Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript explores the mechanistic insights underlying the actions of the LXR modulator 1E5 actions in ER-positive MFC-7 breast cancer cells. The findings suggest that the anti-proliferative and pro-apoptotic effects of this LXR inverse agonist may be mediated not only through an LXR-dependent pathway but also via an independent mechanism. The latter pathway, however, remains to be fully elucidated.

Although it is a relevant study to the scope of the journal some mayor issues should be addressed.

1. Page 2, M&M, Cell Culture and Treatments: MCF-7 cells were cultured in DMEM (Gibco, #12430054) which contain phenol red and supplemented with 10% FBS (Gibco, #A5670701). Therefore, it can be assumed that ER is activated in these assays. In order to rule out any differences, the authors could provide a control experiment conducted on cells cultured in a medium without phenol red and charcoal-stripped FBS.

2. Information regarding statistical significance is not provided in Figures 1A, 1D, 2A, 3A, 3E, 4A, 4D, 4E, 6A, 6D, 6E, 7A, 7B, 7E, 7H, 8B, 8E, 8H, and 8K.

3. Supplementary Table S2 predicts that the RARγ, RXRα, ERRα, and ERα receptors may also serve as potential targets of 1E5. Since these receptors are expressed in MCF-7 cells, it would be valuable to perform docking and receptor selectivity analyses for the other two ligands in order to provide deeper insights into those mechanisms independent of LXR.

4. Discussion: The authors could strengthen their discussion by analyzing the structural similarities and differences between 1E5 and the other two LXR inverse agonists. Reviewing the current state of the art regarding the molecular targets of these three ligands would provide a valuable context.

Author Response

Author Response: 

1. Page 2, M&M, Cell Culture and Treatments: MCF-7 cells were cultured in DMEM (Gibco, #12430054) which contain phenol red and supplemented with 10% FBS (Gibco, #A5670701). Therefore, it can be assumed that ER is activated in these assays. In order to rule out any differences, the authors could provide a control experiment conducted on cells cultured in a medium without phenol red and charcoal-stripped FBS. 

We thank the reviewer for this comment. MCF-7 cells were cultured under standard conditions using phenol red-containing DMEM supplemented with 10% FBS. The objective of this study was to evaluate the mechanism of action of LXR ligand GAC0001E5 (1E5), including its effects on the expression of multiple receptors, such as ERα and AR, under standardized growth conditions, rather than to test estrogen-mediated ER activation. As all experimental conditions were kept consistent across vehicle and treatment groups in our study, any potential estrogenic activity associated with phenol red or FBS supplementation would be consistent across all groups and would therefore not affect interpretation of the effects of ligand treatment. We agree that hormone-depleted conditions are usually included in studies investigating direct ligand-induced ER activation, however, this was not the main focus of the present study.  

 

2. Information regarding statistical significance is not provided in Figures 1A, 1D, 2A, 3A, 3E, 4A, 4D, 4E, 6A, 6D, 6E, 7A, 7B, 7E, 7H, 8B, 8E, 8H, and 8K. 

We thank the reviewer for this comment. The qPCR data in this study were analyzed using the standard ΔΔCt method and presented in the figures as relative fold-change expression, normalized to vehicle DMSO-treated samples. The primary objective of the analysis was to assess relative fold change in gene expression upon treatment with LXR ligands. Thus, the data in the main figures are presented as relative expression values (log fold change). The data from three independent replicates are represented with mean ± SEM. The main conclusions are supported by additional protein-level and proliferation studies. This has been addressed in section 2.2 in the revised manuscript (lines 105-107). 

However, in response to this comment, we have plotted additional graphs showing statistical analysis of qPCR data using ΔCt values. These graphs have been included in a supplementary document with appropriate labels. 

 

3. Supplementary Table S2 predicts that the RARγ, RXRα, ERRα, and ERα receptors may also serve as potential targets of 1E5. Since these receptors are expressed in MCF-7 cells, it would be valuable to perform docking and receptor selectivity analyses for the other two ligands in order to provide deeper insights into those mechanisms independent of LXR. 

The reverse docking analysis in this study was performed to specifically characterize the receptor selectivity of 1E5 to study possible off-target interactions with other members of the nuclear receptor family. With regards to the reviewer’s helpful comments about docking the other inverse agonists to nuclear receptors and possible insights into LXR-independent mechanisms, these ligands are not known to act through LXR-independent mechanisms since they have been experimentally characterized for their binding to nuclear receptor family proteins and exhibited high selectivity for LXR, with minimal or no binding to other nuclear receptors (Flaveny et al., Cancer Cell, 2015). Rather than performing additional docking as suggested, we have included these experimental insights in our discussion of 1E5’s mechanisms of action (lines 531-535). 

 

4. Discussion: The authors could strengthen their discussion by analyzing the structural similarities and differences between 1E5 and the other two LXR inverse agonists. Reviewing the current state of the art regarding the molecular targets of these three ligands would provide a valuable context.   

