DOI: 10.5061/dryad.kkwh70sgd
title: HOXD11 drives NMDAR signaling activation and the neuroendocrine transition in prostate cancer epithelium

description: Prostate cancer frequently develops resistance to androgen deprivation
 therapy (ADT), with adenocarcinoma-neuroendocrine prostate cancer (NEPC)
 lineage plasticity representing a critical yet mechanistically unclear
 driver of therapeutic failure. Here, we employ single-cell transcriptomic
 analysis of 29,436 cells from transgenic adenocarcinoma of mouse prostate
 (TRAMP), recapitulating the full clinical progression of human prostate
 cancer-NEPC evolution, to dissect the molecular dynamics underlying
 lineage transition. We identify Hoxd11 as a key transcriptional driver of
 adenocarcinoma-NEPC plasticity, with its expression and regulatory
 activity markedly upregulated during early NEPC transformation. Genetic
 suppression of Hoxd11 reversed lineage plasticity and delayed ADT-induced
 neuroendocrine differentiation. Mechanistically, HOXD11 directly activates
 the NMDAR signaling pathway (via GRIN1/GRIN3A), which is hyperactivated in
 NEPC and correlates with poor prognosis. Pharmacological inhibition of
 NMDAR using the clinically available antagonist memantine significantly
 suppressed tumor growth in murine models. These findings position the
 HOXD11-NMDAR axis as a druggable pathway in NEPC, with memantine
 repurposing offering near-term therapeutic potential.
description: <strong>Sequencing Protocol:</strong>
 scRNA-seq for TRAMP and human PCa tissue: Fresh tumor specimens
 were preserved in GEXSCOPE Tissue Preservation Solution under ice-cold
 transport conditions. Subsequent processing involved sequential steps: (1)
 washing in Hanks Balanced Salt Solution, (2) mechanical mincing coupled
 with enzymatic digestion using GEXSCOPE Tissue Dissociation Solution, (3)
 centrifugation and resuspension, followed by (4) erythrocyte depletion
 with GEXSCOPE Red Blood Cell Lysis Buffer. Single-cell suspensions derived
 from this workflow were converted into barcoded libraries employing the
 Chromium Single Cell Kit (10x Genomics). Final libraries were prepared
 with 10x Genomics Library Kits and subjected to paired-end 150 bp (PE150)
 sequencing on an Illumina NovaSeq 6000 platform, strictly adhering to
 manufacturer protocols. RNA-seq for HEK-293T and LNCaP
 cells: Total RNA was isolated from HEK-293T cells using Trizol reagent
 according to the manufacturer's protocol, followed by purification
 and DNase I treatment to eliminate genomic DNA contamination. RNA
 concentration and purity were assessed spectrophotometrically using a
 NanoDrop ND-1000 instrument. Subsequently, 1 µg of high-quality RNA
 (A260/A280 ratio ≥ 1.8; A260/A230 ratio ≥ 2.0) underwent poly(A) selection
 using oligo-dT magnetic beads. Stranded RNA sequencing libraries were
 constructed from the enriched mRNA utilizing the KAPA Stranded mRNA-Seq
 Kit (Roche Sequencing Solutions, USA), incorporating unique dual indexing
 adapters. Library fragment size distribution was verified by capillary
 electrophoresis, and quantification was performed via qPCR (KAPA Library
 Quantification Kit). Equimolar pools of validated libraries were sequenced
 on an Illumina NovaSeq 6000 platform (Illumina, USA) employing a 150 bp
 paired-end (PE) read configuration to a target depth of approximately 30
 million reads per sample. Assay for
 Transposase-Accessible Chromatin with high throughput sequencing
 (ATAC-seq)：Library preparation for ATAC-seq was conducted according to the
 protocol of the Vazyme TD711 kit. Briefly, 5×10<sup>4</sup>
 viable cells per reaction were washed twice with ice-cold PBS and then
 lysed in 50 μL of cold lysis buffer for 10 minutes on ice. A transposition
 reaction was performed in 50 μL of TTBL buffer containing 2.5 μL TTE Mix
 V50 for 30 minutes at 37 °C. The resulting DNA fragments were extracted
 with VAHTS DNA Clean Beads, involving two consecutive 80 % ethanol washes.
