Splicing Factor 3a Subunit 1 Promotes Colorectal Cancer Growth via Anti-Apoptotic Effects of Syntaxin12

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by Sasaki et al., found that SF3A1 KD suppressed cell proliferation of CRC cells. They also observed tumor promoting function of SF3A1 in xenograft mouse model. SF3A1 inhibited apoptotic signaling,. RIP analysis demonstrated that STX12as a effector of SF3A1. They conclude that SF3A1 contributes to CRC progression by stabilizing STX12 mRNA and selectively inhibiting apoptosis in CRC cells. This study underlies comprehensive mechanisms for the SF3A1 regulation in CRC cells. Overall, this is an interesting study with novel findings together with clear and convincing data. The manuscript is also well-written. The following are the concerns that need to be improved before publication:

 

1. What are the effects of SF3A1 on HEK293 cells (Fig 1)?
2. Figure 2, Does SF3A1 also affect apoptosis of SW480?
3. The authors claim that (Integrative analysis of RIP and RNA-seq data identified 144 mRNAs that were both physically associated with SF3A1 and significantly downregulated upon its silencing, indicating that SF3A1 contributes to their mRNA stability). The authors need to be cautious for "mRNA stability". Reduced mRNA levels may be caused by reduced transcription.

 

 

 

Author Response

This manuscript by Sasaki et al., found that SF3A1 KD suppressed cell proliferation of CRC cells. They also observed tumor promoting function of SF3A1 in xenograft mouse model. SF3A1 inhibited apoptotic signaling,. RIP analysis demonstrated that STX12as a effector of SF3A1. They conclude that SF3A1 contributes to CRC progression by stabilizing STX12 mRNA and selectively inhibiting apoptosis in CRC cells. This study underlies comprehensive mechanisms for the SF3A1 regulation in CRC cells. Overall, this is an interesting study with novel findings together with clear and convincing data. The manuscript is also well-written. The following are the concerns that need to be improved before publication:

Response:  We thank the reviewer for the thorough evaluation of our manuscript and for the positive comments regarding the novelty, clarity, and overall quality of our study. We are particularly grateful for the reviewer’s recognition of the comprehensive mechanistic insights into SF3A1-mediated regulation in colorectal cancer (CRC) cells and the robustness of our experimental data. In response to the reviewer’s concerns, we have carefully revised the manuscript and performed additional analyses where appropriate. All comments have been addressed in detail below. Corresponding revisions have been incorporated into the revised manuscript, and changes are highlighted in red accordingly. We believe that these revisions have significantly strengthened the manuscript and improved its clarity.

 

1. What are the effects of SF3A1 on HEK293 cells (Fig 1)?

Response: Thank you for this important comment. We agree that assessing the effects of SF3A1 in additional cell lines could provide further biological insights. However, the current study is designed specifically to elucidate the oncogenic role of SF3A1 in CRC. HEK293 cells are non-colorectal, embryonic kidney–derived cells and differ substantially from CRC cells in terms of tissue origin, genetic background, and apoptotic signaling context. Because our mechanistic focus is directed toward SF3A1–STX12 axis of CRC, extending the analyses to HEK293 cells would deviate from the scope and objectives of this study. For this reason, we did not include HEK293 experiments in the present work. We have clarified this rationale in the revised manuscript.

 

1. Figure 2, Does SF3A1 also affect apoptosis of SW480?

Response: Thank you for pointing this out. We have now examined whether SF3A1 knockdown induces apoptosis in SW480 cells. Consistent with our observations in HCT116 cells, SF3A1 knockdown significantly increased the proportion of TUNEL-positive cells, indicating induction of apoptosis in SW480 cells as well. These results have been added to the revised manuscript (Supplemental Fig. 4) and described in the updated text.

1. The authors claim that (Integrative analysis of RIP and RNA-seq data identified 144 mRNAs that were both physically associated with SF3A1 and significantly downregulated upon its silencing, indicating that SF3A1 contributes to their mRNA stability). The authors need to be cautious for "mRNA stability". Reduced mRNA levels may be caused by reduced transcription.

