Roles of STAT3 and STAT Family Proteins and Their Signaling Pathways in Thyroid Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by C. Masaki and colleagues provides a comprehensive overview of the involvement of the proteins of the STAT family, and STAT3 in particular, in thyroid cancer. The authors explain in detail the dual role of STAT3 as a metastasis-promoting factor when upregulated, and as a tumor suppressor when localized in the mitochondrion, and review the currently available therapeutic strategies targeting STAT3.

STAT proteins are of considerable interest in cancer biology; however, their roles in the initiation and progression of specific tumor types remain heterogeneous and not yet fully defined.  The review by Masaki and colleagues provides a remarkable contribution to the field, offering a clear and well-documented overview of the current state of research on STAT proteins, and STAT3 in particular, in thyroid cancer. 

Overall, the authors synthesize a broad and complex body of literature into a coherent narrative that will be valuable to both specialists and non-specialists in the field of thyroid cancer research. The organization of the review is clear, as it presents a balanced coverage of foundational and recent studies, and provides a clear picture of the selected topic. The figures are well designed and informative, and support the text effectively, particularly given the intrinsic complexity of the signaling pathways described. The referencing appears thorough and up to date.

Only a few minor issues should be addressed before the manuscript can be considered for publication.

- Lines 211 and 239: I suggest removing the references to the figures from the sub-header and moving them in the body of the text

- Line 321: substitute “personalities” with “activities”

- Table 1: make the font of the header uniform across columns

Author Response

We would like to express our sincere gratitude to Reviewer #1 for their highly positive and encouraging feedback. We have carefully addressed your minor formatting and phrasing suggestions to further improve the manuscript for publication.

Comment 1.1: Lines 211 and 239: I suggest removing the references to the figures from the sub-header and moving them in the body of the text.
Response: We thank the reviewer for this suggestion. We have removed the figure callouts (e.g., Fig. 4) from the sub-headers throughout the manuscript and seamlessly integrated them into the relevant paragraphs in the main body of the text.

Comment 1.2: Line 321: substitute “personalities” with “activities”.
Response: We agree with the reviewer and have replaced the word “personalities” with “activities” in Section 4.2 (STAT5A and STAT5B) to maintain a rigorous academic tone.

Comment 1.3: Table 1: make the font of the header uniform across columns.
Response: We apologize for the formatting inconsistency. The header fonts in Table 1 have been standardized for uniform size, weight, and style.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting manuscript that covers the role of STATs in thyroid oncogenesis, the writing I very direct, and sometimes it is too straight and could be more developed in terms of extension. Nevertheless, I think the authors did a great work in compiling all these references and aspects, but I think that including another topic about “transcriptional and posttranscriptional regulation” could improve the coverage of the topic (see description below).

 

• Page1, line25: remove “of STATS” because it is redundant
• Page1, line 40-41: this sentence is confusing. It seem to lack a verb.
• Page2, line 60: it lacks to mention that STAT is part of the JAK/STAT signaling and provide some context about the canonical signaling, and then mention the noncanonical roles in metabolism, etc.
• Page2 , line 86: cooperativity and cooperative. Please change one of these words to improve understanding
• Figure1 legend: indicate what is the S and Y in the TAD domain
• Figure 2 legend: it is not clear what is pS, pY
• Item 2.2.2. Acetylation should be combined with the subsequent item. Authors should better explore the references cited and provide an introduction to each post-translational modification.
• Also, how unphosphorylated STAT goes to the nucleus? Line 144, Please clarify the mechanism and provide more references.
• Line 242: STAT3 should be in italic
• Overall, authors should pay attention to figure legends so they can describe everything that is drawn in the graphs.
• Also, pay attention to gene, mRNA ou shRNA (that should be in italic form)
• I think this is an interesting review, very direct to the point, that should be improved in terms of including a section called “transcriptional and posttranscriptional regulation” to cover what is know as transcriptional activators of STAT genes, talking about TFs that bind to the promoter region, usually activated by other oncogenic signaling. Moreover, in the posttranscriptional section, include the role of microRNAs, describing what is known about the microRNAs that target STAT protein, maybe indicating a table of microRNAs (not necessarily in thyroid cancer), and what is known in thyroid cancer. If these miRNAs are downregulated and the role in STAT levels.
•  

Author Response

We sincerely thank the reviewer for their positive appraisal of our work and for recognizing the effort we put into compiling literature on STATs in thyroid oncogenesis. We greatly appreciate your constructive suggestions, particularly regarding the expansion of post-translational modifications (PTMs) and the inclusion of transcriptional/post-transcriptional regulation (Section 2.3). These additions substantially improved the depth and coverage of our review. Below is a point-by-point response to your comments.

