Checkpoint Kinase 1 (CHK1) Functions as Both a Diagnostic Marker and a Regulator of Epithelial-to-Mesenchymal Transition (EMT) in Triple-Negative Breast Cancer

Round 1

Reviewer 1 Report

The manuscript uses public databases to address genes expressed in TNBC that could be useful in prognosis and treatment in these patients subtype.

Although the subject of the manuscript is a very important subject authors have not taken in to account that TNBC nowadays can be subdivided in at least 4 subtypes with different characteristics and outcome. I propose that they introduce this issue in the introduction and discuss it at the discussion section. This is an important point as authors have considered  TNBC in almost all comparison analysis and at the end they used not TNBC but Basal BC saying the difference between them but discarding two TNBC subtypes, one of good prognosis but the other with the worst prognosis.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

1.       In the Abstract, please introduce abbreviations for Genomic Spatial Event (GSE), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI).

2.       Lines 17 and 18: just to confirm and check – a total of 13 genes (not 11) were selected as hub genes, of which 7 (not 11) were correlated with survival rate?

3.       Please describe in the Introduction the in vitro part of the research with more information. It looks like a relatively large part of the results is summed into one sentence, while the previous in silico results are properly described.

 

4.       Line 66: It is quite bold to say the presented results, interesting as they may be, are enough for suggesting a therapy and diagnosis target gene, especially since you haven’t mentioned what kind of therapy would be possible, regarding the CHK1 gene expression in breast cancer patients. I suggest rephrasing the sentence.

5.       Line 71: I suggest “…were searched by these researching words…” instead of “some words”.

6.       Line 73: Either “we selected” or “the datasets selected included” – please, check the grammar of the sentence.

7.       Sometimes you use full stops in section names (e.g. 2.2. Identification of gene expression data in TNBC patients.), sometimes not (most of the other sections) – if the regulative of the journal supports both, please choose one or the other, but be consistent along the way.

8.       Sections 2.6 to 2.12 need to be proofread. Language constructions are inaccurate.

9.       In Figure 5D it can be perceived you investigated CHK1 expression in 6 breast cancer cell lines (MCF-7, T47D, MDA-MB-453, MDA-MB-231, BT549, Hs578T), but in Section 2.6 you mentioned only MCF-7 and MDA-MB-231. Please add information on the other 4 cell lines used in Section 2.6, as well as the origin of all cell lines (where were they acquired). Also, please write the cell line names consistently and correctly throughout the manuscript (MCF-7 and MDA-MB-231).

10.   Line 113: It appears the catalogue number (or manufacturer) for Penicillin-Streptomycin is not correct – please check and provide the correct information.

11.   Line 117: “through” should be changed into “according to”.

12.   The primers are represented in a table – this should be Table 1, with proper caption. And numerations of the following tables should be corrected accordingly.

13.   Please add how you designed the primers used in RT-PCR. The primer sequence for IL6ST does not correspond to the gene, according to the databases I’ve searched.

14.   I suggest you add in Section 2.9 that MCF-7 were knocked-in with CHK1 and in MDA-MB-231 CHK1 expression was silenced with specific siRNA, and add the manufacturer of siRNA.

15.   Was the DNA transfection with plasmid or linear DNA? Please add the manufacturer.

16.   Did you use negative controls in transfections (non-coding DNA sequence and scrambled siRNA)?

17.   Did the transfection last for 48 h, or have you lysed the cells 48 h following transfection?

18.   Manufacturer and catalogue number for the blocking reagent (line 135) can’t be found online.

19.   The serial number provided for vimentin (line 139) corresponds to the Cell Signaling serial number of the vinculin antibody.

20.   Throughout Sections 2.6 to 2.12: “the cells were washed in PBS”, not “to PBS”.

21.   Line 143: Was the 4% formaldehyde (FA) diluted in PBS? As in, you had the stock solution of FA and you diluted it by PBS? The sentence is a bit unclear, please rephrase it.

22.   Line 145: The catalog number listed does not correspond to the product IGEPAL. Please add the correct number.

23.   Line 147: hours = h, as used previously.

24.   Section 2.11: Please add the time frame, after inserting the scratch, how much time passed before obtaining the pictures.

25.   Pay attention to manufacturer names, they should be with the capital first letter, e.g. Corning, Invitrogen etc. Also, Matrigel is a registered trademark (Matrigel®)

26.   Line 161: Please emphasize the medium in the upper chamber did not contain FBS and that FBS in the lower chamber is a chemoattractant.

27.   Line 162: Did you actually keep cells at room temperature for 48 h or is that an error? Please explain why you chose 48 h as the duration time of the invasion assay since 24-h assay is the most common.

