Review Reports - Emerging Protein Targets in Triple-Negative Breast Cancer: Beyond Conventional Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review summarizes current and emerging therapeutic strategies for triple-negative breast cancer (TNBC). The manuscript is well-structured and thoroughly describes the most recent publications and clinical trials in this area.

-However, bearing in mind dozens of reviews on the treatment of triple-negative breast cancer published recently, it is therefore recommended that in the introduction the authors more clearly explain the novelty of the current study as compared to recently published reviews. The none-overlapping recent papers should be properly cited so that interested readers can consult them. Novel aspects of the present review should be also emphasized in the Abstract and in the Simple Summary.

-Table 1 can be moved to Appendix or Supplementary material.

-In addition, the inclusion of graphical illustrations, summarizing current and emerging treatment strategies for triple-negative breast cancer is strongly recommended.

Author Response

REVIEWER 1

Q: However, bearing in mind dozens of reviews on the treatment of triple-negative breast cancer published recently, it is therefore recommended that in the introduction the authors more clearly explain the novelty of the current study as compared to recently published reviews. The none-overlapping recent papers should be properly cited so that interested readers can consult them. Novel aspects of the present review should be also emphasized in the Abstract and in the Simple Summary.

R: The following has been added to the Abstract:

Collectively, this review provides an updated and novel overview of the evolving TNBC therapeutic landscape and highlights promising directions for the development of next-generation, biomarker-driven treatment strategies aimed at improving patient outcomes maintaining a broad perspective on a very large class of targets.

The following has been added to the Introduction:

The poor prognosis of TNBC patients with the current therapies and the heterogeneity of this tumor drive the research towards new therapeutical strategies.

Here we provide a comprehensive and up-to-date overview of potential novel protein targets for TNBC treatment. Target selection was primarily guided by known tumor vulnerabilities, whose expression levels in TNBC were collected from original research articles and omics databases. To our knowledge, no previous review has integrated such a wide range of proteomic evidence and tumor vulnerability data from both original research and multi-omics databases in the context of TNBC.

Q: Table 1 can be moved to Appendix or Supplementary material.

R: We thank the reviewer for the suggestion, but we would prefer to keep Table 1 in the main text as it is a relevant part of the presented work.

Q: In addition, the inclusion of graphical illustrations, summarizing current and emerging treatment strategies for triple-negative breast cancer is strongly recommended.

R: A graphical abstract is already included in the submitted manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

You bring together a ton of data in this paper and it is nice to see it all in one place. Congrats on a nice effort.

I would like to understand more about section 3 and in particular how much manual checking went into the AI research assistant's work. I don't have a good way to assess the validity of the targets shown here and I would encourage the authors to better explain how they generated the final table.

At the end of this paper or each section, it would be immensely valuable if the authors could comment on their final conclusions based on the data presented. I think that the greatest value comes from a review where clear conclusions are presented as part of the summary of reports.

Author Response

REVIEWER 2

You bring together a ton of data in this paper and it is nice to see it all in one place. Congrats on a nice effort.

Q: I would like to understand more about section 3 and in particular how much manual checking went into the AI research assistant's work.

R: The AI tool was used exclusively for literature search and to extract the most relevant studies on the query subject, as stated at the beginning of section 3. Following this extraction, for each section, data were manually revised and commented on. Moreover, literature data were implemented by manual search.

Q: I don't have a good way to assess the validity of the targets shown here and I would encourage the authors to better explain how they generated the final table.

R: The text was expanded with more details as follows (pg. 16):

Deposited omics data yield unique TNBC marker combinations, with integrative analyses spanning transcriptomics, genomics, proteomics, epigenomics, and, in one instance, metabolomics identifying cancer-specific signatures. Across deposited omics studies, predictive performance metrics refer either to the ability of multi-marker signatures to classify triple-negative breast cancer samples from other breast cancer subtypes or normal tissue, or to stratify TNBC patients according to survival outcomes. Unless otherwise specified, reported performance metrics were extracted from the original studies and obtained using cross-validation or independent validation cohorts based on public datasets such as TCGA and GEO. In this context, gene panels ranging from three to 100 markers achieve classification accuracies of up to 98.9% for TNBC discrimination, while survival-oriented models report concordance indices up to 0.7573; additionally, a 29-protein signature reached 88% accuracy in distinguishing TNBC from luminal tumors and showed strong correlations with patient survival. Validation approaches remain heterogeneous and include experimental methods (cell lines, CRISPR screens, western blot) as well as in silico analyses (public datasets, survival curves).

