State-Dependent Decoupling of a CREM-Centric cAMP-Responsive Regulon and Survival in Hepatocellular Carcinoma.

Background: The cAMP-responsive element modulator (CREM) has emerged as a cAMP-responsive transcriptional node associated with dysfunctional lymphocyte states in chronic hepatic infection and some immunosuppressive tumor microenvironments, but its regulatory output and isoform usage by specific cell-types remain insufficiently defined in hepatocellular carcinoma (HCC). Because CREM produces activating isoforms and repressive inducible cAMP early repressor (ICER) isoforms through alternative promoter usage, bulk or single-cell CREM mRNA abundance may not faithfully describe the net direction of downstream CRE-dependent transcriptional programs. Methods: Single-cell RNA-seq datasets spanning 18,665 T cells from two independent, pathologically validated primary liver cancer cohorts (32 HCC and 5 iCCA patients) were integrated, with HCC-only sensitivity analyses confirming the primary findings. Using prior-knowledge transcriptional regulon frameworks cross-referenced with CRE-family genomic occupancy data (ENCODE), CREM mRNA expression was decoupled from its inferred downstream regulon activity at single-cell resolution. Validation was performed using a third-generation long-read direct RNA sequencing hepatic model and bulk TCGA clinical survival cohorts. Results: Across multiple regulon definitions, single-cell CREM mRNA and regulon activity were characterized by prominent low-coupling at the single-cell level (meta-Spearman ρ < 0.20, explaining <4% of variance). Crucially, this low-coupling was not uniform but followed a continuous T-cell state gradient, where the correlation between CREM expression and target activation progressively tightened as T cells transitioned from a resting state toward an activated or exhausted phenotype. Long-read direct RNA sequencing in a hepatic lineage reference model revealed that the activation-associated P1 promoter (CREMτ) predominated over the repressor-associated P2 promoter (ICER) by a 14:1 ratio under steady states. In bulk TCGA-LIHC data, elevated CREM-centric regulon scores, but not raw mRNA levels, correlated with favorable overall survival (HR = 0.787, p = 0.026), operating primarily as a T-cell activation- and infiltration-associated prognostic feature rather than an independent cell-autonomous biomarker. Conclusions: Single-cell CREM mRNA abundance is not a reliable proxy for its transcriptional output in HCC tumor-infiltrating lymphocytes. Instead, the coupling between CREM expression and its regulon activity is dynamically modulated by the T-cell activation state, a phenomenon partially explained by the usage of alternative isoforms in reference models. These findings caution against the routine practice of translating crude transcription factor mRNA metrics directly into functional regulatory inputs within a complex tumor immune microenvironment.