ERK: A Double-Edged Sword in Cancer. ERK-Dependent Apoptosis as a Potential Therapeutic Strategy for Cancer
Round 1
Reviewer 1 Report
In this work, the authors provide a review of the therapeutic strategy targeting ERK MAPK. This review article especially focuses on the pro-apoptotic function of the ERK signaling pathway. The compounds inducing ERK-dependent apoptosis are well summarized and informative for readers. They also mentioned the mechanism underlying ERK-induced apoptosis, including the relationship to p53, ROS, DUSPs, and other factors.
This review provides much-needed information to the experimentalist, especially in its summary of the compounds that induce ERK-dependent apoptosis for cancer therapy and the mechanism underlying the pro-apoptotic function of ERK signaling.
For me, the paper does not require a major revision. However, I have one suggestion that the additional figure summarizing the mechanisms underlying the pro-apoptotic function of ERK, via p53 and ROS, would be helpful for readers to understand.
MInor comments
- line 220, mu tated
Author Response
We are grateful to Reviewer 1 for providing useful suggestions that helped us considerably improve our manuscript. As indicated in the following responses, we have incorporated these comments and suggestions in our revised manuscript.
The reviewer made the following observations: In this work, the authors provide a review of the therapeutic strategy targeting ERK MAPK. This review article especially focuses on the pro-apoptotic function of the ERK signaling pathway. The compounds inducing ERK-dependent apoptosis are well summarized and informative for readers. They also mentioned the mechanism underlying ERK-induced apoptosis, including the relationship to p53, ROS, DUSPs, and other factors. This review provides much-needed information to the experimentalist, especially in its summary of the compounds that induce ERK-dependent apoptosis for cancer therapy and the mechanism underlying the pro-apoptotic function of ERK signaling. For me, the paper does not require a major revision. However, I have one suggestion that the additional figure summarizing the mechanisms underlying the pro-apoptotic function of ERK, via p53 and ROS, would be helpful for readers to understand.
Re: We thank the Reviewer for these pertinent comments. We have followed this reviewer’s excellent suggestion and incorporated p53 and ROS in the revised figure summarizing the mechanisms underlying the pro-apoptotic function of ERK.
The reviewer also stated, “MInor comments line 220, mu tated”
Re: Our apologies for the carelessness. We correct it by removing the space.
Reviewer 2 Report
This is a well written review supported by instructive figures about the role of ERK signaling in apoptosis combined with an extensive pharmacological survey of compounds mediating apoptosis. However, I am missing a discussion of ERK signaling regulators (with the exception of DUSP of which some info is provided). Nevertheless, several important studies have been published supporting the cellular threshold theory for active ERK1/2 in mediating apoptosis in some cancers and possible therapeutic strategies have been suggested, e.g. down-regulation of Spry proteins (for example, see https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/pubmed/29635363).
Author Response
We are grateful to Reviewer 2 for providing useful suggestions that helped us considerably improve our manuscript. As indicated in the following responses, we have incorporated these comments and suggestions in our revised manuscript.
Reviewer 2 Comments for the Author:
The reviewer mentioned, “This is a well written review supported by instructive figures about the role of ERK signaling in apoptosis combined with an extensive pharmacological survey of compounds mediating apoptosis. However, I am missing a discussion of ERK signaling regulators (with the exception of DUSP of which some info is provided). Nevertheless, several important studies have been published supporting the cellular threshold theory for active ERK1/2 in mediating apoptosis in some cancers and possible therapeutic strategies have been suggested, e.g. down-regulation of Spry proteins (for example, see https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/pubmed/29635363).”
Re: We thank the Reviewer for these pertinent comments. We have followed this reviewer’s excellent suggestion by incorporating a discussion on Spry proteins, a family of endogenous proteins that negatively regulate the ERK signaling pathway that is activated by receptor tyrosine kinases. Intriguingly, similar to DUSP proteins, some cancer cells are dependent on Spry proteins for their growth by adaptation, which makes Spry proteins play an onco-promoting role contrary to their original role as a negative regulator of the ERK signaling pathway. The authors concluded that an antitumoral effect of SPRY2 inhibition is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in glioblastoma patients. Altogether, compounds targeting negative regulators of the ERK cascade, such as DUSP6 and SPRY proteins, might offer a treatment strategy for certain cancers by inducing the toxic effects of RAS-mediated hyperactive ERK signaling. These discussions have been incorporated in the revised manuscript (page 14, line 1128-1138).
