Metastatic bone colonization represents a formidable clinical challenge in advanced renal cell carcinoma, where patients face a dismal median overall survival of nineteen point seven months and five-year survival rates below twenty percent. Standard clinical interventions, including surgical resection for solitary lesions and systemic therapies, frequently fail to halt skeletal disease progression because metastatic bone deposits exhibit pronounced intrinsic resistance to conventional bone-targeted pharmaceuticals like bisphosphonates and denosumab. Addressing this clinical impasse, researchers from Zhejiang Chinese Medical University, Hangzhou Medical College, and Zhejiang University investigated the molecular mechanisms governing tumor cell survival within bone lesions, focusing on cuproptosis, an atypical copper-dependent form of regulated cell death. Their study demonstrates that metastatic renal cell carcinoma cells systematically evade cuproptosis through the downregulation of leucine-rich pentatricopeptide repeat containing protein, a crucial regulator of mitochondrial messenger RNA stability and cellular copper homeostasis.

To determine the transcriptomic landscape distinguishing secondary skeletal lesions from localized disease, the authors compared RNA sequencing profiles of thirty-one histopathologically verified renal cell carcinoma bone metastasis tissues with one hundred forty-three primary tumor transcriptomes from The Cancer Genome Atlas. Bioinformatic filtering identified pronounced divergence in transcript abundance, where downregulated genes in metastatic bone samples were predominantly concentrated within mitochondrial bioenergetic pathways, accounting for eight of the top twenty Gene Ontology categories and six of the top twenty Kyoto Encyclopedia of Genes and Genomes pathways. This pronounced transcriptomic signature underscored that secondary bone lesions undergo extensive metabolic divergence during metastatic dissemination. Immunohistochemical staining confirmed that LRPPRC expression was substantially diminished in metastatic bone specimens compared to paired primary renal carcinoma tissues. Furthermore, survival analyses corroborated that reduced LRPPRC abundance strongly correlated with shortened overall patient survival, while pairwise co-expression analyses revealed robust positive associations between LRPPRC and canonical cuproptosis machinery genes including FDX1, LIAS, LIPT1, DLAT, DLD, PDHA1, and PDHB.

Functional validation in cultured human renal cell carcinoma models established that manipulating LRPPRC directly alters cellular susceptibility to copper-induced cytotoxicity. In both 786-O and Caki-2 lines, short hairpin RNA knockdown of LRPPRC triggered a sharp reduction across the cuproptosis gene network, with ferredoxin 1 exhibiting the steepest decline. Pharmacological exposure to elesclomol complexed with divalent copper demonstrated that neither copper nor elesclomol alone produced appreciable cell death; however, their combination induced pronounced cytotoxicity. Silencing LRPPRC significantly blunted this response, shifting the half-maximal inhibitory concentration of elesclomol-copper from eighteen point seven nanomolar to twenty-eight point nine nanomolar in 786-O cells, and from twenty-five point three nanomolar to thirty-three point six nanomolar in Caki-2 cells. Proliferation assays tracking cells across three consecutive days verified that LRPPRC-deficient cells maintained elevated viability under copper challenge, confirming that loss of this mitochondrial protein establishes an effective defense against cuproptotic cell death.

Because cuproptosis selectively kills cells reliant on mitochondrial oxidative phosphorylation while sparing glycolysis-dependent cells, the team dissected how LRPPRC deficiency reprograms cellular bioenergetics. Transcriptomic profiling showed that expression of MT-CO1, MT-CO2, and MT-CO3, core mitochondrial subunits of cytochrome c oxidase, was depressed in patient metastases, a finding replicated following LRPPRC silencing in 786-O and Caki-2 cells. Metabolic flux profiling using Seahorse XFp analyzers demonstrated that LRPPRC knockdown suppressed oxygen consumption rates, including basal respiration, maximal respiratory capacity, and ATP-linked production, while concurrently accelerating extracellular acidification rates. Intracellular and supernatant lactate assays revealed elevated lactate production across all LRPPRC-depleted conditions, reflecting a compensatory glycolytic shift. When tumor cells were co-treated with the glycolytic inhibitor 2-deoxyglucose at ten millimolar, lactate production dropped and cuproptosis sensitivity was partially restored, whereas inhibiting ATP synthase with one micromolar oligomycin reinforced glycolytic dependence and reduced cuproptosis lethality, confirming that bioenergetic reprogramming directly contributes to cell death escape.

