G protein-coupled receptor GPR52, an orphan GPCR enriched in brain tissue, is downregulated in many solid tumours and specifically reduced in breast cancer metastases compared with primary tumours. Low tumour GPR52 mRNA is associated with reduced overall survival, with a stronger effect observed in triple-negative breast cancer (TNBC). Using CRISPR–Cas9 GPR52 knockout (KO) models in human breast epithelial lines, the authors report increased intracellular cAMP signalling, enhanced cell–cell clustering, changes in 3D organisation, and an increased propensity for collective invasion. Re-expression of GPR52 or inhibition of cAMP production rescued several KO phenotypes. In vivo, zebrafish xenografts of GPR52 KO cells produced a larger total cancer area than controls. These results implicate loss of GPR52 as a potential mechanism contributing to breast cancer progression and support investigation of GPR52 agonism as a therapeutic approach.
Analysis of transcriptome profiles using the TNMplot webtool showed that GPR52 mRNA expression is lower in tumour samples than in corresponding non-cancerous tissues for the majority of examined tissue types. Within breast cancer datasets, low tumour GPR52 mRNA correlated with decreased overall survival. The prognostic impact of low GPR52 expression was especially pronounced in TNBC, suggesting a potential link between reduced GPR52 levels and more aggressive disease biology.
CRISPR–Cas9–mediated knockout of GPR52 was performed in the TNBC cell lines MDA-MB-468 and MDA-MB-231 and in the non-tumorigenic MCF10A line. In 2D culture, GPR52 KO cells displayed increased cell–cell interactions and formed multicellular clusters across all three cell lines. Transmission electron microscopy (TEM) comparing wild-type and GPR52 KO cells identified differences in cell–cell adhesion properties, including alterations in the length of the cell–cell interface and closer proximity of adjacent cells along this interface. These ultrastructural changes are consistent with the observed increase in multicellular clustering in 2D.
When cultured in 3D Matrigel, GPR52 KO MDA-MB-468 and MDA-MB-231 spheroids exhibited altered organisation and morphology relative to controls. The loss of GPR52 increased the cells’ tendency to organise collectively and to invade as multicellular groups within Matrigel. These findings indicate that GPR52 status affects not only 2D cell–cell interactions but also three-dimensional multicellular architecture and invasive behaviour relevant to tumour progression.
The culture substrate influenced morphology of GPR52 KO cells. On poly-D-lysine or collagen, GPR52-null cells adopted more spindle-like morphologies that are indicative of more mesenchymal behaviour. The authors report features consistent with hybrid or partial epithelial–mesenchymal transition (EMT) states accompanying GPR52 loss, linking altered multicellular organisation with changes in cell phenotype on different extracellular matrices.
Loss of GPR52 was associated with elevated intracellular cAMP levels. RNA sequencing and proteomic analyses of GPR52-null cells identified an increased cAMP signalling signature and upregulation of pathways implicated in breast cancer. Increased phosphorylation of CREB was observed in GPR52 KO cells, consistent with activation of cAMP-dependent transcriptional programmes. Importantly, re-expression of GPR52 or pharmacologic inhibition of cAMP production rescued several GPR52 KO phenotypes, supporting a mechanistic link between GPR52 loss, cAMP elevation, and downstream phenotypic changes.
To assess in vivo consequences of GPR52 loss, the authors used a zebrafish xenograft model. Zebrafish injected with GPR52 KO breast cancer cells developed a greater total cancer area than those injected with control cells, indicating increased tumour burden associated with loss of GPR52 in this model system. This in vivo result aligns with the in vitro observations of enhanced multicellular organisation and collective invasion.
Re-expression of GPR52 in KO cells and inhibition of cAMP production were reported to rescue some phenotypic consequences of GPR52 loss, including aspects of morphology and organisation. Given that GPCRs comprise a major class of drug targets and that GPR52 agonists have been pursued in other fields, the authors propose that GPR52 agonism merits investigation as a potential therapeutic strategy to counteract mechanisms of breast cancer progression linked to GPR52 loss.
Key experimental approaches described include CRISPR–Cas9 gene knockout in human breast epithelial cell lines (MDA-MB-468, MDA-MB-231, MCF10A), 2D and 3D cell culture assays (including Matrigel spheroid assays), transmission electron microscopy for ultrastructural analysis, measurement of intracellular cAMP and phospho-CREB, bulk RNA sequencing and proteomic analyses to define signalling signatures, rescue experiments via re-expression of GPR52 and pharmacologic inhibition of cAMP production, and a zebrafish xenograft model to assess tumour area in vivo. Specific experimental protocols, reagent concentrations, statistical details, and full datasets were reported in the source article but are not reproduced in full here.
The work demonstrates that loss of the orphan GPCR GPR52 alters multicellular organisation, promotes hybrid/partial EMT features and collective invasion in breast cancer cell models, and increases tumour burden in a zebrafish xenograft. Elevated cAMP signalling and CREB phosphorylation are mechanistic features of GPR52-null cells, and reversing cAMP elevation or re-expressing GPR52 rescues some phenotypes. These findings identify GPR52 loss as a candidate mechanism contributing to breast cancer progression and support further investigation of GPR52 agonism as a potential therapeutic approach. Details of experimental parameters and complete datasets are available in the published article.