Targeting SIK2 with GRN-300 Potentiates Paclitaxel Efficacy in Triple-Negative Breast Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Based on the review of the text of the submitted article titled "Targeting SIK2 with GRN-300 Potentiates Paclitaxel Efficacy in Triple-Negative Breast Cancer," placed. The following are the errors and issues that need improvement or clarification:

1. There are several typographical and spacing errors in the text that detract from the professional aspect of the article. In line 45, the word "PLK1therely" is incorrectly pasted (it should be "PLK1, thereby"). In line 166, the word "whileGRN-300" is placed. In some parts, the proper format is not followed; °C" (degrees Celsius), for example, "incubated overnight at 4 oC with" degrees Celsius is different from other places. In line 269, the sentence "performed immunohistochemical (IHC) analysis on a large..." seems incomplete, and a word such as "cohort" or "set" is probably inserted after "large."
2. In the section "Cell Lines and Culture Conditions," PLK1 is mentioned, but it is necessary to mention the country name after the company name. In addition, abbreviations should be spelled out in full before being repeated throughout the text, such as HEPES.

         3- The article states that GRN-300 reduces the                 expression of key proteins such as CDC27, CDK1, and PLK1, but does not specify whether this regulation is direct (protein-protein) or through mediated signaling pathways.

4- While synergy was seen in most TNBC cell lines, the HCC 1937 cell line showed less resistance or response than the others. The article does not fully analyze the biological reason for this difference.

4- In the section on Western blotting, the phrase 4 to 15%" is ambiguous as to whether the gel was discontinuous or different concentrations were used depending on the type of protein.

5- In the results section, the intensity of SIK2 staining is scored between 0 and 3. This type of assessment can be subjective. It would have been better to state whether this scoring was performed by two independent pathologists in a blinded manner.

6- In Figure 2, the detailed data for all 8 TNBC cell lines that were claimed to have been tested are not fully presented in the main text, and some of them are referred to in the Supplementary Material. To better understand the heterogeneity of responses, the presence of these data in the main text would have been helpful.

7- The article emphasizes the increased expression of SIK2 in TNBC samples, but the correlation between the expression level of this protein and patient survival indicators (overall survival) (stage) or disease stage (stage) in human samples has not been analyzed in detail.

8- Given that GRN-300 was effective in paclitaxel-resistant models, a more detailed investigation of the mechanism of this effect (e.g., the effect on drug efflux pumps or changes in microtubule dynamics) is necessary.

9- In the virus production section, details regarding confirmation of CDC27 overexpression after transduction are not fully mentioned in the preliminary results.

Author Response

Thank you for your review and comments. Please find our responses below. Corresponding revisions have been highlighted  in the revised manuscript.

Comments 1: There are several typographical and spacing errors in the text that detract from the professional aspect of the article. In line 45, the word "PLK1therely" is incorrectly pasted (it should be "PLK1, thereby"). In line 166, the word "whileGRN-300" is placed. In some parts, the proper format is not followed; °C" (degrees Celsius), for example, "incubated overnight at 4 oC with" degrees Celsius is different from other places. In line 269, the sentence "performed immunohistochemical (IHC) analysis on a large..." seems incomplete, and a word such as "cohort" or "set" is probably inserted after "large."

Response 1: Agreed. We carefully reviewed the entire manuscript and corrected these errors, which are highlighted in the revised manuscript.

Comments 2: In the section "Cell Lines and Culture Conditions," PLK1 is mentioned, but it is necessary to mention the country name after the company name. In addition, abbreviations should be spelled out in full before being repeated throughout the text, such as HEPES.

Response 2:  There is no mention of “PLK1” in the “Cell Lines and Culture Conditions” section. However, we have inserted the country name after each company name and spelled out the full term before introducing each abbreviation.

Comments 3: The article states that GRN-300 reduces the expression of key proteins such as CDC27, CDK1, and PLK1, but does not specify whether this regulation is direct (protein-protein) or through mediated signaling pathways.

Response 3: Thank you for this comment. Our study demonstrates that GRN-300 treatment is associated with reduced expression of key cell cycle regulatory proteins, including CDC27, CDK1, and PLK1. However, the current study was not designed to determine whether these effects result from direct protein-protein interactions or are mediated indirectly through downstream signaling pathways. A more detailed mechanistic investigation of these regulatory interactions is beyond the scope of the present manuscript and will be the focus of future studies.

Comments 4.  While synergy was seen in most TNBC cell lines, the HCC 1937 cell line showed less resistance or response than the others. The article does not fully analyze the biological reason for this difference.