To extract the most significant proteins and targets from these datasets, we used the Elicit AI research assistant to perform a literature search and identify candidates based on semantic similarity to the query: “Among the deposited omics data, is it possible to identify unique combinations of markers for triple-negative breast cancer? Select also targets which are not known to be drugged,” across over 138 million academic papers indexed by Elicit, including Semantic Scholar and OpenAlex. The AI retrieved a list of potentially relevant proteins, which were then manually checked one by one to determine whether they had been previously studied in TNBC, with their relevance and novelty discussed individually. Additional proteins not retrieved by Elicit were incorporated through manual literature searches. The final table summarizes only those targets supported by explicit evidence in the primary literature.

Q: At the end of this paper or each section, it would be immensely valuable if the authors could comment on their final conclusions based on the data presented. I think that the greatest value comes from a review where clear conclusions are presented as part of the summary of reports.

R: We thank the reviewer for the suggestion. A “Conclusions” section was added as last paragraph of the Review. Pg. 28

Conclusion

The aim of this review is to present a broad overview of possible new targets for the treatment of TNBC. While well-defined and clinically applicable guidelines have been successfully established for breast cancers characterized by the expression of specific receptors, this is not the case for TNBC. This subtype is primarily defined by the absence of known receptors, and to date, no specific protein target has been clearly identified as a hallmark that could be exploited therapeutically. The purpose of this review is therefore to explore, primarily through the analysis of omics databases, potential protein targets expressed either at the cellular level or within the tumor microenvironment that could be defined as molecular signatures characteristic of TNBC. This approach aims to lay the groundwork for future studies focused on the development of molecules capable of counteracting tumor growth. Ultimately, the objective is to move from a classification based on exclusion, defining TNBC as a tumor that does not belong to other breast cancer subtypes, toward a more precise characterization based on the expression of specific targets. Clearly, this review cannot address this challenge in an exhaustive manner, as the multifactorial nature of TNBC is extremely complex, and its classification is not univocal but rather depends on phenotypic features, genetic alterations, and interactions with the diverse components of the tumor microenvironment. Further in situ transcriptomic and proteomic studies will be required to achieve a more refined subtyping of TNBC and, consequently, to better define potential therapeutic strategies that can be evaluated in clinical trials.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In their review titled "Emerging Protein Targets in Triple Negative Breast Cancer: Beyond Conventional Therapy”, the authors provide an updated overview of the evolving therapeutic landscape of TNBC. It is an interesting topic. Overall, the review is satisfactory. However, several aspects could be improved to enhance the quality of the work.

For example, summaries of each chapter would be helpful. More figures are needed to synthesize information.

The authors should summarize the key points, highlight major themes and research gaps, and discuss the broader implications of the findings in a "Conclusion and Future Directions" section.

The section on molecular, histological, and clinical classification should be updated, and recent literature should be critically discussed, as systematic classification reveals important molecular characteristics for TNBC patients.

The author should also discuss the differences between comprehensive molecular classifications developed in research and practical approaches adopted in clinical settings. Many of these approaches remain theoretical, and standard chemotherapy continues to be the primary treatment.

Overall, the authors should update the literature review by adding more recent references.

Author Response

REVIEWER 3

Q: For example, summaries of each chapter would be helpful. More figures are needed to synthesize information.

R: Thank you for the suggestion. Indeed, a concluding paragraph had not originally been planned; however, it has now been added in order to provide greater clarity regarding the significance and potential impact of the work. To further clarify the conceptual framework, we included a graphical abstract.

