Multi‐omics Analysis Reveals Comprehensive Aberrant Protein and Phosphorylation Characteristics in Breast Cancer and Paired Metastatic Lymph Nodes

Breast cancer is the most frequently diagnosed cancer, with metastasis accounting for the majority of cancer-related deaths. The mechanisms of early-stage breast cancer metastasis to regional immune sites like lymph nodes remain elusive. Here, we performed an in-depth proteomic and phosphoproteomic analysis of a substantial series of breast cancer samples, alongside genomic and transcriptomic evaluations. This cohort encompasses 195 specimens: 65 primary breast tumors, their corresponding normal tissues, and metastatic axillary lymph nodes. We offer an overview of the molecular alterations at the transcriptomic, proteomic, and phosphoproteomic levels during lymph node metastasis. Notably, the findings indicate that regional lymph node metastasis is primarily influenced by proteomic and phosphoproteomic alterations, rather than genomic or transcriptomic changes. Moreover, alternative splicing plays a substantial role in modulating protein and phosphorylation profiles. The key phosphorylation sites, including MARCKSL1-S104 and FKBP15-S320, as well as the upstream kinase PKC, were identified as playing crucial roles in breast cancer lymph node metastasis. Targeted intervention of the kinase PRKCB has been shown to effectively suppress the proliferation and metastasis of breast cancer tumor cells. Immune profiling analysis reveals a positive correlation between phosphorylation of MARCKSL1 and FKBP15 with immune checkpoints PD-1, PD-L1, and CTLA4, suggesting that metastasis may also be mediated through immune evasion mechanisms. This study systematically characterizes the molecular landscape and features of primary breast tumors and their matched metastatic lymph nodes. These insights enhance our understanding of early-stage breast cancer metastasis and may pave the way for improved diagnostic tools and targeted therapeutic strategies.