Response 4: We agree that the differential response observed in HCC1937 cells warrants further biological investigation. However, because HCC1937 cells were only analyzed in the cell cycle studies and were not included in the combination cell viability analyses with GRN-300 and paclitaxel, we believe that the current dataset is insufficient to support a comprehensive mechanistic interpretation of this differential response. Therefore, to avoid overinterpretation, we have removed the HCC1937 data from the revised manuscript. (remove HCC1937 cell cycle data)

Comments 5: In the section on Western blotting, the phrase 4 to 15%" is ambiguous as to whether the gel was discontinuous or different concentrations were used depending on the type of protein.

Response 5: The “4–15%” notation refers to 4–15% gradient SDS-PAGE gels. We have revised the Methods section to explicitly indicate “4–15% gradient gels” to avoid ambiguity.

Comments 6: In the results section, the intensity of SIK2 staining is scored between 0 and 3. This type of assessment can be subjective. It would have been better to state whether this scoring was performed by two independent pathologists in a blinded manner.

Response 6: Thank you for this comment. SIK2 staining intensity was independently evaluated by two pathologists in a blinded manner using the predefined 0–3 scoring criteria. The corresponding information has now been added to the Methods section to clarify the assessment procedure.

Comments 7. In Figure 2, the detailed data for all 8 TNBC cell lines that were claimed to have been tested are not fully presented in the main text, and some of them are referred to in the Supplementary Material. To better understand the heterogeneity of responses, the presence of these data in the main text would have been helpful.

Response 7. Thank you for this comment. We apologize for any confusion. The data for all eight TNBC cell lines tested are included in Figure 2 in the main text and were not placed in the Supplementary Materials. We have revised the figure legend/text to make this point clearer and to facilitate interpretation of the heterogeneous responses across the TNBC cell line panel.

Comment 8.  The article emphasizes the increased expression of SIK2 in TNBC samples, but the correlation between the expression level of this protein and patient survival indicators (overall survival) (stage) or disease stage (stage) in human samples has not been analyzed in detail.

Response 8. Thanks for this important suggestion. We agree that correlating SIK2 expression with clinical outcome parameters such as overall survival and disease stage would provide additional clinical insight. However, the tissue microarray (TMA) used in this study was a commercially purchased, de-identified cohort and does not include linked clinical outcome data (survival information or detailed staging). Consequently, such correlative analyses cannot be performed with the current dataset. We have clarified this limitation in the revised manuscript. Importantly, our analysis was focused on comparing SIK2 expressions across breast cancer subtypes, where we observed significantly higher expression in TNBC compared to non-TNBC samples, supporting its subtype-associated upregulation.

Comment 9.  Given that GRN-300 was effective in paclitaxel-resistant models, a more detailed investigation of the mechanism of this effect (e.g., the effect on drug efflux pumps or changes in microtubule dynamics) is necessary.

Response 9. While the present study demonstrates that GRN-300 effectively enhances paclitaxel sensitivity, including in paclitaxel-resistant models, the precise molecular mechanisms underlying this effect remain to be fully defined. In particular, potential contributions from altered drug efflux (ABC transporter activity), modulation of microtubule dynamics, or changes in stress-response signaling pathways have not yet been systematically investigated. These mechanisms represent important and biologically plausible avenues for future studies to further delineate how SIK2 inhibition potentiates taxane efficacy in TNBC. We have revised the discussion to note these possibilities.

Comment 10.  In the virus production section, details regarding confirmation of CDC27 overexpression after transduction are not fully mentioned in the preliminary results.

Response 10. CDC27 overexpression following viral transduction was confirmed experimentally and is clearly shown in Figure 7. Specifically, successful overexpression was validated by increased CDC27 protein levels compared with control cells after transduction.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments:

This manuscript investigates the therapeutic potential of the SIK2 inhibitor GRN-300 in combination with paclitaxel for triple-negative breast cancer (TNBC), presenting in vitro experiment and in vivo xenograft efficacy, and mechanistic analyses by the combination treatment. The topic is clinically relevant, and the proposed strategy of enhancing paclitaxel efficacy through SIK2 inhibition is potentially of interest for TNBC treatment. However, several key issues currently limit the rigor and interpretability of the work to be published in Cancers, particularly regarding target specificity, the validation of CDC27-mediated signaling, in vivo pharmacodynamic and toxicity assessment, and the evidence supporting activity in paclitaxel-resistant models. The reviewer offers the following major comments to help strengthen the manuscript.

 

⦿ Major comments:

1. The authors examined SIK2 expression in clinical samples and cell lines in Figure 1 and showed that SIK2 is highly expressed in TNBC. However, it is unclear whether SIK2 expression levels are associated with sensitivity to GRN-300 or with the response to GRN-300/paclitaxel combination treatment. 