Conclusion

The aim of this review is to present a broad overview of possible new targets for the treatment of TNBC. While well-defined and clinically applicable guidelines have been successfully established for breast cancers characterized by the expression of specific receptors, this is not the case for TNBC. This subtype is primarily defined by the absence of known receptors, and to date, no specific protein target has been clearly identified as a hallmark that could be exploited therapeutically. The purpose of this review is therefore to explore, through the analysis of omics databases, potential protein targets expressed either at the cellular level or within the tumor microenvironment that could be defined as molecular signatures characteristic of TNBC. This approach aims to lay the groundwork for future studies focused on the development of molecules capable of counteracting tumor growth. Ultimately, the objective is to move from a classification based on exclusion, defining TNBC as a tumor that does not belong to other breast cancer subtypes, toward a more precise characterization based on the expression of specific targets. Clearly, this review cannot address this challenge in an exhaustive manner, as the multifactorial nature of TNBC is extremely complex, and its classification is not univocal but rather depends on phenotypic features, genetic alterations, and interactions with the diverse components of the tumor microenvironment. Further in situ transcriptomic and proteomic studies will be required to achieve a more refined subtyping of TNBC and, consequently, to better define potential therapeutic strategies that can be evaluated in clinical trials.

Q: The authors should summarize the key points, highlight major themes and research gaps, and discuss the broader implications of the findings in a "Conclusion and Future Directions" section.

R: We thank the reviewer for this observation and have added a Conclusions section, also highlighting how we hope our contribution may support the scientific community by advancing knowledge toward improved clinical practice.

Q: The section on molecular, histological, and clinical classification should be updated, and recent literature should be critically discussed, as systematic classification reveals important molecular characteristics for TNBC patients.

R: We thank the reviewer for this observation and agree that an updated classification of triple-negative breast cancer would be desirable, ideally based on the expression of specific targets rather than on the absence of others. The manuscript, in fact, reports several studies in which TNBC is subtyped and classified in a complex manner based on specific characteristics. The studies cited for this classification are recent and reflect the clinical practice currently in use.

Q: The author should also discuss the differences between comprehensive molecular classifications developed in research and practical approaches adopted in clinical settings. Many of these approaches remain theoretical, and standard chemotherapy continues to be the primary treatment.

R: This point is clarified by the distinction between Section 2 (Current Approaches and Clinical Trials) and Section 3 (New Possible Targets Identified by Omics Approaches) and has also been addressed in the Conclusions section. Also, as declared in the Introduction, this review was conceived with the aim of laying the groundwork by collecting molecular data that may serve the scientific community as a basis for initiating the development of targeted therapeutic approaches. This work cannot be considered exhaustive, nor does it intend to draw conclusions sufficient to define a new therapeutic strategy; rather, it aims to support researchers in developing research lines toward potential novel TNBC targets that are still poorly explored or unexplored.

Q: Overall, the authors should update the literature review by adding more recent references.

R: We thank the reviewer for the suggestion, but, to the extent of our knowledge, the most recent published data were cited. For example, epidemiological data are the most recent available (2022/2025), we report ongoing or recently completed phase II–III clinical trials (ClinicalTrials.gov database was consulted in late 2025), and 95 references published in the last 5 years.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Previtali et al review emerging and unconventional targets for the treatment of triple negative breast cancer (TNBC). This is an up-to-date, comprehensive, and clearly written review of recently developed and innovative therapies for TNBC. The summaries of development of each treatment (i.e. PD-1/PDL1 inhibitors) are especially strong. The review should be of interest to both specialists and researchers entering this field.

There are only a few minor comments.

Although the review is extremely well-written, there are instances where English usage could be improved. i.e., line 80 “familiar history” should be familial history, line 348 etc.

Can Table 1 be added as supplemental data?
Lines 399 and 507 need references.

The overall summary should be expanded under another heading, rather than being attached to the section on radiotherapy.

Comments on the Quality of English Language

There are only a few instances where English usage could be improved. Overall, the paper is extremely well-written.

Author Response

REVIEWER 4

Previtali et al review emerging and unconventional targets for the treatment of triple negative breast cancer (TNBC). This is an up-to-date, comprehensive, and clearly written review of recently developed and innovative therapies for TNBC. The summaries of development of each treatment (i.e. PD-1/PDL1 inhibitors) are especially strong. The review should be of interest to both specialists and researchers entering this field.

