Protein MKRN1 Shields Colorectal Cancer by Degrading Key Copper Influx Transporter

A study published in Biological Procedures Online demonstrates that the E3 ubiquitin ligase MKRN1 suppresses cuproptosis, a regulated form of cell death driven by copper overload, in colorectal cancer cells by promoting the ubiquitin-dependent degradation of the copper influx transporter SLC31A1. Cuproptosis represents a specialized modality of regulated cell death that is copper-dependent and characterized by the accumulation of lipoylated proteins alongside the loss of iron-sulfur cluster proteins, differing fundamentally from apoptotic, pyroptotic, and ferroptotic pathways. While colorectal cancer ranks as the third leading cause of cancer-related mortality in the United States and continues to exhibit rising incidence worldwide, chemoresistance remains a severe barrier to long-term clinical efficacy. Because resistance to conventional therapy is often tightly linked to the evasion of regulated cell death mechanisms, targeting alternative death pathways like cuproptosis offers a prospective avenue for therapeutic intervention. Prior to this investigation, the specific functional relationship among MKRN1, SLC31A1, and cuproptosis modulation in colorectal cancer progression remained unresolved.

Initial bioinformatics assessments conducted by the research team indicated an upregulation of copper transport and homeostasis machinery within colorectal cancer datasets, reflecting an elevated cellular demand for copper during malignant progression. An evaluation of the Cancer Genome Atlas and related public repositories revealed a marked discordance between transcriptional and translational profiles of the high-affinity copper transporter SLC31A1, characterized by elevated SLC31A1 messenger RNA expression alongside paradoxically low or undetectable protein levels in clinical specimens documented within the Human Protein Atlas. Although drug sensitivity analysis from the Genomics of Drug Sensitivity in Cancer database demonstrated a weak positive correlation between MKRN1 messenger RNA expression and sensitivity to the copper ionophore elesclomol, the functional direction of MKRN1 in modulating copper toxicity remained unknown. Based on these observations, the authors hypothesized that an E3 ubiquitin ligase, specifically MKRN1, mediates the post-translational destruction of SLC31A1 to tightly restrict intracellular copper accumulation and shield malignant cells from copper-mediated cytotoxicity.

To evaluate this regulatory mechanism experimentally, the researchers established stable MKRN1-knockdown and MKRN1-overexpressing cell lines in human colorectal cancer models, specifically utilizing HCT15 and HT29 cell lines cultured in Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum and antibiotics. Cuproptosis was induced in vitro by co-treating cells with the copper ionophore elesclomol and copper sulfate following pre-incubation with the copper chelator ammonium tetrathiomolybdate. Cell viability assays, including Cell Counting Kit-8 colorimetric measurements and trypan blue exclusion staining, demonstrated that MKRN1 overexpression significantly mitigated the cytotoxic effects triggered by cuproptosis agonists, whereas MKRN1 silencing markedly enhanced cell sensitivity and increased cell death. Furthermore, nuclear morphology examinations using Hoechst 33258 staining confirmed that MKRN1 depletion substantially elevated the frequency of condensed and brightly stained apoptotic nuclei following exposure to cuproptosis-inducing conditions compared to control cultures.

Subsequent biochemical analyses established that MKRN1 directly governs the expression of canonical molecular hallmarks of cuproptosis in colorectal cancer cells. In Western blot experiments, colorectal cancer cells overexpressing MKRN1 exhibited reduced levels of the cytotoxic heat shock protein HSP70 and a marked decrease in the abundance of the copper transporter SLC31A1, while simultaneously maintaining higher stability of the critical iron-sulfur cluster protein FDX1 and monomeric DLAT. Conversely, short hairpin RNA-mediated depletion of MKRN1 reversed these profiles, promoting HSP70 induction, destabilizing FDX1, and elevating SLC31A1 protein abundance. By separating cellular proteins into detergent-soluble and detergent-insoluble fractions, the authors observed that MKRN1 overexpression suppressed the formation of insoluble DLAT oligomeric complexes, which represent a primary structural signature of copper-dependent mitochondrial proteotoxicity, whereas MKRN1 knockdown markedly accelerated DLAT aggregate accumulation.

Direct quantification of intracellular metal concentrations clarified the physiological basis of this protective response. Using a dedicated cell-permeable copper ion detection assay, the authors determined that colorectal cancer cells overexpressing MKRN1 harbored significantly lower intracellular copper concentrations than control counterparts following exposure to copper overload. Parallel measurements revealed that these MKRN1-overexpressing cells maintained elevated levels of intracellular glutathione, an endogenous copper chelator that buffers against toxic free copper ions. In contrast, MKRN1-knockdown cells displayed pronounced intracellular copper accumulation coupled with a severe reduction in intracellular glutathione reserves. Validating these cellular findings in vivo, intestinal tissue isolated from conditional Mkrn1-knockout mice exhibited elevated copper content along with increased protein levels of SLC31A1 and HSP70 and diminished FDX1 abundance, confirming that endogenous MKRN1 restricts copper accumulation in gastrointestinal tissues.