Beyond broad metabolic alterations, the authors delineated a direct post-transcriptional mechanism linking LRPPRC loss to suppression of the cuproptosis effector cascade. Knowing that LRPPRC stabilizes mitochondrial polyadenylated transcripts, the researchers measured ferredoxin 1 messenger RNA decay following actinomycin D transcription inhibition. In 786-O cells, LRPPRC knockdown shortened FDX1 transcript half-life from two point five three hours to zero point eight zero hours, with a parallel reduction from three point zero four hours to two point four nine hours in Caki-2 cells. Molecular modeling supported direct transcript stabilization, calculating an in silico binding docking score of minus two hundred ninety-four point nine four and a confidence score of zero point nine four seven eight between LRPPRC and FDX1 mRNA. Western blotting and immunofluorescence validated that loss of FDX1 protein expression crippled the lipoylation of dihydrolipoamide S-acetyltransferase. Notably, while exogenous FDX1 overexpression restored lipoylated DLAT levels, it only partially rescued cuproptosis sensitivity in cell viability assays, indicating that additional regulatory arms must operate alongside FDX1.

To identify these uncharacterized pathways, the investigators stratified patient bone metastasis samples into predicted cuproptosis-sensitive and resistant subgroups based on median expression of FDX1 and LRPPRC. Differential transcript analysis revealed that sensitive specimens were enriched for metal ion transport genes, highlighting solute carrier family 31 member 1, solute carrier family 40 member 1, six-transmembrane epithelial antigen of prostate 1, and solute carrier family 39 member 8. Experimental perturbation demonstrated that LRPPRC knockdown significantly reduced STEAP1, SLC31A1, SLC40A1, and SLC39A8 abundance, whereas LRPPRC overexpression markedly elevated their expression. Actinomycin D chase assays confirmed that LRPPRC deficiency accelerated the mRNA decay rates of SLC31A1, SLC40A1, and STEAP1 transcripts, while computational docking confirmed strong favorable binding affinities across all three targets. Functional assays revealed that LRPPRC depletion severely suppressed the chemical reduction of divalent copper to monovalent cuprous ions. Reconstitution experiments across ten distinct cell groups confirmed that restoring SLC31A1, SLC40A1, and STEAP1 collectively provided the most pronounced rescue of cuproptosis sensitivity, although rescue remained incomplete without upstream LRPPRC restoration.

The study subsequently uncovered an intricate biochemical coupling between cellular iron regulation and copper-dependent cytotoxicity mediated jointly by SLC40A1 and STEAP1. While STEAP1 functions as a transmembrane metalloreductase reducing extracellular divalent copper to monovalent cuprous ions in a heme-dependent manner, SLC40A1 acts as a ferrous iron exporter rather than a direct copper transporter. In patient bone metastases, both proteins were markedly downregulated. Examining intracellular ion pools revealed that LRPPRC silencing increased ferrous iron levels while paradoxical heme concentrations dropped sharply, indicating a defect in mitochondrial heme biosynthesis downstream of organelle dysfunction rather than substrate depletion. Restoring SLC40A1 relieved iron accumulation and rescued heme production. Crucially, in copper conversion assays, neither STEAP1 nor SLC40A1 alone fully restored cuprous ion levels in LRPPRC-depleted cells; however, co-overexpression of SLC40A1 and STEAP1, or combining STEAP1 overexpression with exogenous hemin supplementation at zero point five micromolar, effectively restored cuprous pools, proving that SLC40A1-driven iron homeostasis supplies essential heme cofactor for STEAP1 reductase activity.