Reviewer 3 Report
The ERK signaling cascade is a central signaling pathway that induces and regulate essentially all stimulated cellular processes. In particular, this cascade is known to induce cell proliferation, but is involved also in the regulation of differentiation, migration and more. Importantly, dysregulation of the cascade often leads to several diseases, including cancer, developmental disorders, neurological problems and others. As for cancer, it was shown that the ERK cascade is activated in more of 85% of the cases, and inhibitors of this cascade serve as therapeutic drugs for melanoma and other cancers. However, aside from its function in proliferation, it was shown that the ERK cascade plays a role in inducing apoptosis, which is the focus of the current review. Overall, this is a well-written review that covers an understudied part of the field that has not been properly reviewed in the past. However, the suitability of the papers cited in table 1 is not clear, particularly due to the side effects of the inhibitors used. This may jeopardize the working hypothesis of the authors and therefore may prevent the publication of the review. In addition, the mechanisms by which the ERK cascades are involved in cell death and how can it be used in combating the diseases are not clear form this review. These points as well as other comments listed below should be addressed before publication in Cells.
Comments
1) The biggest problem with the working hypothesis raised here is the quality of the ERK inhibitors used in the papers cited in table 1. It should be noted that the use of old generation inhibitors of MEK, that are not as specific as the recently developed drugs might be problematic. Importantly, it was shown that PD98059 is able to directly affect mitochondria and induce production of reactive oxygen species (PMID: 12860392). Moreover, U0126 was shown to act as a direct ROS scavenger (PMID: 25544156). These results raise the caution and call for reinterpretation of many former results. Moreover, even the papers that used SiRNA to reduce ERK activity are not very convincing, as the authors of these papers did not establish that they have no off-target effects. Finally, the only paper that used an appropriate inhibitor is cited in Ref 154, but even there the information is not complete. These points may jeopardize the working hypothesis of the authors, and therefore may preclude the publication of the current review.
2) As mentioned in the review, the main effect of the ERK cascade is the induction of proliferation and not apoptosis. Moreover, as far as I know, there are no reports on apoptosis that is induced by overexpression of constitutively active components of the cascade. Therefore, it seems to me that the effects listed in Table 1, are probably indirect, and may indicate that ERK activation supports or cooperate with other apoptosis-inducing processes, but it can’t induce apoptosis by itself. These reservation should be better discussed.
3) The authors should note that direct activation of the ERK cascade is always transient, peaking at 5-10 min after stimulation and reduce back to basal level 30-90 min thereafter. Second and third wave of activation may appear at later time points, due to autocrine loops or other ERK-required processes (e.g. mitosis). Unfortunately, the publications listed in Table 1 followed ERK activation in hours and even days, missing the short term effects. This may support the indirect effect of the ERK in inducing apoptosis. This point should be better discussed as well.
4) It is not clear from this review how can the ERK-induced apoptosis be used as anti-cancer target, as activation of ERK usually induce proliferation. This issue should be clarified.
5) Overall the section entitled “Overview of the ERK signaling cascade” is well-written. However, some information on its regulation is missing. For example, it would be nice to add information on scaffold proteins, the number of components in the MAP3K (Raf-A,B,C, Mos MEKK1/3, etc) level, the alternative spliced isoforms in many of the isoforms, and the localization/stimulated nuclear translocation. In addition, it is recommended to include information on MAPKAPKs (e.g. RSK) in the cascade. Finally, it is suggested to provide a better description of intrinsic vs extrinsic apoptosis.
6) Some of the terms used are not consistent throughout the article. In particular the name of the cascade, which should be the ERK cascade (that lies within the MAPK pathway). There is no need to mention every time ”Ras/Raf/MEK ERK” or “ERK MAPK pathway”, as the term ERK cascade is sufficient. In addition, Raf kinases, are usually used for kinases that phosphorylate Raf.
7) Fig. 1: As far as I know, MSK, MNK and CREB are mostly nuclear substrates. RSK is almost only cytosolic, so it is not clear why it is attached to the chromatin here. Another MEK inhibitor in clinical use is “Mektovi”. Finally, it is suggested to include the importin (Imp7) and exportins (Exp1) involved in the nuclear translocation.
8) Fig. 2: The negative feedback loop presented is not only on Raf. I suggest to mention it and give more information in the text. This figure can benefit from showing some information on the mechanisms that induce apoptosis. It is not clear why is it only nuclear; what about the mitochondrial effect.
