. 2023 Dec 11;41(12):2066-2082.e9.

doi: 10.1016/j.ccell.2023.10.009. Epub 2023 Nov 22.

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation

Chia-Chun Chen¹, Wendy Tran², Kai Song³, Tyler Sugimoto², Matthew B Obusan², Liang Wang², Katherine M Sheu², Donghui Cheng⁴, Lisa Ta¹, Grigor Varuzhanyan², Arthur Huang¹, Runzhe Xu⁵, Yuanhong Zeng¹, Amirreza Borujerdpur¹, Nicholas A Bayley¹, Miyako Noguchi², Zhiyuan Mao¹, Colm Morrissey⁶, Eva Corey⁶, Peter S Nelson⁷, Yue Zhao⁸, Jiaoti Huang⁹, Jung Wook Park⁹, Owen N Witte¹⁰, Thomas G Graeber¹¹

Affiliations

¹ Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, CA, USA.
² Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA.
³ Department of Bioengineering, UCLA, Los Angeles, CA, USA.
⁴ Eli and Edythe Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA.
⁵ Department of Biological Chemistry, UCLA, Los Angeles, CA, USA.
⁶ Department of Urology, University of Washington School of Medicine, Seattle, WA, USA.
⁷ Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA, USA; Division of Clinical Research, Fred Hutchinson Cancer Center, Seattle, WA, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA.
⁸ Department of Pathology, Duke University School of Medicine, Durham, NC, USA; Department of Pathology, College of Basic Medical Sciences and the First Hospital, China Medical University, Shenyang, China.
⁹ Department of Pathology, Duke University School of Medicine, Durham, NC, USA.
¹⁰ Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA; Eli and Edythe Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA; Molecular Biology Institute, UCLA, Los Angeles, CA, USA; Parker Institute for Cancer Immunotherapy, UCLA, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA. Electronic address: owenwitte@mednet.ucla.edu.
¹¹ Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA; Crump Institute for Molecular Imaging, UCLA, Los Angeles, CA, USA; California NanoSystems Institute, UCLA, Los Angeles, CA, USA; Metabolomics Center, UCLA, Los Angeles, CA, USA. Electronic address: tgraeber@mednet.ucla.edu.

PMID: 37995683
PMCID: PMC10878415
DOI: 10.1016/j.ccell.2023.10.009

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation

Chia-Chun Chen et al. Cancer Cell. 2023.

. 2023 Dec 11;41(12):2066-2082.e9.

doi: 10.1016/j.ccell.2023.10.009. Epub 2023 Nov 22.

Authors

Affiliations

¹ Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, CA, USA.
² Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA.
³ Department of Bioengineering, UCLA, Los Angeles, CA, USA.
⁴ Eli and Edythe Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA.
⁵ Department of Biological Chemistry, UCLA, Los Angeles, CA, USA.
⁶ Department of Urology, University of Washington School of Medicine, Seattle, WA, USA.
⁷ Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA, USA; Division of Clinical Research, Fred Hutchinson Cancer Center, Seattle, WA, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA.
⁸ Department of Pathology, Duke University School of Medicine, Durham, NC, USA; Department of Pathology, College of Basic Medical Sciences and the First Hospital, China Medical University, Shenyang, China.
⁹ Department of Pathology, Duke University School of Medicine, Durham, NC, USA.
¹⁰ Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA; Eli and Edythe Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA; Molecular Biology Institute, UCLA, Los Angeles, CA, USA; Parker Institute for Cancer Immunotherapy, UCLA, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA. Electronic address: owenwitte@mednet.ucla.edu.
¹¹ Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA; Crump Institute for Molecular Imaging, UCLA, Los Angeles, CA, USA; California NanoSystems Institute, UCLA, Los Angeles, CA, USA; Metabolomics Center, UCLA, Los Angeles, CA, USA. Electronic address: tgraeber@mednet.ucla.edu.