Downstream of copper reduction, the high-affinity importer SLC31A1 governs physical entry of cuprous ions across the plasma membrane into tumor cytoplasm. Immunohistochemistry verified significant suppression of SLC31A1 in bone metastatic lesions compared to primary tumor tissues. Quantitative fluorometric ion assays confirmed that LRPPRC knockdown decreased cytoplasmic cuprous concentrations, whereas restoring SLC31A1 reversed this reduction and stimulated downstream DLAT accumulation. Furthermore, simultaneous overexpression of SLC31A1, SLC40A1, and STEAP1 in LRPPRC-silenced cells successfully restored DLAT protein expression toward control levels. These data demonstrate that SLC31A1 does not act as a direct transcriptional transactivator of DLAT; instead, SLC31A1-mediated cuprous ion influx indirectly fosters DLAT lipoylation, stability, and oligomerization potential, directly cooperating with mitochondrial metabolism to establish the molecular threshold required for cuproptotic cell death.

To evaluate whether restoring LRPPRC expression confers therapeutic benefit in vivo, the authors established an orthotopic bone metastasis model by injecting one million 786-O cells intratibially into six-week-old male BALB/c nude mice. Animals were allocated into four arms: vehicle control, LRPPRC overexpression alone, elesclomol-copper alone, and combination therapy, receiving intraperitoneal elesclomol-copper at five milligrams per kilogram every three days for twenty-one days following initial tumor formation. In monotherapy cohorts, elesclomol-copper failed to impede tumor expansion relative to control animals, reflecting cuproptosis resistance characteristic of metastatic lesions. Conversely, combining LRPPRC restoration with elesclomol-copper significantly suppressed tumor burden compared to vehicle controls (p = 0.00286) and exhibited a marked trend toward tumor reduction relative to elesclomol-copper alone (p = 0.0726). Crucially, combination treatment significantly prolonged animal survival over both vehicle controls and elesclomol-copper monotherapy. Quantitative real-time PCR and western blots of harvested tibial tissues confirmed that combination therapy elevated FDX1, DLAT, SLC31A1, SLC40A1, and STEAP1 expression, while significantly increasing lipoylated DLAT protein within bone lesions.

Despite these encouraging preclinical results, the authors explicitly noted several critical methodological caveats and translational hurdles that must be addressed before clinical implementation. Looking toward clinical translation, no pharmacological small-molecule agonists currently exist to directly reactivate LRPPRC expression in metastatic lesions, suggesting that future approaches will likely depend on modified lipid nanoparticle vectors delivering LRPPRC messenger RNA directly to bone sites. Furthermore, systemic delivery of copper-ionophore complexes poses potential risks of off-target toxicity in vulnerable organs, highlighting the necessity for bone-seeking conjugates or microenvironment-responsive delivery formulations.

Subject of Research: Mechanisms of cuproptosis evasion and metabolic reprogramming in renal cell carcinoma bone metastasis

Article Title: LRPPRC deficiency drives cuproptosis evasion via metabolic reprogramming and dysregulated copper homeostasis in RCC bone metastasis

Article References: Lu, S., Liu, J., Wang, J., Ye, Z., & Gao, J. (2026). LRPPRC deficiency drives cuproptosis evasion via metabolic reprogramming and dysregulated copper homeostasis in RCC bone metastasis. Frontiers in Cell and Developmental Biology, 14, Article 1966341. https://doi.org/10.3389/fcell.2026.1966341

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1966341

Keywords: renal cell carcinoma, bone metastasis, LRPPRC, cuproptosis, FDX1, DLAT lipoylation, SLC31A1, SLC40A1, STEAP1, elesclomol, mitochondrial metabolism, glycolytic reprogramming, copper homeostasis, oxidative phosphorylation, 786-O cell line, Caki-2 cell line, intratibial xenograft