Response: We appreciate the reviewer’s thoughtful comment. In our study, we did not rely solely on steady-state mRNA levels to infer mRNA stability. Instead, we performed a luciferase reporter assay in which the STX12 mRNA was cloned downstream of the luciferase gene. As described in the manuscript (“To further confirm that SF3A1 stabilizes STX12 mRNA …”, Figure 4C), SF3A1 knockdown significantly reduced luminescence from the STX12 reporter but not from the empty vector, supporting that SF3A1 modulates STX12 mRNA stability. We have clarified this point in the revised text to avoid any misunderstanding.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting article wherein the authors report role and potential mechanism of SF3A1 in promoting colorectal cancer. The study design is meticulous and data support the conclusions. The manuscript is drafted well. My comments are below:

Merits

1. Clear mechanistic insight: The study identifies a specific SF3A1-STX12 regulatory axis and provides mechanistic evidence linking SF3A1-mediated RNA stabilization to apoptosis resistance in colorectal cancer cells.
2. Multi-level validation: The authors support their conclusions using complementary in-vitro assays (cell proliferation, apoptosis assays, RIP, transcriptomics) and in-vivo xenograft data, strengthening the overall reliability of the findings.
3. Selectivity toward cancer cells: Demonstrating minimal effects in non-cancerous epithelial cells highlights the potential therapeutic relevance and specificity of targeting SF3A1 or STX12.

Scope for improvement

1. Limited generalizability across CRC models: The study relies heavily on HCT116 cells; additional CRC cell lines or patient-derived models would improve confidence that the SF3A1–STX12 axis is broadly relevant.
2. Incomplete mechanistic depth: While STX12 is identified as a downstream effector, the work does not fully dissect how STX12 suppresses apoptosis pathways, leaving parts of the signaling mechanism unresolved.
3. Potential off-target or compensatory effects not explored: The study does not evaluate whether other RBPs or RNA-processing pathways compensate when SF3A1 is suppressed, which may impact therapeutic feasibility.

Please address this in light of the data or the literature.

Author Response

This is an interesting article wherein the authors report role and potential mechanism of SF3A1 in promoting colorectal cancer. The study design is meticulous and data support the conclusions. The manuscript is drafted well. My comments are below:

Merits

1. Clear mechanistic insight: The study identifies a specific SF3A1-STX12 regulatory axis and provides mechanistic evidence linking SF3A1-mediated RNA stabilization to apoptosis resistance in colorectal cancer cells.
2. Multi-level validation: The authors support their conclusions using complementary in-vitro assays (cell proliferation, apoptosis assays, RIP, transcriptomics) and in-vivo xenograft data, strengthening the overall reliability of the findings.
3. Selectivity toward cancer cells: Demonstrating minimal effects in non-cancerous epithelial cells highlights the potential therapeutic relevance and specificity of targeting SF3A1 or STX12.

Response: We thank the reviewer for the positive evaluation of our work. We appreciate the recognition of the mechanistic insights, multi-level experimental validation, and cancer-cell selectivity demonstrated in this study. These points strengthen the significance of the SF3A1–STX12 axis as a potential therapeutic target, and we are encouraged that the reviewer found these aspects clearly supported by our data.

Scope for improvement

1. Limited generalizability across CRC models: The study relies heavily on HCT116 cells; additional CRC cell lines or patient-derived models would improve confidence that the SF3A1–STX12 axis is broadly relevant.

Response: We appreciate the reviewer’s thoughtful comment regarding the generalizability of our findings. While HCT116 cells served as our primary model for characterizing the SF3A1–STX12 axis, we also examined the effect of SF3A1 knockdown in an additional CRC cell line, SW480. Consistent with our observations in HCT116, SF3A1 suppression in SW480 cells similarly reduced cell proliferation in vitro, supporting the notion that SF3A1 contributes to CRC cell growth beyond a single cell line.

