Ap4A in Cancer: A Multifaceted Regulator and Emerging Therapeutic Target

Summary

Thank you very much for taking the time to review our review. Please find the detailed responses below and the corresponding revisions/corrections. Minor revisions as was suggested have been made.

Comments 1: The Introduction section is too brief and does not adequately cover the research background. For instance, the current status of tumor therapy could be expanded, with references to relevant literature such as Chem. Commun., 2025, 61, 5333-5336; Chem. Commun., 2025, 61, 2484-2495; and Chin. Chem. Lett., 2023, 34, 108088.

Response 1: We thank the reviewer for the insightful suggestion to expand the introduction section and for pointing out highly relevant and interesting literature. For instance, the Introduction was amplified, and all the modifications can be tracked in red ‘’ Ap4A functions as both a signalling molecule and a regulator of cellular homeostasis, …. in leveraging Ap4A’s biology for cancer treatment.’’ (page 1-2, line 35-71). However, we found the proposed references to be quite specific, especially considering that our manuscript does not address or mention antitumor compounds or nanomaterials in any section. For this reason, we have opted not to expand the literature in that specific direction to maintain the focus and coherence of the manuscript.

Comments 2: The section on AP4A structural conformation (currently a separate subsection) could be integrated into the Introduction to reduce the number of subheadings.

Response 2: This is a good consideration. Nevertheless, since the introduction section was amplified, now the text and the separate heading is integrated more harmonically, so we preferred to maintain it as a separate subheading in order to preserve clarity and emphasize its specific relevance within the manuscript.

Comments 3: Page 3, Line 96: The correct subheading should be "3.2 Degradation of Ap4A" (currently appears to be 1.2 Degradation of Ap4A").

Response 3: Thank you to pointing this out, the numeration of subheading has been corrected.

Comments 4: The text in Figure 2 is too small and illegible. Please enlarge the font size or improve the resolution for clarity.

Response 4: We are completely agreed, thus, the text inside the figure was enlarged.

Summary

Thank you very much for taking the time to review this manuscript. Your comments are really insightful and useful in order to clarify and harmonize the manuscript. Please find the detailed responses below and the corresponding revisions and corrections highlighted in red in the re-submitted files. We hope these revisions meet your expectations and contribute to a clearer and more robust scientific discussion.

Comments 1: Conceptual Framework Needs Strengthening

The review would benefit from a clearer conceptual structure early on. Consider outlining the physiological role of Ap4A in normal cells versus its dysregulation in cancer, and then building on that foundation to address mechanistic and therapeutic relevance.

Response 1: We thank the reviewer for their valuable feedback. In response to the suggestion to enhance the manuscript's depth and clarity, we have substantially amplified and extended the contextual content throughout the text. These additions provide further background, detail, and scientific rationale to support the discussed mechanisms and observations. To ensure full transparency and facilitate the review process, all newly added or expanded paragraphs have been clearly highlighted in red within the revised manuscript. We hope these improvements address the reviewer's concerns and contribute to a more comprehensive and informative version of the manuscript.

Comments 2: Expand Mechanistic Insights

The discussion of Ap4A's role in apoptosis, DNA repair, and stress responses remains somewhat descriptive. Incorporate specific mechanistic details and relevant pathways—for instance, how Ap4A levels modulate PARP1 activity, or its role in nucleotide excision repair and replication stress. Including more mechanistic insight will significantly strengthen the review’s value.

Response 2: We thank you for your constructive comment. As per your suggestion, we have expanded and deepened several sections of the manuscript, particularly those related to the mechanisms involving Ap4A. These additions can be traced throughout the text and are highlighted in red for easier identification. For example, in section 4.4, page 7-8, line 303-314 “Cell cycle progression is a tightly regulated process… ‘’; and line 331-335 ‘’DNA ligase III synthesises Ap4A…’’. Additionally, different amplifications and insights can be found in section 4.5.4, page 9, line 387-393 ‘’Activation of P2X7 in tumour cells has been shown to trigger…’’  and line 400-409 of the same section ‘’P2X7 receptor is well-known for its ability to form membrane pores…’’. This addition aims to better clarify the central role of P2X7 in Ap4A-mediated processes, particularly in signal transduction and the regulation of cellular metabolism.

Comments 3: Cancer-Specific Relevance Needs Focus

While the authors mention several cancer hallmarks, the connection between Ap4A signaling and cancer progression is not always clearly articulated. It would be helpful to include cancer-specific examples or data where Ap4A levels have been directly measured in tumor tissues or linked to prognosis, therapy resistance, or immune evasion.

Response 3: As suggested, different cancer-specific examples were cited in the text. For example, section 4.4, page 8, line 315 ‘’Thus, in breast carcinoma cell lines demonstrated that knocking down NUDT2 led to reduced cell proliferation and an accumulation of cells in the G0/G1 phase of the cell cycle, further highlighting Ap4A's role in regulating cancer cell growth.’’ Section 5.1, page 13, line ‘’ Ap4A has been reported to disrupt the formation of the HINT1-MITF complex, a critical step in MITF's transcriptional activation. This disruption leads to attenuation of MITF-mediated gene expression, thereby suppressing melanoma cell growth [77].’’ Section 5.1, page 12 line 531 ‘’In chronic myelogenous leukemia cells lacking the NUDT2 hydrolase—an enzyme re-sponsible for degrading Ap4A—a dramatic increase in intracellular Ap4A (up to 175-fold) led to transcriptional changes in over 6,000 genes.