 Amplification of the library was achieved through 12 cycles of PCR. The
 final libraries were size-selected for fragments between 100 and 700 bp
 using VAHTS DNA Clean Beads, quantified with a Qubit 4.0 Fluorometer, and
 the fragment distribution was assessed on an Agilent 2100 Bioanalyzer
 prior to paired-end sequencing (150-300 bp) on the
 Illumina NovaSeq™ X Plus platform. HOXD11-Flag
 CUT&Tag assay: The CUT&Tag assay was conducted with the
 Vazyme CUT&Tag Kit in accordance with the manufacturer's
 protocol. In brief, 1×10<sup>5</sup> cells were harvested,
 washed twice with 1× PBS, and subsequently immobilized on activated
 ConA-coated magnetic beads. After permeabilization using Dig-wash buffer
 containing 0.05 % digitonin, the cells were incubated with a primary
 antibody against HOXD11-Flag overnight at 4 °C. This was followed by
 sequential incubations: first with a secondary antibody (1:50 dilution)
 for one hour at room temperature, and then with the pA/G-Tnp transposase
 complex for another hour. Tagmentation was then performed in the
 designated buffer at 37 °C for one hour. The resulting DNA fragments were
 purified using magnetic beads and amplified via a 12-cycle PCR with
 indexed primers. The final libraries were quantified on a Qubit 4.0
 Fluorometer, and their quality was assessed with an Agilent 2100
 Bioanalyzer to confirm insert sizes of 200-500 bp before paired-end
 sequencing on the Illumina NovaSeq™ X Plus platform.
   <strong>Data processing to generate
 matrix:</strong> RNA sequencing reads were
 processed via <em>Cell Ranger</em> (v3.0.3; 10x Genomics) for
 gene expression quantification, with alignment to the GRCm38 reference
 genome and Ensembl 92 gene annotation. Raw RNA-seq
 reads were processed and aligned to the human reference genome (GRCh38)
 using <em>HISAT2</em> (v2.2.1), and gene expression counts
 were generated with <em>featureCounts</em> (v2.0.6) based on
 GENCODE annotation. Reads of ATAC-seq and
 CUT&Tag are aligned to human reference genome (GRCh38) using
 Bowtie2. The resulting BAM files are then processed to remove PCR
 duplicates. For ATAC-seq data, mitochondrial reads are filtered out. Both
 datasets are normalized and converted to BigWig format using
 deepTools' bamCoverage with RPKM/CPM normalization.
description: # HOXD11 drives NMDAR signaling activation and the neuroendocrine
 transition in prostate cancer epithelium Dataset DOI:
 [10.5061/dryad.kkwh70sgd](10.5061/dryad.kkwh70sgd) ## Description of the
 data and file structure This dataset includes a single-cell RNA sequencing
 (scRNA-seq) count matrix of prostate tissues from Transgenic
 Adenocarcinoma of Mouse Prostate (TRAMP) mice across four distinct disease
 stages (one at 8 week, one at 20 week, one at 25 week and one at 30 week)
 in a csv file, generated to resolve lineage dynamics during neuroendocrine
 prostate cancer (NEPC) progression, as well as four standard formatted
 matrices for scRNA-seq data corresponding to these four ages, relatively.
 This dataset also includes scRNA-seq of two *de novo* human NEPC samples
 from clinical biopsy. Furthermore, this dataset contains the bulk RNA-seq
 count matrix and bigwig files of ATAC-seq from HEK-293T cells following
 HOXD11 knockdown, as well as the bulk RNA-seq count matrix and bigwig
 files of CUT&Tag from LNCaP cells following enzalutamide induction.
