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