ORC6 marks a replication-active malignant epithelial state and is associated with immune-low features in oral...

Oral squamous cell carcinoma (OSCC) shows marked biological heterogeneity, but markers that connect malignant epithelial states with the tumor immune context remain limited. Origin recognition complex subunit 6 (ORC6) participates in DNA replication licensing, but its disease-specific role in OSCC is not well defined.

We integrated anatomically filtered bulk transcriptomic data, external validation cohorts, single-cell transcriptomic profiling, immune-context analyses, proliferation-adjusted sensitivity analyses, virtual perturbation, and in vitro ORC6-knockdown assays to evaluate the clinical and biological relevance of ORC6 in OSCC.

After reconstruction of a strictly defined TCGA oral cavity OSCC cohort excluding base of tongue and other non-oral cavity subsites, ORC6 remained upregulated in OSCC tissues and cell lines and was associated with adverse clinicopathological features and poorer survival. Exploratory analysis in the HPV-negative GSE41613 cohort further supported the adverse survival association of ORC6. Single-cell analysis showed preferential enrichment of ORC6 in malignant epithelial cells. Within this compartment, high ORC6 expression marked a replication-active state characterized by activation of DNA-replication and cell-cycle programs and weaker transcriptional signatures of interferon response and antigen presentation. These differences were directionally preserved in donor-matched pseudobulk comparisons and after cell-cycle-score regression. At the bulk-tumor level, proliferation-adjusted models showed that ORC6 retained directionally inverse associations with selected immune-related features, including cytolytic activity, IFN-γ response, and MHC-I/antigen-presentation signatures. ORC6 knockdown in CAL27 cells reduced cell growth, migration, invasion, and S-phase fraction while increasing antigen-presentation-associated transcripts and surface HLA-A/B/C expression.

These findings support ORC6 as a marker of a replication-active malignant epithelial state associated with attenuated immune-related features in OSCC, particularly antigen-presentation-related programs. Further functional immune-recognition studies and independent clinical validation are warranted.