Nonetheless, we agree that broader validation, using multiple molecular subtypes or patient-derived models, would further strengthen confidence in the universality of the SF3A1–STX12 axis. We have now explicitly acknowledged this point as a limitation in the revised manuscript and highlighted the need for future work to evaluate the extent to which this regulatory axis applies across diverse CRC contexts.

1. Incomplete mechanistic depth: While STX12 is identified as a downstream effector, the work does not fully dissect how STX12 suppresses apoptosis pathways, leaving parts of the signaling mechanism unresolved.

Response: We appreciate the reviewer’s insightful comment regarding the mechanistic role of STX12. In this study, we identified STX12 as a downstream effector of SF3A1 whose suppression contributes to reduced apoptosis signaling. However, we acknowledge that the precise molecular mechanisms by which STX12 regulates apoptotic pathways remain incompletely resolved in the present work. We now explicitly describe this as a limitation in the revised manuscript and have emphasized the need for future studies to dissect the downstream signaling cascade in greater detail.

1. Potential off-target or compensatory effects not explored: The study does not evaluate whether other RBPs or RNA-processing pathways compensate when SF3A1 is suppressed, which may impact therapeutic feasibility.

Response: We appreciate the reviewer's attention to this important point. To address the possibility of compensatory changes in other RNA-binding proteins or RNA-processing pathways following SF3A1 suppression, we refer to our transcriptome analysis, which did not reveal notable suppression of alternative RNA-processing pathways or upregulation of other RBPs. Instead, the dominant transcriptional changes included the downregulation of pathways associated with cancer cell survival, such as the Wnt signaling pathway, consistent with the observed reduction in cell viability. These results suggest that the phenotypic effects induced by SF3A1 knockdown are unlikely to be driven by major compensatory RNA-processing mechanisms.

Nevertheless, we acknowledge that more subtle post-transcriptional compensatory effects cannot be fully excluded. We have therefore added this point to the revised Discussion section, noting it as a limitation and emphasizing that future work employing proteomic and RNA-protein interaction analyses will be required to comprehensively assess potential compensatory mechanisms.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, this manuscript presents an interesting study exploring the role of SF3A1 in colorectal cancer through the stabilization of STX12 mRNA. The experiments are generally well organized, and the findings have potential translational relevance. However, several aspects of the study require clarification, additional methodological detail, or further mechanistic support to strengthen the conclusions. The comments below outline specific concerns that should be addressed before the manuscript can be considered for publication.

1.The criteria used to integrate the RIP-seq and RNA-seq datasets require clearer explanation. While the authors identified 144 mRNAs as both directly associated with SF3A1 and downregulated upon its knockdown, it remains unclear how the thresholds for fold enrichment, p-values, and noise reduction were established. In addition, it is not described whether potential high-abundance background transcripts were filtered out, nor whether the authors performed any sequence motif or binding-site analysis to support the specificity of SF3A1–mRNA interactions. Greater methodological transparency is essential for ensuring reproducibility of the bioinformatic analyses.

2.The functional distinction of STX12 between colorectal cancer cells and non-cancerous epithelial cells warrants deeper mechanistic exploration. Although the knockdown of STX12 induces apoptosis selectively in cancer cells, the manuscript does not provide evidence explaining why STX12 is indispensable in malignant but not normal cells. A comparison of downstream apoptotic regulators or pathway activation between the two cell types would be valuable. Without such data, the selective vulnerability of cancer cells remains observational and lacks mechanistic substantiation.

3.The in vivo xenograft experiment relies solely on local intratumoral siRNA injection, which limits the translational relevance of the findings. Local delivery may not accurately reflect the pharmacodynamic and pharmacokinetic behavior of systemic therapeutic approaches. It is also unclear whether the authors assessed the stability, tissue distribution, or potential toxicity of siRNA in vivo. These limitations should be acknowledged, and additional discussion or supplementary experiments are recommended to contextualize the relevance of the in vivo findings.