There are only a few minor comments.

Q: Although the review is extremely well-written, there are instances where English usage could be improved. i.e., line 80 “familiar history” should be familial history, line 348 etc.

R: We thank the reviewer, the text was updated and the typo was corrected.

Q: Can Table 1 be added as supplemental data?

R: We thank the reviewer for the suggestion, but we would prefer to keep Table 1 in the main text as it is a relevant part of the presented work.

Q: Lines 399 and 507 need references.

R: The clinical study NCT06851299 (l. 399) and NCT03218826 (l. 507) are still recruiting and have not yet published any results.

Q: The overall summary should be expanded under another heading, rather than being attached to the section on radiotherapy.

R: A “Conclusions” section with header has been added to the manuscript.

Pg. 28

Conclusions

The aim of this review is to present a broad overview of possible new targets for the treatment of TNBC. While well-defined and clinically applicable guidelines have been successfully established for breast cancers characterized by the expression of specific receptors, this is not the case for TNBC. This subtype is primarily defined by the absence of known receptors, and to date, no specific protein target has been clearly identified as a hallmark that could be exploited therapeutically. The purpose of this review is therefore to explore, through the analysis of omics databases, potential protein targets expressed either at the cellular level or within the tumor microenvironment that could be defined as molecular signatures characteristic of TNBC. This approach aims to lay the groundwork for future studies focused on the development of molecules capable of counteracting tumor growth. Ultimately, the objective is to move from a classification based on exclusion, defining TNBC as a tumor that does not belong to other breast cancer subtypes, toward a more precise characterization based on the expression of specific targets. Clearly, this review cannot address this challenge in an exhaustive manner, as the multifactorial nature of TNBC is extremely complex, and its classification is not univocal but rather depends on phenotypic features, genetic alterations, and interactions with the diverse components of the tumor microenvironment. Further in situ transcriptomic and proteomic studies will be required to achieve a more refined subtyping of TNBC and, consequently, to better define potential therapeutic strategies that can be evaluated in clinical trials.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

This manuscript is an admirable attempt to summarize current and potential therapeutic approaches for the treatment of TNBC. Like similar attempts, it is ultimately dissatisfying because it sacrifices analyses of possible causes or consequences in order to simply cover the breadth of clinical and experimental findings. While it might serve as a primer for those entering the field, important questions are unaddressed. The most blaring omission is that of a concluding paragraph which covers the progress that has been made and that remains to be made. A diagram summarizing current and potential first, second, and third line treatment strategies for localized and metastatic TNBC would be helpful. Interest might also be stimulated by a discussion of the impediments to development of new targeted therapies, e.g., the exorbitant costs of clinical trials, difficulties in recruiting from a limited patient pool, no clear rationales for prioritization of different strategies, the inherent heterogeneity of the TNBC indication, lack of incentives for side-by-side testing of competing alternatives, etc.

Other comments:

The last sentence in the section on Anti-Angiogenic Agents needs to be reconciled with the rest of the section. Is it better to disrupt or augment tumor angiogenesis?
The last sentence in the section on CDK 4/6 inhibitors is incomplete and not comprehensible. The entire section needs to be reorganized.
At the start of the Omics Approaches section, classification accuracies are not defined and therefore the cited statistics are meaningless.
The conclusion of the section on Metabolic Vulnerabilities in TNBC is ambiguous. If systemic toxicity and off target effects limit the therapeutic use of metabolic inhibitors, how is this approach promising?
The Soluble Factors section is confusing - are they or are they not potential therapeutic targets?

Author Response

REVIEWER 5

R: We thank the Reviewer for the useful comments. A concluding paragraph was included (pg. 28), but we opted not to include the suggested diagram, as the Review is not focused on clinical treatment guidelines. The suggested discussion is, in our opinion, more apt for an editorial than for a Review, but we are ready to introduce extra text if needed.

Other comments:

Q: The last sentence in the section on Anti-Angiogenic Agents needs to be reconciled with the rest of the section. Is it better to disrupt or augment tumor angiogenesis?