Comments 4: Therapeutic Targeting: Overlooked Opportunity

The therapeutic potential of modulating Ap4A metabolism or signaling is only briefly discussed. Please expand this section:

- Mention inhibitors/activators of Ap4A-related enzymes (e.g., NUDT2, Ap4A hydrolases);

- Discuss feasibility of Ap4A analogs or small molecules modulating its pathways;

- Highlight whether Ap4A might serve as a biomarker in cancer diagnostics or treatment stratification.

Response 4: As requested, we have substantially expanded the section discussing Ap4A as a therapeutic target. In particular, we elaborated on its dual role in angiogenesis and tumor proliferation, highlighting the context-dependent effects of Ap4A via purinergic receptor signaling—especially its interaction with P2X7 and P2X1 receptors. The updated text now discusses how Ap4A influences endothelial cell migration and proliferation, its ability to modulate the tumor microenvironment, and the implications for targeted therapies—including the use of Ap4A analogs or modulators of its metabolic enzymes (e.g., NUDT2, FHIT). All the newly added content can be found in the section 6 and is highlighted in red to facilitate identification and review.

-          Section 6, page 16, line 652 ‘’ Thus, inhibitors of P2X7 or strategies aimed at disrupting Ap₄A-mediated signaling may hold potential for controlling tumor progression, immune suppression, and therapeutic resistance by modulating purinergic signaling dynamics within the TME’’.

-          Section 6, page 16, line 664 ‘’ Recent studies have also synthesized Ap₄A analogues with dual inhibitory activity at P2Y1 and P2Y12 receptors, demonstrating synergistic inhibition of platelet aggregation and underscoring Ap₄A’s potential as an antithrombotic agent.’’

-          In this section, Ap4A’s potential as both a biomarker and therapeutic target, particularly in strategies aiming to modulate its synthesis, degradation, or receptor-mediated signaling was discussed.

Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions and changes evidenced in red inside the in the resubmitted text.

Comments 1: The introduction section is too simple; the authors should rewrite the introduction section to 3-4 paragraphs.

Response 1: We agree with this comment. Therefore, the introduction was amplified and improved. All the changes are highlighted in red.  

Comments 2: Is there any difference between the synthesis and degradation of Ap4A in normal and tumor tissues? And the difference in Ap4A level in normal and tumor tissues should be stated.

Response 2: We have, accordingly, integrated some parts in the manuscript to answer to this question, thus in section 3.2, page 4, line 168, we added the statement about ap4A’s degradation:’’ In cancer cells, degradation is often reduced due to downregulation or mutation of hydro-lases, leading to accumulation of Ap4A, which can interfere with tumour suppressor (e.g., HINT1) and modulate gene expression.’’

Summary

Thank you very much for your thoughtful and encouraging comments on our manuscript. We truly appreciate you taking the time to review our work and are grateful for your recognition of the novelty and relevance of our focus on Ap4A, particularly in the context of cancerogenesis. It is very motivating to hear that you found our review both interesting and meaningful among the existing literature.

Please find below our detailed responses to your comments and suggestions, as well as the corresponding revisions highlighted in track changes in the re-submitted files. We hope the changes address your feedback effectively.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lines 126-128 ‘A significant function of Ap4A lies in cell cycle regulation. By interacting with key cell cycle proteins, Ap4A influences cell division and growth, ensuring proper progression through the different phases of the cell cycle’.

I think it would be beneficial to shed some light on the mechanisms underlying cell cycle progression.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, some insight about cell cycle progression was added in section 4.4, page 7, line 303-315 ‘’ Cell cycle progression is a tightly regulated process orchestrated by a sequential activation of CDKs and their regulatory partners… tumour cells with defective checkpoints continue to divide, accumulating mutations that drive onco-genesis’’

Comments 2: The authors place a strong emphasis on the interaction between Ap4A and purinergic receptors. Although the roles of different receptors are reviewed in detail, I would suggest expanding the introduction to this section with a brief overview of the connection between purinergic receptors and cancer in general.

Response 2: Agree. Thus, the introduction section was widely revised and expanded in order to provide an overview about purinergic receptors and cancer in general. All new paragraphs are highlighted in red. 

Comments 3: In my opinion, it would be helpful to include information about which cells, organs, or tissues have shown a particular property of Ap4A. For instance, the authors mention the names of certain cell lines on line 333, but this information is not always provided for other cases.

Response 3: Thank you for this helpful suggestion. We agree that specifying the cells in which particular properties of Ap4A have been observed adds valuable context and clarity for the reader. Thus, we have revised the manuscript to include additional examples of specific cancer cell lines associated with the described effects of Ap4A. These additions can now be found throughout the revised text, particularly in the relevant sections discussing Ap4A's role in cancer-related processes. The new information is highlighted in track changes for your convenience.

-          In Section 4.4, page 8, line 315-317: ‘’Thus, in breast carcinoma cell lines demonstrated that knocking down NUDT2 led to re-duced cell proliferation and an accumulation of cells in the G0/G1 phase of the cell cycle, further highlighting Ap4A's role in regulating cancer cell growth [36].’’

-          In Section 4.4, page 8, line 325-327: ‘’This delay is believed to prevent replication, giving cells time to repair potential DNA damage, particularly in ovarian cancer cell lines [3].’’

-          In Section 4.5.4, page 10, line 409-411:’’ For instance, inhibiting P2X1R and P2X7R has been observed to reduce mitochondrial ac-tivity, intracellular calcium levels, and proliferation in various cancer cell lines, such as leukemia cell lines [46].’’

-          In Section 5.1, page 13, line 525-527: ‘’ Ap4A has been reported to disrupt the formation of the HINT1-MITF complex, a critical step in MITF's transcriptional activation. This disruption leads to attenuation of MITF-mediated gene expression, thereby suppressing melanoma cell growth [77].’’