The authors should clarify whether SIK2 expression correlates with GRN-300 single-agent activity, paclitaxel IC50, combination index, G2/M arrest, or apoptosis induction across the tested cell lines. In addition, response data from SIK2-low or SIK2-negative cell lines would help support the claim that the observed cytotoxic effects are related to SIK2 inhibition. Since SUM159 is used as a major model in this study, its SIK2 expression level should also be clearly shown.

2. In the Simple Summary and Abstract, the authors state that GRN-300 remains effective in paclitaxel-resistant TNBC models and may help overcome acquired chemoresistance. However, the main results presented in the manuscript appear to be largely based on parental TNBC cell lines and xenograft models.

To substantiate the role of SIK2 inhibition in overcoming paclitaxel resistance, the authors should provide data using paclitaxel-resistant TNBC cell lines. Specifically, they should show the anticancer effects of GRN-300 alone and in combination with paclitaxel in paclitaxel-resistant models, along with validation of the resistant phenotype and, if possible, analysis of relevant resistance mechanisms.

3. The in vivo xenograft results in Figure 3 are promising, as the combination therapy reduced tumor growth and improved survival in both MDA-MB-231 and SUM159 models. However, the current in vivo data mainly focus on efficacy, and additional information is needed to assess the translational relevance of this combination therapy.

The authors should provide tolerability and toxicity data, such as body weight changes and hematologic toxicity, during treatment. In addition, pharmacodynamic validation in xenograft tumors would strengthen the study. For example, showing whether the SIK2 pathway is actually inhibited in tumor tissues after GRN-300 treatment, including changes in SIK2-related downstream markers, would provide stronger support for the proposed mechanism and therapeutic benefit of the combination therapy..

4. The authors in the result in Figure 6 show that CDC27 overexpression restores the levels of p-PLK1 and p-CDK1 reduced by GRN-300 treatment, and suggest that CDC27 acts as a downstream mediator of SIK2. However, the rescue experiment in Figure 6 is mainly limited to western blot markers.

It remains unclear whether CDC27 overexpression functionally rescues the biological effects of GRN-300/paclitaxel combination treatment, such as reduced cell viability, decreased clonogenic survival, G2/M arrest, or increased apoptosis. The authors should provide at least one functional rescue experiment to support the claim that CDC27 is a key downstream mediator of SIK2 in this setting..

 

⦿ Minor comments:

1. The manuscript requires careful revision for consistency in terminology, numerical reporting, sentence structure, and figure descriptions. There are several grammatical and formatting issues that reduce the professionalism and readability of the manuscript.

For example, the number of TNBC cell lines is described inconsistently as “all eight TNBC cell lines” and “eight of nine TNBC cell lines.” The terms “hrs” and “hours” are also used inconsistently. In addition, there are clear title errors, such as “in clinical TNBC clinical specimens” in the subtitle on page 7 and “GRN-300 300. 300. inhibits colony formation” in the subtitle on page 9.

2. The authors should revise the figure titles so that they are presented as concise phrase-style headings rather than full sentence-style titles. These issues are not only editorial but may also affect the interpretation of some results; therefore, they should be carefully addressed during revision.

3. Please remove the analysis date “20240620” from the double-staining dot plot results shown in Figure 4B.

Comments on the Quality of English Language

The manuscript requires careful revision for consistency in terminology, numerical reporting, sentence structure, and figure descriptions. There are several grammatical and formatting issues that reduce the professionalism and readability of the manuscript.

Author Response

Thank you for your review and comments. Please find our responses below. Corresponding revisions have been highlighted in the revised manuscript.

 

Point by point response to Comments and Suggestions for authors.

Comment 1. The authors examined SIK2 expression in clinical samples and cell lines in Figure 1 and showed that SIK2 is highly expressed in TNBC. However, it is unclear whether SIK2 expression levels are associated with sensitivity to GRN-300 or with the response to GRN-300/paclitaxel combination treatment. 

The authors should clarify whether SIK2 expression correlates with GRN-300 single-agent activity, paclitaxel IC50, combination index, G2/M arrest, or apoptosis induction across the tested cell lines. In addition, response data from SIK2-low or SIK2-negative cell lines would help support the claim that the observed cytotoxic effects are related to SIK2 inhibition. Since SUM159 is used as a major model in this study, its SIK2 expression level should also be clearly shown.