Author Response
Reviewer 3
The reviewer mentioned “The ERK signaling cascade is a central signaling pathway that induces and regulate essentially all stimulated cellular processes. In particular, this cascade is known to induce cell proliferation, but is involved also in the regulation of differentiation, migration and more. Importantly, dysregulation of the cascade often leads to several diseases, including cancer, developmental disorders, neurological problems and others. As for cancer, it was shown that the ERK cascade is activated in more of 85% of the cases, and inhibitors of this cascade serve as therapeutic drugs for melanoma and other cancers.
However, aside from its function in proliferation, it was shown that the ERK cascade plays a role in inducing apoptosis, which is the focus of the current review. Overall, this is a well-written review that covers an understudied part of the field that has not been properly reviewed in the past. However, the suitability of the papers cited in table 1 is not clear, particularly due to the side effects of the inhibitors used. This may jeopardize the working hypothesis of the authors and therefore may prevent the publication of the review. In addition, the mechanisms by which the ERK cascades are involved in cell death and how can it be used in combating the diseases are not clear form this review. These points as well as other comments listed below should be addressed before publication in Cells.”
Re: We are grateful to Reviewer 3 for providing useful and constructive suggestions that helped us to considerably improve our manuscript. As indicated in the following responses, we have incorporated these general issues in our revised manuscript by re-evaluating and updating the compounds and references listed in Table1 as well as by addressing concerns and suggestions listed in the reviewer’s comments as below.
Comments
- The reviewer mentioned “The biggest problem with the working hypothesis raised here is the quality of the ERK inhibitors used in the papers cited in table 1. It should be noted that the use of old generation inhibitors of MEK, that are not as specific as the recently developed drugs might be problematic. Importantly, it was shown that PD98059 is able to directly affect mitochondria and induce production of reactive oxygen species (PMID: 12860392). Moreover, U0126 was shown to act as a direct ROS scavenger (PMID: 25544156). These results raise the caution and call for reinterpretation of many former results. Moreover, even the papers that used SiRNA to reduce ERK activity are not very convincing, as the authors of these papers did not establish that they have no off-target effects. Finally, the only paper that used an appropriate inhibitor is cited in Ref 154, but even there the information is not complete. These points may jeopardize the working hypothesis of the authors, and therefore may preclude the publication of the current review.”
Re: We thank this reviewer for the pertinent comments. Accordingly, we incorporated the Reviewer’s comments to attract the reader’s attention regarding the possible side effects of the inhibitors of MEK, particularly the use of old generation MEK inhibitors. We also add special remarks on the papers using SiRNA to reduce ERK activity due to the lack of experiments to rule out the possible off-target effects. Furthermore, to address the reviewer’s concern, we searched for papers using an appropriate inhibitor PD 184352, which is a potent, highly selective ATP non-competitive MEK1/2 inhibitor, and updated Table1 by incorporating the new papers with adequate information. Finally, we re-evaluated the papers listed in the original Table1 whether the compounds or stimuli (in Table1) can induce ROS and the validation of the ERK activation-dependent apoptosis is achieved by U0126. These compounds include Derrone, Ginsenoside Rg5, PhysalinB, Paraquat, and Cadmium. We also add comments to call the reader’s attention that if the mechanism of apoptosis induction by these compounds involves ROS, the MEK inhibitor U0126 can serve as a direct ROS scavenger, and therefore, U0126 could cancel apoptosis directly via ROS elimination and indirectly via ERK inhibition (ref. 83). Notably, however, a paper by Szydlowska et al, (ref. 81) confirmed that the apoptosis induced by glutamate was mediated through ERK activation by using the MEK inhibitor U0126 as well as by DUSP overexpression, providing a further validation that apoptosis is a direct consequence of ERK activation.
These discussions, special cautions, and reservations suggested by this reviewer have been incorporated in the revised manuscript (page 7-).
2) The reviewer mentioned “As mentioned in the review, the main effect of the ERK cascade is the induction of proliferation and not apoptosis. Moreover, as far as I know, there are no reports on apoptosis that is induced by overexpression of constitutively active components of the cascade. Therefore, it seems to me that the effects listed in Table 1, are probably indirect, and may indicate that ERK activation supports or cooperate with other apoptosis-inducing processes, but it can’t induce apoptosis by itself. These reservation should be better discussed.”
