Marija Lazić*, Marija Cumbo Stikić, Aleksandra Vitkovac, Igor Davidović, Mateja Ilić, Marija Schwirtlich, Martina Mia Mitić, Branko Tomić and Valentina Đorđević
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
marijal3009 [at] gmail.com
Abstract
Emerging evidence highlights the non-hemostatic roles of the coagulation proteins in shaping the immune and stromal landscape of various cancers. However, despite the prevalence of coagulation factors in the tumor microenvironment, the precise gene expression profiles induced by prothrombin remain poorly understood.
To elucidate these mechanisms, we systematically profiled the temporal transcriptional response of Caco-2 colorectal cancer cells to prothrombin treatment. Using a robust experimental design, we generated an in-house RNA-seq dataset (DNBseq technology) comprising cells treated with prothrombin, glycerol-vehicle controls, and untreated counterparts at 24 and 48 hours, thereby allowing us to distinguish genuine treatment-induced signals from time-dependent culture drift. Raw counts were analyzed with DESeq2 using Benjamini–Hochberg false discovery rate control, followed by pre-ranked Gene Set Enrichment Analysis (GSEA) of Gene Ontology Biological Process terms.
GSEA showed that, under control conditions, the 48 h versus 24 h contrasts were characterized by a marked shift toward reduced biosynthetic and proliferative programs: ribosome biogenesis, rRNA metabolism, and DNA replication were negatively enriched (NES ≈ −2 to −3). In untreated cells, autophagy regulation and Golgi/vesicle transport were positively enriched at 48 h, consistent with a time-dependent adaptive shift toward autophagy and vesicular trafficking. In contrast, prothrombin-treated cells displayed a distinct 48 h versus 24 h signature with positive enrichment for endoplasmic reticulum to Golgi and vesicular transport, protein quality control and proteostasis, translational initiation and regulation, microtubule-dependent organelle transport, and cell-cycle regulation (NES ≈ +1.6 to +2.3). The broadly similar decline observed across control conditions supports the interpretation that prothrombin-associated enrichments are not explained by vehicle exposure or time in culture alone.
These results demonstrate that prothrombin can act as a modulator of cellular state, redirecting the transcriptional landscape toward pathways critical for cellular fitness and proliferation. This distinct signature provides a foundation for future studies investigating how prothrombin-mediated signaling influences colorectal cancer.
Keywords: prothrombin, colorectal cancer, RNA-seq, GSEA