Response 1. Thank you for this important comment. In this study, we demonstrated that SIK2 expression is elevated in TNBC clinical samples and cell lines compared with non-TNBC models, and that TNBC models generally showed greater sensitivity to the GRN-300/paclitaxel combination treatment. However, we did not perform a formal correlation analysis between baseline SIK2 expression levels and GRN-300 single-agent activity, paclitaxel IC50, combination index, G2/M arrest, or apoptosis induction across the tested cell lines. In addition, SIK2-low or SIK2-negative models were not systematically evaluated in this study.

SUM159 cells were incorporated during the later stages of the project based on their robust response to the combination treatment and suitability for mechanistic studies; therefore, they were not included in the initial SIK2 expression analysis shown in Figure 1. We agree that inclusion of SUM159 SIK2 expression data and broader evaluation across models with varying SIK2 expression levels would further strengthen the study.

We have clarified these points in the revised manuscript and added the following statement to the Discussion section: “Although TNBC models exhibited both higher SIK2 expression and greater sensitivity to GRN-300/paclitaxel treatment, additional studies using a broader panel of models, including SIK2-low or SIK2-negative cell lines, are needed to determine whether SIK2 expression directly correlates with therapeutic response or may serve as a predictive biomarker.”

Comment 2: In the Simple Summary and Abstract, the authors state that GRN-300 remains effective in paclitaxel-resistant TNBC models and may help overcome acquired chemoresistance. However, the main results presented in the manuscript appear to be largely based on parental TNBC cell lines and xenograft models.

To substantiate the role of SIK2 inhibition in overcoming paclitaxel resistance, the authors should provide data using paclitaxel-resistant TNBC cell lines. Specifically, they should show the anticancer effects of GRN-300 alone and in combination with paclitaxel in paclitaxel-resistant models, along with validation of the resistant phenotype and, if possible, analysis of relevant resistance mechanisms.

Response 2: Thank you for this comment. We agree that the current study does not include experiments using established paclitaxel-resistant TNBC models and therefore does not directly demonstrate that GRN-300 overcomes acquired paclitaxel resistance. The majority of the experiments in this manuscript were performed using parental TNBC cell lines and xenograft models.

In response to the reviewer’s concern, we have revised the Simple Summary, Abstract, and Discussion sections to remove statements suggesting that GRN-300 overcomes paclitaxel resistance or remains effective in paclitaxel-resistant TNBC models. We have clarified that our findings support enhanced antitumor activity of the GRN-300/paclitaxel combination in TNBC models tested in this study.

Comment 3: The in vivo xenograft results in Figure 3 are promising, as the combination therapy reduced tumor growth and improved survival in both MDA-MB-231 and SUM159 models. However, the current in vivo data mainly focus on efficacy, and additional information is needed to assess the translational relevance of this combination therapy.

The authors should provide tolerability and toxicity data, such as body weight changes and hematologic toxicity, during treatment. In addition, pharmacodynamic validation in xenograft tumors would strengthen the study. For example, showing whether the SIK2 pathway is actually inhibited in tumor tissues after GRN-300 treatment, including changes in SIK2-related downstream markers, would provide stronger support for the proposed mechanism and therapeutic benefit of the combination therapy.

Response 3: Thank you for this helpful comment. We have now added mouse body weight data to the Results section. Body weights remained stable throughout the treatment period across all groups, with no significant changes observed during GRN-300 and paclitaxel combination therapy.

 

We also agree that pharmacodynamic assessment of SIK2 pathway inhibition in tumor tissues would further strengthen the mechanistic interpretation of the in vivo findings, but these analyses were not performed in the current study.

Comment 4. The authors in the result in Figure 6 show that CDC27 overexpression restores the levels of p-PLK1 and p-CDK1 reduced by GRN-300 treatment, and suggest that CDC27 acts as a downstream mediator of SIK2. However, the rescue experiment in Figure 6 is mainly limited to western blot markers.

It remains unclear whether CDC27 overexpression functionally rescues the biological effects of GRN-300/paclitaxel combination treatment, such as reduced cell viability, decreased clonogenic survival, G2/M arrest, or increased apoptosis. The authors should provide at least one functional rescue experiment to support the claim that CDC27 is a key downstream mediator of SIK2 in this setting.

Response 4: Thank you for this important comment. In Figure 6, the CDC27 overexpression experiments were designed to assess whether CDC27 can rescue GRN-300–induced changes in key mitotic signaling markers, including p-PLK1 and p-CDK1. These data demonstrate a partial restoration of signaling downstream of SIK2 inhibition at the molecular level.

We agree that functional rescue experiments (cell viability, clonogenic survival, cell cycle distribution, or apoptosis assays) would further strengthen the conclusion that CDC27 is a key functional downstream mediator of SIK2 in this context. However, such functional assays were not performed in the current study.