We thank the reviewer for this valuable suggestion. We have expanded the Discussion section to include additional information on the structural differences between 1E5, SR9238, and SR9243. Specifically, we discuss the reported differences in His435-Trp457 interactions within the ligand-binding pocket, which are disrupted following SR9238 and SR9243 binding (Elgendy et al., Bioorganic Chemistry, 2021), but appears to be maintained with 1E5 (Karaboga et al., ACS Chemical Biology, 2020). Additionally, we briefly describe the influence of conformational properties and molecular size on ligand-receptor interactions to provide more context. The changes are included in the revised manuscript in section 4 (lines 502-511).

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This study aims to investigate the mechanism of action of the small molecule liver X receptor (LXR) modulator GAC0001E5 (1E5) in countering endocrine resistance in estrogen receptor (ER)-positive breast cancer cells. The authors provide the evidence to show that IE5 reduces the expression level of ERα, Her2, and AR. The study also demonstrated the function of IE5 on the sensitivity of ER+ breast cancer cells to anti-estrogen treatment. However, several points require further validation, and the discussion section could be expanded to make the audiences better understand.

1. Currently, “Statistical Analysis” and “Reverse Docking” are both listed under section 2.8. It is recommended that statistical methods should be presented as a separate section, and the reverse docking methodology should be moved to an independent subsection or supplementary materials. In addition, inconsistent abbreviations are observed, such as the interchangeable use of “SR9238” and “SR38,” as well as “SR9243” and “SR43.” It is recommended to standardize the use of full abbreviations (e.g., SR9238) throughout the text to avoid confusion. Multiple figure legends fail to clearly specify data presentation formats (e.g., mean ± SEM) or the comparisons corresponding to statistical significance markers (e.g., *p < 0.05). It is recommended to standardize the legend format, explicitly indicating the statistical comparisons and the number of replicates for each figure.
2. Nearly all experiments were performed using a single concentration (10 μM) without providing dose–response curves or IC50 values. It is recommended to perform additional experiments using a range of concentrations (e.g., 0.1, 1, 5, 10, 20 μM) to assess cell viability and protein expression, thereby evaluating the concentration-dependent effects of the compound. Moreover, only a single siRNA transfection was performed, with no evaluation of long-term knockdown effects, nor were LXRα/β double-knockout experiments attempted. It is recommended to generate stable LXRβ knockout cell lines using CRISPR technology to assess the dependency of 1E5’s antiproliferative effects on LXR.
3. The results in Figure 3E showed that ERBB1–4 transcription is downregulated, but no corresponding protein-level data are provided. It is recommended to perform Western blot analysis to assess changes in EGFR, HER3, and HER4 protein expression. In addition, the study does not evaluate whether long-term treatment induces drug resistance, nor does it assess potential synergistic effects with existing endocrine therapies (e.g., tamoxifen, fulvestrant). These points could be appropriately addressed and discussed.
4. It has been recognized that AR plays the different roles in breast cancer. AR mainly inhibits ER+ breast cancer progression. In this study, IE5 suppresses AR expression. How to understand the function of IE5 on expression regulation of the different receptors?

Author Response

Author Response: 

1. Currently, “Statistical Analysis” and “Reverse Docking” are both listed under section 2.8. It is recommended that statistical methods should be presented as a separate section, and the reverse docking methodology should be moved to an independent subsection or supplementary materials. In addition, inconsistent abbreviations are observed, such as the interchangeable use of “SR9238” and “SR38,” as well as “SR9243” and “SR43.” It is recommended to standardize the use of full abbreviations (e.g., SR9238) throughout the text to avoid confusion. Multiple figure legends fail to clearly specify data presentation formats (e.g., mean ± SEM) or the comparisons corresponding to statistical significance markers (e.g., *p < 0.05). It is recommended to standardize the legend format, explicitly indicating the statistical comparisons and the number of replicates for each figure. 

We thank the reviewer for bringing these points to our attention. We have revised the manuscript accordingly. Specifically, statistical analysis and reverse docking have been separated into sections 2.8 and 2.9. We have also standardized ligand nomenclature throughout the manuscript to include the full abbreviations (SR9238 and SR9243) in both figures and the text. Additionally, we have edited the figure legends to incorporate information on data presentation and statistical comparison. 

 

2. Nearly all experiments were performed using a single concentration (10 μM) without providing dose–response curves or IC50 values. It is recommended to perform additional experiments using a range of concentrations (e.g., 0.1, 1, 5, 10, 20 μM) to assess cell viability and protein expression, thereby evaluating the concentration-dependent effects of the compound. Moreover, only a single siRNA transfection was performed, with no evaluation of long-term knockdown effects, nor were LXRα/β double-knockout experiments attempted. It is recommended to generate stable LXRβ knockout cell lines using CRISPR technology to assess the dependency of 1E5’s antiproliferative effects on LXR. 

We appreciate the opportunity to clarify the experimental design. The present study builds on our previously published work (Premaratne et al., Biomolecules, 2023) in which dose-response analysis across multiple concentrations was performed and IC50 values were in the range of 7-8 μM. Based on these findings, the present study used 10 μM as a representative concentration. 

