Proteomic analysis of extracellular vesicles identified PI3K pathway as a potential therapeutic target for
Seiji Hishida1, Kyojiro Kawakami2, Yasunori Fujita3
1Department of Urology, Gifu University Graduate School of Medicine, Gifu, Japan.
Background:
Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines.
Methods:
Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis.
Results:
Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated.
Conclusions:
Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.
Insights
Cabazitaxel resistance in prostate cancer (PC) can be targeted by inhibiting the PI3K pathway. Extracellular vesicles (EVs) from resistant PC patients and cell lines revealed elevated proteins linked to PI3K signaling, suggesting a new therapeutic strategy.
Area of Science:
- Oncology
- Proteomics
- Biochemistry
Background:
- Cabazitaxel (CBZ) is a key treatment for docetaxel (DOC)-resistant prostate cancer (PC).
- Acquired resistance to CBZ remains a significant clinical challenge, necessitating novel therapeutic targets.
- Understanding the molecular mechanisms of CBZ resistance is crucial for developing effective treatments.
Purpose of the Study:
- To identify potential therapeutic targets for CBZ-resistant prostate cancer.
- To analyze proteomic changes in extracellular vesicles (EVs) associated with CBZ resistance.
- To investigate the role of the PI3K signaling pathway in CBZ resistance.
Main Methods:
- Purification of EVs from serum of PC patients and PC cell lines using Tim-4 magnetic beads.
- Proteomic analysis of EVs via nanoLC-MS/MS and comparative expression profiling.
- Evaluation of a PI3K inhibitor (ZSTK474) on CBZ-resistant cells and assessment of PI3K/PTEN signaling.
Main Results:
- Six proteins, including CD63 and CD151, were consistently elevated in EVs from CBZ-resistant cell lines and patients.
- Network analysis highlighted the PI3K signaling pathway as significantly associated with CBZ resistance.
- The PI3K inhibitor ZSTK474 reduced proliferation and resensitized CBZ-resistant cells, correlating with PI3K activation and PTEN deactivation.
Conclusions:
- Proteomic analysis of serum EVs, facilitated by Tim-4 isolation, successfully identified markers of CBZ resistance.
- The PI3K signaling pathway is implicated in CBZ resistance in prostate cancer.
- Combination therapy with CBZ and PI3K inhibitors shows promise for treating CBZ-resistant PC patients.
