Orphan GPCR GPR52 Governs Collective Invasion and Multicellular Organization in Breast Cancer

Loss of the orphan G protein-coupled receptor GPR52 (搜索)搜索该靶点 drives breast cancer (搜索)搜索该疾病 cells to assemble into tightly packed multicellular clusters and invade surrounding tissue collectively, according to a study published in the British Journal of Cancer. The research, led by Hanif, Kutz, Au and colleagues, identifies GPR52 as a molecular switch that controls cell–cell adhesion, extracellular matrix (ECM) remodeling, and tumor burden in vivo, with the strongest clinical relevance in triple-negative breast cancer (搜索)搜索该疾病 (TNBC).

GPR52 (搜索)搜索该靶点 expression is reduced in cancerous tissue compared to normal tissue and is inversely associated with breast cancer (搜索)搜索该疾病 prognosis and metastatic potential. Using the TNMplot webtool, the authors found that GPR52 mRNA expression was lower in tumors than in non-cancerous samples across the majority of solid tumor tissue types examined (P < 0.05). In breast cancer specifically, low tumor GPR52 mRNA expression was associated with reduced overall survival, an effect that was more pronounced for TNBC patients. GPR52 mRNA expression was also lower in metastatic lymph nodes than in the primary resected tumor (p = 4.45e-17).

GPR52 Loss Promotes Mesenchymal Features and Collective Invasion

To dissect the functional role of GPR52 (搜索)搜索该靶点, the researchers used CRISPR-Cas9 to generate indels in GPR52 in two human breast cancer (搜索)搜索该疾病 cell lines (MDA-MB-231 and MDA-MB-468) and one non-cancerous breast epithelial cell line (MCF10A), employing two different guide RNAs. GPR52 knockout (KO) cells formed clusters of tightly packed cells with clearly defined borders in monolayer culture. Transmission electron microscopy of MDA-MB-468 cells cultured in suspension showed that GPR52 KO cells had a shorter cell–cell interface than wild-type cells but interacted more closely along the length of that interface.

When cultured on poly-D-lysine or collagen, GPR52 (搜索)搜索该靶点 KO MDA-MB-468 cells elongated and adopted a mesenchymal phenotype, which was not observed in wild-type cells. Western blotting revealed upregulation of mesenchymal markers Snai1 and vimentin, while the cells continued to express E-cadherin. Gene expression analysis showed a 1.93-fold increase in SNAI1 mRNA (P = 0.003) and a 1.34-fold decrease in CDH1 mRNA (P < 0.001) relative to wild-type cells.

In 3D Matrigel culture, GPR52 (搜索)搜索该靶点 KO cells produced more spheroids that were disorganized with irregular borders and significantly reduced roundness. In Matrigel invasion assays, GPR52 KO cells tended to organize and invade collectively in large clusters (MDA-MB-468) or sheets (MDA-MB-231). Quantifying cancer foci with an area of at least 3140 μm² (approximately 10 cells), the authors found that the fraction of area occupied by these larger collective structures was increased in both GPR52 KO groups, indicating a greater propensity to organize collectively.

Focal Extracellular Matrix Degradation

Using dye-quenched (DQ)-collagen IV, which releases sequestered fluorophores upon proteolysis of the basement membrane component collagen IV, the team characterized how collective invasion alters ECM digestion. At the 24-hour timepoint, the DQ signal colocalized strongly with GPR52 (搜索)搜索该靶点 KO cells, whereas it was more diffuse and less colocalized with vector control cells. When normalized to the area occupied by cancer cells, the DQ signal tended to be lower in GPR52 KO groups, suggesting a focal area of matrix degradation rather than broad proteolysis.

cAMP Signaling Links GPR52 to MCAM Expression

Proteomic and transcriptomic analyses pointed to cAMP signaling as a central mediator. GPR52 (搜索)搜索该靶点 KO MDA-MB-468 cells had significantly higher basal cAMP levels than control cells, an effect reversible upon GPR52 rescue. Ingenuity Pathway Analysis identified upregulation of the cAMP response element-binding protein (CREB1) signature in both GPR52 KO cell lines and in TCGA-BRCA tumors lacking GPR52 expression. Phosphoproteomic analysis revealed the greatest increase in the rank of VRK1, a kinase that regulates cell cycle progression via phosphorylation and activation of CREB.

RNA-sequencing showed that GPR52 (搜索)搜索该靶点 loss was associated with differential expression of many cell adhesion molecules, including melanoma cell adhesion molecule (MCAM (搜索)搜索该靶点). MCAM and GPR52 mRNA were inversely correlated in resected breast tumors, and increased MCAM expression was associated with reduced overall survival. Immunofluorescence demonstrated that MCAM and DSCAML1 were expressed at higher levels at the cell membrane of GPR52 KO cells, with signals enriched at cell–cell junctions. Treatment with 1.4 mM adenylyl cyclase inhibitor (ACi) reduced MCAM expression, confirming that MCAM is regulated by cAMP.

Increased Tumor Burden and Residual Disease in Zebrafish

To assess in vivo consequences, the researchers used a zebrafish xenograft model with the TG(flk1:EGFP-NLS) strain, which expresses green fluorescent protein in endothelial cells. At 30 hours post-injection, the trunk and total cancer area were significantly greater in zebrafish injected with GPR52 (搜索)搜索该靶点 KO MDA-MB-468 cells than with wild-type cells, although the number of cancer foci did not differ significantly.

Because increased clustering of cancer cells is associated with reduced sensitivity to cytotoxic chemotherapeutics, the team conducted a therapeutic study incorporating 8 μM doxorubicin查看药品详情 or vehicle. Doxorubicin caused a significant reduction in breast cancer (搜索)搜索该疾病 cell area in the head of the zebrafish for both wild-type and GPR52 (搜索)搜索该靶点 KO groups, but with noticeable residual disease in animals xenografted with GPR52 KO cells. In the trunk, doxorubicin decreased cancer area in GPR52 KO-xenografted zebrafish, but this effect was not significant.

The authors note that GPR52 (搜索)搜索该靶点 is described as an "orphan" receptor because its endogenous ligand has not been firmly established, a biological mystery that has made the receptor difficult to place within conventional cancer signaling maps. The findings suggest that GPR52 may serve as a molecular interface through which cancer cells interpret their environment and choose whether to remain attached, reorganize, or invade. Any therapeutic implications remain at an early stage, as disrupting cell organization does not automatically eliminate cancer, and tumor cells can reroute signals through parallel pathways or switch between collective and single-cell invasion.