NEDDylation of Insulin Receptor Substrates as Regulators of Insulin Signaling and Its Effect on Cancer Cell Migration

Academic Background

In recent years, the association between cancer and diabetes has garnered significant attention due to their overlapping pathological mechanisms and epidemiological links. Epidemiological studies have shown that patients with type 2 diabetes (T2DM) have a significantly higher risk of developing various cancers, including pancreatic cancer, colorectal cancer, prostate cancer, and ovarian cancer. Dysregulation of insulin, which is a prominent feature of T2DM, interferes with the insulin signaling pathway and is considered a risk factor for the development of various cancers.

In insulin signaling, insulin receptor substrates 1 and 2 (IRS1 and IRS2) play a critical role as mediators of insulin effects into cellular processes. In cancer cells, IRS proteins are activated when insulin binds to its receptor, triggering a complex signaling cascade. Compared to normal tissues, higher levels of IRS proteins are observed in cancers, suggesting their involvement in tumorigenesis. Most types of cancers also exhibit high expression of IRS proteins, indicating their diverse and context-dependent roles in cancer.

As the understanding of the activation mechanisms of IRS proteins deepens, post-translational modifications (such as phosphorylation, ubiquitination, O-GlcNAcylation, and acetylation) are thought to be key factors in regulating IRS protein activity and interaction with downstream effectors. To gain deeper insight into the regulatory functions of these modifications, further research into the post-translational modifications of IRS proteins is particularly important.

Research Origin

This research was conducted in collaboration with researchers from Seoul National University College of Medicine and Yokohama National University, with related findings published in the journal Cancer Gene Therapy.

Research Methods

Human Tissue and Cell Culture

In accordance with the Helsinki Declaration, tissue samples from highly differentiated and poorly differentiated ovarian cancer patients were collected. Specifically, samples were obtained during surgical resection procedures carried out between 2019 and 2022 from the pathology department at SNUH. For cell culture, cell lines such as HEK293, SKOV3, U373, and RCC4 were used.

Differential Gene Expression and Enrichment Analysis

Using a combination of volcano plots and GSEA, differentially expressed genes between low-grade and high-grade ovarian cancer patients were investigated to identify key differential genes and pathways. The results indicated that the mRNA levels of the NEDDylation enzyme NAE1 were significantly higher in low-grade ovarian cancer patients compared to high-grade patients. Enrichment analysis of the cell response to insulin stimulation and low-grade ovarian cancer downregulated gene sets supported this finding.

Protein Identification and Immunohistochemistry Analysis

Using LC-MS/MS analysis, differentially expressed proteins (DEPs) were identified, and pathway analysis using the KEGG database confirmed the key role of IRS1 in biological function enrichment. Further immunohistochemistry analysis showed that treatment with insulin and NEDDylation inhibitors significantly increased the expression levels of IRS1 and IRS2.

Migration Experiment and Quantitative PCR (RT-qPCR)

Cell migration experiments (including transwell and wound healing assays) were conducted to evaluate the effects of NEDDylation inhibition and insulin treatment on cell migration. Additionally, RT-qPCR methods analyzed IRS1 and IRS2 mRNA levels, revealing that although protein levels increased, combined NEDDylation inhibition and insulin treatment did not affect the mRNA expression levels of IRS1 and IRS2.

Stability Experiment and Protein Precipitation Experiment

The effects of insulin on the stability of IRS1 and IRS2 proteins under NEDDylation blockage were studied using cycloheximide (CHX) treatment, showing that combined treatment significantly increased the stability of IRS1 and IRS2. Moreover, protein precipitation experiments verified the sequential relationship between NEDDylation and ubiquitination in the regulation of IRS1 and IRS2 protein homeostasis.

Research Results

Negative Correlation Between NEDDylation and Insulin Stimulation in Ovarian Cancer

The study found a negative correlation between NEDDylation and the malignancy of ovarian cancer, with low expression of the specific NEDDylation enzyme NAE1 associated with increased response to insulin stimulation, suggesting the important regulatory role of NEDDylation in insulin signaling.

Relationship Between IRS Protein Levels and Poor Prognosis in Ovarian Cancer

Protein identification results showed that under high levels of insulin stimulation, NEDDylation inhibition significantly increased the protein expression levels of IRS1 and IRS2. Furthermore, in ovarian cancer patient samples, high expression of IRS1 and IRS2 was associated with lower overall survival rates, particularly in ovarian cancer patients with T2DM, where IRS1 and IRS2 expression levels were significantly increased.

Regulation of Cell Migration and Glucose Intolerance

In three cancer cell lines, the study demonstrated that combined NEDDylation inhibition and insulin treatment significantly enhanced cell migration capability through activation of the PI3K/AKT signaling pathway by IRS1 and IRS2. The upregulation of IRS proteins further promoted cancer cell migration, with IRS1 and IRS2 potentially functioning through independent mechanisms.

Role of C-CBL as E3 Ligase

The study confirmed the role of C-CBL as the NEDDylation E3 ligase for IRS1 and IRS2, promoting their NEDDylation and regulating their degradation and stability through interaction with IRS proteins. Knockdown experiments suggested that the loss of C-CBL significantly increased the expression of IRS1 and IRS2 and cancer cell migration, whereas co-knockdown of IRS1 and IRS2 reduced these effects.

Conclusion

This study elucidates the regulatory role of NEDDylation in insulin signaling and cancer cell migration, emphasizing the crucial role of C-CBL in this process. The findings indicate that insulin signaling dysregulation significantly affects the stability of IRS proteins, with the regulation of IRS1 and IRS2 degradation through NEDDylation offering new potential targets for cancer therapy. However, caution is needed when using NEDDylation inhibitors in patients with T2DM or hyperinsulinemia due to their anti-cancer effects.

Research Highlights

1. Negative Correlation Between NEDDylation and Insulin Signaling: The study found that low expression of NAE1 was associated with increased insulin response, highlighting the regulatory role of NEDDylation in insulin signaling.
2. Association of IRS Proteins with Cancer Prognosis: High expression of IRS1 and IRS2 was associated with poorer prognosis in ovarian cancer patients, particularly significant in those with T2DM.
3. Critical Role of C-CBL: C-CBL acts as an E3 ligase for IRS proteins’ NEDDylation, regulating their stability and significantly affecting cancer cell migration capacity.
4. Methodological Innovations: Combining various experimental techniques, including LC-MS/MS, immunohistochemistry, cell migration experiments, and protein precipitation experiments, the study comprehensively analyzed the NEDDylation mechanism of IRS proteins.

Research Significance

This study not only reveals the crucial role of NEDDylation in insulin signaling but also provides new perspectives and potential targets for the treatment of diabetes and cancer. In-depth research on NEDDylation and its regulatory molecules (e.g., C-CBL) may assist in developing new cancer treatment strategies, especially for cancer patients with disrupted insulin signaling.