Re: We thank this reviewer for the insightful comments. Indeed, there are only limited numbers of reports on apoptosis that is induced by overexpression of constitutively active components of the ERK cascade. These include papers describing that the expression of constitutively active forms of Raf1 (ref. 99, 100), RasV12T35S (ref.100), or ΔRaf1:ER induction (ref.102) was sufficient to promote cell death.
Notably, Leung et al, reported that hyperactivation of MAPK signaling by ERK2 overexpression is toxic in BRAF-mutant melanoma, by inducing stress responses and apoptotic signaling (ref. 109). Furthermore, a large-scale pooled shRNA screen revealed that only BRAF or MAP2K silencing could rescue the toxic effects associated with ERK2 overexpression in the BRAF mutant background. Importantly, the authors concluded that the toxic effects achieved by ERK2 overexpression is specific for RAS/RAF mutant context, but not to the RAS/RAF wild-type melanoma, proposing that the vulnerability to MAPK hyperactivation raises the novel therapeutic approaches for RAS/RAF-mutant cancers (ref. 109).
In line with this publication, a recent paper by Unni et al reported that hyperactivation of ERK is toxic to the receptor-tyrosine kinase or the Ras mutation-driven lung adenocarcinoma (LUAD) cells (ref. 226). The authors further demonstrated that DUSP6, which negatively regulates phosphorylation of ERK is up-regulated in EGFR- or KRAS-mutant signaling. In addition, knockdown or pharmacological inhibition of DUSP6 elevates P-ERK and reduces the viability of LUAD cells when combined with KRAS or EGFR oncogenic mutations. These results imply that tumors with mutant oncogenes in the RAS pathway restrain the activity of ERK1/2 by upregulating DUSP6 expression to avoid toxicities and enable tumor growth. Thus, targeting DUSP6 or other negative regulators such as Spry proteins might offer a treatment strategy for certain cancers by inducing the toxic effects of RAS-mediated signaling. These data are consistent with our findings obtained with DUSP6 and ACA-28 in HER2-overexpressing cells. This paper is important in that (as suggested by the reviewer) a single oncogenic mutation of either EGFR- or KRAS-mutant is not sufficient to reduce cell viability in LUAD and hyperactivation of p-ERK achieved by KRAS or EGFR mutation combined with DUSP6 inhibition can cause cell death. A similar situation was reproduced by the paper by Leung et al, that the ERK activation by the oncogenic mutation in the upstream components of the ERK cascade combined with ERK2 overexpression causes toxicity. We assume that these findings support the hypothesis that hyperactivation of the ERK cascade above a certain threshold is required to cause cell death, although a special caution is needed in that DUSP6 inhibition or knockdown can affect other apoptosis-inducing processes such as p38 or JNK signaling activation.
Altogether, as suggested by the reviewer, rigorous verification of these cell death examples concerning the requirement of the Raf/ERK pathway activation in apoptosis induction using appropriate MAPK inhibitors and/or add-back experiments to corroborate siRNA experiments will be future studies.
These discussions and reservations have been incorporated in the revised manuscript (page 7).
3) The reviewer mentioned “The authors should note that direct activation of the ERK cascade is always transient, peaking at 5-10 min after stimulation and reduce back to basal level 30-90 min thereafter. Second and third wave of activation may appear at later time points, due to autocrine loops or other ERK-required processes (e.g. mitosis). Unfortunately, the publications listed in Table 1 followed ERK activation in hours and even days, missing the short term effects. This may support the indirect effect of the ERK in inducing apoptosis. This point should be better discussed as well.”
Re: We thank this reviewer for the insightful comments. We then re-evaluated the references listed in Table1. First, we have to apologize for the erroneous labeling of the column showing “Duration of ERK activation”, which actually indicates the timing of evaluating the apoptosis induction by the compound in each paper. We then amended the label of the column and added a new column, showing the time course wherein the ERK activation was investigated in each paper. Consequently, we found several references investigating the ERK activation at 5-15 min or a longer period after compound stimulation (refs. 16, 119, 120, 129, 135). These include H. pylori secreted protein HP1286, Etoposide, Qizhen capsule, Perfluorooctane sulfonate, Perfluorohexanesulfonate, and Cypermethrin. The ERK activation at an early stage in these papers was abolished by U0126, which leads to the cancellation of apoptosis. Therefore, the short-term effects of ERK activation by these compounds and cancellation of apoptosis by U0126 suggest that some compounds exert a direct effect to induce ERK activation-induced apoptosis. However, as suggested by this reviewer, since a large number of studies evaluate ERK activation at a time point when the authors investigate apoptosis (for example, 24hr), the information regarding short-term effects is missing in these papers. So, we mentioned special remarks to readers that future work to eliminate the possible indirect effect of the ERK in apoptosis induction should be performed.