Confocal microscopy and biochemical assays subsequently unraveled the molecular interaction between MKRN1 and the copper transporter. Immunofluorescence co-localization assays showed that MKRN1 resides within both the nuclear and cytoplasmic compartments of colorectal cancer cells, while SLC31A1 is localized predominantly at the plasma membrane. Knockdown of MKRN1 enhanced the accumulation of SLC31A1 at the plasma membrane, indicating that MKRN1 regulates the trafficking and abundance of the surface transporter. Endogenous and exogenous co-immunoprecipitation experiments confirmed a physical interaction between MKRN1 and SLC31A1 in colorectal cancer lines and human embryonic kidney cells. Quantitative real-time polymerase chain reaction confirmed that genetic alteration of MKRN1 caused no significant alteration in SLC31A1 messenger RNA levels, confirming that MKRN1 modulates SLC31A1 exclusively at the post-translational level.

Dose-dependent ectopic expression of MKRN1 in HCT15 cells produced a progressive reduction in SLC31A1 protein levels, which was fully blocked upon treatment with the proteasome inhibitor MG132. Cycloheximide chase assays indicated that MKRN1 knockdown significantly extended the half-life of SLC31A1 protein, whereas MKRN1 overexpression accelerated its turnover. In vivo ubiquitination assays in human embryonic kidney cells expressing linkage-specific ubiquitin constructs revealed that MKRN1 promotes both Lys48-linked and Lys63-linked polyubiquitination of SLC31A1. Notably, introducing a lysine-to-arginine mutation at position 63 (K63R) markedly attenuated the overall ubiquitination of SLC31A1, demonstrating that MKRN1 predominantly orchestrates Lys63-linked ubiquitin chains on SLC31A1 to drive its proteasomal degradation and suppress cell-surface copper transport activity.

Functional profiling of mitochondrial dynamics demonstrated that MKRN1 shields the organelle from structural and energetic collapse under copper challenge. Differential gene expression and functional enrichment analyses after MKRN1 knockdown revealed significant alterations in pathways governing mitochondrial respiratory chain complexes, cytochrome c oxidase, and the tricarboxylic acid cycle. Measurements of mitochondrial membrane potential via JC-1 dye fluorescence demonstrated that MKRN1 overexpression preserved membrane polarization during cuproptosis challenge, whereas MKRN1 knockdown exacerbated potential dissipation. Ultrastructural imaging through transmission electron microscopy confirmed that MKRN1 overexpression prevented mitochondrial swelling and preserved cristae architecture under copper stress. Immunoblotting demonstrated that MKRN1 maintains protein levels of Tomm20, a central mitochondrial import receptor that was otherwise depleted following MKRN1 knockdown or cuproptosis agonist treatment.

Comprehensive genetic rescue experiments confirmed that the anti-cuproptotic activity of MKRN1 depends specifically on the degradation of SLC31A1. Restoring SLC31A1 expression via plasmid transfection into MKRN1-overexpressing HCT15 cells counteracted the preservation of mitochondrial membrane potential, diminished Tomm20 levels, re-established HSP70 accumulation, and restored sensitivity to elesclomol-induced cell death. Conversely, simultaneous short hairpin RNA knockdown of SLC31A1 in MKRN1-depleted HT29 cells reversed mitochondrial depolarization, prevented excessive cell mortality, and blunted HSP70 induction, proving that SLC31A1 down-regulation is the direct downstream mechanism through which MKRN1 confers cellular resistance against cuproptosis.

The oncogenic role of the MKRN1/SLC31A1 regulatory axis was further validated in vivo through subcutaneous xenograft models in NOD-SCID mice injected with stably transfected HCT15 cells. Over the two-week monitoring period, tumors derived from MKRN1-overexpressing cells exhibited significantly enhanced growth rates, resulting in markedly higher excised tumor weights and volumes compared to vector controls. Immunohistochemical staining and Western blotting of tumor lysates revealed that MKRN1-overexpressing xenografts displayed reduced SLC31A1 protein levels alongside preserved FDX1 expression, demonstrating in vivo inhibition of copper-mediated proteotoxic stress and corroborating the tumor-promoting effect of MKRN1-driven cuproptosis suppression.

The authors note several boundaries to the current investigation, pointing out that their mitochondrial assessments are largely correlative and warrant direct metabolic measurements of oxygen consumption rates, adenosine triphosphate production, tricarboxylic acid cycle enzyme activity, and reactive oxygen species generation in future studies. Furthermore, while subcutaneous xenografts demonstrated accelerated tumor growth upon MKRN1 overexpression, validating cuproptosis resistance in response to combination therapies using spontaneous adenomatous polyposis coli mutant colorectal models remains necessary. Nonetheless, these findings establish the MKRN1/SLC31A1 axis as a pivotal post-translational regulator of copper metabolism and suggest that disrupting this interaction or deploying targeted degraders against MKRN1 could sensitize colorectal cancers to copper-based therapeutic regimens.

Subject of Research: MKRN1-mediated ubiquitination of SLC31A1 and cuproptosis evasion in colorectal cancer

Article Title: MKRN1 suppresses cuproptosis in colorectal cancer through ubiquitin-dependent regulation of SLC31A1

Article References: Luo, H., Hu, J., Lin, X., Zhao, S., Li, M., & Li, Q. (2026). MKRN1 suppresses cuproptosis in colorectal cancer through ubiquitin-dependent regulation of SLC31A1. Biological Procedures Online, 28(1), Article 37. https://doi.org/10.1186/s12575-026-00349-x

Image Credits: AI Generated

DOI: 10.1186/s12575-026-00349-x

Keywords: colorectal cancer, cuproptosis, MKRN1, SLC31A1, ubiquitination, copper homeostasis, proteasomal degradation, mitochondrial integrity, cancer biology, targeted therapy, suppresses, colorectal