DRD1 suppresses cell proliferation and reduces EGFR activation and PD-L1 expression in NSCLC

Abstract

Dopamine (DA) acts in various key neurological and physiological processes as both a neurotransmitter and circulating hormone. Over the past several decades, the DA signaling network has been shown to regulate the progression of several types of solid tumors, and considerable evidence has shown it is a druggable pathway in the cancer cell context. However, the specific activity and effect of these pathway components appears to be tissue-type and cell-context-dependent. In the present study, expression and methylation of dopamine receptor D1 (DRD1) were measured using RNA sequencing (RNAseq) and reverse transcription polymerase chain reaction (RT-PCR) in non-small cell lung cancer (NSCLC) samples, and validated using publicly available datasets, including The Cancer Genome Atlas (TCGA). In vitro and in vivo functional experiments were performed for cell proliferation and tumor growth, respectively. Mechanistic analyses of the transcriptome and kinome in DRD1-modulated cells informed further experiments, which characterized the effects on the epidermal growth factor receptor (EGFR) pathway and programmed cell death 1 ligand 1 (PD-L1) proteins. Through these experiments, we identified the DRD1 gene as a negative regulator of disease progression in NSCLC. We show that DRD1, as well as other DA pathway components, are expressed in normal human lung tissue, and that loss of DRD1 expression through promoter hypermethylation is a common feature in NSCLC patients and is associated with worse survival. At the cellular level, DRD1 affects proliferation by inhibiting the activation of EGFR and mitogen-activated protein kinase 1/2 (ERK1/2). Interestingly, we also found that DRD1 regulates the expression of PD-L1 in lung cancer cells. Taken together, these results suggest that DRD1 methylation may constitute a biomarker of poor prognosis in NSCLC patients while other components of this pathway could be targeted to improve response to EGFR- and PD-L1-targeted therapies.

Keywords: DRD1; EGFR; PD‐L1; dopamine; non‐small cell lung cancer.

Fig. 1

DRD1 expression in normal and malignant lung tissue. (A) Immunohistochemistry‐based expression of dopamine receptors 1–5 in normal human bronchial tissues and levels of dopamine produced by lung cell lines. Scale bars = 50 μm for images captured at 200× magnification. Enlarged area captured at 400× magnification. Yellow boxes denote non‐transformed cells, gray boxes denote cancer cell lines. (B) Expression of DRD1 mRNA by RNAseq data in the NCI‐MD study in normal (non‐involved adjacent) and tumor tissues. (C) Methylation levels of probes in the DRD1 promoter in the NCI‐MD study in normal (non‐involved adjacent) and NSCLC tumor tissues. (D) Methylation levels of probes in the DRD1 promoter in preinvasive lesions that progress to LUSC or regress. (E) Relationship between DRD1 mRNA expression and lung cancer‐specific survival in stage I LUAD patients in the NCI‐MD study. Statistical significance in panels B, C, and D determined using two‐tailed t‐tests; significance in E determined using Cox regression. LUAD denotes lung adenocarcinoma, LUSC denotes lung squamous cell carcinoma, and RSEM denotes RNASeq by Expectation–Maximization. Graphs in panels A, B, C, and D show mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2

DRD1 inhibits cell proliferation through EGFR. (A) Cell proliferation assay using xCELLigence real‐time cell analysis system comparing growth of H727 DRD1 knockout (KO) cells and control cells (n = 4, two‐tailed t‐test, **P < 0.001). (B) Cell proliferation assay using xCELLigence real‐time cell analysis system comparing growth of H1299 DRD1 overexpression (OE) cells and empty vector (EV) control cells (n = 4, ordinary one‐way ANOVA with Dunnett's multiple comparisons test, ***P < 0.0001). (C) Tumor growth volume over time following subcutaneous injection of 1 × 10⁵ H727 DRD1 KO cells and 1 × 10⁵ H727 Pooled Control (Pooled Ctrl) cells in the left and right flanks, respectively. Graph shows mean ± SD for n = 10 carrying one of each DRD1 genotype (two‐tailed t‐tests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (D) Ingenuity pathway analysis following transcriptomic analysis of H1299 DRD1 OE cells and H727 DRD1 knockdown (KD) cells compared to respective controls. (E) Representative images of western blot analysis, accompanied by graphs of densitometry analysis, of EGFR pathway proteins in two H1299 DRD1 OE cell lines (n = 3, two‐tailed paired t‐tests, *P < 0.05, **P < 0.01 compared to vector control) and H727 DRD1 KO cells (n = 8, two‐tailed paired t‐test, *P < 0.05 compared to pooled control). (F) Representative images of proximity ligation assay showing proximal interactions (PLA signals) between EGFR and EGFR (assay positive control) and between V5‐tagged DRD1 and EGFR in H1299 cells. Images representative of three independent experiments. Scale bars = 20 μm. (G) Quantification of V5‐EGFR and EGFR‐EGFR PLA signals in H1299 vector (V) and DRD1‐expressing (OE) cells, shown as box‐and‐whisker plots of results pooled from three independent experiments with at least 20 cells quantified per experiment per group (*P < 0.0001, Mann–Whitney U, two‐tailed). Bar graphs in panels A, B, and E show mean + SD.

Fig. 3

DRD1 modulates PD‐L1 expression in NSCLC cells. (A) Western blot analysis of PD‐L1 expression in DRD1 OE H1299 cells, with 15 μg of total protein loaded for each sample. Blot is representative of three independent experiments. (B) Immunofluorescent staining of PD‐L1 on permeabilized H1299 DRD1 OE cell clones. Images are representative of three independent experiments. Images were captured using confocal microscopy. Scale bars = 50 μm. (C) Western blot analysis of PD‐L1 expression in H727 DRD1 KO cells, with 15 μg of total protein loaded for each sample. Blot is representative of two independent experiments. (D) Immunofluorescent staining of PD‐L1 on non‐permeabilized H727 DRD1 KO cell clones. Images are representative of two independent experiments. Scale bars = 50 μm. (E) Western blot analysis of PD‐L1 expression in H727 cells treated with 10 μm SKF‐38393 or 50 μm SCH‐23390 for 24 h, accompanied by graph results of densitometry analysis of band intensity. (F) Survival analyses of ipilimumab‐pretreated patients treated with second line nivolumab in two clinical trials [49, 50], stratified into DRD1 high or DRD1 low expression groups. Graphs were generated using TIDE. (G) Western blot analysis of H727 cells treated with 10 μm DRD4 antagonist PNU96415E for 24 h, accompanied by graphs of densitometry analysis. (H) Western blot analysis of H727 cells treated with 10 μm DRD4 agonist PD168077 for 24 h, accompanied by graphs of densitometry analysis. Bar graphs in panels E, G, and H show mean ± SD for n = 3, and statistical significance was determined using two‐tailed paired t‐tests, *P < 0.05, **P < 0.01 compared to respective controls.