RNA-seq profiling identifies FOX genes as potential CRC biomarkers

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide, including Puerto Rico, highlighting the urgent need for reliable biomarkers to enable earlier and more accurate detection. The FOX family of transcription factors regulates key cellular processes, including cell proliferation, apoptosis, metabolism, and immune regulation. However, their expression patterns and diagnostic utility in Hispanics individuals living in Puerto Rico (HPR) have not been characterized. The aim of this study is to characterize FOX gene expression profiles across healthy and CRC individuals and evaluate their prognostic and diagnostic performance in HPR. Tissue samples from 28 participants in the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR) cohort (13 controls-healthy normal mucosa, 7 early-stage, and 8 advanced-stage CRC tumors) were used for RNA extraction and bulk RNA sequencing. Differential gene expression was performed using DESeq2 (|log2 fold change|>2, adjusted p<0.05). Four FOX signature scores were constructed from significant non-collinear FOX genes (VIF<10) using Gene Set Variation Analysis. Kaplan Meier survival analysis was performed for each differentially expressed FOX gene using publicly available colon cancer data. Gene associations with CRC status were evaluated using binomial generalized linear models (GLM), unadjusted and adjusted for age and BMI. Diagnostic performance was assessed using sensitivity, specificity, and AUC-ROC. Significance was set at 0.05. Statistical analyses were performed using R, version 4.3.3.Expression profiles of 35 FOX genes separated samples into distinct clusters by early- and advanced-stages of CRC and controls. Of 12 differentially expressed genes, 5 genes were upregulated (FOXC1, FOXC2-AS1, FOXH1, FOXN2, FOXQ1), while 7 were downregulated (FOXA1, FOXA3 FOXD2, FOXD3-AS1, FOXE3, FOXF2, FOXO1) in CRC relative to controls. Kaplan-Meier analysis showed that higher expression of 4 genes (FOXA3, FOXC1, FOXF2, FOXQ1) was associated with reduced survival probabilities in CRC patients, whereas higher expression of FOXA1 and FOXN2 was associated with improved survival (p<0.05). In unadjusted analysis, FOXQ1 expression and a 6-gene FOX signature, including FOXC1 and FOXQ1, significantly increased CRC odds (p<0.05), while 7 genes significantly decreased CRC odds. After adjusting for age and BMI, protective associations were observed for FOXE3 (OR:0.19), and FOXD2 (OR:0.42), while FOXQ1 (OR:1.45) and FOXC1 (OR:2.83) showed the largest increased odds of CRC. Multiple FOX genes were differentially expressed in CRC, with FOXC1 and FOXQ1 showing significant associations with both survival of CRC patients and disease risk. A FOX gene signature further improved diagnostic performance. These findings support the potential of FOX transcription factors as clinically relevant biomarkers for HPR.