These discussions and reservations have been incorporated in the revised manuscript (page 14-15).
4) The reviewer mentioned “It is not clear from this review how can the ERK-induced apoptosis be used as anti-cancer target, as activation of ERK usually induce proliferation. This issue should be clarified.”
Re: We thank this reviewer for the constructive comments. For the strategy to utilize ERK-induced apoptosis as an anti-cancer target, the hurdle to overcome include, how to achieve selective apoptosis induction in cancer cells. One way to address this issue would be a drug delivery system targeting cells expressing high-ERK phosphorylation levels by using high DUSP6 or SPRY proteins as a marker. This idea is based on the reports cited in this review, showing that inhibition/silencing of DUSP6 protein or SPRY2 protein is toxic to the oncogenic mutant lung cancer cells or glioblastoma cells, respectively, but not to the normal cell background. Furthermore, these negative regulators are highly expressed in cancer cells compared with normal cells. Then, how to achieve drug delivery targeting cells expressing high DUSP6 or high SPRY proteins? For example, Ota et al, reported selective targeting cancer cells by utilizing lysine-specific demethylase 1 (LSD1) to trigger the controlled release of anticancer drug tamoxifen in cancer cells, wherein LSD1 is highly expressed. Conjugates of the LSD1 inhibitor PCPA were used as prodrugs to selectively release tamoxifen by LSD1 inhibition. Importantly, the pro-drug inhibited the growth of breast cancer cells by the simultaneous inhibition of LSD1 enzymatic activity and the estrogen receptor by tamoxifen without hampering growth in normal cells, wherein only a low amount of LSD1 is expressed with the almost undetectable release of tamoxifen. Thus, the selective release of tamoxifen in cancer cells utilizing the high expression of LSD1 in cancer cells as compared with the normal cells can be an excellent prototype for future study to achieve cancer-cell selective delivery of compounds such as ACA-28 derivatives to induce ERK-dependent apoptosis in cancer cells, but not to normal cells.
These discussions and reservations have been incorporated in the revised manuscript (page 19).
5) The reviewer mentioned, “Overall the section entitled “Overview of the ERK signaling cascade” is well-written. However, some information on its regulation is missing. For example, it would be nice to add information on scaffold proteins, the number of components in the MAP3K (Raf-A,B,C, Mos MEKK1/3, etc) level, the alternative spliced isoforms in many of the isoforms, and the localization/stimulated nuclear translocation. In addition, it is recommended to include information on MAPKAPKs (e.g. RSK) in the cascade. Finally, it is suggested to provide a better description of intrinsic vs extrinsic apoptosis.”
Re: We thank this reviewer for the instructive comments. To address the reviewer’s suggestions, we incorporated the information on scaffold proteins, the number of components in the MAP3K (Raf-A, B, C, Mos MEKK1/3, etc) level, the alternative spliced isoforms in many of the isoforms, and the localization/stimulated nuclear translocation and MAPKAPKs. Furthermore, we incorporated a more detailed description of intrinsic vs extrinsic apoptosis (page 2-3).
6) The reviewer mentioned “Some of the terms used are not consistent throughout the article. In particular the name of the cascade, which should be the ERK cascade (that lies within the MAPK pathway). There is no need to mention every time ”Ras/Raf/MEK ERK” or “ERK MAPK pathway”, as the term ERK cascade is sufficient. In addition, Raf kinases, are usually used for kinases that phosphorylate Raf.”
Re: We thank this reviewer for the instructive comments. Accordingly, we replaced“Ras/Raf/MEK ERK” or “ERK MAPK pathway”, as the term ERK cascade” uniformly. We also corrected Raf kinases as Raf.
7) The reviewer mentioned “Fig. 1: As far as I know, MSK, MNK and CREB are mostly nuclear substrates. RSK is almost only cytosolic, so it is not clear why it is attached to the chromatin here. Another MEK inhibitor in clinical use is “Mektovi”. Finally, it is suggested to include the importin (Imp7) and exportins (Exp1) involved in the nuclear translocation.”
Re: Accordingly, we revised Figure 1 as suggested by the Reviewer, and incorporated another MEK inhibitor “Mektovi” and the importin (Imp7) and exportins (Exp1) involved in the nuclear translocation.
