Mimicking a Negative Elongation Factor-E (NELF-E) Single Nucleotide Polymorphism (SNP) in Hepatocellular Carcinoma (HCC)
Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths in the US and worldwide. Negative elongation factor E (NELF-E) promotes tumor progression in more than 38% of all HCC tumors and patients with high NELF-E levels have significantly lower overall survival. Our previous work identified a germline single nucleotide polymorphism (SNP) of NELF-E, rs79208225 (G>A), which conferred an overall survival benefit of 22 months and disease-free survival benefit of 28 months. We sought to determine, first, how the SNP affected NELF-E to cause the phenotype and, second, how to harness that to combat the hallmarks of cancer. Our hypothesis was that by mimicking the NELF-E SNP phenotype we could decrease NELF-E protein and mRNA levels, as well as slow colony formation, proliferation, and migration in vitro and in vivo.
Methods: To determine the effect of the NELF-E SNP on mRNA and protein, we performed quantification and sequencing via patient sample and polymerase chain reaction (PCR) and Western blot (WB) as well as HCC cell line mini-gene assays. We designed two anti-sense oligonucleotides (ASOs) to mimic the effects of the SNP in vitro. Three cell lines, Huh1, Hep3b, and HLE, were electroporated to deliver the ASOs. PCR and WB were performed to confirm the ASO effect on NELFE. Xcelligence and colony formation assays were performed to determine ASO effects on proliferation. Finally, NOD-SCID mice were injected with Huh1 cells to create orthotopic in vivo HCC tumor models. These mice subsequently received intraperitoneal ASO injections. T-tests were performed to compare treatment endpoints. All statistics were performed on STATA 17.
Results:NELFE SNP affects splicing to decrease its mRNA and protein expression. Specifically, the SNP causes exon-10 skipping, mRNA truncation, and subsequent deletion leading to decreased protein translation. Treatment ASOs, i9 and e10, caused decreased NELF-E mRNA and protein. Xcelligence assays showed significantly decreased proliferation and (p
Conclusions: The NELF-E SNP causes exon 10 skipping, creating truncated mRNA and protein product. ASOs were successfully designed to mimic this phenotype pharmacologically and substantially reduced protein and mRNA levels, as well as slowed colony formation and proliferation in vitro. Utilizing NODSCID mice, we successfully created a model for tumor growth and ASO treatment in vivo and showed preliminary reduction in tumor volume following treatment. With success, these findings could lead to a pharmacological treatment of HCC which confers a level of overall and disease-free survival currently unavailable to patients.
Lamm, MD, Ryan, "Mimicking a Negative Elongation Factor-E (NELF-E) Single Nucleotide Polymorphism (SNP) in Hepatocellular Carcinoma (HCC)" (2022). Department of Surgery Resident Research Day. Paper 54.