Epigenetic and 3D Genome Changes Drive Primary Trastuzumab Resistance in HER2+ Breast Cancer

Reviewer #1 (Public review):

Summary:

This study investigates epigenetic and three-dimensional chromatin alterations associated with primary trastuzumab resistance in HER2-positive breast cancer using integrated CUT&Tag, RNA-seq, and Micro-C analyses in JIMT1 (resistant) and SKBR3 (sensitive) cell models. The authors identify widespread remodeling of histone modification landscapes, chromatin compartment organization, and promoter-enhancer looping, highlighting SGK1 as a candidate epigenetically activated mediator associated with intrinsic resistance. The manuscript provides a technically solid and extensive multi-omic resource for the study of HER2-positive breast cancer resistance states.

Strengths:

The study integrates multiple state-of-the-art epigenomic and chromatin conformation approaches, including CUT&Tag, RNA-seq, and Micro-C, generating a comprehensive dataset that will likely be valuable to the field. The analyses are generally technically rigorous and well executed, and the manuscript is overall clearly written. The integration of chromatin architecture, enhancer activity, transcriptional regulation, and histone modification profiling provides an informative overview of large-scale epigenomic remodeling associated with resistant versus sensitive HER2-positive breast cancer states. The identification of SGK1-associated chromatin activation and enhancer rewiring is particularly interesting and supported by multiple orthogonal datasets.

The inclusion of both intrinsic and acquired trastuzumab resistance models also strengthens the study conceptually, even if the biological interpretation remains somewhat complex.

Weaknesses:

The major limitation of the study is that many of the central mechanistic conclusions remain largely correlative. Although coordinated changes in chromatin architecture, histone modifications, enhancer activity, and SGK1 expression are observed, direct evidence demonstrating that these epigenetic alterations causally drive SGK1 activation or trastuzumab resistance is currently lacking.

In addition, the interpretation of SGK1 as a broader trastuzumab-resistance driver is somewhat weakened by the analyses in the acquired resistant SKBR3_HR model, where SGK1-associated chromatin and transcriptional changes appear largely absent. This raises the possibility that SGK1 dependency may reflect a lineage- or model-specific vulnerability intrinsic to JIMT1 cells rather than a generalizable resistance mechanism.

The study also remains descriptive in several sections. Numerous chromatin interactions and compartment changes are cataloged without sufficient biological contextualization or mechanistic integration. As a result, parts of the manuscript currently read more as a comprehensive epigenomic profiling resource than a fully mechanistic study of resistance biology.

Finally, the translational impact is limited by the lack of patient-level validation linking SGK1 activation to trastuzumab response or clinical outcome in HER2-positive breast cancer cohorts.

Reviewer #2 (Public review):

Summary:

Duan, Hua et al. used CUT&Tag and Micro-C to investigate that in primary trastuzumab-resistant HER2+ breast cancer cells, promoter H3K4me3 rather than H3K27me3 is strongly correlated with transcriptional activity. Resistant cells also exhibited more abundant promoter-enhancer loops and enriched cohesin at loop anchors, accompanied by shifts in A/B compartment status. Through multi-omics integration, the authors identified SGK1 as a key gene showing elevated promoter H3K4me3 levels, enhancer activation, strengthened chromatin loops, and upregulated transcription in resistant cells, and validated SGK1 as a potential therapeutic target. These findings reveal the coordinated interplay between three-dimensional chromatin architecture and epigenetic modifications, offering important insights into trastuzumab resistance in HER2+ breast cancer.

Strengths:

Previous investigations into trastuzumab resistance have largely focused on genetic mutations or individual epigenetic modifications. In contrast, this study moves beyond genetic or single epigenetic views by integrating histone modifications and 3D chromatin architecture into a unified framework, proposing a synergistic model of promoter H3K4me3, enhancer activation, and chromatin looping that underlies non-genetic resistance. It provides a new conceptual basis for understanding non-genetic resistance mechanisms. Secondly, using high-resolution epigenomic and conformational mapping together with bidirectional in vitro and in vivo functional validation, it establishes a solid link between epigenetic changes and phenotypes, and demonstrates that SGK1 inhibition suppresses tumor growth in a xenograft model, revealing clear translational potential.

Weaknesses:

(1) All findings are based on a single pair of cell lines, JIMT1 and SKBR3, which does not allow exclusion of cell line‑specific effects. The authors did not examine SGK1 expression levels, promoter H3K4me3 status, or relevant chromatin loops in tumor tissues from patients with clinical trastuzumab resistance. Consequently, whether the conclusions can be extrapolated to actual patient populations remains unclear, which limits the clinical relevance of the findings. It is recommended that the authors directly validate the key findings using tumor samples from patients with clinical trastuzumab resistance or analyze the correlation between SGK1 expression levels and disease-free survival or pathological complete response using data from public databases for HER2+ breast cancer patients, which would help address the current limitation of lacking clinical sample validation and the uncertainty regarding the association of SGK1 with patient prognosis and treatment response.

(2) In the Discussion, the authors propose that SGK1 may assume the role of AKT to sustain mTOR activation, thereby bypassing the dependence on HER2 signaling following trastuzumab inhibition. Although this hypothesis is supported by published literature, the present study provides no direct signaling evidence, such as examining phosphorylation changes of SGK1, AKT, mTOR, or their downstream effectors.