Mechanism of DAPK1 for Regulating Cancer Stem Cells in Thyroid Cancer
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsMechanism of DAPK1 regulating cancer stem cells in thyroid cancer
The review provides a comprehensive overview of the role of Death-associated protein kinase 1 (DAPK1) in cancer, highlighting its downregulation in metastatic cancer and its involvement in both early and late stages of cancer progression. The discussion extends to the complex regulatory mechanisms controlling DAPK1 expression, including epigenetic, transcriptional, post-transcriptional, and post-translational processes. Notably, the review delves into DAPK1's influence on not just cancer cells but also stromal cells, and its critical role in tumor suppression, EMT (Epithelial to Mesenchymal Transition), and CSC (Cancer Stem Cells) formation in colon and thyroid cancers. The exploration of DAPK1's impact on CSCs is particularly relevant, given their significant role in cancer aggressiveness and metastasis. Despite these insights, the review acknowledges the current gaps in understanding the molecular mechanisms of DAPK1 regulation in cancer cells, emphasizing ongoing research efforts. The focus on CSC regulation by DAPK1 in thyroid cancer, specifically in papillary thyroid carcinoma (PTC), adds valuable context to the current state of knowledge, addressing an area where information is notably sparse.
The overall review was interesting to read and provided lots of details, however, the following comments need to be addressed to make the review have a clearer structure:
(1) The paragraph in the introduction section (starting with ‘DAPK1 is abundant not only in the lung but also’) is currently redundant as the authors are not going to talk about the function in lung or brain.
(2) For section 2.1 (the Discovery of DAPK1): the author didn’t talk much about the actual discover, whereas talked a lot about the phosphorylation of DAPK1 (which should be in a section that describe DAPK1 regulation) and what can be phosphorylated by DAPK1 (which can be combined with DAPK1 functions).
(3) The structure of DAPK1 needs to be introduced first before the author talks about the detailed phosphorylation sites and also the DAPK family, as a lot of the protein domains are currently introduced in this section without even letting the readers to know where are these domains.
(4) It would be more helpful for the author to provide some sort of figure indication where are the DAPK1 phosphorylation sites and what are the potential functions etc. This could potentially be combined with the current Figure 1.
Comments for author File: 
Comments.pdf
Author Response
Response to reviewer 1
Responses to Reviewer1 are marked in yellow in the manuscript.
Additionally, in order to complete the manuscript more thoroughly, errors were corrected through document editing service.
The review provides a comprehensive overview of the role of Death-associated protein kinase 1 (DAPK1) in cancer, highlighting its downregulation in metastatic cancer and its involvement in both early and late stages of cancer progression. The discussion extends to the complex regulatory mechanisms controlling DAPK1 expression, including epigenetic, transcriptional, post-transcriptional, and post-translational processes. Notably, the review delves into DAPK1's influence on not just cancer cells but also stromal cells, and its critical role in tumor suppression, EMT (Epithelial to Mesenchymal Transition), and CSC (Cancer Stem Cells) formation in colon and thyroid cancers. The exploration of DAPK1's impact on CSCs is particularly relevant, given their significant role in cancer aggressiveness and metastasis. Despite these insights, the review acknowledges the current gaps in understanding the molecular mechanisms of DAPK1 regulation in cancer cells, emphasizing ongoing research efforts. The focus on CSC regulation by DAPK1 in thyroid cancer, specifically in papillary thyroid carcinoma (PTC), adds valuable context to the current state of knowledge, addressing an area where information is notably sparse.
The overall review was interesting to read and provided lots of details, however, the following comments need to be addressed to make the review have a clearer structure:
- The paragraph in the introduction section (starting with ‘DAPK1 is abundant not only in the lung but also’) is currently redundant as the authors are not going to talk about the function in lung or brain.
Thank you for your good comment.
Since it was first discovered in cancer, a lot of research has been done on the role of the brain, but as you mentioned, this review focuses on cancer, so I summarized it briefly in about 5 lines like below.