8) The reviewer mentioned “Fig. 2: The negative feedback loop presented is not only on Raf. I suggest to mention it and give more information in the text. This figure can benefit from showing some information on the mechanisms that induce apoptosis. It is not clear why is it only nuclear; what about the mitochondrial effect.”
Re: Accordingly, we revised Figure 2 and text as suggested by the Reviewer, and incorporated an additional negative feedback loop on Ras and MEK. In addition, we also added mitochondria as a possible localization wherein ERK functions to apoptosis.
Round 2
Reviewer 3 Report
The authors addressed nicely the comments. Although I still do not trust many of the results demonstrated in Table 1 sue to the lack of specificity of the MEK inhibitors, I agree with the authors that there are enough studies that provide convincing evidence that ERK-dependent apoptosis does exist. In addition, the authors incorporated a word of caution in the description of Table 1 that clarify the problems in the field. At this stage I have only a few minor comments that I suggest to address.
- In line 92, please replace isoforms of Raf with MAP3K isoforms.
- In lines 101-103 I suggest to replace the current wording with: "Thus, these various isoforms and spliced variants exert various cellular and pathophysiological functions, which may be mediated by antagonizing their full-length counterparts [41]. Alternative spliced isoforms of MEK (ERK1b) and ERK (ERK1c) exist as well (PMID: 34189435), extending the signaling specificity of the cascade even further.
- In line 316, I suggest to change the current wording with "Special caution should be taken when considering the results in Table 1 that use old generation inhibitors of MEK that are not as specific as the recently developed drugs were used." This would clarify why was the discussion in the rest of the paragraph necessary.
- I suggest to include a mentioning of a possible non-apoptotic cell death by the ERK cascade (PMID: 32340261).
Author Response
The reviewer mentioned “The authors addressed nicely the comments. Although I still do not trust many of the results demonstrated in Table 1 due to the lack of specificity of the MEK inhibitors, I agree with the authors that there are enough studies that provide convincing evidence that ERK-dependent apoptosis does exist. In addition, the authors incorporated a word of caution in the description of Table 1 that clarify the problems in the field. At this stage I have only a few minor comments that I suggest to address.”
Re: We are grateful to Reviewer 3 for providing useful and constructive suggestions that helped us to further improve our manuscript. As indicated in the following responses, we have incorporated comments and a reference as suggested by the Reviewer.
Comments and Suggestions for Authors
- The reviewer mentioned, “In line 92, please replace isoforms of Raf with MAP3K isoforms.”
Re: Accordingly, we replaced isoforms of Raf with MAP3K isoforms.
2) The reviewer mentioned “In lines 101-103 I suggest to replace the current wording with: "Thus, these various isoforms and spliced variants exert various cellular and pathophysiological functions, which may be mediated by antagonizing their full-length counterparts [41]. Alternative spliced isoforms of MEK (ERK1b) and ERK (ERK1c) exist as well (PMID: 34189435), extending the signaling specificity of the cascade even further.”
Re: We thank this reviewer for the insightful comments. Accordingly, we replaced the current wording with the sentences as suggested by the Reviewer.
- The reviewer mentioned, “In line 316, I suggest to change the current wording with "Special caution should be taken when considering the results in Table 1 that use old generation inhibitors of MEK that are not as specific as the recently developed drugs were used." This would clarify why was the discussion in the rest of the paragraph necessary.”
Re: We thank this reviewer for the insightful comments. Accordingly, we replaced the current wording with the sentences as suggested by the Reviewer.
3) The reviewer mentioned “In line 316, I suggest to change the current wording with "Special caution should be taken when considering the results in Table 1 that use old generation inhibitors of MEK that are not as specific as the recently developed drugs were used." This would clarify why was the discussion in the rest of the paragraph necessary.”
Re: We thank this reviewer for the insightful comments. Accordingly, we replaced the current wording with the sentences as suggested by the Reviewer.
4) The reviewer mentioned, “I suggest to include a mentioning of a possible non-apoptotic cell death by the ERK cascade (PMID: 32340261).”
Re: Accordingly, we included a mentioning of the paper suggested by the Reviewer.
We hope that the aforementioned new findings would satisfy your requirements. If you would still find issues to be addressed and suggest better descriptions or phrases appropriate to further strengthen our conclusion, we would respectfully take your advice.
Your generous consideration is highly appreciated.