 ### Files and variables #### File: TRAMP_scRNAseq_counts.csv
 **Description:** The single-cell RNA sequencing (scRNA-seq) count matrix
 in csv file, which is generated from integrated 10X Genomics-typed matrix
 files. #### File: TRAMP_8w.zip **Description:** Single-cell RNA sequencing
 files of a sample from a TRAMP mouse at 8 weeks of age, which are
 sequenced with the 10x genomics platform and generated from the Cell
 Ranger pipeline: matrix.mtx.gz, features.tsv.gz, and barcodes.tsv.gz,
 which can be loaded by Seurat in R. The expression levels are measured in
 counts. #### File: TRAMP_20w.zip **Description:** Single-cell RNA
 sequencing files of a sample from a TRAMP mouse at 20 weeks of age, which
 are sequenced with the 10x genomics platform and generated from the Cell
 Ranger pipeline: matrix.mtx.gz, features.tsv.gz, and barcodes.tsv.gz,
 which can be loaded by Seurat in R. The expression levels are measured in
 counts. #### File: TRAMP_25w.zip **Description:** Single-cell RNA
 sequencing files of a sample from a TRAMP mouse at 25 weeks of age, which
 are sequenced with the 10x genomics platform and generated from the Cell
 Ranger pipeline: matrix.mtx.gz, features.tsv.gz, and barcodes.tsv.gz,
 which can be loaded by Seurat in R. The expression levels are measured in
 counts. #### File: TRAMP_30w.zip **Description:** Single-cell RNA
 sequencing files of a sample from a TRAMP mouse at 30 weeks of age, which
 are sequenced with the 10x genomics platform and generated from the Cell
 Ranger pipeline: matrix.mtx.gz, features.tsv.gz, and barcodes.tsv.gz,
 which can bewhich can be loaded by Seurat in R. The expression levels are
 measured in counts. #### File: NEPC1_filtered_feature_bc_matrix.zip
 **Description:** Single-cell RNA sequencing files of a sample from a
 neuroendocrine prostate cancer patient, which are sequenced with the
 SeekOne DD platform and generated from the SeekSoulTools pipeline:
 matrix.mtx.gz, features.tsv.gz, and barcodes.tsv.gz, which can be loaded
 by Seurat in R. The expression levels are measured in counts. #### File:
 NEPC2_filtered_feature_bc_matrix.zip **Description:** Single-cell RNA
 sequencing files of a sample from a neuroendocrine prostate cancer
 patient, which are sequenced with the SeekOne DD platform and generated
 from the SeekSoulTools pipeline: matrix.mtx.gz, features.tsv.gz, and
 barcodes.tsv.gz, which can be loaded by Seurat in R. The expression levels
 are measured in counts. #### File:
 HEK293t_siHOXD11vsNC_RNA_seq_gene_count_mtx.csv **Description:** The bulk
 RNA-seq count matrix from HEK-293T cells following HOXD11 knockdown, which
 is generated from raw data from Illumina NovaSeq, containing three
 reduplicates of control group and three reduplicates of knockdown group. 
 #### File: LNCaP_ENZA_OEHOXD11vsNC_RNA_seq_gene_count_matrix.csv
 **Description:** The bulk RNA-seq count matrix from enzalutamide-treated
 LNCaP cells following HOXD11 overexpression, which is generated from raw
 data from Illumina NovaSeq, containing four reduplicates of control group
 and four reduplicates of ENZA_OE group. #### File:
 HEK293t_siHOXD11vs_NC_ATACseq_bigwig.zip **Description:** The ATAC-seq
 bigwig files from HEK-293T cells following HOXD11 knockdown, which is
 generated from raw data from Illumina NovaSeq, including one of the
 control group (NC) and one of the si-HOXD11 group (si). #### File:
 LNCaP_ENZA_OEHOXD11vsNC_CUTTAG_bigwig.zip **Description:** The CUT&tag
 bigwig files from enzalutamide-treated LNCaP cells following HOXD11
 overexpression, which is generated from raw data from Illumina NovaSeq,
 including two reduplicates of control group (NC), two reduplicates of
 overexpression group (OE), and corresponding IgG files. ### Code/software
 After unpacking, the scRNA-seq data can be loaded by the Seurat package in
 R with the "Read10X" function. Other matrices can be viewed as
 tables and be loaded in R through the "read.table" function.