PMID: 37995683
PMCID: PMC10878415
DOI: 10.1016/j.ccell.2023.10.009

Abstract

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

Keywords: ASCL1; ASCL2; POU2F3, TFAP4; cancer; lineage plasticity; neuroendocrine; prostate; small cell; stem-like; trans-differentiation.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests E.C. served as a paid consultant to DotQuant and received Institutional sponsored research funding unrelated to this work from AbbVie, Gilead, Sanofi, Zenith Epigenetics, Bayer Pharmaceuticals, Forma Therapeutics, Genentech, GSK, Janssen Research, Kronos Bio, Foghorn Therapeutics, and MacroGenics. P.S.N. has served as a paid consultant for Janssen, Merck, Bristol Myers Squibb and received research funding from Janssen for work unrelated to the present study. O.N.W. currently has consulting, equity, and/or board relationships with Trethera Corporation, Kronos Biosciences, Sofie Biosciences, Breakthrough Properties, Vida Ventures, Nammi Therapeutics, Two River, Iconovir, Appia BioSciences, Neogene Therapeutics, 76Bio, and Allogene Therapeutics. None of these companies contributed to or directed any of the research reported in this article. T.G.G. reports receiving an honorarium from Amgen, having consulting and equity agreements with Auron Therapeutics, Boundless Bio, Coherus BioSciences and Trethera Corporation. The lab of T.G.G. has completed a research agreement with ImmunoActiva. A provisional patent application related to this study was submitted.

Figures

**Figure 1.. Temporal gene expression programs of the PARCB transformation model reveal SCNPC trans-differentiation pathways.**
(A) Schematic summary of PARCB time course study and representative Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining of neuroendocrine markers (SYP and NCAM1) on sequential tumors from the tissue microarray. Time point (TP1-6) samples were sequenced using bulk RNA sequencing (green circle), bulk ATAC-sequencing (red circle) and/or single cell RNA sequencing (blue circle, tumors only). (B) Projection of the PARCB time course samples onto the PCA framework defined by pan-cancer clinical tumor datasets ^,,–. LUAD: Lung adenocarcinoma. LUAD norm: lung adenocarcinoma adjacent normal tissue. SCLC: small cell lung cancer. PRAD: prostate adenocarcinoma. PRAD norm: prostate adenocarcinoma adjacent normal tissue. CRPC: castration resistant prostate cancer. SCNPC: small cell neuroendocrine prostate cancer. (C) Arage gene expression of selected SCNPC-associated proteins and markers. (D) Heatmap of hierarchical clusters (HC) of samples (columns) and corresponding differentially upregulated gene modules (rows). Differential expression defined by one HC vs all other HCs). (E) PCA of the PARCB time course samples and trans-differentiation trajectories including primary arc and secondary bifurcation. A 3-dimensional rotatable version of this figure is available on the PARCB Multi-omics Explorer website. [For review, a 3D rotatable version is included as Data S1.] (F) Selected enriched GO terms across HC. See also Figure S1.

**Figure 2.. Sequential transcription regulators modulate reprogramming and neuroendocrine programs through a highly entropic and accessible chromatin state.**
(A) Overall differential chromatin accessibility across HC. (B) PCA of chromatin accessibility of PARCB time course samples with entropy analysis using ATAC sequencing. (C) Overall mean accessible peaks near TSS of each HC in PARCB time course study. (D) Enriched motifs from suites of transcription factors in each HC using ATAC-sequencing. Top 5 motif suites for each comparison are shown, with additional analysis in Figure S2B, and full results in Table S1D. (E) Top ranked transcription factors and known neuroendocrine transcription factors across PARCB time course using bulk RNA sequencing. HOXC TFs avg: Average expression of HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12 and HOXC13. (F) Expression of ASCL1, ASCL2, NEURDO1 and POU2F3 in each HC. See also Figure S2.

