Author response:
Reviewer #1 (Public review):
Summary:
The study by Fernandes et al used multiple types of experiments to study the increased metastatic activity conferred by p95-HER2 and presented evidence that biased downstream signaling was engaged by p95-HER2. Further studies demonstrated crucial phosphorylation sites and downstream transcription targets. Overall, this study will contribute to increased understanding of HER2 biology and breast cancer metastasis.
Strengths:
Multiple types of experiments to assess the increased invasion property by p95-HER2. Solid evidence was provided that p95-HER2 elicits a different transcription program, enhancing EMT. Convincing results were achieved in knockdown studies on crucial genes such as MRTFA and TGFB1I1.
We thank the reviewer for this positive feedback on our manuscript.
Weaknesses:
To enhance the quality of this study, several points need to be addressed.
(1) First, as the background to this study, the authors stated in the introduction that different isoforms of HER2 lead to different invasive properties. Is this conclusion based on clinical observation or animal models? This needs to be specified to help readers grasp the importance of the paper.
We thank the reviewer for bringing this to our attention; we will add additional language to emphasize and explain prior findings referenced in the introduction establishing HER2 isoforms’ different invasive properties. The increased invasiveness of both d16 and p95 isoforms of HER2 has been established both in animal models and in clinical studies (1-6). We summarize these studies below:
(1) Expression of the delta16 splice variant isoform has been shown in human cell lines to be more oncogenic than the WT HER2 isoform (7), and associated with different transcriptional profile including increased expression of cancer metastasis and cancer stem cell-related genes (4). Clinically, patients with d16HER2-positive breast cancer have higher rates of lymph node metastases than those with d16HER2-negative breast cancer (8). Castagnoli et al (9) provides an overview of preclinical and clinical data on d16 in HER2+ cancers.
(2) The p95 isoform of HER2 is associated with increased metastasis (2) and worse outcomes (1, 10) in HER2+ breast cancer patients. Preclinical models show that p95-HER2 (also known as 611-CTF) activates a transcriptional program that promotes invasion and metastasis (3), part of the basis for our interest in HER2 isoform biology.
Moreover, our group has previously detailed different tumor growth and dissemination characteristics of WT, d16, and p95 HER2 isoforms in our HER2 Crainbow mouse model which expresses all 3 of these isoforms in the mammary epithelium (5).
Both d16 and p95 tumors in Her2Crainbow mice have the ability to progress to overt metastasis, unlike WT HER2 in this model (6). During early stages of malignant transformation prior to palpable tumor formation, p95 cells disseminated to the lung were 5x more numerous than d16 cells in the lung (despite more proliferation of d16 cells in the mammary gland at that timepoint). This is the foundation on which we built this current work, to study what could promote these different proliferation, dissemination, and metastasis behaviors in different HER2 isoforms.
(2) Second, this study is mostly performed on three cell lines generated from three tumor models, WT, d16, and p95. Were multiple cell lines generated from the same tumor model (e.g., p95), and how similar are these cell lines if they are from the same tumor model?
Our lab has generated a number of tumor cell lines derived from individual tumors developed in different HER2 Crainbow mice. We hypothesize, based on preliminary data in these cell lines as well as our previously reported data examining phenotypes of WT, d16, and p95 tumors (5, 6), that different clones will share core features such as invasive properties. In order to strengthen the generalizability of our conclusions, we intend to perform transwell assays, matrix degradation assays, and western blots to examine expression of Mrtfa and Tgfb1i1 in additional HER2 Crainbow-derived cell lines to validate if our findings hold true across cell lines derived from different tumors.
(3) Third, Figure 2 presented data from a very interesting experiment. However, the overall conclusion of this experiment is hard to grasp from the text.
We thank the reviewer for this comment. We will revise the text accompanying Figure 2 to provide further detail on the interpretation of the PCA plots and clarify the overall conclusions by adding the following text:
Vectors indicate variable loadings, where vector direction corresponds to increasing values of the variable and vector length reflects the magnitude of the variable’s contribution to the displayed principal components. For example, HCR-WT populations cluster toward lower values of PC1 and higher values PC2, notably in the direction of higher self-correlation indicating an association with greater spatial clustering. HCR-16 and HCR-95 cells cluster similarly and are driven by their similarity in speed, MSD, persistence, and individual behavior (low self-correlation and high proportions of cells with zero neighbors), whereas HCR-WT is distinct for its collective cell behavior (high self-correlation) (Figure 2E). This separation indicates potentially distinct kinematic and spatial organization compared to HCR-16 and HCR-95 cells.
Reviewer #2 (Public review):
Summary:
This is a significant study with implications for signal transduction, breast cancer, and metastasis research. The study uses cell lines from the innovative HER2BOW model mouse to compare and contrast properties of mouse mammary tumors induced by wt HER2, dex16 HER2, and p95 HER2. The linkage between p95 Y1139 and TGFB1I1, as well as MRTFA, is very interesting. The study involves mechanistic approaches to distinguish signaling from dex16 HER2 and p95 HER2 that will be well received by the field.
Strengths:
The study describes the development of novel cell lines for mouse mammary tumors induced by different forms of HER2. In vitro analysis includes a wide variety of assays, including high-resolution fluorescent imaging and tracing of cell movement/ECM degradation, and single-cell RNA-seq analysis. Several approaches are used to confirm p95 HER2 pY1139-induced signaling through nuclear MRTFA and TGFB1I1. shRNA and CRISPR/Cas9 were used to confirm signaling through this pathway.
