The adenosine A_2A receptor (A_2AAR) is a class A G-protein-coupled receptor (GPCR). It is an immune checkpoint in the tumor micro-environment and has become an emerging target for cancer treatment. In this study, we aimed to explore the effects of cancer-patient-derived A_2AAR mutations on ligand binding and receptor functions. The wild-type A_2AAR and 15 mutants identified by Genomic Data Commons (GDC) in human cancers were expressed in HEK293T cells. Firstly, we found that the binding affinity for agonist NECA was decreased in six mutants but increased for the V275A mutant. Mutations A165V and A265V decreased the binding affinity for antagonist ZM241385. Secondly, we found that the potency of NECA (EC₅₀) in an impedance-based cell-morphology assay was mostly correlated with the binding affinity for the different mutants. Moreover, S132L and H278N were found to shift the A_2AAR towards the inactive state. Importantly, we found that ZM241385 could not inhibit the activation of V275A and P285L stimulated by NECA. Taken together, the cancer-associated mutations of A_2AAR modulated ligand binding and receptor functions. This study provides fundamental insights into the structure–activity relationship of the A_2AAR and provides insights for A_2AAR-related personalized treatment in cancer.

Overexpression of the adenosine A₁ receptor (A₁AR) has been detected in various cancer cell lines. However, the role of A₁AR in tumor development is still unclear. Thirteen A₁AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. We have investigated the pharmacology of the mutations located at the 7-transmembrane domain using a yeast system. Concentration–growth curves were obtained with the full agonist CPA and compared to the wild type hA₁AR. H78L^3.23 and S246T^6.47 showed increased constitutive activity, while only the constitutive activity of S246T^6.47 could be reduced to wild type levels by the inverse agonist DPCPX. Decreased constitutive activity was observed on five mutant receptors, among which A52V^2.47 and W188C^5.46 showed a diminished potency for CPA. Lastly, a complete loss of activation was observed in five mutant receptors. A selection of mutations was also investigated in a mammalian system, showing comparable effects on receptor activation as in the yeast system, except for residues pointing toward the membrane. Taken together, this study will enrich the view of the receptor structure and function of A₁AR, enlightening the consequences of these mutations in cancer. Ultimately, this may provide an opportunity for precision medicine for cancer patients with pathological phenotypes involving these mutations.

The serotonin 5-HT2C receptor is a G protein-coupled receptor (GPCR) mainly expressed in the central nervous system. Besides regulating mood, appetite, and reproductive behavior, it has been identified as a potential target for cancer treatment. In this study, we aimed to investigate the effects of cancer patient-derived 5-HT2C receptor mutations on ligand binding and receptor functionality. By filtering the sequencing data from the Genomic Data Commons data portal (GDC), we selected 12 mutations from multiple cancer types. We found that the affinity of the endogenous agonist serotonin (5-HT) and inverse agonist mesulergine were both drastically decreased by mutations L209HECL2 and F328S6.52, which are located in the orthosteric binding pocket. In the calcium-flux assay, the potency of 5-HT was decreased at F328S6.52, while a trend of increased efficacy was observed. In contrast, 5-HT displayed higher affinity at E306K6.30 and E306A6.30, while a trend of decreased efficacy was observed. These two mutations may disrupt the conserved ionic interaction between E6.30 and R3.50, and thus increase the constitutive activity of the receptor. The inhibitory potency of mesulergine was increased at E306A6.30 but not E306K6.30. Lastly, P365H7.50 decreased the expression level of the receptor by more than tenfold, which prevented further functional analyses. This study shows that cancer-associated mutations of 5-HT2C receptor have diverse effects on ligand binding and function. Such mutations may affect serotonin-mediated signaling in tumor cells as well as treatment strategies targeting this receptor.

Breast cancer cells undergo metabolic reprogramming to support proliferation and metastasis, but these alterations are heterogeneous. To systematically assess this heterogeneity under controlled conditions, we profiled 31 intracellular polar metabolites and 50 amines, together with exchange rates of 57 amines, in a panel of 51 breast cancer cell lines spanning diverse phenotypes. These data were integrated with lipidomics and transcriptomics. Multi-omics factor analysis identified metabolic signatures linked to proliferation and to differences between luminal, HER2-positive, basal A, and basal B cell lines, overlapping with an epithelial-to-mesenchymal transition phenotype. Fast-proliferating, aggressive cell lines showed increased uptake of essential amino acids and altered nucleotide levels, while heterogeneity in glutamine metabolism was mainly driven the subtype. This was partially associated with heterogeneous expression of metabolite transporters. To test the functional relevance, 67 metabolic genes were silenced in Hs578T using siRNA, and effects on proliferation and migration were measured by a sulforhodamine B assay, live-cell imaging, and a random cell migration assay. This resulted in 34 knockdowns that reduced proliferation and 20 reduced migration, with strong effects for the glycosyltransferases EXT1 and EXT2, nucleotide metabolism genes GART and HPRT1, and metabolite transporters SLC7A1, SLC7A11, and SLC16A3. These findings highlight phenotype-specific metabolic dependencies and identify candidate drug targets in aggressive breast cancer.