Targeting Growth Hormone Receptor to Overcome Therapy Resistance in Non-Small Cell Lung Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Ahmad et al. have examined the role of GH and GHR signaling in therapy resistance and tumor aggressiveness in NSCLC demonstrating that high GHR expression was associated with poorer overall survival and with gene signatures related to drug resistance. The experiments in vitro have shown that GH promotes proliferation, chemoresistance to cisplatin and doxorubicin, migration, invasion, and upregulation of ABC transporters and EMT markers. The pegvisomant has reversed these effects and sensitized cells to chemotherapy, pointing towards GHR antagonism as a potential strategy to overcome therapy resistance in NSCLC.

This study has clear mechanistic framework and combines large patient transcriptomic datasets with functional assays in four NSCLC cell lines. Manuscript is clear and well written. However, there are several points that should be considered and adequately addressed:

 

1. The manuscript would benefit from multivariable Cox regression including standard clinical covariates (e.g., stage, age, sex, smoking status…) to test whether GHR remains an independent prognostic factor. This would substantially strengthen the prognostic claims derived from the transcriptomic datasets
2. The core conclusion that GHR signaling promotes aggressive, therapy-resistant phenotypes in NSCLC and that GHR antagonism may sensitize tumors to chemotherapy is well supported by the presented in vitro However, the translational claims should be phrased more cautiously. In the current study, there is no in vivo model demonstrating that the combination of pegvisomant with cisplatin and/or doxorubicin improves tumor control, survival, or tolerability compared with chemotherapy alone. Without such in vivo and further mechanistic validation, it is too strong to claim that GHR antagonism will improve clinical outcomes or that the identified gene signatures are causal mediators of resistance
3. The authors should discuss inter-cell line heterogeneity in more detail and comment explicitly on why some cell lines show limited or no changes in certain ABC transporters or EMT markers. It would be helpful to consider whether these differences relate to underlying mutational, molecular, or phenotypic characteristics of the individual NSCLC models
4. The limitations section should be expanded to more clearly acknowledge the in vitro nature of the work and the absence of in vivo chemotherapy data. The authors should also outline specific next steps for clinical translation, such as in vivo validation of pegvisomant/chemotherapy combinations and deeper mechanistic dissection of downstream pathways.

 

 

Author Response

We appreciate reviewers for their time and constructive assessment. Each concern has been addressed objectively and point by point below, with our responses following each comment.

Comment 1:The manuscript would benefit from multivariable Cox regression including standard clinical covariates (e.g., stage, age, sex, smoking status…) to test whether GHR remains an independent prognostic factor. This would substantially strengthen the prognostic claims derived from the transcriptomic datasets.

Response 1:

We thank the reviewer for this important recommendation. We agree that multivariable Cox regression incorporating standard clinical covariates would strengthen the prognostic assessment of GHR.

However, in the publicly available transcriptomic datasets analyzed in this study, complete and clinical annotation (particularly Age, smoking status and tumor stage) was not available for a sufficient number of samples to permit a robust multivariable analysis. To address this limitation, we now explicitly acknowledge this issue in the Discussion section and clarify that the current findings establish a strong univariable association between GHR expression and survival, which warrants further validation in clinically well-annotated cohorts.

We emphasize that future studies incorporating comprehensive clinical variables are needed to definitively establish GHR as an independent prognostic biomarker.

Comment 2: The core conclusion that GHR signaling promotes aggressive, therapy-resistant phenotypes in NSCLC and that GHR antagonism may sensitize tumors to chemotherapy is well supported by the presented in vitro However, the translational claims should be phrased more cautiously. In the current study, there is no in vivo model demonstrating that the combination of pegvisomant with cisplatin and/or doxorubicin improves tumor control, survival, or tolerability compared with chemotherapy alone. Without such in vivo and further mechanistic validation, it is too strong to claim that GHR antagonism will improve clinical outcomes or that the identified gene signatures are causal mediators of resistance.

Response 2:
We thank the reviewer for this important and insightful comment. We agree that, in the absence of in vivo validation and additional mechanistic studies, our conclusions should be framed more cautiously with respect to translational and clinical implications. We have revised the manuscript to temper the translational claims throughout the Abstract, and Discussion sections. Specifically, we now emphasize that our findings demonstrate a proof-of-concept role for GHR signaling in promoting aggressive and therapy-resistant phenotypes in NSCLC in vitro, rather than asserting direct clinical benefit. We have also clarified that the observed sensitization to chemotherapy upon GHR antagonism is limited to in vitro models and should be interpreted as hypothesis-generating.

Comment 3: The authors should discuss inter-cell line heterogeneity in more detail and comment explicitly on why some cell lines show limited or no changes in certain ABC transporters or EMT markers. It would be helpful to consider whether these differences relate to
underlying mutational, molecular, or phenotypic characteristics of the individual NSCLC models.