We thank the Reviewer for the question. The answer is complex. Targeting tumor angiogenesis has always been considered a possibility to shut down oxygen supply to cancer cells, but the recent data we report, along with the low success achieved by anti-VEGF drugs, suggest that stimulating angiogenesis can be a counterintuitive approach that need to be explored. We clarified this concept in the text.

2.5. Anti-Angiogenic Agents

Angiogenesis is the process of development of new immature and disorganized blood vessels within tumorigenesis that let cancer cells get the oxygen and nutrients they need to proliferate. The expansion of the tumor increases oxygen consumption and generates regions of the malignant mass that lie far from blood vessels, leading these areas to experience hypoxic conditions. This crucial step in tumor development is called angiogenic switch. Hypoxia triggers the nuclear translocation of Hypoxia-Inducible Factors (HIFs) in cancer cells. HIF-1a dimerizes with HIF-1b and the complex binds the Hypoxia Response Element (HRE) on the DNA, acting as a transcriptional factor for pro-angiogenic mediators like Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PlGF), Fibroblast Growth Factor (FGF) and Platelet-Derived Growth Factor (PDGF). When these factors reach their receptors on tip and stalk cells, specialized endothelial cells that emerge during angiogenesis to form sprouts, their activated pathway crosstalk and determine the sprouting of blood vessels towards the hypoxic regions of the tumor. The persistent and deregulated presence of pro-angiogenic factors marks a difference with the normal genesis of arteries, veins and capillaries, and causes the formation of abnormal, disorganized and chaotic vessels: tumor-induced vessels, in fact, are characterized by heterogenous size and caliber and irregular blood flow, which fail to provide oxygen and nutrients to all cancer cells, leaving hypoxic areas. The abnormal structure of the tumor vessels and the impaired blood perfusion block the infiltrations of immune cells, which creates an immunosuppressive tumor microenvironment, and interferes with drug delivery. Therefore, in a counterintuitive approach, targeting angiogenesis to restore a normal blood flow in the TME would permit the perfusion of drugs and immune cells in the microenvironment [60–62], going against the classical view about anti-angiogenesis approaches.

Bevacizumab, commercialized as Avastin and used to treat metastatic breast cancer, NSCLC, glioblastoma, renal cell carcinoma, ovarian cancer and cervical cancer, is an anti VEGF mAb (32335505). Its safety and efficacy in TNBC have been tested in phase II clinical trials NCT03577743 (RIBBON-2) and NCT06817525. While the first one showed positive results (22415477), the outcomes of the latter have not been published yet.

Other anti-angiogenic agents are under investigation. Lenvatinib (commercialized as Lenvima) is a powerful antiangiogenic agent, tyrosine kinase inhibitor, already in use to treat medullary, anaplastic thyroid, adenoid cystic and endometrial cancer [63]. The phase II clinical trial MK-7902-005/E7080-G000-224/LEAP-005 (NCT03797326) tested its efficacy and safety in combination with anti-PD-1 mAb Pembrolizumab on a cohort of patients with solid tumors, including TNBC, and demonstrated antitumor activity with a manageable safety profile [64].

Another tyrosine kinase inhibitor anti-angiogenic agent is anlotinib (commercial name FOCUS V, AL3818), already approved by China Food and Drug Administration (CFDA) for the treatment of NSCLC [65]. Its efficacy in combination with anti-PD-1 mAb sintilimab on TNBC is under investigation in the context of phase II clinical trial NeoSACT (NCT04877821). The combined treatment exhibited a very rapid response, with 96.6% of patients achieving tumor reduction by ³30% after just one cycle of treatment [66].

An innovative anti-angiogenic molecule is B1962, a bispecific antibody against PD-L1 and VEGF with a high anti-angiogenic activity, which is being studied in phase II clinical trial NCT06724263, after phase I demonstrated excellent safety and promising therapeutic effects.

Q: The last sentence in the section on CDK 4/6 inhibitors is incomplete and not comprehensible. The entire section needs to be reorganized.

We checked the text of the last sentence and we cannot detect incompleteness. We reformulated the text of the paragraph to increase clarity.

