Bulk and single-cell analysis of FAM72D reveals its prognostic and functional relevance in hepatocellular carcinoma

Family with sequence similarity 72 member D (FAM72D) has been implicated in proliferative programs in several malignancies, but its clinical significance, cellular distribution and underlying biological programs in hepatocellular carcinoma (HCC) remain insufficiently characterized. FAM72D expression and its clinical relevance were evaluated using The Cancer Genome Atlas-liver hepatocellular carcinoma (TCGA-LIHC) cohort and three independent GEO cohorts. Cox regression, patient-level bootstrap validation, calibration analysis and decision curve analysis were used to assess its prognostic contribution. Single-cell and spatial transcriptomic analyses were performed to define the cellular distribution of FAM72D. Differential-expression, functional-enrichment, protein-protein interaction and SCENIC analyses were used to characterize FAM72D-centered transcriptional programs. Immune-related analyses were conducted using tumor immune estimation resource and tumor immune dysfunction and exclusion (TIDE) and the functional effects of FAM72D silencing were examined in HuH7 cells. FAM72D was consistently upregulated in HCC tissues in TCGA and three independent GEO cohorts and was associated with adverse clinicopathological characteristics. Higher FAM72D expression was independently associated with unfavorable overall survival in the multivariable TCGA model (hazard ratio 2.02; 95% confidence interval 1.45-2.81; P<0.001). Adding FAM72D increased the optimism-corrected C-index from 0.611 to 0.662, while independent tissue-level validation yielded area under the curve of 0.861, 0.926 and 0.832 in GSE76427, GSE39791 and GSE54236, respectively. Single-cell analysis identified a FAM72D-high malignant-cell population with pronounced G2/M, mitotic, DNA-replication and DNA-repair activity. Bulk and single-cell transcriptomic analyses converged on a coordinated proliferative program characterized by enhanced cell-cycle signaling and forkhead box M1 (FOXM1) regulon activity. High FAM72D expression was also coupled to increased immune-checkpoint expression, reduced estimated CD8+ T-cell abundance and higher TIDE scores. Silencing FAM72D reduced HuH7-cell viability, colony formation and migration and increased apoptosis. FAM72D expression is associated with unfavorable prognosis and a proliferative malignant-cell state in HCC. The combined clinical, transcriptomic, single-cell, immune-related and functional findings support further evaluation of FAM72D as a candidate prognostic marker and a feature of proliferative HCC-cell programs.