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

Breast cancer is a leading cause of female cancer mortality worldwide, exhibiting heterogeneity with subtypes including HER2-positive (HER2+), accounting for 25% of cases. HER2 overexpression and gene amplification are associated with poor prognosis. While HER2-targeted therapy (e.g., trastuzumab) has improved survival, resistance remains a major clinical challenge. Developing new molecular targets is crucial. HER2, encoded by the oncogene ERBB2, belongs to the epidermal growth factor receptor (EGFR) family, functioning as a receptor tyrosine kinase (RTK). Upon growth factor binding, it phosphorylates, activating downstream signaling cascades involved in cell proliferation and survival. Regulating HER2 phosphorylation is a therapeutic strategy. Protein degradation through the ubiquitin proteasome system (UPS) is another promising approach. The UPS is a post-translational modification (PTM) central to protein stability. Besides ubiquitination, various PTMs, including phosphorylation, sumoylation, neddylation, acetylation, glycosylation, and methylation, exist, often cross-talking with ubiquitination. While HER2 stability regulation by the UPS is known, the influence of other PTMs on HER2 ubiquitination remains unclear. Neddylation, the attachment of ubiquitin-like protein NEDD8 to target substrates, is a novel PTM similar to ubiquitination, primarily activating Cullin-RING ubiquitin ligases but with growing identified targets. Neddylation is reversible and its inhibitor, pevonedistat/MLN4924, is in clinical trials. Aberrant neddylation upregulation is linked to diseases like cancer, making it a therapeutic target. This study investigates the role of HER2 neddylation in breast cancer.

The introduction thoroughly reviews the existing literature on breast cancer subtypes, the role of HER2 in breast cancer progression and prognosis, the current therapeutic strategies targeting HER2, the limitations of current therapies due to resistance mechanisms, and the importance of identifying new therapeutic targets. It also discusses the ubiquitin proteasome system (UPS), various protein post-translational modifications (PTMs) involved in regulating protein stability and function, and the emerging role of neddylation in cancer development. The authors highlight the lack of understanding regarding the interplay between different PTMs, particularly the influence of neddylation on HER2 ubiquitination and degradation.

The study employed a multi-faceted approach combining bioinformatic analyses, in vitro and in vivo experiments, and clinical data analysis. Initially, they used multi-omics analysis (LinkedOmics) to identify genes associated with NEDD8, revealing ErbB2 (HER2) as a potential candidate. Next, they analyzed published microarray datasets comparing NEDD8 expression in healthy, breast cancer tumor, and adjacent tissues, along with survival analysis. Immunohistochemistry was performed on breast cancer patient samples to evaluate the expression of NEDD8 and HER2 proteins and their correlation. RNA interference (RNAi) was used to silence NEDD8 expression in HER2-positive breast cancer cell lines (BT474 and SK-BR3), and the effects on HER2 protein expression were assessed via western blot. The neddylation inhibitor MLN4924 was also used to assess its impact on HER2 protein expression and mRNA levels. To study the role of NEDD8 in HER2 protein stability, cycloheximide (CHX) chase assays were conducted to measure HER2 protein half-life. Immunofluorescence was used to examine the subcellular localization of HER2. Co-immunoprecipitation (Co-IP) assays were performed to investigate the interaction between NEDD8-cullins and HER2. To further study the mechanism, they generated HER2 truncated mutants to pinpoint the NEDD8 interaction sites. The role of neddylation in HER2 ubiquitination was investigated using MG132 (proteasome inhibitor), and Co-IP assays were performed to assess K48-linked polyubiquitination. HER2 mutants were generated to determine the role of specific ubiquitination sites. Cell viability, colony formation, cell proliferation (EdU staining), cell cycle analysis, and apoptosis assays were performed to evaluate the effects of neddylation inhibition on cell growth and survival. The synergistic effects of MLN4924 and trastuzumab were investigated. Finally, the expression of NAE1 (neddylation E1 enzyme) was analyzed using microarray data and immunohistochemistry, including correlation with HER2 expression and functional studies using NAE1 siRNA. Overexpression studies of HER2 and NEDD8 were also conducted.

The study found a positive correlation between NEDD8 and HER2 expression in breast cancer tissues, with higher NEDD8 expression associated with poorer patient survival. Silencing NEDD8 or using the neddylation inhibitor MLN4924 decreased HER2 protein levels but not mRNA levels, suggesting that neddylation regulates HER2 protein stability. CHX chase assays confirmed that neddylation inhibition reduced HER2 protein half-life, promoting its degradation. Co-IP experiments showed that NEDD8-cullins directly interacted with HER2. The interaction was mapped to all four domains of HER2, suggesting a complex regulatory mechanism beyond just ubiquitination. Neddylation inhibition increased K48-linked polyubiquitination of HER2, targeting it for proteasomal degradation. Furthermore, a specific tyrosine residue (Y1112) on HER2 was identified as crucial for this process. Functionally, neddylation inhibition (using MLN4924 or NEDD8 siRNA) suppressed cell viability, colony formation, proliferation, and induced cell cycle arrest (G2/M phase) and apoptosis in HER2-positive breast cancer cells. Importantly, MLN4924 synergistically enhanced the anti-cancer effects of trastuzumab. The study also revealed that NAE1, the neddylation activating enzyme, is overexpressed in HER2+ breast cancer and interacts with HER2, and its knockdown inhibited HER2 expression and cell growth. Finally, they showed that HER2 overexpression is necessary for neddylation-mediated breast cancer progression, as HER2 overexpression counteracted the anti-cancer effects of MLN4924.

The study establishes a novel mechanism by which HER2 is regulated in breast cancer through neddylation. The findings demonstrate that neddylation of HER2 stabilizes the protein, preventing its degradation via the ubiquitin-proteasome system. Inhibition of neddylation disrupts this stability, leading to increased HER2 ubiquitination and degradation, thereby suppressing breast cancer cell growth. The synergistic effect of neddylation inhibition with trastuzumab suggests a potential combined therapeutic strategy. The clinical relevance is supported by the positive correlation between NEDD8, NAE1, and HER2 expression in breast cancer patient samples and the association with poor prognosis. The identification of Y1112 as a key residue in the neddylation-mediated ubiquitination process provides a potential target for future therapeutic interventions. The research highlights the complexity of PTM crosstalk in cancer biology, suggesting that targeting multiple PTMs may be a more effective therapeutic approach.

This study reveals a novel role for neddylation in regulating HER2 stability and oncogenic activity in breast cancer. The findings demonstrate that NEDD8-dependent neddylation of HER2 protects it from degradation, promoting tumor growth. Inhibition of neddylation represents a potential therapeutic strategy, either alone or in combination with existing HER2-targeted therapies. Future research could focus on developing more specific neddylation inhibitors, exploring the precise mechanism of NEDD8/NAE1 interaction with HER2 and investigating the clinical implications of these findings in larger patient cohorts and preclinical models.

The study primarily used in vitro cell line models and tissue microarrays. Further in vivo studies in animal models are needed to confirm these findings. The mechanistic studies focused on specific interactions; more research is needed to fully understand the complexity of neddylation regulation of HER2 and the potential involvement of other PTMs. Additionally, the generalizability of the findings to all HER2+ breast cancer subtypes needs further investigation.