Single-cell transcriptional regulation and genetic evolution of neuroendocrine prostate cancer

Abstract

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer, with a 10% five-year survival rate. However, little is known about its origin and the mechanisms governing its emergence. Our study characterized ADPC and NEPC in prostate tumors from 7 patients using scRNA-seq. First, we identified two NEPC gene expression signatures representing different phases of trans-differentiation. New marker genes we identified may be used for clinical diagnosis. Second, integrative analyses combining expression and subclonal architecture revealed different paths by which NEPC diverges from the original ADPC, either directly from treatment-naïve tumor cells or from specific intermediate states of treatment-resistance. Third, we inferred a hierarchical transcription factor (TF) network underlying the progression, which involves constitutive regulation by ASCL1, FOXA2, and selective regulation by NKX2-2, POU3F2, and SOX2. Together, these results defined the complex expression profiles and advanced our understanding of the genetic and transcriptomic mechanisms leading to NEPC differentiation.

Keywords: Cancer; Cancer systems biology; Transcriptomics.

Graphical abstract

Figure 1

Overview of 36,036 single cells from seven prostate cancers (A) UMAP of the 36,036 cells profiled in seven patients. Two HSPC, three CRPC, and two pure small cell prostate cancers were depicted here. Different cell types were color-coded. For samples with parallel WES data (P1, P3, and P6), cells with at least one mutant read detected were colored black; P4, P5, and P7 come from published data (GSE137829) (also see Figure S1, S2, Tables S1, S2, and S3, and S5). (B) Distribution of cancer score in TCGA and CPGEA, respectively (STAR Methods) for cells categorized as malignant or normal, data are represented as boxplot (Wilcoxon rank-sum test, ∗∗∗∗: p ≤ 0.0001) (also see Table S4).

Figure 2

cNMF algorithm reveals distinct expression signatures (A) Pairwise correlation clustering of intra-tumoral GEPs. 71 GEPs were derived by cNMF from seven tumors and formed ten consensus modules, whose biological significance was predicted by GO analysis (top). GEPs in two NE modules were annotated (right) (also see Table S6). (B) GO analysis of genes over-represented in NE1 and NE2, respectively (hypergeometric distribution test adjusted by FDR <5%) (also see Figure S3, Table S7). (C) UMAP plot of malignant cells from P3. Cells are colored by their NE subtype (right) and the min-max normalized activity score of the corresponding GEPs (left, middle). (D) Volcano plot of the differentially expressed genes (DE-Gs) between NE1 and NE2 cells of P3 (Wilcoxon rank-sum test adjusted by FDR <5%). Blue, upregulated in NE1; Red, upregulated in NE2; gray, | log 2-fold change <1 | or adjusted P-value > 0.05. Expression of marker genes of note, ASCL1 and CHGB, for NE1 and NE2, respectively, were shown in the UMAP plot. TPM, edtranscript per million (normalized value). (also see Figure S4, Table S8).

Figure 3

NE trans-differentiation in P5 (A) The min-max normalized activity score of one NE1 GEP and two NE2 GEPs were colored. UMAP Plots were arranged in the assumed chronological order of differentiation. Expression of marker genes of note, ASCL1 and CHGB, for NE1 and NE2, respectively, were shown. In the right panel, the arrows denote the transition direction inferred from velocity analysis by Dong et al. (2020). TPM, transcript per million (normalized value). (B) Dynamic expression along the trajectory identified 100 genes that vary significantly over trans-differentiation pseudo-time (likelihood ratio test of nested models adjusted by FDR <5%). Cells were arranged in column by pseudo-time series. Hierarchical clustering of these genes at row via Ward D2’s method recovered three nonredundant groups that covary over the trajectory. Cluster analysis indicated large shifts in gene expression occurred as NE progenitor progressed toward maturation. TPM, transcript per million (scaled value). (C) inferCNV heatmap with hierarchical clustering of P5. The oncogenes or tumor suppressor genes in Cancer Gene Census were depicted in CNV regions. The top panel indicates a lack of CNV events in reference cells. The bottom panel is the malignant cells. Different types of NE cells were annotated on the sidebar (also see Figure S6).

Figure 4

Clonal evolution of P3 (A) inferCNV heatmap with hierarchical clustering of P3. The oncogenes or tumor suppressor genes in Cancer Gene Census were depicted in CNV regions. The top panel indicates a lack of CNV events in reference cells. The bottom panel is the malignant cells. Different types of NE cells were annotated on the sidebar. Genes of note in NEPC studies were highlighted in red (also see Figure S6). (B) Cells are colored according to the amplification status of chr2 and chr12 in the UMAP embedding (also see Figure S5). (C) Evolution model of P3 extrapolated from inferCNV results.

Figure 5

Clonal evolution of P1 (A) UMAP plot of malignant cells from P1. The relative expression of NE2 GEP was colored. Expression of marker genes of note, ASCL1 and CHGB, for NE1 and NE2, respectively, were shown in the UMAP plot. TPM, transcript per million (normalized value). (B) inferCNV heatmap with hierarchical clustering of P1. The oncogenes or tumor suppressor genes in Cancer Gene Census were depicted in CNV regions. The top panel indicates a lack of CNV events in reference cells. The bottom panel is the malignant cells. NE types, chr3 amplification, and KLK3 expression levels were annotated on the sidebar (also see Figure S6). TPM, transcript per million (normalized value). (C) Cells were colored by chr3 amplification status (left) and expression levels of KLK3 (middle) and AR (right) in the UMAP embedding. (D) Evolution model of P1 extrapolated from inferCNV results.

Figure 6

TF regulation of NEPC (A) Heatmap showed three types of TF (Common, NE1 specific, and NE2 specific) predicted by pySCENIC. The black block represents turn "on" status while the white block represents turn "off" status. (B) The summarized model of transcriptional regulation and genetic evolution of NEPCs in this study.