Comment 2.1: Page 1, line 25: remove “of STATS” because it is redundant.
Response: We appreciate your comments. We have removed the redundant phrase “of STATs” from the Introduction.

Comment 2.2: Page 1, line 40-41: this sentence is confusing. It seem to lack a verb.
Response: We apologize for the confusion. We have completely rewritten this sentence to ensure that it is grammatically correct and that its meaning is clear.

Comment 2.3: Page 2, line 60: it lacks to mention that STAT is part of the JAK/STAT signaling and provide some context about the canonical signaling, and then mention the noncanonical roles in metabolism, etc.
Response: Thank you for pointing out this structural gap. We have revised this section (Section 2) to clearly introduce the STAT family as the downstream transcription factor component of the canonical JAK/STAT signaling cascade first by adding a following sentence to line 60; “STAT proteins serve as the primary downstream transcription factors of the canonical Janus kinase (JAK)/STAT (JAK/STAT) signaling cascade, mediating essential physiological responses to cytokines and growth factors.”
In addition, we also added a following sentence to line 69, introducing their non-canonical roles in metabolism; “Beyond its canonical nuclear functions, STAT3 engages in non-canonical pathways, most notably regulating cellular energy metabolism.”

Comment 2.4: Page 2, line 86: cooperativity and cooperative. Please change one of these words to improve understanding.
Response: We have revised the description of the N-terminal domain (NTD) to read: "The N-terminal domain (NTD) facilitates dimer formation and cooperative binding to tandem DNA elements," thereby eliminating repetitive phrasing.

Comment 2.5: Figure 1 legend: indicate what is the S and Y in the TAD domain.
Response: We have updated Figure 1 legend to explicitly define 'S' as serine and 'Y' as tyrosine.

Comment 2.6: Figure 2 legend: it is not clear what is pS, pY.
Response: Figure 2 and 3 legends have been updated to clearly state that 'pS' stands for phosphorylated serine and 'pY' stands for phosphorylated tyrosine.

Comment 2.7: Item 2.2.2. Acetylation should be combined with the subsequent item. Authors should better explore the references cited and provide an introduction to each post-translational modification.
Response: As suggested, we have restructured Section 2.2. We combined acetylation with the subsequent PTM items (SUMOylation, Methylation) into a more cohesive subsection. Furthermore, we have expanded the text to provide a clear biological introduction to each PTM and explored the cited references in greater depth to explain how these modifications dictate STAT3 function.

Comment 2.8: Also, how unphosphorylated STAT goes to the nucleus? Line 144, Please clarify the mechanism and provide more references.
Response: We appreciate this important question. We have updated the text to clarify that unphosphorylated STAT3 (uSTAT3) relies on specific proteins for nuclear transport, specifically interacting with importin-α3 (as opposed to importin-α5/β1 used by pY-STAT3), which constitutively shuttles uSTAT3 between the cytoplasm and nucleus independently of tyrosine phosphorylation. We have added the corresponding foundational references (e.g., Liu et al., PNAS, 2005 [Ref.35 in the revised version]) to support this proposed mechanism.

Comment 2.9: Line 242: STAT3 should be in italic.
Response: We have carefully reviewed the manuscript and corrected Line 242 to properly italicize STAT3, where it refers to the gene/mRNA.

Comment 2.10: Overall, authors should pay attention to figure legends so they can describe everything that is drawn in the graphs.
Response: We have thoroughly reviewed and expanded all figure legends to ensure that every molecular component, pathway, and symbol depicted in the illustrations is accurately and comprehensively described.

Comment 2.11: Also, pay attention to gene, mRNA ou shRNA (that should be in italic form).
Response: We have rigorously proofread the entire manuscript to ensure that all gene names, mRNA, and shRNA designations are consistently and correctly italicized in accordance with the standard genetic nomenclature.

Comment 2.12: I think this is an interesting review, very direct to the point, that should be improved in terms of including a section called “transcriptional and posttranscriptional regulation” to cover what is know as transcriptional activators of STAT genes, talking about TFs that bind to the promoter region, usually activated by other oncogenic signaling. Moreover, in the posttranscriptional section, include the role of microRNAs...
Response: We agree that this is a highly valuable addition that vastly improves the depth of our review. We have added a new, dedicated section titled "2.3. Transcriptional and Post-transcriptional Regulation of STAT3."

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript provides a broadly informative overview of JAK/STAT biology in thyroid cancer. It is well-written and has nice figures. Unfortunately, several essential mechanistic and translational studies are currently missing. Their omission substantially limits the completeness and contemporary relevance of the review. Therefore, a thorough major revision is required. If the authors can do this thoroughly, it will be a truly valuable review. I am convinced that the authors can fulfill this in time.

