Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer

by Selleckchem | Nov 10 2023

Background: Increasing evidence suggests that immunotherapy, especially immune checkpoint inhibitors (ICIs), has the potential to facilitate long-term survival in various cancer besides prostate cancer. Emerging evidence indicated that pyroptosis, an immunogenic form of cell death, could trigger an anti-tumor immune microenvironment and enhance the effectiveness of immunotherapy. Nevertheless, the mechanism underlying the regulation of pyroptosis signaling in prostate cancer remains unclear.

Methods: The differential expression of human E3 ligases in prostate cancer was integratedly analyzed from five independent public datasets. Moreover, the immunohistochemistry analysis of a tissue microarray derived from prostate cancer patients confirmed the results from the bioinformatic analysis. Furthermore, prostate cancer cell lines were evaluated via the next-generation RNA sequencing to assess transcriptomic profile upon CDC20 depletion. Next, qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay were employed to explore the correlation and interaction between CDC20 and GSDME. Both immune-deficient and immune-competent murine models were utilized to examine the anti-tumor efficacy of CDC20 inhibition with or without the anti-PD1 antibodies, respectively. To analyze the immune microenvironment of the xenografts, the tumor tissues were examined by immunohistochemistry and flow cytometry.

Results: The analysis of multiple prostate cancer cohorts suggested that CDC20 was the most significantly over-expressed E3 ligase. In addition, CDC20 exerted a negative regulatory effect on the pyroptosis pathway by targeting GSDME for ubiquitination-mediated proteolysis in a degron-dependent manner. Knockdown of CDC20 leads to increased GSDME abundance and a transition from apoptosis to pyroptosis in response to death signals. Furthermore, in our syngeneic murine models, we found that depletion of CDC20 significantly enhances the anti-tumor immunity by promoting the infiltration of CD8+ T lymphocytes dependent on the existence of GSDME, as well as reducing myeloid immune cells. More importantly, Apcin, a small molecular inhibitor that targets CDC20, exhibited synergistic effects with anti-PD1-based immunotherapy in murine models of prostate cancer.

Conclusions: Overall, these findings provide new insights into the upstream regulation of GSDME-mediated pyroptosis by CDC20, which specifically interacts with GSDME and facilitates its ubiquitination in a degron-dependent manner. Importantly, our data highlight novel molecular pathways for targeting cellular pyroptosis and enhancing the effectiveness of anti-PD1-based immunotherapy.

Comments:

The provided background, methods, results, and conclusions outline a comprehensive study focused on the role of CDC20, an E3 ligase, in regulating pyroptosis and its implications for prostate cancer and immunotherapy. Here's a breakdown of the key elements in the study:

**Background:**
1. **Immunotherapy and Prostate Cancer:** Immunotherapy, particularly immune checkpoint inhibitors (ICIs), has shown promise in various cancers, including prostate cancer.
2. **Pyroptosis and Immune Microenvironment:** Pyroptosis, a type of cell death, has immunogenic properties and can enhance the effectiveness of immunotherapy. However, its regulation in prostate cancer is not well understood.

**Methods:**
1. **Data Analysis:** The study integrated data from multiple public datasets to analyze the expression of E3 ligases in prostate cancer.
2. **Immunohistochemistry:** Tissue microarrays from prostate cancer patients were used for immunohistochemistry to validate the bioinformatic analysis.
3. **RNA Sequencing:** Prostate cancer cell lines were subjected to next-generation RNA sequencing to assess changes in the transcriptomic profile upon CDC20 depletion.
4. **Experimental Assays:** Various experimental assays, including qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay, were employed to investigate the interaction between CDC20 and GSDME.
5. **Mouse Models:** Both immune-deficient and immune-competent mouse models were used to evaluate the effects of CDC20 inhibition on tumor growth and the immune microenvironment.
6. **Immunohistochemistry and Flow Cytometry:** These techniques were used to analyze the immune microenvironment in xenografts.

**Results:**
1. **CDC20 Overexpression:** The analysis of multiple prostate cancer datasets revealed that CDC20 was significantly overexpressed compared to other E3 ligases.
2. **CDC20 Regulation of Pyroptosis:** CDC20 was found to negatively regulate the pyroptosis pathway by targeting GSDME for ubiquitin-mediated proteolysis in a degron-dependent manner.
3. **Effect of CDC20 Knockdown:** Depletion of CDC20 increased GSDME levels and shifted cell death responses from apoptosis to pyroptosis.
4. **Anti-Tumor Immunity:** CDC20 depletion in murine models enhanced anti-tumor immunity, with increased CD8+ T lymphocyte infiltration and reduced myeloid immune cells.
5. **Synergistic Effect with Immunotherapy:** Apcin, a CDC20 inhibitor, showed synergistic effects with anti-PD1-based immunotherapy in prostate cancer mouse models.

**Conclusions:**
The study concludes that CDC20 plays a critical role in regulating GSDME-mediated pyroptosis in prostate cancer. It specifically interacts with GSDME and promotes its ubiquitination in a degron-dependent manner. Importantly, the data suggest that targeting cellular pyroptosis pathways, such as CDC20 inhibition, can enhance the effectiveness of anti-PD1-based immunotherapy in prostate cancer.

This research provides valuable insights into the molecular mechanisms underlying pyroptosis and its potential application in cancer immunotherapy, specifically in the context of prostate cancer.