28.   Section 2.12: 100x magnification is too high, it is necessary to take (five to ten) random microscopic fields at 10x or 20x magnification. This is probably why in the representative photos (Figure 6 E, I) you have very few cells per field. In Figures 6E and I - what exactly are "invasion folds", how did you calculate it?

29.   In Materials and Methods “Statistics” section needs to be added, where you will describe data distribution and tests you performed in statistical analysis. Also, please add in Materials and Methods how many times you repeated the experiments.

30.   HA-CHK1 abbreviation was not introduced or explained in the text, nor in the figure legends. Please explain what it signifies.

31.   Line 168: DEG was previously introduced as abbreviation.

32.   All Tables and Figures should be inserted after the first mention in the text. Figure legends need to be explained more clearly and with more information on what is presented in the picture.

33.   Line 180: In the text, it says 168 genes, and in Figure 1D it says 170 (same goes for the up-regulated genes, 74 (text) vs 75 (figure), and down-regulated genes, 94 (text) vs 95 (figure).

34.   Line 184: “that showing” grammar error should be corrected.

35.   I suggest Table 1 be more clearly designed, maybe with lines between rows.

36.   Figure 1B and C: I suggest adjusting the y-axis range, or explaining why you chose PV 10^6 as the max cut-off.

37.   Line 199: “uses” appears to be a grammar error.

38.   Line 204: It appears as if some text is missing after ‘cell cycle’ and before ‘mitotic’.

39.   Figure 1E – please provide a better quality figure, the text can’t be read.

40.   Line 238: It says 13 genes were found to correlate in total, but then 7 genes are part of up-regulated and 4 are down-regulated (7+4 = 11) – the two genes (out of 13) are not in that group of up-regulated and down-regulated ones? Please express this more clearly.

41.   Please sort the text in the first row of Table 4 so it is clear what describes what column.

42.   Line 252: It says you analyzed the correlation of TNBC patient survival with 11 gene candidates previously mentioned using KM plots – could you please add to supplementary material the analysis of all 11 genes?

43.   Line 292: her2 should be in all caps (HER2-positive).

44.   Line 296: Please refer to the (pathological classification) database you used.

45.   Line 307: “We can found…” is a grammar error.

46.   Line 317: The cells are not CHK1-reducing – it would mean the cell reduces CHK1 somehow. The cells have reduced/decreased content of CHK1. Please rephrase the sentence.

47.   In Figure 5B, why is the y-axis scale bar for HER2-positive vs. basal like different from the other two graphs?

48.   Can you please provide a larger image for Figure 5C? Also, if I’m not mistaken, you missed mentioning which database you used for this KM plot in this figure.

49.   Figure 5D – MDA-MB-453 are triple-negative breast cancer cells as well, why are they in a non-TNBC group? Even if you got a result CHK1 is not expressed in them, as much as in other TNBC cell lines, it is still an interesting result and should be commented on accordingly. Certainly, not all TNBC are completely genetically homogenous.

50.   Figure 5E – please state clearly that, in the figure, you quantified the western blot with CHK1 expression in cell lines. Did you normalize the CHK1 expression to the endogenous control (GAPDH)? If so, state it in the y-axis label and figure legend.

51.   In Figures 6D and H, please insert high-quality larger pictures of graphs.

52.   Line 376: PI3K, the “k” should be in caps.

53.   Line 382: “…cells, converted from the primary tumour to the mesenchymal type, move…” – this is a suggestion, to use commas for clearer expression.

 

54.   Line 386: I do not think here you proved that increased expression of CHK1 induces EMT since you haven’t demonstrated it in a live tumour/organism. The correlation is not the final proof, it placed the hypothesis. You proved CHK1 induces protein expression changes characteristic of EMT, in silico and in vitro. Please rephrase the sentence. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors responded constructively to most remarks made. The most important concerns, regarding methodology applied, were cleared out in version 2 of the manuscript. However, minor error remain in text.

The authors did not adequately describe in vitro experiments in the introduction, it remained as in the previous version.

Some of the suggestions on text consistency and minor corrections of text errors were implemented, and some were not (in spite of the authors saying in the letter they were). In general, the text needs to be read carefully and corrected for minor errors (e.g. hr written instead of h (Figure 6d and h), inserted dashes were not needed (hemagglu-tinin), wrong sentence construction etc.).

In 2.6 Cell culture, although all cell lines were listed in the new version, it was not mentioned where they were acquired.

Sufficient information was added in section 2.9, but on the whole, it is poorly written. The authors say where DNA was purchased. Then they write about siRNA negative control. Then they describe the methodology for plasmid transfection. Then they mention where siRNA was purchased and describe the siRNA transfection.

Line 215: Table 1 should be Table 2 (because Table 1 now is the one with primer sequences). It was correctly mentioned in the text as Table 2, Table caption should be adjusted. 

In Supp. material MDA-MB453 was not listed as TNBC.

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx