Endometrial cancer is among the most common malignancies of the female reproductive system, and its global incidence has climbed markedly in recent years. Roughly ninety percent of cases are caught early and respond well to surgery and chemoradiotherapy, but patients with locally advanced, recurrent, or metastatic disease often resist standard treatment and face poor outcomes. That clinical gap has pushed researchers to hunt for the molecular drivers that separate indolent tumors from aggressive ones. The new study focuses on the epitranscriptome, the layer of chemical modifications deposited onto RNA after transcription, which has emerged in the past decade as a major regulator of gene expression in health and disease.

Among these modifications, 5-methylcytosine, abbreviated m5C, is one of the most abundant marks on eukaryotic messenger RNA. Like other RNA modifications, m5C operates through a cast of enzymes and binding proteins: writer enzymes such as NSUN2 and NSUN6 install the methyl group, erasers can remove it, and reader proteins such as ALYREF recognize the mark and determine what happens to the tagged transcript. ALYREF, historically known for its role in ferrying mature mRNA out of the nucleus, binds m5C-modified sites and influences RNA stability, export, and metabolic fate. Previous work had implicated ALYREF in nasopharyngeal carcinoma, glioma, lung cancer, and bladder cancer, but its role in endometrial cancer remained uncharted.

The team began by mining several independent public datasets, including the Gene Expression Omnibus, The Cancer Genome Atlas, GTEx, and the Human Protein Atlas. Across every source, ALYREF was significantly elevated in tumor tissue compared with normal endometrium, at statistical thresholds below p = 0.001. The pattern held in paired comparisons of tumors and adjacent normal tissue from the same patients, and immunohistochemistry on clinical specimens confirmed the protein-level difference. Most strikingly, survival analysis of the TCGA endometrial carcinoma cohort showed that patients with high ALYREF expression had significantly shorter overall survival and relapse-free survival, and multivariate Cox regression established ALYREF as an independent prognostic factor after adjustment for clinicopathological variables. High ALYREF also tracked with aggressive histology, including grade 3 tumors and serous or mixed subtypes.

To test whether ALYREF actively drives the disease rather than merely marking it, the researchers manipulated its expression in endometrial cancer cell lines. Silencing ALYREF with short hairpin RNAs in HEC-1A and Ishikawa cells, which naturally express the protein at high levels, sharply reduced colony formation, cell proliferation measured by CCK-8 assays, and DNA synthesis measured by EdU incorporation. Wound healing and Transwell experiments showed impaired migration and invasion, while flow cytometry revealed G2/M cell cycle arrest and increased apoptosis. Conversely, forcing ALYREF expression in KLE cells, which normally produce little of the protein, enhanced every one of those malignant behaviors. In nude mice bearing subcutaneous xenografts, ALYREF knockdown significantly slowed tumor growth, reducing both tumor volume and final tumor weight over a 42-day observation period.

The mechanistic hunt began with an unbiased, three-pronged sequencing strategy. RNA sequencing of ALYREF-knockdown cells identified 1,890 differentially expressed genes. RNA immunoprecipitation sequencing captured 703 transcripts physically bound by ALYREF. And bisulfite sequencing tailored to detect m5C mapped 4,084 modified transcripts, revealing that the marks cluster mainly within the coding sequence of mRNAs. Intersecting all three datasets narrowed the field to 28 candidate targets, and among the ten most downregulated after ALYREF depletion, one gene stood out: XRCC6, which encodes the Ku70 subunit of the non-homologous end joining DNA repair complex. The correlation was robust at every level, from a Pearson coefficient of 0.749 between ALYREF and XRCC6 mRNAs in the TCGA cohort to a Spearman coefficient of 0.82 between the two proteins in 50 clinical tumor samples.

Rescue experiments then confirmed that XRCC6 is the functional effector of ALYREF. Restoring XRCC6 expression in ALYREF-deficient cells reversed nearly every malignant phenotype: proliferation recovered, migration and invasion returned, G2/M arrest eased, and apoptosis subsided. In xenografted mice, XRCC6 overexpression largely canceled the tumor-suppressive effect of ALYREF knockdown. The regulation itself proved to be a matter of mRNA stability rather than export. Actinomycin D chase assays showed that XRCC6 mRNA degraded much faster without ALYREF, its half-life shrinking significantly, while nuclear-cytoplasmic fractionation demonstrated that cytoplasmic levels of the transcript fell even as nuclear levels stayed constant, indicating that ALYREF protects the mRNA in the cytoplasm rather than shuttling it out of the nucleus.