New text pg. 15

2.8. CDK 4/6 Inhibitors

Cyclin-dependent kinases (CDKs) are the primary enzymes regulating cell cycle progression. Their activation depends on binding with specific cyclins. Specifically, CDK 4/6 acts as a critical regulator of the G1/S transition. In this process, Cyclin D1 binds to CDK 4 and 6; once this complex is activated by CDK-activating kinases (CAKs), it phosphorylates the tumor suppressor Retinoblastoma protein (Rb). In its dephosphorylated state, Rb binds to and inhibits the E2F transcription factor. However, phosphorylation by CDK 4/6 releases E2F, which then promotes the expression of DNA synthesis genes and Cyclin E, driving the cell cycle forward. Dysregulation of this pathway causes the uncontrolled proliferation characteristic of cancer. Consequently, inhibiting CDK 4/6 can arrest the cell cycle in the G1 phase, effectively blocking tumor growth.

In TNBC, targeting this pathway is complicated by frequent Rb dysfunction, occurring in approximately 30% of cases [77–80]. While TNBC cell lines exhibit sensitivity to CDK 4/6 inhibition, they are generally less responsive than ER+ lines [81]. This partial resistance is often attributed to the loss of Rb (seen in 7–20% of TNBC) and the overexpression of Cyclin E [78]. Several CDK inhibitors are currently under clinical investigation for TNBC. Trilaciclib is a CDK 4/6 inhibitor primarily used to mitigate chemotherapy-induced bone marrow suppression [85]. Its efficacy in TNBC combination therapy was evaluated in Phase II (NCT05112536) and Phase III (PRESERVE 2, NCT04799249) trials. Phase II data suggested a positive correlation between favorable outcomes and higher levels of Tumor-Infiltrating Lymphocytes (TILs), specifically a high CD8+/T regulatory cell ratio. Phase III results are currently undergoing quality control review [82]. Although already FDA-approved for HR+ and HR+/HER2- breast cancer respectively [83, 84], Ribociclib (Kisqali) and Palbociclib (Ibrance) are being investigated for TNBC in the CHARGE (NCT04315233) and CAREGIVER (NCT05067530) trials. Emerging studies are also exploring CDK2 as a target. Two molecules, AVZO-021 (NCT05867251) and NKT3964 (NCT06586957), are currently in Phase I/II trials to assess their safety and pharmacokinetics in solid tumors, including TNBC. Notably, while four clinical trials involving CDK 4/6 inhibitors were recently terminated, these closures were not due to concerns regarding safety or efficacy.

Q: At the start of the Omics Approaches section, classification accuracies are not defined and therefore the cited statistics are meaningless.

R: The text was updated as follows (pg. 16):

To extract the most significant proteins and targets, we used the Elicit AI research assistant to perform a literature search and identify candidates based on semantic similarity to the query: “Among the deposited omics data, is it possible to identify unique combinations of markers for triple-negative breast cancer? Select also targets which are not known to be drugged,” across over 138 million academic papers indexed by Elicit, including Semantic Scholar and OpenAlex. The AI retrieved a list of potentially relevant proteins, which were then manually checked one by one to determine whether they had been previously studied in TNBC, with their relevance and novelty discussed individually. Additional proteins not retrieved by Elicit were incorporated through manual literature searches. The final table summarizes only those targets supported by explicit evidence in the primary literature.

Q: The conclusion of the section on Metabolic Vulnerabilities in TNBC is ambiguous. If systemic toxicity and off target effects limit the therapeutic use of metabolic inhibitors, how is this approach promising?

A: In red the explanation for our initial comment.

All in all, targeting metabolism in TNBC is a promising route for improved therapy, but systemic toxicity and off-target effect limit the use of this approach into the clinics, which requires further effort in improving targeting, selectivity and localization.

Q: The Soluble Factors section is confusing - are they or are they not potential therapeutic targets?

A: From the review text at pg. 27, l. 1027.

Therefore, soluble factors cannot currently be considered effective therapeutic targets but could rather be used as diagnostic or prognostic markers to monitor the progression of the disease. In this context, extracellular vesicles are also considered a promising diagnostic biomarker and a novel drug delivery system among the prospects for immunotherapy in TNBC [241].

We clearly state that soluble factors, at the moment, are more suitable as prognosis markers than as targets for therapy.