreased, while GPC-4 is increased in tumour vs normal. It is not possible to tell from Figure 1 what the expression levels of GPC-5 are like in tumour vs normal.

Lines 192-200. While the differences in GPC-1 expression for tumour stage are statistically significant, the relative fold changes are small.

Line 196 – referring to Figure 2 western blots. The western blot for GPC-1 is very dark and the signal quite weak. The fold change in expression between normal and cancer lines shows error bars but it is unclear how many times these blots were performed to generate the densitometry. There is also no control for GPC-1 (e.g. recombinant GPC-1 or a cell line known to overexpress GPC-1).

Under the Methods section line 169 it is unclear if NCM460, RKO, CACO2 cells were also lysed in RIPA buffer and their growth conditions are not described in the Methods section.

Line 219 – Figure 3. GPC1 mRNA expression levels were knocked down by up to 60% but not 100%. This should be commented on. It is also not clear from the text or the figure legends what si-GPC-1-1 and si-GPC1-2 refer to (although this is mentioned in the methods). For 3B and D, it is not clear when the transient transfection of the siRNAs occurred relative to the start of the proliferation assay.

There is insufficient detail in Apoptosis and Cell Cycle methods to describe Fig 4. Also no information about number of times the experiment was repeated to generate the error bars is provided.

Figure 4 shows relatively minor changes in Apoptosis and Cell Cycle effects, although they appear from the data to be statistically significant.

Line 233 – cell migration. Again it is unclear when the GPC-1 knockdown was performed relative to the assay being performed.

Line 253 seems to have missed completion of a sentence.

Line 260 – protein levels of TGFb1. Presumably these are cellular levels as secreted TGFb1 would not be collected and analysed via the western blot protein extraction method described?

Line 271 – “after treatment with” rather then “after treated with”

Reviewer #3: Summary

This paper introduces the promoting role of GPC1 in CRC, and provides evidence that GPC1 may be used as an early biological detection standard and therapeutic target for CRC in the future, which can help doctors better treat CRC patients and has very high clinical significance.

Advantages

1、With clear logic and extensive data, this paper provides sufficient evidence that GPC1 promotes the development of CRC

2、In this paper, the functional pathway of GPC1 has also been confirmed.

Disadvantages

1、The picture is not clear enough. For example, the legend in the survival analysis of B in figure1 is not clear.

2、The expression levels of GAPDH in Western blot were significantly different, such as K in figure2

3、Since there are many figures in this paper, each figure can be placed under the corresponding results for easy reading

4、The language needs to be more refined

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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GPC1 promotes the growth and migration of colorectal cancer cells through regulating the TGF-β1/SMAD2 signaling pathway

PONE-D-21-26390R1

Dear Dr. Liu,

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.

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Kind regards,

Hamidreza Montazeri Aliabadi

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: 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: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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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: (No Response)

Reviewer #2: Yes

Reviewer #3: 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)

Reviewer #2: (No Response)

Reviewer #3: After a rigorous revison on this manuscript, the manuscript is suitable for pubulication with sound quality on science.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No