Inhibition of dopamine receptor D1 signaling promotes human bile duct cancer progression via WNT signaling

Abstract

Bile duct cancer (BDC) frequently invades the nerve fibers, making complete surgical resection difficult. A single tumor mass contains cells of variable malignancy and cell-differentiation states, with cancer stem cells (CSCs) considered responsible for poor clinical outcomes. This study aimed to investigate the contribution of autosynthesized dopamine to CSC-related properties in BDC. Sphere formation assays using 13 commercially available BDC cell lines demonstrated that blocking dopamine receptor D1 (DRD1) signaling promoted CSC-related anchorage-independent growth. Additionally, we newly established four new BDC patient-derived organoids (PDOs) and found that blocking DRD1 increased resistance to chemotherapy and enabled xenotransplantation in vivo. Single-cell analysis revealed that the BDC PDO cells varied in their cell-differentiation states and responses to dopamine signaling. Further, DRD1 inhibition increased WNT7B expression in cells with bile duct-like phenotype, and it induced proliferation of other cell types expressing Wnt receptors and stem cell-like signatures. Reagents that inhibited Wnt function canceled the effect of DRD1 inhibition and reduced cell proliferation in BDC PDOs. In summary, in BDCs, DRD1 is a crucial protein involved in autonomous CSC proliferation through the regulation of endogenous WNT7B. As such, inhibition of the DRD1 feedback signaling may be a potential treatment strategy for BDC.

Keywords: bile duct cancer; cancer stem cells; dopamine D1 receptors; organoids; single-cell analysis.

FIGURE 1

Sphere formation assay depicting increased spheres with DRD1 inhibition. (A) Schematic picture of sphere formation assay, enriching cancer stem cells (CSCs) (blue dots) out of a mixture of CSC (blue dots) and non‐CSC cells (red dots) (top). Examples of a convertible line (TYGBK1), an unconvertible line (KKU‐100) (down), and a typical “sphere” (arrowhead). Scale bar, 100 μm. (B) Change in mRNA expression of dopamine receptors from monolayer culture to sphere culture quantified via qRT‐PCR. Each value is calculated as the median of the triplicate. Comparison using Wilcoxon rank‐sum test. DRD1: median log2Foldchange (FC) of 2.8 in convertible and of 0.14 in “unconvertible,” p = 0.035. (C) Schematic picture of sphere formation assay (left). Examples of microscopic images of spheres (right). Scale bar, 100 μm. (D) Sphere formation assay with D1 inhibitor (SKF‐83566 0.4 μM, 1 μM or DMSO) using TYGBK1, NOZ, and TFK1. Tukey's test is used. n = 4. (E) Sphere formation assay with D1 inhibitor (SKF‐83566 1 μM, 4 μM or DMSO) using vector‐induced or DRD1‐knockdown (KD) TFK1s n = 4

FIGURE 2

Distinct patient‐derived cancer organoid (PDO) formation with D1 inhibitor or DRD1 KD. (A) Schematic picture of PDO establishment and organoid formation assay (left). Examples of microscopic images of PDOs cultured for 17 days (right). Scale bar, 100 μm. (B) Organoid formation assay with D1 inhibitor (SKF‐83566 1 μM, 4 μM, or DMSO) using Sph18‐08, 16, 29. Tukey's test is used. n = 4. (C) Organoid formation assay with D1 inhibitor using vector‐induced or DRD1‐KD Sph18‐16 n = 4

FIGURE 3

ScRNA‐seq of PDO Sph18‐16. (A) Schema of scRNA‐seq (left) and clusters shown in Uniform Manifold Approximation and Projection (UMAP) (right). (B) Dot plot of enrichment analysis for each cluster. Gene sets related to hepato‐biliary development or stem cells derived from MSigDB:C8 are listed. The color of the dots represents the adjusted p‐value, and the diameter represents the count of enriched genes. Results of adjusted p < 0.05 are shown. (C) Feature plots of each gene signature score and expression of TROP2 and MAOB. (D) Expressions of genes or gene signature scores of the five clusters. Table S6 shows the details of the gene sets

FIGURE 4

ScRNA‐seq analysis comparing control (DMSO) and D1 inhibitor (SKF‐83566 4 μM) treatment. (A) Heatmap visualization of the 50 most frequently appearing upregulated genes in D1 inhibitor‐treated Sph18‐16 sample by enrichment analysis using all GOBP terms. The details are provided in Table S5. (B) Violin plots depicting the expression of genes compared with control (red) and D1 inhibitor‐treated sample (green). Bar indicates the median value. Adjusted p‐value for B1 of WNT7A, 0.00058; B1 of WNT7B, 0.0029; INT1 of WNT7B, 0.0013; B1 of DKK1, 0.0018. (C) Proliferating cells of each cluster in control or D1 inhibitor‐treated sample (top). Listed percentages of cells in S and G2/M phases of each cluster (down). (D) Trajectories showing INT1 as the center in DMSO and INT2 in D1 inhibitor treatment

FIGURE 5

Autosignaling of Wnt as downstream of D1 inhibition. (A) Sphere formation assay with D1 inhibitor (SKF‐83566 0.4 μM for NOZ and TFK1, 1 μM for TYGBK1, or DMSO) and porcupine inhibitor (LGK974 1 nM for NOZ and TFK1, 10 nM for TYGBK1 or DMSO). Tukey's test is used, n = 4. D1i, D1 inhibitor; Pi, porcupine inhibitor. (B) Organoid formation assay of Sph18‐16 with D1 inhibitor (SKF‐83566 4 μM or DMSO) and porcupine inhibitor (LGK974 1 nM,10 nM or DMSO). Tukey's test is used, n = 4. Results of Sph18‐08 and 29 are shown in Figure S11A.

FIGURE 6

D1 inhibition enhancing cancer stem cell (CSC)‐related capacities in the absence of exogenous niches. (A) Peri‐sciatic nerve xenograft, subcutaneous xenograft, and splenic injection assay of Sph18‐29. Vector or two independent DRD1‐targeting shRNA is transduced to Sph18‐29. Table of the result (right). (B) Gross images of xenografted tumor in the peri‐sciatic nerve (left). Control patient‐derived cancer organoid (PDO tumor forming only along the nerves (arrowhead). Scale bar, 5 mm. The tumor size vertical to the nerve (mm) is measured (right). Wilcoxon rank‐sum test is used. (C) Macroscopic and HE‐stained histological images of a subcutaneously xenografted tumor and lung metastasis of DRD1‐KD sph18‐29. Scale bar, 5 mm in macroscopic images and 100 μm in histological images. (D) CCK8 proliferation assay of Sph18‐16, 29 with 5FU in the niche‐enriched or niche‐deficient medium, n = 4. The result of cisplatin and gemcitabine is shown in Figure S12C. Two‐tailed unpaired Student's t‐test is used at each concentration

FIGURE 7

Schematic illustrations depicting variations of B, INT, H clusters, and WNT7B as endogenous cancer stem cell (CSC) niche downstream of DRD1‐ERK‐FOS