Response 3: We thank the reviewer for this thoughtful and important comment. We agree that inter–cell line heterogeneity is a critical consideration when interpreting our findings.
In response, we have expanded the Discussion section to explicitly address the observed variability in ABC transporter and EMT marker regulation across NSCLC cell lines. We now emphasize that NSCLC is a highly heterogeneous disease and that differential responses to GHR antagonism likely reflect intrinsic molecular and phenotypic differences among the cell models used. Specifically, the variability may be influenced by differences in oncogenic driver mutations (e.g., KRAS, TP53 status), baseline epithelial versus mesenchymal states, and distinct dependencies on GHR/JAK2/STAT5 signaling across cell lines. In addition, we note that some ABC transporters and EMT markers may already be maximally or minimally expressed in certain models, thereby limiting their dynamic range of modulation following GHR inhibition. For example, in H1299 cells, vimentin does not show any changes following GHR inhibition because it reflects a pre-existing resistant phenotype rather than insensitivity to GHR signaling. TP53-null status is associated with enhanced EMT and drug resistance. Similarly, H1734 is KRAS-mutant with partial EMT characteristics and also exhibit non-uniform ABC transporter modulation. It is important to note that partial EMT states often show pathway-specific rather than global transcriptional changes.

Comment 4: The limitations section should be expanded to more clearly acknowledge the in vitro nature of the work and the absence of in vivo chemotherapy data. The authors should also outline specific next steps for clinical translation, such as in vivo validation of pegvisomant/chemotherapy combinations and deeper mechanistic dissection of downstream pathways

Response 4: We thank the reviewer for this constructive suggestion. We agree that the limitations and future directions of the study should be more explicitly articulated. We have expanded the limitations section to clearly acknowledge that the present study is restricted to in vitro NSCLC models and does not include in vivo evaluation of pegvisomant in combination with chemotherapy, including effects on tumor control, survival, or treatment tolerability. We now explicitly state that these limitations preclude definitive conclusions regarding translational or clinical efficacy.

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Altogether the work presented in this manuscript by Ahmad et al summerizes timely contribution with interesting, important and novel data. It provides also new insight into the pathogenesis and potential novel therapy of NSCLC.

The topic of the manuscript is especially highly valued since it is focused on human lung cancer that is key important since lung cancer cancer remains one of the leading causes of cancer-related death in the world despite new advances in therapy.

Growth hormone (GH) and even growth hormone-releaseing hormone (GH-RH) functions and actions have been investigated in the past decade in various human cancers, but the potential  role of GH in lung cancer, particularly non-small cell lung cancer (NSCLC), remains poorly defined.

The manuscript contains a valuable collection of data which were generated from the series of cell culture experiments and from studies on human tissue samples. The research data collected both form cell culture experiments and human tissues further increases its value.

 In general, the manuscript is well written and provides a lot of interesting data supported by several experiments and methods. In the well structured and designed study various state-of-the art methods were used. The introduction and background sections are reasonable, logically structured and easily readable. The manuscript follows the most recent literature and implies feasible and updated information on GH and NSCLC reasearch. The results presented here do not only bring us one step closer to further clarify the mechanisms of GH action in lung cancer, but their findings also provide us with a promising potential target (GH receptor) for future treatment of this malignancy. In addition, the manuscript provides further support for novel therapeutic approaches and opens new avenues in drug development and therapeutic strategies of the disease.

In conclusion the manuscript is undoubtedly interesting and well written and the conceptual advance is pretty high thus it is a welcome addition to the literature.

I would highly support to accepting the manuscript.

Author Response

Comment: 

Altogether the work presented in this manuscript by Ahmad et al summerizes timely contribution with interesting, important and novel data. It provides also new insight into the pathogenesis and potential novel therapy of NSCLC.

The topic of the manuscript is especially highly valued since it is focused on human lung cancer that is key important since lung cancer cancer remains one of the leading causes of cancer-related death in the world despite new advances in therapy.

Growth hormone (GH) and even growth hormone-releaseing hormone (GH-RH) functions and actions have been investigated in the past decade in various human cancers, but the potential  role of GH in lung cancer, particularly non-small cell lung cancer (NSCLC), remains poorly defined.

The manuscript contains a valuable collection of data which were generated from the series of cell culture experiments and from studies on human tissue samples. The research data collected both form cell culture experiments and human tissues further increases its value.

 In general, the manuscript is well written and provides a lot of interesting data supported by several experiments and methods. In the well structured and designed study various state-of-the art methods were used. The introduction and background sections are reasonable, logically structured and easily readable. The manuscript follows the most recent literature and implies feasible and updated information on GH and NSCLC reasearch. The results presented here do not only bring us one step closer to further clarify the mechanisms of GH action in lung cancer, but their findings also provide us with a promising potential target (GH receptor) for future treatment of this malignancy. In addition, the manuscript provides further support for novel therapeutic approaches and opens new avenues in drug development and therapeutic strategies of the disease.

In conclusion the manuscript is undoubtedly interesting and well written and the conceptual advance is pretty high thus it is a welcome addition to the literature.

I would highly support to accepting the manuscript.

Response: We thank the reviewer for their careful assessment of our work and for their positive feedback. We are pleased that the reviewer has no further suggestions and is satisfied with the current version of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no further comments.