Joint Mapping of Chromatin Accessibility and Targeted Proteomics in HER2-expressing Breast Cancer Systems
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HER2 proteoforms promote therapeutic resistance and aggressiveness in HER2-positive breast cancer, yet their epigenetic consequences remain poorly defined. Here, we establish EpiBlot, a joint assay incorporating a customized plateATAC-seq workflow that minimizes sample inputs with single-cell western blotting to concurrently profile chromatin accessibility with protein and proteoform expression in breast cancer MCF7 cells. Engineered MCF7 cells expressing HER2 proteoforms - full-length p185HER2 or truncated 611-CTF - were evaluated on the impact of such proteoforms after lapatinib or doxorubicin exposure. Expression of 611-CTF elicits pervasive chromatin remodeling, whereas p185HER2 provokes only modest accessibility shifts under the same treatments. Lapatinib treatment produces limited global effects but unmasks proteoform-specific responses, while treatment with doxorubicin drives extensive genome-wide accessibility changes. Concordance between chromatin accessibility and protein abundance is moderate, underscoring complex regulatory coupling. Extending this dual-modality approach to HER2-low patient-derived organoids uncovers distinct chromatin states and reveals a subpopulation of triple-negative breast-cancer cells expressing truncated HER2 proteoforms. These findings highlight the value of multimodal profiling with proteoform identification for dissecting tumor heterogeneity and therapeutic response in HER2-positive breast cancer.
Highlights
To evaluate the impact of chromatin accessibility on HER2 proteoform expression, we introduce EpiBlot, a novel workflow combining a plateATAC-seq assay (i.e., mini-96 well-plate requiring 5-500 nuclei) with single-cell Western blot.
Increased chromatin accessibility and significantly larger nuclei were detected in a breast cancer cell line (MCF7) engineered to express the truncated HER2 proteoform (611-CTF) versus a line expressing only the full-length HER2 protein.
Lapatinib-targeted treatment did not induce widespread changes in chromatin accessibility.
Doxorubicin reshapes chromatin accessibility in HER2-proteoform cell lines, producing both gains and losses across the genome.
Chromatin accessibility and protein abundance are moderately correlated, with a wide range of protein abundance measured across single cells.
HER2-low patient-derived breast cancer organoids exhibit decreased accessibility in the ERBB2 region despite increased HER2 protein expression, as compared to normal breast cells.