**Figure 3.. Transcription factor-defined cell populations contribute to lineage divergence and tumor heterogeneity.**
(A) Dimension reduction UMAP analysis of four patient series (P2, P5, P6 and P7) over time (TP3-6) using single cell RNA sequencing. (B) Temporal UMAP analysis of all the samples. (C) Expression of selected markers and transcription factors. KRT5 marks basal cells. KRT15 marks luminal cells. The expression is presented in log normalized counts. (D) Top enriched inferred cell types from the Human Cell Type Database using SingleR . (E) Projection of single cell RNA-sequencing samples on PCA framework by bulk RNA-sequencing samples (top panel) and the expression of selected markers and transcription factors (bottom panel). Each data point is a single cell colored by their corresponding HC. (F) Expression of ASCL1 (top) and ASCL2 (middle) and percentage of ASCL1/2 positive cells (cells with expression value >0) (bottom) in human biopsy and GEMM model tumors from five single cell RNA-sequencing datasets ^,–. Other: prostatic intraepithelial neoplasia. NMYC_RB_M: *Pten*^f/f; Rb1^f/f;MYCN + (PRN) and RB_M: *Pten*^f/f; Rb1^f/f (PR) mouse model in Brady et al. See also Figure S3.

**Figure 4.. ASCL1 and ASCL2 specify independent transcriptional programs and sub-lineages in SCNPC.**
(A) Inferred clonal tracing analysis of the PARCB time course samples using Monocle 2 . (B) Relative expression of KRT5, ASCL1 and ASCL2 in the inferred clonal tracing analysis (pseudo-time). (C) Percentages of ASCL1 or ASCL2 positive, double positive and double negative cell populations over time. (D) Volcano plot of differential gene expression in high ASCL1+ vs high ASCL2+ cell populations. (E) Representative genes from the predicted transcriptional programs of ASCL1 and ASCL2 trained on data from patient and model prostate cancer tumors (^,, including TCGA), as determined by the ARACNE algorithm . (F) Western blot of panel of genes in the PARCB tumor derived cell lines from different tissue of origin (prostate, bladder and lung) ^,. (G) Representative images of *in situ* hybridization of ASCL1 and ASCL2 mRNA analysis on transitional tumors (P7-TP5 and P9-TP4). See also Figure S4.

**Figure 5.. ASCL1 and ASCL2 as pan-cancer classifiers.**
See also Figure S5. (A) Projection of the PARCB time course samples on the PCA framework defined by the CRPC subtypes using RNA sequencing (left) and ATAC-sequencing (right) . SCL: stem-cell like. NEPC: Neuroendocrine prostate cancer. 3-dimensional rotatable versions of these figures are available on the PARCB Multi-omics Explorer website. [For review, 3D rotatable versions are included as Data S2 and Data S3.] (B) Projection of the PARCB time course samples on the PCA framework defined by the SCLC subtypes ^,. (C) mRNA expression of ASCL1 and ASCL2 in the PARCB time course samples and multiple sets of clinical CRPC-PRAD and SCNPC samples including TCGA and different research groups ^,–. (D) Representative images of *in situ* RNA hybridization of ASCL1 and ASCL2 in clinical SCNPC tissues. (E) mRNA expression of ASCL1 and ASCL2 in pan cancer cell lines (CCLE). (F) mRNA expression of ASCL1 and ASCL2 in pan cancer tumors from TCGA.

**Figure 6.. Alternating ASCL1 and ASCL2 expression through reciprocal interaction and TFAP4 epigenetic regulation.**
See also Figure S6. (A) Western blot analysis of exogenously expressing either V5 tagged ASCL2 in ASCL1+ cell lines (left) or V5-tagged ASCL1 in ASCL2+ cell lines (right). (B) Schematic of putative cis regulatory elements (CREs) of ASCL1 and ASCL2 (top) and the heatmap of open chromatin accessibility across CREs of ASCL1 and ASCL2 using the PARCB time course ATAC-sequencing (bottom). Red box: CREs containing predicted TFAP4 binding sites by HOMER motif enrichment analysis . (C) Top 8 ranked transcription factor motifs in ASCL1 promoter and ASCL2 enhancer regions, ranked by p-values. (D) Western blot analysis of doxycycline-inducible knockout of TFAP4 and proteins of interest in P7-TP6 (ASCL1+) and P3-TP5 (ASCL2+) cell lines. DOX: doxycycline. (E) Cell proliferation analysis of P7-TP6 (ASCL1+) and P3-TP5 (ASCL2+) cell lines with doxycycline-inducible knockout of TFAP4. Ctrl: no addition of doxycycline. TFAP4: with addition of doxycycline. (G) Schematic summary of the PARCB time course study.

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Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation

Affiliations

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

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Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases