Abstract
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(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. 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.