DAPK1 is abundant in developing brain tissue [14]. It gradually decreases after birth, but is highly expressed in the cerebral cortex and hippo-campus in adult rats and mice [14]. Several studies have shown that DAPK1 knockout and inhibition of several studies have shown that DAPK1 knockout and inhibition of DAPK1 function protects the brain, and conversely, overexpression of DAPK1 has various adverse effects on the brain [15-17]. A large number of studies have proven that DAPK1 might play an important role in acute and chronic neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, traumatic brain injury, and stroke [12,15-26].
- For section 2.1 (the Discovery of DAPK1): the author didn’t talk much about the actual discover, whereas talked a lot about the phosphorylation of DAPK1 (which should be in a section that describe DAPK1 regulation) and what can be phosphorylated by DAPK1 (which can be combined with DAPK1 functions).
Thank you for your good comment.
Thank you for the appropriate comment.
After DAPK1 discovery, DAPK1 phosphorylation was a different topic, so I divided it into sections. Also, as I mentioned, I changed it because I thought it would be more appropriate to go with DAPK1 structure with DAPK1 discovery (briefly) (2-1), and DAPK1 family (2-2). Additionally, DAPK1 phosphoyrlation was performed afterwards
.
(3) The structure of DAPK1 needs to be introduced first before the author talks about the detailed phosphorylation sites and also the DAPK family, as a lot of the protein domains are currently introduced in this section without even letting the readers to know where are these domains.
Thank you for your great suggestion. As you mentioned, I changed the order of DAPK1 structure to DAPK1 family.
- It would be more helpful for the author to provide some sort of figure indication where are the DAPK1 phosphorylation sites and what are the potential functions etc. This could potentially be combined with the current Figure 1.
Thank you for your good comments. As you mentioned, Figure 1 has been changed as follows.
Please check the main picture again.
Additionally, content has also been added.
DAPK1 is phosphorylated by several different substrates and its function is regulated (Figure 1). It was indicated which structure of DAPK1 each substrate attaches to and acts on, and those that enhance the function of DAPK1 include NDRG2, protein phosphatase 2A (PP2A), p90 ribosomal S6 kinases 1/2 (RSK 1/2), UNC5H2, and extracellular signal-regulated kinase (ERK). Other substances that inhibit DAPK1 function include Pin1 (Figure 1).
2.3. Phosphosrylation of DAPK1
DAPK1 is phosphorylated by several different substrates and its function is regulated (Figure 1). The figure indicates which structure of DAPK1 each substrate attaches to and acts on, and those that enhance the function of DAPK1 include NDRG2, protein phosphatase 2A (PP2A), p90 ribosomal S6 kinases 1/2 (RSK 1/2), UNC5H2, and extracellular signal-regulated kinase (ERK). Other substances that inhibit DAPK1 function include Pin1 (Figure 1).
Several dephosphorylated phosphatases that regulate DAPK1 have been reported, among which PP2A is a well- known example [29]. PP2A is known to dephosphorylate DAPK1 at Ser308 and activate DAPK1 as a result, thereby inducing autophagy, cell proliferation, and cell apoptosis in vitro [11]. UNC5H2 has also been studied to bind to the death domain of DAPK1 and activate it by dephosphorylating Ser308 of DAPK1. In this way, it activates the apoptosis function of DAPK1 [29]. It is also well known that phosphorylation of Ser735 of DAPK1 by ERK can result in cell apoptosis [30]. The Phosphorylation of Ser289 by RSK can reduce cell apoptosis of DAPK1 [31]. Since DAPK1 is known to regulate brain aging through phosphorylation of Tau and APP as well as the isomerase Pin1. It has been shown that DAPK1 can phosphorylate 'N-myc downstream regulatory gene 2 (NDRG2)' through phospho-peptide library screening [12,18,19]. DAPK1 can directly phosphorylate Ser350 of NDRG2 and the NDRG2 domain can specifically bind to DAPK1 through the ROC-COR domain [12]. NDRG2 has been shown to have the ability to promote the death of neurons in a mitochondria-dependent manner in both in vitro and in vivo human samples [12]. Interestingly, NDRG2 is overexpressed in AD. Since NDRG2 was originally known as a tumor suppressor, additional studies are needed to determine the role Ser350 of NDRG2 plays in cancer metastasis.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors reviewed DAPK1, focusing on metastasis and cancer stem cells. DAPK1 downregulation is correlated with cancer metastasis and might also be involved in cancer stemness. The review provides a comprehensive overview of DAPK1, from basic to advanced knowledge. However, there are the following concerns:
#1. The author's English contains numerous incorrect, unclear or redundant expressions. This paper requires extensive proofreading. Examples are shown below.
