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

Abstract

Most metastatic prostate cancers (PCa) initially depend on androgen for survival and proliferation. Thus, antiandrogen or castration therapies are the mainstay treatment. Although effective at first, androgen dependent PCa (ADPC) universally develops therapy resistance, thereby evolving to the 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 how a therapy-naive ADPC cell line LNCaP responds to the anti-androgen drug, enzalutamide. We found that most cells expressed the drug target androgen receptor (AR+), while a small subpopulation (~12%) expressed low or no AR (ARlow/none). Gene set enrichment analysis (GSEA) revealed that AR+ and ARlow/none cells were enriched with significantly different gene expressions and signaling pathways. Unexpectedly, ARlow/none cells displayed robust transcriptional response, including upregulations of genes and pathways involved in clinical CRPC. Next, we isolate ARlow/none and AR+ cells from the LNCaP cell line, and functionally confirmed the enzalutamide resistant phenotype of ARlow/none cells in vitro and in xenograft models in vivo. Finally, to explore a therapeutic option for ARlow/none cells, we found that ARlow/none cells expressed low levels of NAD+ biosynthesis genes, notably NAPRT, indicating a possible vulnerability to inhibitors blocking NAD+ synthesis. Indeed, treating ARlow/none cells with NAD+ synthesis inhibitors, FK866 and OT82, significantly inhibited the survival and proliferation of ARlow/none cells, thus suggesting a possible novel therapeutic option for ADT and enzalutamide resistant PCa.