Regarding knockdown studies, transient siRNA-mediated knockdown of both LXR isotypes (LXRα and LXRβ) was performed as the standard approach to study the role of LXR signaling and compare its effects with those of LXR ligand GAC0001E5 (1E5). This strategy was used for initial characterization in this study. LXR knockdown resulted in a modest reduction in cell viability in our experimental design, indicating a potential role for LXR in maintaining cellular homeostasis and viability. We acknowledge that additional validation using stable knockout models and in vivo models would further strengthen the assessment of LXR dependency. However, these experiments were beyond the scope of the present study but are important directions for future work. This limitation has been included in section 5 (lines 556-557). 

 

3. The results in Figure 3E showed that ERBB1–4 transcription is downregulated, but no corresponding protein-level data are provided. It is recommended to perform Western blot analysis to assess changes in EGFR, HER3, and HER4 protein expression. In addition, the study does not evaluate whether long-term treatment induces drug resistance, nor does it assess potential synergistic effects with existing endocrine therapies (e.g., tamoxifen, fulvestrant). These points could be appropriately addressed and discussed. 

We thank the reviewer for this constructive comment. We agree that protein-level validation of HER family members would further strengthen interpretation of downstream signaling effects. In the present study, HER2 protein analysis was prioritized due to its well-established role as a key driver of endocrine resistance and its relevance as a therapeutic target in breast cancer. Transcript-level analysis was performed to provide an overview of changes in HER family expression following 1E5 treatment, while protein validation of additional HER receptors was beyond the scope of the present study. This limitation has been addressed in section 4 (lines 484-487).  

Regarding long-term effects, the present study was designed to assess the molecular and cellular responses of LXR inverse agonist 1E5 in ER-positive breast cancer models. Evaluation of the effects of prolonged exposure was beyond the scope of the current study design but represents an important direction for future in vivo studies once the pharmacodynamics and pharmacokinetics of 1E5 have been characterized. Similarly, combination treatment with well-established endocrine therapies was not performed in this study, however the downregulation of ERα expression following 1E5 treatment provides a rationale for further investigation. These limitations have been included in the revised manuscript (lines 552-556).  

 

4. It has been recognized that AR plays the different roles in breast cancer. AR mainly inhibits ER+ breast cancer progression. In this study, IE5 suppresses AR expression. How to understand the function of IE5 on expression regulation of the different receptors? 

We thank the reviewer for this comment. We acknowledge that AR plays a complex role in breast cancer, with both tumor-suppressive and tumor-promoting functions reported in different contexts. AR has also been implicated in endocrine resistance, with previous studies reporting that AR loss can restore tamoxifen sensitivity in resistant models, suggesting potential non-canonical AR activity in endocrine resistance (Chia et al., Endocrine-Related Cancer, 2019).  Consistent with this, we observed that 1E5 reduced AR expression and inhibited endocrine-resistant cells. It was also observed in the previously published study that the endocrine-resistant MCF-7-TamR cells showed higher AR expression compared to parental MCF-7 cells. Thus, AR downregulation by 1E5, together with its broader antiproliferative effects and proapoptotic effects, may contribute to the reduced cell viability observed in this study. 

Additionally, the observed antiproliferative effects following 1E5 treatment are likely associated with the broad disruption of compensatory signaling networks instead of direct receptor-specific targeting alone. This includes changes at both transcript and protein levels for ERα, AR, and HER2. These effects may also be due to multiple regulatory mechanisms, including both transcript-level changes as well as post-transcriptional modifications and protein stability. Potential genetic and epigenetic changes induced by 1E5 may also contribute to these effects and represent additional directions for future research. Taken together, our findings suggest that 1E5 impacts receptor signaling networks at different regulatory levels, thereby contributing to the overall antiproliferative effects. 

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The study addressed mechanisms of action of novel LXR ligand GAC0001E5 (1E5) in responsive and endocrine-resistant ER-positive breast cancer cells. Treatment with 1E5 resulted in downregulation of LXR and its target genes, reducing expression of ERα and ER-responsive genes ( such as AR and HER2). Knockdown of LXR expression only partially recapitulated the actions of 1E5, suggestive of the involvement of LXR-dependent and independent mechanisms. The data reveals potential crosstalk between LXR and genetic and epigenetic regulation of genes involved in endocrine response and alternative signaling mechanisms. The study is interesting. However, there are several issues to address.

1. Abstract should indicate which cell lines were used in this study. Authorsshould provide more details about experiments in the Abstract. For instance, the knock-down method can be indicated.
2. Why the level of ERbeta was not investigated? This should be addressed. ERbeta was shown as an important contributor of drug resistance in breast cancer cells. What is the expression of ERbeta before and after 1E5 treatments?
3. Figure 1B; it is shown that MCF-TamR cells express high level of ERalpha, comparable or nearly the same level as in parental MCF-7 cells. It is unclear why MCF-TamR cells are resistant to tamoxifen. What is the main mechanism of resistance in these cell line? Author should indicate the relevant published papers for this (if available), and explain the mechanism of resistance ( for this cell line) in the Methods and/or in the Discussion section. What are the main mechanisms of the resistance to Tamoxifen in this cell line?
4. Professional editing is required. Line 227:it should be “…were examined [33-35]”. Why authors cited other papers if it is their own data shown below on Figure 1? The whole sentence should be rephrased. My guess is that authors tell us that PGR, TFF1 etc are genes regulated by ERalpha. It is good. Then you can cite relevant papers. And after that authors can say, “Therefore, we tested the expression of these genes etc”.
5. Discussion section is very plain. Author can include a diagram of signalling pathway to support their hypothesis. What kind of LXR-independent mechanisms can be involved? Authors can provide more insights.

