Understanding the development of enzalutamide resistance based on a functional single-cell approach

Area of Science:

• Oncology
• Genomics
• Molecular Biology

Background:

• Metastatic prostate cancer (PCa) initially responds to androgen deprivation therapies (ADT).
• Prostate cancer universally develops resistance to ADT, evolving into incurable castration-resistant prostate cancer (CRPC).
• Mechanisms driving the transition from androgen-dependent PCa (ADPC) to CRPC remain unclear.

Purpose of the Study:

• To investigate the cellular heterogeneity of ADPC in response to enzalutamide using single-cell RNA-sequencing (scRNA-Seq).
• To identify and characterize potential therapy-resistant subpopulations within ADPC.
• To explore therapeutic vulnerabilities of identified resistant cells.

Main Methods:

• Single-cell RNA-sequencing (scRNA-Seq) of the LNCaP cell line treated with enzalutamide.
• Gene Set Enrichment Analysis (GSEA) to compare gene expression profiles of different cell subpopulations.
• In vitro and in vivo functional assays using isolated cell subpopulations and xenograft models.
• Assessment of NAD+ biosynthesis gene expression and response to NAD+ synthesis inhibitors.

Main Results:

• scRNA-Seq revealed two distinct subpopulations: androgen receptor-positive (AR+) and androgen receptor-low/negative (AR^low/-).
• AR^low/- cells (∼12%) exhibited distinct transcriptional profiles and upregulated pathways associated with clinical CRPC.
• Isolated AR^low/- cells demonstrated functional enzalutamide resistance in vitro and in vivo.
• AR^low/- cells showed low expression of NAD+ biosynthesis genes, particularly NAPRT.
• Treatment with NAD+ synthesis inhibitors (FK866, OT-82) significantly inhibited AR^low/- cell survival and proliferation.

Conclusions:

• Single-cell RNA-sequencing can identify functionally significant tumor cell subpopulations.
• A distinct AR^low/- subpopulation within ADPC exhibits inherent resistance to enzalutamide.
• This resistant subpopulation presents a therapeutic vulnerability to NAD+ synthesis inhibition, offering a potential new treatment strategy for advanced prostate cancer.