4.The manuscript notes that STX12 expression is not significantly elevated in colorectal cancer tissues based on GEPIA analysis. This observation conflicts with the claim that STX12 functions as a key tumor-promoting gene. If expression levels are not increased, the authors should provide a clearer explanation of why STX12 is functionally important specifically in cancer cells. It would be helpful to discuss whether post-translational modifications, altered subcellular localization, or differences in upstream regulatory pathways account for STX12’s context-dependent role. This clarification is necessary to reconcile the apparent discrepancy between expression data and functional relevance.

5.Several figures and datasets lack sufficient statistical details. In multiple Western blot and quantitative analyses, the number of biological replicates is not clearly reported, and it is not stated whether multiple-comparison corrections were applied following ANOVA. Additionally, some graphs do not display error bars or measures of data variability, making it difficult to assess the robustness of the results. The authors should standardize statistical reporting across the manuscript and provide complete information on sample size, variability, and statistical methodology.

Author Response

Overall, this manuscript presents an interesting study exploring the role of SF3A1 in colorectal cancer through the stabilization of STX12 mRNA. The experiments are generally well organized, and the findings have potential translational relevance. However, several aspects of the study require clarification, additional methodological detail, or further mechanistic support to strengthen the conclusions. The comments below outline specific concerns that should be addressed before the manuscript can be considered for publication.

Response: We sincerely thank the reviewer for the positive evaluation of our work. We appreciate the constructive comments and have revised the manuscript extensively to address all points raised. Additional methodological details, expanded discussion of mechanistic implications, and clearer explanations of experimental limitations have been incorporated as recommended. We believe these revisions have strengthened both the clarity and scientific rigor of the manuscript.

 

1.The criteria used to integrate the RIP-seq and RNA-seq datasets require clearer explanation. While the authors identified 144 mRNAs as both directly associated with SF3A1 and downregulated upon its knockdown, it remains unclear how the thresholds for fold enrichment, p-values, and noise reduction were established. In addition, it is not described whether potential high-abundance background transcripts were filtered out, nor whether the authors performed any sequence motif or binding-site analysis to support the specificity of SF3A1–mRNA interactions. Greater methodological transparency is essential for ensuring reproducibility of the bioinformatic analyses.

Response: We appreciate the reviewer’s thoughtful comments regarding the transparency of our integrative analysis. In the revised Methods section, we have included a more detailed description of the thresholds used for both the RIP-seq and RNA-seq datasets. For RNA-seq, ribosomal RNA was depleted before library construction. We applied fold-enrichment (fold change >2 for RIP-seq, Absolute fold change >2 for RNA-seq) and adjusted p-value thresholds (p<0.01) that are widely used in the identification of mRNA changes.

We also agree with the reviewer that motif or binding-site analysis would further support the specificity of SF3A1–mRNA interactions. Although SF3A1 contains canonical SURP RNA-binding domains, a defined RNA-recognition motif for SF3A1 has not been established to date. Therefore, it remains unclear whether STX12 mRNA contains a sequence motif that could mediate preferential binding. We have added a statement to the Discussion acknowledging this limitation and noting that future studies employing CLIP-based mapping and de novo motif discovery will be essential to elucidate the binding specificity of SF3A1 and to determine whether STX12 is a primary, motif-dependent target.

 

2.The functional distinction of STX12 between colorectal cancer cells and non-cancerous epithelial cells warrants deeper mechanistic exploration. Although the knockdown of STX12 induces apoptosis selectively in cancer cells, the manuscript does not provide evidence explaining why STX12 is indispensable in malignant but not normal cells. A comparison of downstream apoptotic regulators or pathway activation between the two cell types would be valuable. Without such data, the selective vulnerability of cancer cells remains observational and lacks mechanistic substantiation.

