Neddylation of HER2 Inhibits its Protein Degradation and promotes Breast Cancer Progression

The study addresses how post-translational modifications other than phosphorylation and ubiquitination regulate HER2 stability and function in HER2-positive breast cancer. HER2 is overexpressed in approximately 25% of breast cancers and is associated with aggressive disease and poor prognosis. Although HER2-targeted therapies like trastuzumab have improved outcomes, primary and acquired resistance remain major challenges. The ubiquitin–proteasome system (UPS) is known to regulate HER2 degradation, but the impact of crosstalk with other PTMs is unclear. The authors hypothesize that neddylation, a ubiquitin-like modification mediated by NEDD8 and its activating enzyme NAE1, modulates HER2 stability and oncogenic signaling, thereby contributing to breast cancer progression and therapeutic response.

- Patient specimens and TMA: Immunohistochemistry (IHC) for HER2, NEDD8, and NAE1 was performed on breast cancer tissue microarrays (129 cancers, 77 adjacent tissues). Expression correlations and survival analyses (Kaplan–Meier) were conducted using public datasets (bc-GenExMiner, survival transcriptome datasets) and tissue IHC scoring. - Cell lines and culture: HER2-positive BT474 and SK-BR3 breast cancer cells, and HEK293T cells were cultured under standard conditions (RPMI-1640, McCoy’s 5A, or DMEM with 10% FBS). - Neddylation/ubiquitination modulation: Pharmacologic inhibition of neddylation with MLN4924 (pevonedistat); proteasome inhibition with MG132; protein synthesis inhibition with cycloheximide (CHX, 50 μg/ml). Genetic depletion using siRNAs targeting NEDD8 and NAE1 (two independent siRNAs each). - Plasmids and mutagenesis: Overexpression of NEDD8 (His-tagged), HER2 (FLAG-tagged), HER2 mutant Y1112F, and HER2 truncations (1–653 aa; 654–1255 aa; 720–987 aa; 988–1255 aa). Transfections performed with Lipofectamine 3000 or RNAiMAX. - Protein assays: Western blotting for HER2, NEDD8-cullins, K48-linked ubiquitin, and cell-cycle/apoptosis markers (CDK1, p21, p27, PARP, Bcl-2). Co-immunoprecipitation (Co-IP) using anti-HER2, anti-NEDD8, or anti-NAE1 to examine interactions and modification states. - In vitro neddylation: Purified HER2 was incubated with NEDD8 pathway components (E1, E2, NEDD8, ATP) to detect neddylation by immunoblot. - Confocal immunofluorescence: Localization and co-localization of HER2 with His-NEDD8; membrane HER2 levels with/without MLN4924 or NEDD8 knockdown. - mRNA analysis: RT-qPCR for HER2 and GAPDH to assess transcriptional effects. - Protein stability: CHX chase assays to determine HER2 half-life after MLN4924 treatment or NEDD8 knockdown. - Proliferation and viability: MTS assays (48–96 h), colony formation assays, EdU incorporation assays. - Cell cycle and apoptosis: Flow cytometry for PI staining (cell cycle) and Annexin V-FITC/PI staining (apoptosis) after MLN4924 or siRNA treatments. - Bioinformatics: LinkedOmics multi-omics analyses to identify genes associated with NEDD8 and ERBB2; KEGG pathway enrichment of 504 overlapping genes. - Statistics: t-test or ANOVA for group comparisons, Kaplan–Meier survival analysis, Pearson correlation for expression correlations; significance at P<0.05.

