Bipotent Progenitors Do Not Require Androgen Receptor for Luminal Specification during Prostate Organogenesis

Abstract

Androgen receptor (AR) plays a fundamental role in most aspects of adult prostate homeostasis, and anti-androgen therapy represents the cornerstone of prostate cancer treatment. However, early prostate organogenesis takes place during pre-pubertal stages when androgen levels are low, raising the possibility that AR function is more limited during prostate development. Here, we use inducible AR deletion and lineage tracing in genetically engineered mice to show that basal and luminal epithelial progenitors do not require cell-autonomous AR activity during prostate development. We also demonstrate the existence of a transient bipotent luminal progenitor that can generate luminal and basal progeny, yet is also independent of AR function. Furthermore, molecular analyses of AR-deleted luminal cells isolated from developing prostates indicate their similarity to wild-type cells. Our findings suggest that low androgen levels correlate with luminal plasticity in prostate development and may have implications for understanding how AR inhibition promotes lineage plasticity in prostate cancer.

Keywords: androgen receptor; lineage tracing; luminal progenitor; plasticity; prostate organogenesis.

Graphical abstract

Figure 1

AR Is Not Required in Bipotent Basal Progenitors in the Prostate Epithelium (A–E) AR and E-cadherin (Ecad) expression in wild-type mouse prostates. Arrows in (C) indicate epithelial cells with low AR expression (yellow) and high AR expression (white) in prostate cells facing the lumen. (F–J) Immunofluorescence (IF) staining for luminal (CK8) and basal (p63 and CK5) markers. Red arrow in (F) indicates co-expression of CK5 and CK8 at P0. Pre-basal cells (CK5^highCK8^lowp63⁺) and pre-luminal cells (CK5^lowCK8^highp63^–; red arrows and insets in G) were observed at P2. (K–V) AR deletion in basal cells during postnatal prostate development. (K) Analysis timeline. (L) Quantitation of YFP⁺ cells in the luminal layer that express CK8 at 4 weeks after lineage marking (n = 4 mice and n = 3 mice). (M) Quantitation of AR-negative YFP⁺ cells at 4 weeks after lineage marking (n = 3 mice each). (N–V) IF staining at 3 days (P2 to P5) or 4 weeks (P2 to 4 weeks) after lineage marking. Arrowheads indicate YFP⁺ basal cells, and arrows indicate YFP⁺ luminal cells. bas, basal; lum, luminal; P, postnatal day; UGS, urogenital sinus; n.s., not significant. Scale bars, 50 μm. Error bars represent standard deviation. The p values were calculated by t tests. See also Figures S1–S3, and Tables S1A–S1E.

Figure 2

Identification of Bipotent Luminal Cells during Postnatal Prostate Development (A) Analysis timeline for P2 lineage marking. (B–I) IF staining at 2 or 3 days (P2 to P4 or P5), 2 weeks (P2 to 2 weeks), or 4 weeks (P2 to 4 weeks) after lineage marking. (B′) Two-channel and single-channel images. (J) Quantitation of YFP⁺ cells in the basal layer that express CK5 at P5, 2 weeks, and 4 weeks (n = 5, n = 3, and n = 4 mice). (K) Analysis timeline for P7 lineage marking. (L–N) IF staining at 3 days (P7 to P10) or 4 weeks (P7 to 4 weeks) after lineage marking. (O) Quantitation of YFP⁺ cells in the basal layer that express CK5 at P10 and 4 weeks (n = 4 and n = 5 mice). Arrowheads point to YFP⁺ basal cells, and arrows point to YFP⁺ luminal cells. AP, anterior prostate, DLP, dorsal lateral prostate; VP, ventral prostate. Scale bars, 50 μm. Error bars represent standard deviation. The p values were calculated by t tests. See also Figures S2 and S4, and Table S1F–S1L.

Figure 3

Cell Autonomous AR Is Not Essential for Luminal Prostate Progenitor Cells during Postnatal Prostate Development (A) Timeline for lineage marking and analysis. (B) Quantitation of YFP⁺ cells in the basal layer that express CK5 at 4 weeks after lineage marking (n = 4 and n = 3 mice). (C) Quantitation of AR-negative YFP⁺ cells at 4 weeks after lineage marking (n = 2 and n = 4 mice). Error bars in (B and C) represent standard deviation. The p values were calculated by t tests. n.s., not significant. (D–G) Immunofluorescence staining at 4 weeks after lineage marking. Arrowheads point to YFP⁺ basal cells, and arrows point to YFP⁺ luminal cells. Scale bars, 50 μm. (H–N) RNA sequencing (RNA-seq) analysis of AR-deleted luminal cells. (H) Principal component analysis of RNA-seq samples from YFP⁺ luminal cells. (I–N) GSEA of an AR-deleted luminal signature comparing AR-deleted YFP⁺CD49f^low/– luminal cells and wild-type YFP⁺CD49f^low/– luminal cells with a signature of (I) AR-deleted adult luminal cells (Zhang et al., 2016), (J) AR-regulated genes (Carver et al., 2011), (K) neuroendocrine prostate cancer (Beltran et al., 2016), or (L) AR-negative non-neuroendocrine prostate cancer (Bluemn et al., 2017). (M) A signature defined between profiles of AR-deleted YFP⁺CD49f^low/– luminal cells and bulk wild-type prostate shows significant enrichment with a signature defined between wild-type YFP⁺CD49f^low/– luminal cells and bulk wild-type prostate. (N) Significant enrichment between basal-origin (CK5-trace versus bulk wild-type prostate) and luminal-origin (CK8-trace versus bulk wild-type prostate) signatures. NES, normalized enrichment score; p value was calculated using 1,000 gene permutations. See also Figures S2–S4, and Tables S1M–S1O.

Figure 4

Schematic Time Course of Basal and Luminal Differentiation during Prostate Organogenesis Urogenital epithelial progenitors with mixed basal and luminal characteristics (CK5⁺, CK8⁺, and p63⁺, depicted in purple) give rise to distinct populations of “outer” pre-basal (CK5^high, CK8^low, and p63⁺) and “inner” pre-luminal cells (CK5^low, CK8^high, and p63⁻). Pre-basal cells generate both bipotent and unipotent basal progenitors. Pre-luminal cells generate both bipotent and unipotent luminal progenitors. However, bipotent luminal progenitors are more transient than bipotent basal progenitors. P, postnatal day.