Comments on the Quality of English Language

Professional editing is recommended.

Author Response

Author Response: 

1. Abstract should indicate which cell lines were used in this study. Authorsshould provide more details about experiments in the Abstract. For instance, the knock-down method can be indicated. 

We thank the reviewer for this suggestion. The abstract has been revised to include additional details, including information on the use of cell lines and siRNA-mediated knockdown. However, due to the journal’s 200-word abstract limit, only limited additional information could be included in this section. The allowable changes have been made in the Abstract section (lines 13-30) however.  

 

2. Why the level of ERbeta was not investigated? This should be addressed. ERbeta was shown as an important contributor of drug resistance in breast cancer cells. What is the expression of ERbeta before and after 1E5 treatments? 

We thank the reviewer for highlighting the importance of ERβ as a mediator of estrogen signaling, an area of research where we have previously contributed and published multiple studies in collaboration with Professor Jan-Åke Gustafsson, who discovered ERβ, and his research group. The main focus of this study, however, was to investigate well-established and clinically actionable (established cancer therapeutic markers and targets) mechanisms of breast cancer progression and endocrine resistance. Therefore, we chose to focus on ERα, AR, and HER2, given the constraints on our time and resources. We share the reviewer’s enthusiasm for ERβ and hope to devote a more detailed study on its potential role as a downstream target of 1E5 and LXRs in the future.  

 

3. Figure 1B; it is shown that MCF-TamR cells express high level of ERalpha, comparable or nearly the same level as in parental MCF-7 cells. It is unclear why MCF-TamR cells are resistant to tamoxifen. What is the main mechanism of resistance in these cell line? Author should indicate the relevant published papers for this (if available), and explain the mechanism of resistance ( for this cell line) in the Methods and/or in the Discussion section. What are the main mechanisms of the resistance to Tamoxifen in this cell line? 

We thank the reviewer for this comment. The MCF-7-TamR cells used in this study were generated following long-term exposure of MCF-7 cells to 4-hydroxytamoxifen. ERα expression was not reported in the original characterization. However, these cells have been reported to exhibit increased androgen receptor expression and modification compared to their parental counterparts (Bahnassy et al., Cell Communication and Signaling, 2020). This suggests that tamoxifen resistance in this cell line may be associated with reprogramming of receptor signaling, including AR, and may contribute to compensatory proliferative signaling and reduced ERα dependence. Additionally, as tamoxifen does not directly degrade ERα, the expression of ERα may not fully correlate with endocrine sensitivity. The manuscript has been revised to include information about the cell line in section 2.1 (lines 80-83) and section 4 (lines 478-482).  

 

4. Professional editing is required. Line 227:it should be “…were examined [33-35]”. Why authors cited other papers if it is their own data shown below on Figure 1? The whole sentence should be rephrased. My guess is that authors tell us that PGR, TFF1 etc are genes regulated by ERalpha. It is good. Then you can cite relevant papers. And after that authors can say, “Therefore, we tested the expression of these genes etc”. 

We thank the reviewer for the opportunity to clarify. For the comment regarding the grammar, the reviewer might have been mistaken regarding the subject of the sentence. The subject of the referenced sentence is “expression” a singular noun. Therefore “was examined” is grammatically correct. The cited references were meant to support PGR, TFF1, GREB1, and NRIP1 as ERα target genes. We apologize for the poor placement of the citation and have revised the text for better clarity in section 3.1 (lines 227-230). 

 

5. Discussion section is very plain. Author can include a diagram of signalling pathway to support their hypothesis. What kind of LXR-independent mechanisms can be involved? Authors can provide more insights. 

We thank the reviewer for this suggestion. We have revised our submission to include a graphical abstract illustrating a proposed mechanism of action of 1E5 in ER-positive breast cancer, based on our findings. This includes potential LXR-mediated receptor crosstalk with ERα, AR, and HER2, as well as predicted off-target effects with RARγ, as indicated by the reverse docking analysis. We have also added more context into LXR-independent mechanisms in section 4 (lines 525-535). 

 

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Authors addressed all my comment properly.  Manuscript has been amended. I am satisfied with the current /revised version of the study.

Comments on the Quality of English Language

I could not find any mistakes in English grammar.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents an interesting follow-up on the effects of the Liver X Receptor (LXR) modulator GAC0001E5 on endocrine-related target genes. While many relevant targets are examined, the manuscript lacks sufficient rationale, experimental justification, and appropriate controls clarification. Improved experimental design, clearer justification of conditions, and inclusion of proper controls are required before the work can be considered for acceptance.

 

Figure 1

• What is the rationale for selecting GW3965? No justification or selection criteria are provided.
• Why were the 48-hour time point chosen? A time-course or gradient analysis is recommended.
• Only mRNA expression of ER target genes is reported. why are protein-level validations not included?
• The statement regarding expression of established ER target genes (e.g., progesterone receptor) requires appropriate references.