- Clinical association: NEDD8 mRNA is elevated in breast tumors versus healthy and adjacent tissues; higher NEDD8 associates with worse overall survival. IHC on TMAs shows high HER2 and NEDD8 in tumors with a positive correlation between their protein levels. NEDD8 is an independent poor prognostic factor. - HER2 is regulated post-transcriptionally by neddylation: NEDD8 knockdown or MLN4924 treatment reduces HER2 protein without altering HER2 mRNA, indicating post-translational control. MLN4924 and NEDD8 depletion shorten HER2 protein half-life (CHX chase). - Direct interaction and neddylation of HER2: Co-IP shows NEDD8-cullins and NAE1 interact with HER2 in BT474 and SK-BR3. His-NEDD8 co-localizes with HER2 by confocal microscopy. HER2 acquires higher molecular weight species consistent with neddylation; MLN4924 blocks this. In vitro, purified HER2 is neddylated in an E1/E2/NEDD8/ATP-dependent manner. Multiple HER2 domains (extracellular and cytoplasmic truncations) bind NEDD8. - Crosstalk with ubiquitination: Proteasome inhibitor MG132 rescues MLN4924-induced HER2 degradation, implicating the UPS. Overexpressed NEDD8 decreases K48-linked polyubiquitination of HER2, whereas MLN4924 or NEDD8 siRNA increases HER2 K48 polyubiquitination. The HER2 Y1112F mutant reduces NEDD8 chain association and diminishes K48-linked polyubiquitination upon neddylation inhibition, indicating Y1112 is needed for this regulation. - Functional impact on cancer cells: MLN4924 decreases viability, colony formation, and EdU incorporation in HER2+ cells; induces G2/M arrest (increased p27, decreased CDK1) and apoptosis (Annexin V, PARP cleavage/Bcl-2 changes). NEDD8 knockdown similarly suppresses proliferation. - Therapeutic synergy: MLN4924 synergizes with trastuzumab to further inhibit colony formation and proliferation, enhance cell-cycle arrest, and modulate HER2/CDK1/p21 levels compared with either agent alone. - NAE1 significance: NAE1 is upregulated in HER2+ breast cancer in public datasets and is associated with worse survival. IHC shows higher NAE1 in tumors and positive correlation with HER2. NAE1 knockdown reduces HER2 protein and cell viability. - Dependency on HER2: LinkedOmics identifies 504 genes associated with NEDD8 in HER2+ breast cancer enriched in ErbB2, cell cycle, and breast cancer pathways. NEDD8 overexpression enhances proliferation; HER2 overexpression accelerates growth, reduces p27, and counteracts MLN4924-induced G2/M arrest and p27 upregulation, supporting HER2 dependence of neddylation-driven progression.

The findings demonstrate that HER2 is a bona fide substrate of the neddylation machinery in HER2-positive breast cancer. Neddylation stabilizes HER2 by suppressing its K48-linked polyubiquitination and proteasomal degradation, thereby sustaining oncogenic signaling. Elevated NEDD8/NAE1 correlating with HER2 expression and poor outcomes supports clinical relevance. Mechanistically, direct interactions of NEDD8-cullins and NAE1 with HER2, in vitro neddylation of purified HER2, and modulation of HER2 stability by MLN4924 or NEDD8/NAE1 knockdown establish causality. The observation that HER2 Y1112 is required for neddylation-dependent regulation of ubiquitination links known ubiquitin sites with neddylation crosstalk. Functionally, neddylation inhibition triggers G2/M arrest and apoptosis and enhances trastuzumab efficacy, indicating that targeting the neddylation axis could overcome or delay resistance to HER2-directed therapy. The dependence of MLN4924 effects on HER2 levels underscores HER2 as a key effector of neddylation-mediated tumor progression.

This study identifies neddylation as a novel post-translational modification controlling HER2 stability and activity in breast cancer. NEDD8/NAE1-mediated neddylation inhibits K48-linked polyubiquitination and proteasomal degradation of HER2, promoting proliferation and survival of HER2+ cells. Pharmacologic inhibition of neddylation with MLN4924 reduces HER2 protein, induces cell-cycle arrest and apoptosis, and synergizes with trastuzumab. Clinically, NEDD8 and NAE1 are upregulated and correlate with HER2 expression and poor prognosis. Future work should delineate the precise enzymology and structural determinants of HER2 neddylation, validate efficacy and safety of neddylation inhibitors alone and in combination with HER2-directed therapies in animal models and clinical settings, and investigate the impact of HER2 mutations (e.g., Y1112) on neddylation-ubiquitination crosstalk.

- The mechanistic cooperation and sequence of events between NEDD8 and NAE1 in HER2 modification require further elucidation. - While in vitro and cell-based evidence is strong, in vivo validation (animal models) of MLN4924 efficacy and combination with trastuzumab was not presented and is proposed for future studies. - Although HER2 Y1112 is implicated, comprehensive mapping of HER2 neddylation/ubiquitination sites and the responsible E3 ligases was not fully defined.