The epitranscriptomic logic of the axis came into focus through experiments on the writer enzymes. When NSUN2 was silenced, XRCC6 mRNA levels dropped; knocking down NSUN6 had no effect. Crucially, in cells lacking NSUN2, additional depletion of ALYREF produced no further reduction in XRCC6, showing that the methyl mark is a prerequisite for ALYREF’s action. Bisulfite sequencing and integrative genomics viewer analysis revealed distinct m5C peaks on the XRCC6 transcript that overlapped precisely with ALYREF binding regions. Reporter assays drove the point home: mutating the methylated sites abolished ALYREF’s suppressive effect on luciferase activity driven by the XRCC6 sequence. Mutating either of two adjacent m5C sites in the coding region partially weakened ALYREF binding and mRNA stability, but mutating both together caused a synergistic collapse, suggesting the two sites operate cooperatively as a local recognition module, a model the authors note will require structural biology to fully confirm.

Downstream, the chain terminates at Wnt/β-catenin signaling, a pathway central to development and frequently hijacked in cancer. ALYREF knockdown drained β-catenin from the nucleus of HEC-1A cells and reduced levels of the Wnt target c-Myc, while XRCC6 overexpression restored both. Notably, co-immunoprecipitation found no direct physical interaction between XRCC6 and β-catenin, implying that XRCC6, a multifunctional nuclear protein, promotes β-catenin transcriptional output indirectly, perhaps by shaping nuclear co-activator complexes or chromatin accessibility. TOP/FOP Flash reporter assays confirmed that XRCC6 boosts β-catenin/TCF-dependent transcription, and canonical Wnt target genes such as AXIN2 and CCND1 fell when ALYREF was silenced. The decisive experiment used XAV-939, a pharmacological Wnt inhibitor: the drug largely blocked the proliferative rescue that XRCC6 overexpression conferred on ALYREF-deficient cells, proving the oncogenic phenotype depends on intact Wnt signaling.

The authors are candid about the study’s limits. Clinical protein validation relied on a single-center retrospective cohort of modest size, the cell line models do not capture the full genomic heterogeneity of TCGA molecular subtypes such as POLE-ultramutated or mismatch-repair-deficient tumors, and the work examined cell-autonomous regulation without probing interactions with cancer-associated fibroblasts or immune cells. Even so, the findings delineate a complete oncogenic axis, ALYREF to m5C to XRCC6 to Wnt/β-catenin, and broaden a growing picture in which ALYREF’s downstream targets differ sharply from one cancer type to another. Because XRCC6 plays dual roles in DNA damage repair and Wnt signaling, patients whose tumors show high ALYREF/XRCC6 expression might ultimately benefit from therapies aimed at either pathway, an idea that now rests on a solid mechanistic foundation and awaits prospective, multicenter testing.

Subject of Research: The role of the m5C reader protein ALYREF in promoting endometrial cancer progression through XRCC6-mediated Wnt/β-catenin signaling

Article Title: m5C Reader ALYREF Promotes the Progression of Endometrial Cancer by Activating the XRCC6‐Mediated Wnt/β‐catenin Signaling Pathway

Article References: Hao, L., Zhang, J., Qian, Q., Jiang, M., Liu, Y., Geng, Y., Cui, N., Guo, Q., & Guo, J. (2026). m 5 C Reader ALYREF Promotes the Progression of Endometrial Cancer by Activating the XRCC6‐Mediated Wnt/β‐catenin Signaling Pathway. Advanced Science, Article e77548. https://doi.org/10.1002/advs.77548

Image Credits: AI Generated

DOI: 10.1002/advs.77548

Keywords: endometrial cancer, ALYREF, m5C, epitranscriptomics, XRCC6, Wnt/β-catenin signaling, NSUN2, RNA modification, mRNA stability, Ku70, prognostic biomarker, Advanced Science