 

Figure 2 / Figure 3

• Why was the T-47D cell line not included or tested?

 

Figure 3

• Why are other HER family members not assessed at the protein level (e.g., via Western blot)? This limits interpretation of pathway-level effects.

 

Figure 4

• Again, what is the rationale for using a 48-hour treatment? Justification or time-course data is needed.

 

Figure 5

• The background should include known effects of LXR agonists and inverse agonists.
• Why were SR9238 and SR9243 selected as controls instead of other LXR modulators?
• Given that GW3965, SR9238, and SR9243 show similar effects on suppressing cell proliferation, what distinguishes SR9238 and SR9243 mechanistically to justify their use here?

 

Figure 6

• GW3965-treated conditions are not shown in the Western blot corresponding to panel A and downstream panels (D and E). These data should be included for completeness.

 

Figure 7

• The reported effects of SR9238 and SR9243 could be better integrated into earlier figures (Figures 1–3) to strengthen the overall narrative and comparison.

Author Response

Author Response:

We thank the reviewer for their detailed and constructive evaluation of our manuscript. We appreciate the opportunity to clarify the experimental design and rationale of the study, and the manuscript has been revised to address the points raised.

Figure 1

• What is the rationale for selecting GW3965? No justification or selection criteria are provided.

We thank the reviewer for bringing this to our attention. In this study, GW3965 was selected as a well-characterized synthetic LXR agonist, known to activate LXR signaling. In the present study, it was included to enable comparison with the LXR inverse agonist GAC0001E5 across Figures 1-4.

Previous studies (Nguyen-Vu et al., Breast Cancer Research, 2013, Vedin et al., Carcinogenesis, 2009) have demonstrated that GW3965 treatment can suppress ER-positive breast cancer cells by disrupting signaling pathways involved in cell proliferation, including ER-dependent signaling, supporting its use in this experimental context. This rationale has been included in the revised manuscript in section 3.1 (lines 199-201).

 

• Why were the 48-hour time point chosen? A time-course or gradient analysis is recommended.

The 48-hour treatment time point was selected based on the approximate doubling time of MCF-7 cells, to ensure that observed molecular changes occur within a biologically relevant context without introducing effects associated with resource depletion or cellular overgrowth.

In addition, this time point was consistent with conditions optimized in our previously published work (Premaratne et al., Biomolecules, 2023) in which dose-response (IC₅₀) analyses were performed and 48-hour exposure was identified as an optimal treatment duration for capturing robust antiproliferative effects of LXR modulation with 1E5 and GW3965 treatment.

We agree that a full time-course analysis would provide additional insight, however, this was beyond the scope of the present mechanistic study, which focused on the molecular effects of 1E5 in the context of previously established antiproliferative activity.

 

• Only mRNA expression of ER target genes is reported. why are protein-level validations not included?

We appreciate the opportunity to clarify this point. The aim of the present study in Figure 1 was to assess the expression and activity of ERα following treatment with 1E5. The transcriptional output of ERα signaling is reflected in the expression of ER target genes at the mRNA level, therefore we specifically studied the transcript levels of these established ER target genes.

Given our limited resources, protein-level validation was prioritized for key receptors, including ERα, AR, and HER2, which regulate proliferative signaling mechanisms, rather than all downstream transcriptional targets. While protein-level validation of ER target genes could provide further validation of ER activity, we believe the presented data are sufficient to show a decrease in receptor protein and a reduction in its transcriptional activity to illustrate down-regulation of ER signaling.

 

• The statement regarding expression of established ER target genes (e.g., progesterone receptor) requires appropriate references.

We thank the reviewer for bringing this to our attention. The references for ER target genes studied in this work have now been added in the revised manuscript in section 3.1 (line 227).

 

Figure 2 / Figure 3

• Why was the T-47D cell line not included or tested?

We appreciate the opportunity to clarify this point. T-47D cells were included in Figure 1 to assess the effects of 1E5 in an additional ER-positive breast cancer model. However, the primary focus of the present study was to investigate the mechanistic differences between endocrine-sensitive and endocrine-resistant breast cancer models. Therefore, in the follow-up experiments, we focused on the parental MCF-7 and resistant MCF-7-TamR cell lines where a direct comparison can be conducted. Inclusion of endocrine-sensitive T-47D and endocrine-resistant T-47D-TamR cell lines, as well as other ER-positive cell lines, would have been ideal but such reagents were not available at the time of the reported studies.

 

Figure 3

• Why are other HER family members not assessed at the protein level (e.g., via Western blot)? This limits interpretation of pathway-level effects.

We acknowledge that studying all HER family members at the protein level could provide deeper insight. However, protein-level validation of HER2 was prioritized due to its well-established role in endocrine resistance and its relevance as a key therapeutic target in breast cancer. Transcript level analysis of other HER family members was performed to provide an overview of coordinated changes in HER signaling following 1E5 treatment. However, protein level effects were beyond the scope of the present study. This limitation has been addressed in section 4 (lines 471-474).

 

Figure 4

• Again, what is the rationale for using a 48-hour treatment? Justification or time-course data is needed.