1. Titel is misinformative. “Roles of STAT3 and STAT Family Proteins…” – STAT1 was the first identified STAT protein and why is STAT3 pointed out as STAT3 and STAT family…? Same for STAT5,6,… The title must more precisely state that it is a summary of STAT protein functions on this cancer.
2. there are too many old reviews (some over 10 years!) whereas new reviews have not been cited at all. To correct this, the very recent authoritative review by Mustafa & Krämer (Pharmacol. Rev., 2023; PMID: 36752816) on the intersection between aberrant JAK signaling and HDAC biology must be cited in sections addressing JAK-STAT signaling, JAK structure, JAK inhibitor resistance, pathway plasticity, and rational combinatorial strategies. This review provides the necessary conceptual framework linking cytokine signaling, JAK mutations, post-translational regulation, chromatin state, and therapeutic vulnerabilities.
3. Aberrant activation of JAK/STAT is commonly triggered by inflammatory cytokines, oncogenic kinases, or genetic mutations. I agree on mutations, but the whole review article does not cover this adequately. The authoritative review by Mustafa & Krämer (Pharmacol. Rev., 2023; PMID: 36752816) provides the necessary conceptual framework linking cytokine signaling, post-translational regulation, chromatin state, JAK2 mutations, and therapeutic vulnerabilities.
4. As the present manuscript covers epigenetic signaling only superficially, integrating this work is essential to accurately reflect how chromatin modifiers and ubiquitin ligases shape JAK/STAT signal amplitude, stability, and therapeutic responsiveness. The authors must incorporate the recent seminal work by Mustafa et al. (Signal Transduct Target Ther, 2025, PMID: 40877230) demonstrating that HDAC1/HDAC2 regulate JAK2V617F–STAT signaling through the ubiquitin ligase SIAH2. This study shows that SIAH2 knockout cells are markedly less sensitive to HDAC inhibitors, and global transcriptomics identifies JAK–STAT signaling as a primary SIAH2-dependent pathway.
5. Together with the Mustafa et al. 2025 findings, these studies reveal tumor-context–specific duality in SIAH2 function and underscore that ubiquitin-mediated regulation is integral to JAK/STAT signal modulation. The current manuscript does not discuss ubiquitin ligases in this context, representing a substantial conceptual gap that must be addressed.

Author Response

We sincerely thank Reviewer #3 for their thoughtful evaluation and constructive critique. We agree that incorporating contemporary reviews and deepening the mechanistic discussion—particularly regarding genetic mutations, epigenetic crosstalk, and ubiquitin-mediated regulation—substantially improves the completeness and contemporary relevance of our manuscript. We have carefully addressed all your comments and incorporated the seminal references you suggested.

Comment 3.1: Titel is misinformative. “Roles of STAT3 and STAT Family Proteins…” – STAT1 was the first identified STAT protein and why is STAT3 pointed out as STAT3 and STAT family…? Same for STAT5,6,… The title must more precisely state that it is a summary of STAT protein functions on this cancer. 
Response: We appreciate the reviewer’s perspective and fully agree that STAT1 holds immense historical significance as the first identified STAT protein, and we acknowledge its renowned role in inflammation and cytokine signaling. However, in the specific context of cancer biology, STAT3 is widely recognized as the most prominent, extensively studied, and classically targeted member of the family.
Because the core narrative of our manuscript predominantly revolves around the complex, paradoxical "double-edged sword" role of STAT3 in thyroid cancer, while the other STAT family members are discussed to provide a comprehensive signaling context, we believe that explicitly highlighting STAT3 in the title accurately reflects the primary focus and main draw of the paper for the oncology readership. Therefore, we respectfully prefer to retain the current title: "Roles of STAT3 and STAT Family Proteins and Their Signaling Pathways in Thyroid Cancer" to best represent the specific emphasis of the manuscript.

Comment 3.2: there are too many old reviews (some over 10 years!) whereas new reviews have not been cited at all. To correct this, the very recent authoritative review by Mustafa & Krämer (Pharmacol. Rev., 2023; PMID: 36752816) on the intersection between aberrant JAK signaling and HDAC biology must be cited in sections addressing JAK-STAT signaling, JAK structure, JAK inhibitor resistance, pathway plasticity, and rational combinatorial strategies. This review provides the necessary conceptual framework linking cytokine signaling, JAK mutations, post-translational regulation, chromatin state, and therapeutic vulnerabilities.
Response: We thank the reviewer for highlighting the need for contemporary perspectives. Our rationale for including older references was a deliberate effort to appropriately appreciate and acknowledge the original seminal articles and foundational discoveries that established the current understanding of JAK/STAT biology. We believe that it is crucial to properly credit the primary sources that led to these original findings.
However, we completely agree that integrating modern conceptual frameworks is essential for a comprehensive review. To address this, we retained the foundational citations to honor the original discoveries; however, we additionally incorporated the recent authoritative review by Mustafa & Krämer (2023) in our sections addressing JAK-STAT signaling, pathway plasticity, and rational combinatorial strategies to provide the necessary up-to-date conceptual framework.

