Abstract
Prostate cancer is heterogeneous and patients would benefit from methods that stratify those who are likely to respond to systemic therapy. Here, we employ single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identify pre-existing and treatment-persistent cell subpopulations that possess regenerative potential when subjected to treatment. We find distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent cells are able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal as yet unrecognized molecular predictors of treatment response. This suggests that the application of single-cell methods with high analytical resolution in pre-clinical models may powerfully inform clinical decision-making.
Introduction
Prostate cancer (PC) relies on androgen-receptor (AR) signaling for development and progression. Progression on androgen-deprivation therapy (ADT) or AR signaling inhibitors (ARSIs), such as the second-generation AR antagonist enzalutamide (ENZ), leads to castration-resistant (CRPC) or treatment-induced neuroendocrine prostate cancer (NEPC)1. The most frequently characterized mechanisms of PC or CRPC resistance to ARSIs, ADT, or both revolve around the restoration of AR signaling via AR overexpression or AR mutations2,3,4,5.
PC is profoundly heterogeneous6,7,8,9 and patients would benefit from methods that differentiate between clinically mild and more aggressive forms of the disease. Although evidence of clonal expansion has been shown6, most studies to date have characterized genetic mutations10,11,12 that do not make allowance for tissue complexity or the biological basis for the emergence of treatment resistance. Furthermore, nongenetic changes in transcriptomics, chromatin structure, and DNA accessibility of transcription factor (TF)-binding motifs are more frequent but less understood in PC drug resistance2,3,4,5. DNA accessibility is the first layer of gene regulation and transcriptomic changes are now being used to identify molecular predictors of cancer-treatment response13. However, most RNA sequencing data are obtained from the bulk of the tumors and therefore cannot account for PC heterogeneity. This is because the transcriptome is the result of several biological processes contributing to differential gene regulation and such processes are not necessarily synchronized in all cells within the tumor bulk14,15. The development of single-cell sequencing technologies has enabled a more detailed examination of genomic features in treatment-resistant cancers, but the associated analytical methods are just beginning to reveal their potential.
In this work, we analyze the emergence of resistance in the epithelial-derived component of prostate tumors in ENZ-exposed and -resistant PC cell lines at a single-cell level to explore how heterogeneous PCs respond to ARSIs. Through enrichment analysis of transcriptional signals from molecular gene classifiers derived in this study, we show evidence of treatment-persistent and pre-existing PC cells that can predict treatment response in both primary and advanced patients.
Results
Chromatin reprogramming underpins enzalutamide resistance
To study the molecular consequences of AR signaling suppression and drug-resistance dynamics in PC, we utilized LNCaP parental cell lines, LNCaP-derived ENZ-resistant cell lines RES-A and RES-B generated via long-term exposure to AR-targeting agents16 (see “Methods”), and other independently generated LNCaP- and VCaP-derived models (Fig. 1a, Supplementary Table 1). We hypothesized that chromatin structure would undergo reshaping in ENZ-resistant cells and lead to modification of the transcriptome17,18.