The 48-hour treatment time point was selected based on the doubling time of MCF-7 cells and previously optimized conditions. A detailed justification is provided in response to Figure 1, Question 2.

 

Figure 5

• The background should include known effects of LXR agonists and inverse agonists.

We appreciate the reviewer’s feedback. The manuscript has been revised to include background information on the known effects of LXR agonists and inverse agonists (section 3.5, lines 321-324).

 

• Why were SR9238 and SR9243 selected as controls instead of other LXR modulators?

We included SR9238 and SR9243 in the study as representative LXR inverse agonists that have been previously examined in metabolic and cancer models. This part of the study aimed to determine whether the antiproliferative effects observed with 1E5 earlier are mainly due to LXR inverse agonism. Inclusion of the SR compounds allowed direct comparison of different LXR modulators within the same class to test cellular responses in ER-positive cancer models. The manuscript has been revised to better explain the rationale for including SR9238 and SR9243 in section 3.5 (lines 321-328).

 

• Given that GW3965, SR9238, and SR9243 show similar effects on suppressing cell proliferation, what distinguishes SR9238 and SR9243 mechanistically to justify their use here?

Thank you for highlighting this observation regarding LXR agonist GW3965 and LXR inverse agonists SR9238 and SR9243 in the context of this study. Although all three ligands showed similar effects, they represent mechanistically distinct modes of LXR modulation. LXR agonist GW3965 upregulates LXR target genes such as SREBF1 and FASN. On the other hand, LXR inverse agonists SR9238 and SR9243 downregulates downstream genes under LXR transcriptional control. We have confirmed these opposing effects in figure 6D.

Therefore, in this study, the inclusion of SR9238 and SR9243 was not based on differential antiproliferative potency, but rather on their established use as representative LXR inverse agonists to draw direct comparisons with 1E5. SR9238 and SR9243 are structurally related LXR inverse agonists and both were included in this study to strengthen mechanistic interpretation and to confirm that the observed effects are not specific to a single molecule.

 

Figure 6

• GW3965-treated conditions are not shown in the Western blot corresponding to panel A and downstream panels (D and E). These data should be included for completeness.

We thank the reviewer for this comment. In the present study, GW3965 was used as a reference LXR agonist in figures 1-4 to establish the comparison between LXR activation and LXR inverse agonism. In figure 6, the experimental design specifically focused on comparing LXR inverse agonists 1E5, SR9238 and SR9243 at the signaling level to study effects primarily attributable to inverse agonism.

In figures 6A, D, and E, GW3965 was included as a representative LXR agonist to illustrate the mechanistic contrast between LXR agonism and inverse agonism. As protein-level effects of GW3965 had been established in earlier experiments, this analysis focused on signaling changes associated specifically with inverse agonist treatment.

 

Figure 7

• The reported effects of SR9238 and SR9243 could be better integrated into earlier figures (Figures 1–3) to strengthen the overall narrative and comparison.

 

We thank the reviewer for this suggestion. In the present study, figures 1-4 were designed to establish the primary comparison between LXR activation (with GW3965 treatment) and LXR inverse agonism (with 1E5 treatment). Thereafter, SR9238 and SR9243 were introduced in subsequent experiments as additional representative LXR inverse agonists to further validate class-specific responses and to assess whether the observed effects with 1E5 treatment were mainly associated with LXR inverse agonism.

This stepwise design enabled progression from an initial LXR agonist versus inverse agonist comparison to subsequent validation of inverse agonist-associated effects using functionally similar compounds. The manuscript has been revised to clarify this rationale in section 3.5 (lines 321-328).

Reviewer 2 Report

Comments and Suggestions for Authors

General Assessment: The study presents a well-structured and scientifically sound investigation into the mechanisms of endocrine resistance in ER+ breast cancer cells. The data provided supports the authors' conclusions; however, addressing the following points would further strengthen the manuscript and provide deeper insight into the mechanism of action.

Major Suggestion:

• Selectivity Profile: To provide a more comprehensive understanding of the mechanism of action, the authors should provide data regarding the selectivity of 1E5 against a panel of other nuclear receptors. Establishing the degree of selectivity for LXR versus other related receptors would significantly clarify whether the observed effects are exclusively LXR-dependent.

Minor Revision:

• Citations: Please provide a formal reference for the compound SR9238 where it is first mentioned in the text.

Author Response

Author Response:

Major Suggestion:

• Selectivity Profile: To provide a more comprehensive understanding of the mechanism of action, the authors should provide data regarding the selectivity of 1E5 against a panel of other nuclear receptors. Establishing the degree of selectivity for LXR versus other related receptors would significantly clarify whether the observed effects are exclusively LXR-dependent.

We appreciate the reviewer’s positive feedback and helpful suggestion. We agree that evaluating the receptor selectivity profile of GAC0001E5 would provide valuable insight into its mechanism of action and help clarify the extent to which the observed antiproliferative effects are LXR-dependent.