Line 45. Several studies have shown that DAPK1 knock-out and inhibition of several studies have shown that DAPK1 knockout and inhibition of 46 DAPK1 function protects the brain, and conversely, overexpression of DAPK1 has various 47 adverse effects on the brain [15-17]. (incorrect)
Line 48. Additionally, DAPK1 KO mouse behavioral experiments report that DAPK1 also plays an important role in brain cognitive abilities (learning and memory disorders) [15-17]. It is reported to play an important role in memory disorders. (redundant)
Line 72. Currently, tumor progression in cancer cells depending on phosphorylation of DAPK1 has not been reported yet [3,31]. (unclear)
Line 146. The [35], an isoform of the glycolytic 146 enzyme commonly expressed in cancer, was also studied to bind to this death domain. (no subject)
Line 203. This phosphorylation also inhibits the catalytic activity and tumor suppressor function of DAPK1. (unclear; which phosphorylation?)
Line 221. These cells provide a micro-environment environment for tumor to grow.
#2. Fig. 1 should also summarize DAPK family proteins and compare their protein structures. This will enhance the readers' understanding.
Additionally, the author should indicate ∆CaM site (line 66) in Fig. 1.
#3. The author should cite a reference for the sentence, “However, distant metastasis occurs in 5-10%. In such cases, the prognosis is known to be poor.” (line 246)
#4. I think the references cited in the following text are incorrect.
“A research team's thyroid cancer study has indicated the mechanism by which DAPK1 regulates OCT4 (POU5F1), a master gene that determines cancer stem cell properties (Figure 2) [62].” (line 255)
#5. Including information on the putative intracellular signaling pathways in Figure 2 would enhance understanding.
The description “Poor clinical TNM stage, disease free survival” should be revised. “Higher (or advanced) clinical TNM stage” and “shorter disease-free survival” are preferable.
#6. The sentence “Tail vein injection of DAPK1 into mice reduced lung metastases [10]” (line 159) seems to be incorrect. They injected DAPK1-transfected cells into the tail vein.
#7. There are many typographical errors.
Line 26: intravasion -> intravasation
Line 201: DAPk1 -> DAPK1
Line 209: DaPK1 -> DAPK1
Line 224: Hip1 alpha -> HIF1 alpha
Etc.
Comments on the Quality of English LanguageThe quality of English is very poor, and the manuscript requires extensive editing.
Author Response
Responses to Reviewer1 are marked in green in the manuscript.
The authors reviewed DAPK1, focusing on metastasis and cancer stem cells. DAPK1 downregulation is correlated with cancer metastasis and might also be involved in cancer stemness. The review provides a comprehensive overview of DAPK1, from basic to advanced knowledge. However, there are the following concerns:
#1. The author's English contains numerous incorrect, unclear or redundant expressions. This paper requires extensive proofreading. Examples are shown below.
Line 45. Several studies have shown that DAPK1 knock-out and inhibition of several studies have shown that DAPK1 knockout and inhibition of 46 DAPK1 function protects the brain, and conversely, overexpression of DAPK1 has various 47 adverse effects on the brain [15-17]. (incorrect)
Thank you for the nice comment.
The correction was carried out as you mentioned. Sentences Lin45 and 48 were deleted because they were used to explain the role of DAPK1 in the brain.
Line 48. Additionally, DAPK1 KO mouse behavioral experiments report that DAPK1 also plays an important role in brain cognitive abilities (learning and memory disorders) [15-17]. It is reported to play an important role in memory disorders. (redundant)
Thank you for the nice comment.
The correction was carried out as you mentioned. Sentences Lin45 and 48 were deleted because they were used to explain the role of DAPK1 in the brain.