Response: We agree that the molecular basis of the selective vulnerability remains incompletely resolved. In the Discussion (third paragraph), we have elaborated on the role of post-translational regulation of STX12: Malik et al. reported that STX12 is phosphorylated by SGK3, a kinase downstream of PI3K oncogenic signaling, highlighting that post-translational modification—rather than expression level alone—contributes to its functional importance in CRC progression. This context-dependent phosphorylation could explain why STX12 is indispensable in malignant cells but not in non-transformed epithelial cells.

Nevertheless, we acknowledge that direct comparisons of downstream apoptotic regulators and pathway activity between normal and malignant cells would provide clearer mechanistic clarity. We have therefore added this point as a limitation in the Discussion and noted that future work will include systematic analyses of apoptosis-related signaling and SGK3-dependent phosphorylation events to better define why STX12 is selectively essential in CRC cells.

 

3.The in vivo xenograft experiment relies solely on local intratumoral siRNA injection, which limits the translational relevance of the findings. Local delivery may not accurately reflect the pharmacodynamic and pharmacokinetic behavior of systemic therapeutic approaches. It is also unclear whether the authors assessed the stability, tissue distribution, or potential toxicity of siRNA in vivo. These limitations should be acknowledged, and additional discussion or supplementary experiments are recommended to contextualize the relevance of the in vivo findings.

Response: We agree with the reviewer that intratumoral siRNA delivery has limited translational relevance. Our in vivo experiment aimed specifically to assess the tumor requirement for the SF3A1–STX12 axis rather than therapeutic efficacy. While we did not evaluate systemic pharmacokinetics, biodistribution, or siRNA stability, we have now explicitly acknowledged these limitations in the revised Discussion.

 

4.The manuscript notes that STX12 expression is not significantly elevated in colorectal cancer tissues based on GEPIA analysis. This observation conflicts with the claim that STX12 functions as a key tumor-promoting gene. If expression levels are not increased, the authors should provide a clearer explanation of why STX12 is functionally important specifically in cancer cells. It would be helpful to discuss whether post-translational modifications, altered subcellular localization, or differences in upstream regulatory pathways account for STX12’s context-dependent role. This clarification is necessary to reconcile the apparent discrepancy between expression data and functional relevance.

Response: We appreciate the reviewer’s insightful comment. As noted, STX12 expression is not significantly elevated in colorectal cancer based on GEPIA and TCGA datasets, which may initially seem inconsistent with its functional importance. In the revised Discussion, we clarify that the oncogenic role of STX12 is likely not driven by changes in bulk mRNA abundance, but rather by cancer-specific regulatory contexts. Particularly, prior work has shown that STX12 undergoes post-translational modification relevant to oncogenic signaling. Given that PI3K signaling is frequently activated in colorectal cancer, STX12 function may be enhanced through PTM-dependent mechanisms rather than expression upregulation. We have incorporated these points into the Discussion to more clearly reconcile the absence of overt expression upregulation with the strong functional requirement of STX12 in colorectal cancer cells.

 

5.Several figures and datasets lack sufficient statistical details. In multiple Western blot and quantitative analyses, the number of biological replicates is not clearly reported, and it is not stated whether multiple-comparison corrections were applied following ANOVA. Additionally, some graphs do not display error bars or measures of data variability, making it difficult to assess the robustness of the results. The authors should standardize statistical reporting across the manuscript and provide complete information on sample size, variability, and statistical methodology.

Response: We thank the reviewer for highlighting the inconsistencies in our statistical reporting. In response, we have thoroughly re-evaluated all figures and figure legends and revised them to clearly specify: (i) the exact number of biological replicates (n), (ii) whether Student’s t-tests or ANOVA were used for statistical analyses, (iii) the specific post hoc correction applied (Tukey or Dunnett), and (iv) the use of error bars indicating mean ± SD. Quantitative Western blot data have also been updated to include replicate numbers and corresponding measures of variability. These revisions enhance the accuracy, transparency, and reproducibility of our results.

