Triple-negative breast cancer (TNBC) lacks estrogen receptor, progesterone receptor, and HER2 expression and is associated with poorer outcomes compared with other breast cancer subtypes. Regulated forms of cell death are being explored as therapeutic vulnerabilities in cancer; one such mechanism is cuproptosis, a copper-dependent form of cell death linked to mitochondrial energy metabolism. This study sought to identify cuproptosis-associated genes in TNBC and to characterize regulatory pathways that might mediate tumor progression and resistance to cuproptosis.
The authors applied weighted gene co-expression network analysis (WGCNA) to gene expression data from TNBC patients to identify co-expression modules related to disease biology. Candidate cuproptosis-associated genes were identified by intersecting module genes with a predefined cuproptosis gene dataset. Subsequent experimental validation involved manipulation of selected genes in TNBC cell lines and assessment of cellular behaviors and responses to copper-induced stress. The PubMed abstract provides an overview of methods but does not report detailed experimental parameters, sample sizes, or in vivo study design.
G protein-coupled receptor 68 (GPR68) emerged from the intersection analysis as a cuproptosis-associated gene that is expressed at high levels in TNBC cells. The identification positioned GPR68 as a candidate regulator of both tumor phenotypes and cuproptosis sensitivity in TNBC.
Functional experiments demonstrated that knockdown of GPR68 inhibited key malignant phenotypes of TNBC cells in vitro. Specifically, reducing GPR68 expression curtailed cell growth, migration, and invasion, indicating that GPR68 supports pro-tumorigenic behaviors in TNBC models.
The investigators tested the role of GPR68 in regulating sensitivity to copper-induced cell death. Knockdown of GPR68 enhanced cuproptosis in TNBC cells when stimulated with the copper agent ES-Cu, leading to accumulation of cellular copper. The elevated sensitivity to cuproptosis following GPR68 depletion was reversed by the copper chelator tetrathiomolybdate (TTM), supporting a copper-dependent mechanism for the observed cell death effects.
Correlation analysis in the study linked GPR68 to regulation of glutaminase (GLS) expression in TNBC cells. Functional experiments showed that knockdown of GLS reversed the oncogenic effects attributed to GPR68 (that is, the promoted TNBC progression) and also reversed the inhibitory effect of GPR68 on cuproptosis. These findings indicate that GLS is a downstream effector in the GPR68-associated network that influences both tumor growth and copper-dependent cell death susceptibility.
Pathway enrichment analysis identified significant involvement of the mitogen-activated protein kinase (MAPK) signaling pathway. Mechanistically, GPR68 overexpression increased phosphorylation of ERK, implicating activation of the MEK/ERK signaling axis as a mediator of GPR68's effects in TNBC. The linkage suggests a signaling cascade in which GPR68 modulates GLS and activates MEK/ERK to promote tumorigenic phenotypes and reduce cuproptosis sensitivity.
To test pathway dependency, the authors used the ERK pathway inhibitor Trametinib. Pharmacologic blockade of the MEK/ERK pathway with Trametinib abrogated the pro-tumorigenic effects of GPR68 and reversed its ability to inhibit cuproptosis in TNBC cells. This pharmacologic result supports the conclusion that GPR68 acts, at least in part, through the MEK/ERK signaling cascade.
The study concludes that GPR68 promotes TNBC progression and confers resistance to cuproptosis by regulating GLS and activating the MEK/ERK pathway. Knockdown of GPR68 increases cuproptosis in the presence of copper (ES-Cu), and this effect can be reversed by copper chelation with tetrathiomolybdate. Inhibition of MEK/ERK signaling with Trametinib blocks GPR68-mediated tumor promotion and cuproptosis resistance. These findings identify GPR68, GLS, and the MEK/ERK axis as potential nodes for therapeutic intervention aimed at sensitizing TNBC to cuproptosis-based approaches.
Limitations and notes
The summary is based on the PubMed abstract. Detailed experimental conditions, quantitative effect sizes, numbers of replicates, animal model data (if any), and other methodological specifics were not reported in the abstract and therefore are not described here. For full experimental detail, consult the full text of the cited article.