Line 72. Currently, tumor progression in cancer cells depending on phosphorylation of DAPK1 has not been reported yet [3,31]. (unclear)
Thank you for telling me. After thinking about it, I thought it was an unnecessary sentence, so I deleted it.
Line 146. The [35], an isoform of the glycolytic 146 enzyme commonly expressed in cancer, was also studied to bind to this death domain. (no subject)
Thank you for letting me know. The sentence you provided has been changed as follows.
Pin1 is expressed to varying degrees in cancer, and DAPK1 inhibits the function of Pin1 by binding to Pin1 in the death domain.
Line 203. This phosphorylation also inhibits the catalytic activity and tumor suppressor function of DAPK1. (unclear; which phosphorylation?)
Thank you for telling me. After thinking about it, I thought it was an unnecessary sentence, so I deleted it.
Line 221. These cells provide a micro-environment environment for tumor to grow.
A reference was added later.
Tanabe, S.; Quader, S.; Cabral, H.; Ono, R. Interplay of EMT and CSC in Cancer and the Potential Therapeutic Strategies. Front Pharmacol 2020, 11, 904, doi:10.3389/fphar.2020.00904.
#2. Fig. 1 should also summarize DAPK family proteins and compare their protein structures. This will enhance the readers' understanding.
Thank you for your good comments. We have modified the review paper to make it easier to read by adding DAPK1 types (DAPK1, DAPK2, DAPK3) as follows.
Additionally, the author should indicate ∆CaM site (line 66) in Fig. 1.
The location of CaM is indicated in Figure 1.
#3. The author should cite a reference for the sentence, “However, distant metastasis occurs in 5-10%. In such cases, the prognosis is known to be poor.” (line 246)
Thank you for your good comments. I have added a reference based on your comment.
(You, M.H.; Lee, W.K.; Jin, M.; Song, D.E.; Cheng, S.Y.; Kim, T.Y.; Kim, W.B.; Jeon, M.J.; Kim, W.G. Death-Associated Protein Kinase 1 Inhibits Progression of Thyroid Cancer by Regulating Stem Cell Markers. Cells 2021, 10, doi:10.3390/cells10112994)
#4. I think the references cited in the following text are incorrect.
“A research team's thyroid cancer study has indicated the mechanism by which DAPK1 regulates OCT4 (POU5F1), a master gene that determines cancer stem cell properties (Figure 2) [62].” (line 255)
Thank you for telling me. The reference to paper number 27, which I wrote in 2021, is correct.
(You, M.H.; Lee, W.K.; Jin, M.; Song, D.E.; Cheng, S.Y.; Kim, T.Y.; Kim, W.B.; Jeon, M.J.; Kim, W.G. Death-Associated Protein Kinase 1 Inhibits Progression of Thyroid Cancer by Regulating Stem Cell Markers. Cells 2021, 10, doi:10.3390/cells10112994)
#5. Including information on the putative intracellular signaling pathways in Figure 2 would enhance understanding.
The description “Poor clinical TNM stage, disease free survival” should be revised. “Higher (or” advanced) clinical TNM stage and “shorter disease-free survival” are preferable.
Thank you for your good comments. We have modified it according to your comments.
Please check the main picture again.
#6. The sentence “Tail vein injection of DAPK1 into mice reduced lung metastases [10]” (line 159) seems to be incorrect. They injected DAPK1-transfected cells into the tail vein.
Thank you for the nice comment. I have corrected the sentence you mentioned like below;
They injected DAPK1-transfected cells into the tail vein. [10].
#7. There are many typographical errors.
Line 26: intravasion -> intravasation
Line 201: DAPk1 -> DAPK1
Line 209: DaPK1 -> DAPK1
Line 224: Hip1 alpha -> HIF1 alpha
Etc.
Thank you for the nice comment. I have corrected the part you mentioned.
Additionally, we corrected additional missed parts through English Editing Service. I attached the certification of editing service
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author corrected most of my concerns appropriately. However, the author's English still contains incorrect, unclear, or redundant expressions. This paper requires extensive proofreading by a native English speaker.
Comments on the Quality of English LanguageSame as above.
Author Response
Author Response File: Author Response.pdf