To conduct the important binding studies against all 47 other nuclear receptors, using purified proteins or engineered cell lines, as recommended by the reviewer is not feasible given our resource limitations as an academic lab. However, we alternatively performed silico reverse docking analyses against the human nuclear receptor superfamily in the MCF-7 model. This analysis identified LXR among the top-ranked predicted targets, supporting receptor selectivity patterns of 1E5. Expression data suggested that LXRβ is more abundant in MCF-7 cells and may be more functionally relevant. Additionally, other nuclear receptors, including retinoic acid receptor γ (RARγ), were also identified as potential binding partners, suggesting possible off-target interactions or LXR-independent mechanisms.

We have now incorporated these findings into the revised manuscript (section 2.8, lines 168-194; section 4, lines 496-504; section 5, lines 522-524; supplementary table S2). While these preliminary results provide insight into the potential receptor selectivity profile of 1E5, further validation through binding assays, will be required to define its specificity and mechanism of action. This limitation and the new insights have been incorporated into the revised manuscript in section 4 (lines 496-504) and section 5 (lines 522-530).

 

Minor Revision:

• Citations: Please provide a formal reference for the compound SR9238 where it is first mentioned in the text.

We thank the reviewer for bringing this to our attention. The reference for SR9238 has been added into the text in section 3.5 (line 324).

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, authors reported that the role of small molecule, GAC0001E5 in ER positive breast cancer cells. This small molecule inhibited ERα, AR, HER2 and LXRβ expression and its target gene expressions in endocrine resistant breast cancer cells, thereby acts as an important therapeutic target for endocrine-resistant breast cancers. Authors mentioned that anti-proliferative activity of GAC0001E5 in previous studies, thus, focused on mechanistic studies in this manuscript. Overall, authors presented very good publication quality results at protein levels and transcription levels using small molecules and molecular silencing of LXR. However, this manuscript clearly lacks a specific and detailed mechanistic studies as well as in vivo xenograft mouse models to further strengthen the manuscript.

Author Response

Author Response:

In this study, authors reported that the role of small molecule, GAC0001E5 in ER positive breast cancer cells. This small molecule inhibited ERα, AR, HER2 and LXRβ expression and its target gene expressions in endocrine resistant breast cancer cells, thereby acts as an important therapeutic target for endocrine-resistant breast cancers. Authors mentioned that anti-proliferative activity of GAC0001E5 in previous studies, thus, focused on mechanistic studies in this manuscript. Overall, authors presented very good publication quality results at protein levels and transcription levels using small molecules and molecular silencing of LXR. However, this manuscript clearly lacks a specific and detailed mechanistic studies as well as in vivo xenograft mouse models to further strengthen the manuscript.

 

We thank the reviewer for their positive assessment of our work and for highlighting the importance of further mechanistic investigation and in vivo validation. In our previously published work (Premaratne et al., Biomolecules, 2023), we demonstrated the antiproliferative effects of GAC0001E5 in ER-positive breast cancer models. However, the underlying mechanism of action remained to be studied. This study was designed to address the gap by investigating the molecular effects of LXR inverse agonist 1E5 on LXR signaling as well as key pathways involved in endocrine resistance, including ERα, AR, and HER2.

To further strengthen the mechanistic interpretation, we performed in silico reverse docking analyses against the human nuclear receptor superfamily (section 2.8, lines 168-194; section 4, lines 496-504; section 5, lines 522-524). The analysis identified LXRα and LXRβ as among the top-ranked predicted targets (supplementary table S2). Additionally, RARγ was also identified as a predicted target, suggesting LXR-independent mechanisms may also be involved. Consistent with our experimental findings, the use of pharmacological modulation and molecular silencing approaches indicates a potential involvement of LXR signaling in mediating the observed effects of 1E5.

We agree that in vivo studies will be important to define the therapeutic potential of 1E5 in cancer models. However, such studies require comprehensive characterization of pharmacodynamic and pharmacokinetic properties, as well as additional optimization of the compound. These efforts are currently ongoing and represent an important direction for future research while the current study represents an important step in the characterization of this novel compound.

Finally, while these findings provide important mechanistic insights at the transcript and protein levels, we acknowledge that additional studies, including detailed molecular interaction and receptor binding assays, would further aid our understanding of the mechanism of action of 1E5. These limitations and future directions have been incorporated in the revised manuscript in section 5 (lines 519-530).

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors, my recommendations for the manuscript entitled "Small Molecule Liver X Receptor Modulator GAC0001E5 Targets Mechanisms of Endocrine Resistance in Estrogen Receptor-positive Breast Cancer Cells", biomolecules-4233468

 Reviewer comments to the authors

 The manuscript offers a comprehensive study with significant fundamental importance and potential for application in clinical practice. The research explores the mechanisms of action of ligands of the liver X nuclear receptor (LXR), which are involved in metabolic reprogramming.While acknowledging the relevance and substance of the work conducted, I would like to raise a few points:

• In the "Materials and Methods" section, I suggest indicating the limitations of the conducted research. Additionally, I recommend including a rationale for the chosen cell lines in the study and providing a more detailed characterization of them.
• Describe how the Western Blotting results were
• Can the study findings be extrapolated to luminal B HER-2 positive BC?
• The gold standard for detecting estrogen receptors in clinical practice is immunohistochemistry.Whether the immunohistochemicaldetermination of the level of estrogenreceptors was carried out inthisstudy,andwhyWestern blotting waspreferredinthis
• The paper should specify the criteria the authors used to select the methods of statistical analysis. Was the normality of the distribution of the study samples assessed, and if so, by what method? Was a power analysis conducted in this study? Which software product was used for the statistical analysis?
• Section 3.3, Results. Clarify why the influence of 1E5 and GW3965 on HER-2 expression was investigated using the MCF-7 cell line, which is characterized by a fairlylowornegativeHER-2Were similar studies carried out on cell lines with high HER-2 levels (for example,HCC-1954 (HER-2+))?