Comment 3.3
Aberrant activation of JAK/STAT is commonly triggered by inflammatory cytokines, oncogenic kinases, or genetic mutations. I agree on mutations, but the whole review article does not cover this adequately. The authoritative review by Mustafa & Krämer (Pharmacol. Rev., 2023; PMID: 36752816) provides the necessary conceptual framework linking cytokine signaling, post-translational regulation, chromatin state, JAK2 mutations, and therapeutic vulnerabilities.
Response: We apologize for the inadequate coverage of the genetic mutations driving JAK/STAT activation. Prompted by your comment, we have significantly expanded our discussion on the mutational activation. Utilizing the framework provided by Mustafa & Krämer (2023), we explicitly detail how the intersection of aberrant cytokine signaling, post-translational regulation, and specific JAK mutations generates distinct therapeutic vulnerabilities. We have added a description in Section “2.5. Aberrant Activation of JAK/STAT signaling,” highlighting recent genomic studies demonstrating that thyroid tumors dedifferentiate and acquire resistance to BRAF inhibitors, frequently harboring newly acquired functional missense mutations in JAK1 and JAK2, providing a critical mutational bypass mechanism.

Comment 3.4: As the present manuscript covers epigenetic signaling only superficially, integrating this work is essential to accurately reflect how chromatin modifiers and ubiquitin ligases shape JAK/STAT signal amplitude, stability, and therapeutic responsiveness. The authors must incorporate the recent seminal work by Mustafa et al. (Signal Transduct Target Ther, 2025, PMID: 40877230) demonstrating that HDAC1/HDAC2 regulate JAK2V617F–STAT signaling through the ubiquitin ligase SIAH2. This study shows that SIAH2 knockout cells are markedly less sensitive to HDAC inhibitors, and global transcriptomics identifies JAK–STAT signaling as a primary SIAH2-dependent pathway.
Response: We agree that our previous coverage of epigenetic signaling was superficial. We integrated the seminal work of Mustafa et al. (2025) to accurately reflect the complex crosstalk between chromatin modifiers and signaling networks. We have added dedicated text detailing how HDAC1 and HDAC2 tightly regulate mutation-driven JAK-STAT signaling (such as JAK2V617F) via the ubiquitin ligase SIAH2. We also highlight the crucial finding of this study that targeting SIAH2 renders cells less sensitive to HDAC inhibitors, demonstrating how the epigenetic state and therapeutic responsiveness are fundamentally linked to ubiquitin-mediated regulation.

Comment 3.5:
Together with the Mustafa et al. 2025 findings, these studies reveal tumor-context–specific duality in SIAH2 function and underscore that ubiquitin-mediated regulation is integral to JAK/STAT signal modulation. 
Response: We thank the reviewer for pointing out this substantial conceptual gap. To address the omission of ubiquitin ligases, we have added a completely new subsection titled "Ubiquitin-Mediated Regulation and Epigenetic Crosstalk" (Section 2.3.3). In this section, we deeply discuss how the ubiquitin-proteasome system acts as a fundamental controller of JAK/STAT signal amplitude and protein stability. Drawing on the Mustafa et al. (2025) findings, we specifically underscore the tumor-context-specific duality of ubiquitin ligases, such as SIAH2, emphasizing that they do not merely degrade signaling components but actively regulate the epigenetic landscape and therapeutic vulnerabilities of the tumor.

Reviewer 4 Report

Comments and Suggestions for Authors

This is generally a good review of STATs in cancer with an emphasis on thyroid cancer.  I do suggest adding more on the non-canonical pathway, lines 119-121, introducing the triggering factors here and some other background.  As is, the next few paragraphs seem a bit random.  So, it needs a coherent introduction. 

Author Response

Comment 4.1: This is generally a good review of STATs in cancer with an emphasis on thyroid cancer. I do suggest adding more on the non-canonical pathway, lines 119-121, introducing the triggering factors here and some other background. As is, the next few paragraphs seem a bit random. So, it needs a coherent introduction.

Response: We thank the reviewer for identifying this disjointed transition. We have significantly revised the introduction to the non-canonical pathway (now in Section 2.2). We now properly introduce the specific physiological and microenvironmental triggering factors, such as oxidative stress, metabolic shifts (e.g., hypoxia), and serine kinase activity (e.g., MAPKs), that push STAT3 away from canonical DNA-binding and toward non-canonical compartmentalization (like mitochondria). Providing this background greatly improves the flow and provides a coherent context for the subsequent paragraphs detailing specific post-translational modifications.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The article was good before revision and is now excellent.