Reviewer 4 Report

Comments and Suggestions for Authors The manuscript presents a clear and well-structured study on the SF3A1–STX12 axis in colorectal cancer. The scientific rationale is strong, and the mechanistic data convincingly support the proposed model of mRNA stabilization and apoptosis resistance. The experiments are rigorous, appropriately controlled, and reproducible across cell lines, with adequate integration of transcriptomics, RIP-seq, and functional assays.   However, several minor revisions would improve clarity and strengthen the presentation: 1. Introduction Some background sections are overly detailed and could be streamlined to emphasize the translational relevance of targeting RNA-binding proteins in CRC. 2. Results presentation A few figure legends (particularly Figures 3–5) would benefit from clearer descriptions of the experimental workflow and sample sizes. Minor typographical inconsistencies were noted. 3. Discussion While comprehensive, the discussion occasionally repeats concepts already described in the results. A more concise synthesis would enhance readability. 4. English language The manuscript would benefit from light editing to improve flow and reduce redundancy. No major linguistic issues are present.   Overall, the study is scientifically sound, novel, and of interest to the readership. The requested changes are minor and pertain only to clarity and editorial polishing.

Author Response

The manuscript presents a clear and well-structured study on the SF3A1–STX12 axis in colorectal cancer. The scientific rationale is strong, and the mechanistic data convincingly support the proposed model of mRNA stabilization and apoptosis resistance. The experiments are rigorous, appropriately controlled, and reproducible across cell lines, with adequate integration of transcriptomics, RIP-seq, and functional assays.   However, several minor revisions would improve clarity and strengthen the presentation:

Response: We thank the reviewer for the positive and constructive evaluation of our manuscript. We appreciate the reviewer’s recognition of the clarity of the study design, the strength of the scientific rationale, and the reproducibility of the mechanistic data supporting the SF3A1–STX12 axis in colorectal cancer. In response to the reviewer’s suggestions, we have carefully revised the manuscript to improve clarity and presentation. All comments have been addressed in detail below, and the corresponding changes have been incorporated into the revised manuscript. We believe that these revisions enhance the readability and overall quality of the manuscript without altering its scientific conclusions.

 

1. Introduction Some background sections are overly detailed and could be streamlined to emphasize the translational relevance of targeting RNA-binding proteins in CRC.

Response: We thank the reviewer for this insightful comment. In accordance with the suggestion, we have streamlined the Introduction by removing overly detailed background information and reorganizing the section to more clearly emphasize the clinical and translational relevance of targeting RNA-binding proteins in colorectal cancer. These revisions improve the focus and readability of the Introduction.

 

2. Results presentation A few figure legends (particularly Figures 3–5) would benefit from clearer descriptions of the experimental workflow and sample sizes. Minor typographical inconsistencies were noted.

Response: We appreciate the reviewer’s feedback. We have revised the figure legends for Figures 3–5 to include clearer descriptions of the experimental workflow and specify the exact sample sizes (biological replicates) for each experiment. In addition, we carefully reviewed the entire manuscript and corrected minor typographical inconsistencies. These revisions enhance clarity and ensure consistency throughout the figures.

 

3. Discussion While comprehensive, the discussion occasionally repeats concepts already described in the results. A more concise synthesis would enhance readability.

Response: Thank you for this helpful suggestion. We have revised the Discussion to reduce redundancy by removing repeated descriptions of results and improving the overall logical flow. The updated version provides a more concise synthesis of our findings and their implications while maintaining the necessary scientific context.

 

4. English language The manuscript would benefit from light editing to improve flow and reduce redundancy. No major linguistic issues are present.  

Response: We appreciate the reviewer’s comment regarding the English writing. The manuscript has undergone careful language editing to improve readability, flow, and clarity while preserving the scientific meaning. We believe these refinements contribute to a more polished presentation of our work.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

the authors responded to all of my concerns so that the manuscript is acceptable to ijms.