In general, highly appreciating the quality of the manuscript, I recommend it for publication in the Journal «Biomolecules» with minor revisions.

Best regards, Reviewer

 

 

 

Author Response

Author Response:

• In the "Materials and Methods" section, I suggest indicating the limitations of the conducted research. Additionally, I recommend including a rationale for the chosen cell lines in the study and providing a more detailed characterization of them.

We thank the reviewer for their kind comments and thorough review. The suggested edits regarding cell line details were incorporated into the main text in section 2.1 (lines 71-82). The limitations of the conducted research, including the need for additional models, receptor selectivity studies, and ligand optimization, have also been included in the conclusion (section 5 – lines 519-530).

 

• Describe how the Western Blotting results were

We thank the reviewer for bringing this to our attention. We have now clarified the analysis and interpretation of Western blot data in section 2.3 (lines 121-125). Specifically, band intensities were quantified by densitometric analysis using ImageJ software, normalized to β-actin as a loading control, and expressed as relative fold change compared with control conditions. All experiments were performed in biological triplicates.

 

• Can the study findings be extrapolated to luminal B HER-2 positive BC?

We acknowledge this important point regarding the broader scope of our findings. The present study primarily utilized ER-positive endocrine-sensitive and endocrine-resistant luminal A breast cancer cell lines. Therefore, direct extrapolation to luminal B HER2-positive models is limited due to the distinct characteristics of HER2-driven cancer models. However, in our previously published work (Premaratne et al., Cancers, 2024), we evaluated LXR modulation upon GAC0001E5 treatment in HER2-positive breast cancer models and observed antiproliferative effects as well as downregulation of HER2. Subsequently, in the present study, we found that 1E5 downregulates ERα expression. Taken together, these findings suggest that 1E5 may exert antiproliferative effects in broader breast cancer contexts, including potentially luminal B HER2-positive disease. However, dedicated studies in luminal B models are required to confirm this.

 

•  The gold standard for detecting estrogen receptors in clinical practice is immunohistochemistry.Whether the immunohistochemicaldetermination of the level of estrogenreceptors was carried out inthisstudy,andwhyWestern blotting waspreferredinthis

We fully agree that immunohistochemistry (IHC) represents the gold standard for assessing receptor status and localization in clinical breast cancer tissues. However, the present study is a preclinical in vitro investigation using established cell line models, rather than clinical tissue samples. Therefore, Western blotting, the standard for in vitro ER studies, was used instead, as a quantitative method to assess changes in overall protein expression under controlled experimental conditions. This strategy is widely used in mechanistic studies of estrogen receptor signaling in cell-based models. The limitation regarding the absence of IHC-based tissue validation has been noted in section 5 (lines 519-521).

 

• The paper should specify the criteria the authors used to select the methods of statistical analysis. Was the normality of the distribution of the study samples assessed, and if so, by what method? Was a power analysis conducted in this study? Which software product was used for the statistical analysis?

We appreciate the opportunity to clarify the statistical methods. Statistical analyses were performed in Microsoft Excel. Comparisons between treatment and control groups, as well as between sensitive and resistant cell lines, were conducted using Student’s t-test, with paired or unpaired tests applied as appropriate to the experimental design. Data are presented as mean ± standard error of the mean (SEM) and displayed in graphical form in the figures. This information has been included in section 2.8 (lines 161-166).

Formal tests for normality and power calculation were not performed since the experimental design and statistical power have been determined empirically repeatedly in genetically conditionally homogenous cell line models in previously published studies.

 

• Section 3.3, Results. Clarify why the influence of 1E5 and GW3965 on HER-2 expression was investigated using the MCF-7 cell line, which is characterized by a fairlylowornegativeHER-2Were similar studies carried out on cell lines with high HER-2 levels (for example,HCC-1954 (HER-2+))?

We thank the reviewer for this comment and agree that MCF-7 cells typically exhibit low HER2 expression. The present study was designed to investigate the effects of LXR modulators in the context of ER-positive breast cancer models representing endocrine-sensitive versus endocrine-resistant states. Therefore, MCF-7 cells were used as an ER-positive, low-HER2 expressing baseline model for comparison with the endocrine-resistant MCF-7-TamR cells which express high level of HER2.

We have previously evaluated HER2-positive breast cancer models, including HCC-1954 and SKBR3, and observed antiproliferative effects following LXR modulation with 1E5 treatment, as reported in our previously published work (Premaratne et al., Cancers, 2024).

Taken together, the HER2-related findings in the present study relate specifically to endocrine resistance in ER-positive breast cancer models and is consistent with our previously published findings in HER2-positive breast cancer cells. We have clarified this point in the revised manuscript in section 3.3 (lines 265-274).