Understanding Enzalutamide-Resistance Based on a Functional Single-Cell Approach

Anti-androgen or castration therapies are the mainstay treatment for metastatic prostate cancers (PCa). Although effective at first, androgen-dependent PCa (ADPC) universally develops therapy resistance, thereby evolving into an incurable disease called castration-resistant PCa (CRPC). Currently, mechanisms underlying the emergence of CRPC from ADPC are largely unclear.

We used single-cell RNA-sequencing (scRNA-Seq) to determine the transcription heterogeneity of a therapy-naïve ADPC cell line-LNCaP and how it responded to the anti-androgen drug, enzalutamide. Based on the results, we used single-cell/colony-based cloning to isolate a pre-enzalutamide cell subset, displaying low and/or no expression of androgen receptor (AR^low/-).

We found that most LNCaP cells expressed enzalutamide-target androgen receptor (AR+), while a small subpopulation (~10%) expressed low or no AR (AR^low/-). Gene set enrichment analysis (GSEA) revealed that AR⁺ and AR^low/- cells were enriched with significantly different gene expressions and signaling pathways. Unexpectedly, AR^low/- cells displayed robust transcriptional response, including upregulations of genes and pathways involved in clinical CRPC. Next, we isolated AR^low/- and AR⁺ cells from enzalutamide-naïve LNCaP cells and functionally confirmed the enzalutamide-resistant phenotype of AR^low/- cells in vitro and in xenograft models in vivo. Through xenograft-based single-nucleus RNA-Seq, we further found that the AR^low/- cells were selected, while the AR⁺ cells were de-selected in vivo by enzalutamide. Also, we found that the selection and expansion of AR^low/- clone were recapitulated in another enzalutamide-resistant cell model.

In summary, our single-cell-based sequencing and functional tests suggest a clonal selection and expansion model of enzalutamide resistance, in which the pretreatment AR-low subpopulation is selected and expanded to confer treatment resistance.