Investigating The Role of Activator Protein -1 Subunits (FOSL2, c-Jun and JunB) in Regulating The Hypoxic Response in Colorectal Cancer (CRC) Cells and Identifying Its Role as a Possible Therapeutic Target For Hypoxic Regions in CRC Tumours
Publication Date
July 27, 2026
Creator
Abstract
Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Hypoxia (low oxygen) is frequently (~45%) seen in these tumours where it mediates metastasis, resistance to conventional therapy, immunotherapy, and molecular-targeted therapy, resulting in poor patient survival. While hypoxia-induced transcriptional reprogramming is primarily attributed to HIF transcription factors, we identified Activator protein 1 (AP1) transcription factor as a critical mediator of hypoxic CRC cell viability through functional shRNA screening. AP-1 is a dimeric transcription factor regulating proliferation, differentiation, and invasion under normoxic conditions. However, the specific roles of individual subunits in hypoxic adaptation remain unknown. Herein, this study characterised the roles of AP-1 subunits FOSL2, c-Jun, and JunB in CRC molecular adaptation under hypoxic conditions.
Method: Patient datasets were analysed for AP-1 expression, prognostic value, and hypoxia correlations. Tissue microarray staining assessed JunB clinicopathological significance. HCT116 and LS174T inducible CRISPRCas9 knockouts for FOSL2, c-Jun, and JunB were characterised using proliferation, colony formation, FACS (cell cycle/apoptosis), spheroid growth, and migration/invasion assays. FOSL2 knockouts underwent xenograft studies with histological analysis (Ki67, cleaved caspase-3, TUNEL). c-Jun/JunB knockouts were tested in 3D hydrogel models. RNASeq with qPCR validation identified knockout-induced transcriptional changes. Drug sensitivity to cisplatin and PT2399 was assessed under normoxic/hypoxic conditions. Peptide-based c-Jun inhibitors were evaluated for cellular uptake, viability effects, and AP-1 transcriptional inhibition.
Results: Hypoxia upregulated all three AP-1 subunits expression with enhanced phosphorylation. FOSL2 showed increased dimerization with JunB, and c-Jun. Patient datasets revealed elevated expression in advanced versus early-stage tumours (all, p≤ 0.001) with compartment-specific associations to lymph node metastasis, lymphovascular invasion, and perineural invasion, though lacking prognostic significance (HR=1.10, p=0.34).
Functional studies demonstrated all knockouts reduced proliferation, colony formation, and migration/invasion, with enhanced effects under hypoxia. FOSL2 knockout induced hypoxia-specific cell cycle arrest and apoptosis (increased cleaved caspase-3, p21), confirmed in xenografts showing increased necrosis (H&E), apoptosis (TUNEL) and reduced Ki67.
RNA-Seq revealed FOSL2 regulated metabolic pathways (glycolysis, gluconeogenesis, pentose phosphate pathway), while c-Jun and JunB disrupted ECM organisation and RTK signalling and TP53-mediated cell death (c-Jun knockout only).
Drug sensitivity analysis showed FOSL2 knockout markedly enhanced cisplatin sensitivity under hypoxia, while c-Jun and JunB had minimal effects. All three knockouts increased sensitivity to PT2399, with FOSL2 and JunB showing greater effects. Investigation of AP-1/HIF crosstalk revealed HIF-1α regulated all subunits while HIF-2α selectively controlled FOSL2 and JunB. Reciprocally, FOSL2 knockout inhibited both HIF-1α and HIF-2α in HCT116 cells, JunB knockout specifically reduced HIF-1α in both cell lines, and c-Jun showed no regulatory effect
c-Jun peptide inhibitors demonstrated hypoxia-selective efficacy, reducing AP-1 transcriptional activity and viability while inducing apoptosis, with negligible cell cycle effects.
Conclusion: This study establishes FOSL2 as a primary driver of hypoxic adaptation in CRC, with JunB as a key partner and c-Jun showing contextdependent effects. The bidirectional AP-1/HIF crosstalk reveals targetable vulnerabilities in hypoxic tumours. Future work should elucidate AP-1 activation mechanisms in hypoxic CRC to guide therapeutic development.
Item Type
ethesis
Thesis Type
PhD
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Subjects (LC)
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