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

Most metastatic prostate cancers (PCa) initially depend on androgen for survival and proliferation. Thus, anti-androgen 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-naïve 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 (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 isolate AR ^low/- and AR+ cells from the LNCaP cell line, and functionally confirmed the enzalutamide resistant phenotype of AR ^low/- cells in vitro and in xenograft models in vivo. Finally, to explore a therapeutic option for AR ^low/- cells, we found that AR ^low/- cells expressed low levels of NAD+ biosynthesis genes, notably NAPRT, indicating a possible vulnerability to inhibitors blocking NAD+ synthesis. Indeed, treating AR ^low/- cells with NAD+ synthesis inhibitors, FK866 and OT-82, significantly inhibited the survival and proliferation of AR ^low/- cells, thus suggesting a possible novel therapeutic option for ADT and enzalutamide resistant PCa.

Single-cell RNA-Sequencing reveals heterogeneities of tumor cell populations. In most cases, however, the functional significance of the observed heterogeneity is not tested. In this study, we first identified a possible therapy-resistant prostate cancer cell subpopulation with scRNA-Seq, then confirmed the resistant phenotype with single cell and colony - based cloning and functional testing. In addition, we also identified a therapeutic vulnerability of the resistant cells.