To avoid overinterpretation, we have revised the text to more clearly state that CDC27 overexpression partially rescues SIK2-associated signaling changes rather than fully restoring the biological effects of GRN-300 treatment. We have also moderated the corresponding statements in the Results and Discussion sections.

Comment 5: The manuscript requires careful revision for consistency in terminology, numerical reporting, sentence structure, and figure descriptions. There are several grammatical and formatting issues that reduce the professionalism and readability of the manuscript.

For example, the number of TNBC cell lines is described inconsistently as “all eight TNBC cell lines” and “eight of nine TNBC cell lines.” The terms “hrs” and “hours” are also used inconsistently. In addition, there are clear title errors, such as “in clinical TNBC clinical specimens” in the subtitle on page 7 and “GRN-300 300. 300. inhibits colony formation” in the subtitle on page 9.

Response 6: Thank you for this helpful comment. We have carefully revised the manuscript to improve consistency in terminology, numerical reporting, sentence structure, and figure/title descriptions. Specifically, we corrected inconsistencies such as the description of TNBC cell line numbers (e.g., “all eight TNBC cell lines” vs. “eight of nine TNBC cell lines”), standardized the use of time notation (“hrs” vs. “hours”), and corrected typographical and formatting errors in figure titles and subtitles ( “in clinical TNBC clinical specimens” and “GRN-300 inhibits colony formation”).

These revisions have been made throughout the manuscript to improve clarity and readability.

Comment 7: The authors should revise the figure titles so that they are presented as concise phrase-style headings rather than full sentence-style titles. These issues are not only editorial but may also affect the interpretation of some results; therefore, they should be carefully addressed during revision.

Response 7: We thank this comment. In response, we have revised all figure titles to ensure they are presented as concise phrase-style headings rather than full sentence-style titles. We have carefully checked and updated all figures to improve consistency and readability.

Comment 8:  Please remove the analysis date “20240620” from the double-staining dot plot results shown in Figure 4B.

Response 8: Thank you for this comment. The numbers have been removed from Figure 4B.

Comment 9.  The manuscript requires careful revision for consistency in terminology, numerical reporting, sentence structure, and figure descriptions. There are several grammatical and formatting issues that reduce the professionalism and readability of the manuscript.

Response 9: Thank you for this comment. We have carefully revised the entire manuscript to improve consistency in terminology, numerical reporting, sentence structure, figure descriptions, and overall grammar and formatting.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Although the authors have addressed several of the reviewer’s comments and improved the manuscript, some issues still require minor revision before the manuscript can be considered for acceptance.

1. The issues raised in the previous Comment 6 regarding consistency in terminology and wording have not been fully resolved throughout the revised manuscript. In particular, inconsistent usage of terms such as “hrs” and “hours” still remains in several sections of the text. The authors should carefully re-check the entire manuscript to ensure uniform terminology and style.
2. The concerns raised in the previous Comment 7 also remain only partially addressed. Although the authors stated that all figure titles were revised into concise phrase-style headings, several figure titles and descriptions (particularly Figures 3–5) still require further revision for consistency and clarity. The authors are encouraged to carefully review all figure titles and legends again to ensure that they follow a consistent phrase-style format throughout the manuscript.

These remaining issues are relatively minor but should be corrected to improve the overall readability and professionalism of the manuscript.

Comments on the Quality of English Language

The manuscript requires careful revision for consistency in terminology, numerical reporting, sentence structure, and figure descriptions. 

Author Response

Comment 1. The issues raised in the previous Comment 6 regarding consistency in terminology and wording have not been fully resolved throughout the revised manuscript. In particular, inconsistent usage of terms such as “hrs” and “hours” still remains in several sections of the text. The authors should carefully re-check the entire manuscript to ensure uniform terminology and style.

Response 1. We thank the reviewer for pointing this out. We have carefully re-checked the entire revised manuscript and corrected the remaining inconsistencies in terminology and wording, including standardizing the usage of “hrs”/“hours” throughout the text to ensure uniform style and presentation.

 

Comment 2. The concerns raised in the previous Comment 7 also remain only partially addressed. Although the authors stated that all figure titles were revised into concise phrase-style headings, several figure titles and descriptions (particularly Figures 3–5) still require further revision for consistency and clarity. The authors are encouraged to carefully review all figure titles and legends again to ensure that they follow a consistent phrase-style format throughout the manuscript.

Response 2. We thank the reviewer for this helpful comment. We have carefully re-reviewed all figure titles and legends throughout the manuscript and further revised Figures 3-5 to improve consistency, clarity, and adherence to a uniform phrase-style format. All figure headings have now been standardized across the manuscript, with the revised portions highlighted.