USP6NL knockdown suppresses colorectal cancer progression by inducing CASP9-Mediated apoptosis and disrupting FOXC2/SNAI1-Driven EMT and angiogenesis.

BACKGROUND

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with tumor progression often driven by dysregulated oncogenic pathways. USP6NL, a known regulator of endocytic trafficking, has recently been implicated in tumorigenesis. However, its precise role in CRC remains unclear, and more studies are still needed to deepen our understanding of underlying mechanisms implicated in its oncogenic role. Therefore, silencing USP6NL could provide a novel therapeutic strategy by concurrently disrupting several oncogenic mechanisms, creating a new avenue for CRC management, particularly in patients who develop resistance against conventional therapies. This study investigates the impact of USP6NL knockdown on CRC cell morphology, proliferation, apoptosis, migration, angiogenesis, and metabolic adaptation, providing mechanistic insights into its oncogenic functions.

METHODS

HCT116 colorectal cancer cells were transfected with USP6NL-specific siRNA. Immunocytochemistry was used to confirm successful silencing, functional assays were performed to assess changes in cell morphology using phase-contrast and scanning electron microscopy, and colony formation and wound healing assays were performed to assess cell clonogenic capacity and migration, respectively, in addition to apoptosis assay via flow cytometry, and RT²-Profiler PCR array to measure variation in gene expression of 84 cancer-related genes. Statistical analyses were performed to evaluate significant differences between control and USP6NL-silenced groups.

RESULTS

USP6NL depletion led to profound morphological changes, including membrane blebbing, cell shrinkage, and loss of adhesion, reflecting late apoptotic features of cells. These findings were further supported by flow cytometry, which confirmed increased apoptosis, with a higher proportion of late apoptotic cells (20.99% in USP6NL knockdown vs. 2.69% in control, p = 0.042). Colony formation assays revealed a significant reduction in the clonogenic potential, suggesting a critical role of USP6NL in promoting CRC cell proliferation (p ≤ 0.05). The wound healing assay demonstrated impaired migration in USP6NL-silenced cells, with a marked delay in wound closure (p = 0.0201 at 48 h). Gene expression analysis revealed a significant downregulation of VEGFC (-8.62-fold) and ANGPT2 (-4.03-fold), impairing angiogenesis and suppressing FOXC2, SNAI1, and SNAI2, indicating EMT inhibition. Additionally, CASP9, APAF1, and BCL2L11 were upregulated, confirming the activation of intrinsic apoptosis, while metabolic regulators HIF1A and LDHA were downregulated, suggesting impaired tumor hypoxic adaptation.

CONCLUSION

This study establishes USP6NL as a key modulator of CRC progression, regulating proliferation, apoptosis, migration, angiogenesis, and metabolic pathways. The loss of USP6NL leads to EMT suppression, apoptosis induction, and reduced tumor cell viability, positioning it as a potential therapeutic target in colorectal cancer. Further investigations are warranted to explore USP6NL's interactions in oncogenic signaling networks and its feasibility as a target for CRC therapy. It could serve as a promising therapeutic target in colorectal cancer, potentially enhancing tumor cell death and limiting metastasis. Targeting USP6NL could also provide a novel approach in combination with existing therapies, improving treatment efficacy and reducing side effects.