We thank the reviewer for these positive comments.
Weaknesses:
The authors have only described one cell line for each HER2 form, a weakness which they acknowledge. The fact that these lines mimic characteristics that were described in their previous work on tumors induced by wt HER2 vs. dex16 HER2 vs. p95 HER2 is, however, supportive of their conclusions. In addition, the specific connection between p95 and nuclear MRTFA is confirmed in HEK cells. The manuscript could be improved with some discussion on how p95 p-Y1139 is likely to signal (what binds to this site?)
We thank the reviewer for this assessment, and we agree that adding additional cell lines derived from different HER2 Crainbow mouse tumors would strengthen the conclusions drawn in this manuscript. As mentioned in response to Reviewer #1, we intend to perform transwell assays, matrix degradation assays, and western blots to examine expression of Mrtfa and Tgfb1i1 in additional HER2 Crainbow-derived cell lines to validate if our findings hold true across cell lines derived from different tumors.
With regards to p95 p-Y1139 signaling, previous work by Dankort et al. (11, 12) established that tyrosine autophosphorylation site Y1144 on rat neu (equivalent to Y1139 in human HER2) promoted lung metastasis in animal models, and that this site was necessary and sufficient for direct binding of Grb2.
We intend to clarify and discuss links between Y1139 on p95 HER2 and the nuclear translocation of MRTFA.
Reviewer #3 (Public review):
Summary:
The study aims to explain why different HER2 isoforms produce distinct cancer-cell phenotypes, with a particular focus on the poorly understood mechanisms by which p95-HER2 promotes the invasive and metastatic behaviors of HER2-positive breast cancer. The authors propose that functional selectivity at the HER2 receptor level, particularly signaling through Y1139, preferentially engages an MRTFA-TGFB1I1 transcriptional/cytoskeletal program, thereby shifting p95-HER2-expressing cells toward individual motility, matrix degradation, invasion, and metastasis rather than simply proliferation.
Strengths:
A major strength is the multi-level experimental strategy. Rather than relying on a single invasion assay, the authors integrate phenotypic profiling, single-cell RNA-seq, imaging of MRTFA nuclear localization, protein validation, cytoskeletal analysis, matrix-degradation assays, transwell invasion, 3D spheroid/organoid assays, and orthotopic transplantation. This provides relatively coherent evidence that p95-HER2 cells occupy a distinct biological state characterized by individual motility and invasion, accompanied by a myogenic/contractile transcriptional program and increased MRTFA nuclear localization. Another strong aspect is the attempt to connect the downstream phenotype to a specific HER2 phosphorylation site. The authors identify Y1139 as a candidate determinant of the "go" phenotype and show that mutation of Y1139 to phenylalanine eliminates MRTFA translocation as well as the associated motility and invasion phenotypes
We thank the reviewer for these positive comments on our manuscript.
Weaknesses:
The main limitation is that the evidence for the proposed signaling mechanism is stronger for functional dependence than for direct molecular mechanism. The data establish that Y1139, MRTFA, and TGFB1I1 are required for the p95-HER2 invasive phenotype, but they do not appear to fully establish how phosphorylation at Y1139 mechanistically couples p95-HER2 to MRTFA activation. In particular, a direct biochemical link between Y1139 and the machinery controlling actin polymerization/MRTFA nuclear translocation would make the proposed pathway substantially more convincing.
We thank the reviewer for these insightful comments. We agree that establishing a mechanistic link between p95 Y1139 and MRTFA nuclear translocation would strengthen the conclusions in this study. As such, we intend to perform Rho activation assays and western blotting for proteins in HER2 signaling cascades to explore the signaling link between p95 Y1139 and MRTFA. Additionally, we plan to treat cells with RhoA/ROCK inhibitors to explore our hypothesis that Y1139 acts through RhoA activation to ultimately alter MRTFA translocation.
A second limitation is the model system used in the study. Much of the mechanistic work relies on three particular engineered HER2 isoform-expressing cell lines, while the in vivo experiments use NOD/SCID mice. This is useful for testing tumor-cell intrinsic invasion and metastasis, but it limits conclusions about the generality of the functional regulation across different cell contexts, and also limits the understanding of how the pathway operates in the intact immune microenvironment. This is especially relevant because the manuscript itself places p95-HER2 in the broader context of immune evasion and notes that MRTFA can regulate PD-L1; however, the current models cannot adequately establish whether the proposed pathway contributes to immune-mediated metastatic progression in vivo.
We also acknowledge the limitations of relying on mostly in vitro assays and in vivo experiments in an immunodeficient environment, and will more explicitly address this in our discussion. The interplay between p95-HER2 and the immune system is being explored in a parallel project that will be published separately in the future; the focus of the current manuscript is the cell-intrinsic signaling mechanisms that contribute to p95 invasiveness. Points about immune evasion that were brought up in the introduction will be moved to the discussion section, to make it clear that the current study does not address the immunocompetent environment but that we wish to further investigate the results we report in this manuscript in that context as well.
Finally, the distinction between "go" and "grow" signaling is conceptually attractive, but the extent to which it represents a general property of HER2-positive human breast cancers remains less certain. The study provides strong experimental evidence for functional selectivity in the models examined, but additional validation in independent patient-derived models and clinically relevant tumors would be needed before generalizing this mechanism across HER2-positive disease.
With respect to validating findings in human samples, we intend to utilize human HER2+ breast cancer cell lines and/or patient-derived xenografts to investigate the extent to which our described mechanism is relevant in human HER2+ breast cancer.
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