CCSG Pilot: NRP1 Exon 4-Skipping Variant and Colorectal Cancer Prognosis in Appalachian Kentucky

Lay Abstract Colorectal cancer (CRC) is a serious disease that affects the colon or rectum. In Appalachian Kentucky, more people develop and die from CRC compared to other parts of the state and country. The main reason people die from CRC is because the cancer spreads to other parts of the body, a process called metastasis. Unfortunately, doctors and scientists still don’t fully understand how this happens, and there are not enough tools to detect or stop it. One important protein in cancer is called Neuropilin-1 (NRP1). It helps cancer cells grow and spread. Our research team recently discovered a new version of this protein, called NRP1-ΔE4, which is found in many CRC cases. This new version of the protein makes cancer spread more aggressively than the regular form of NRP1. We also found that NRP1-ΔE4 moves between cells inside tiny bubble-like structures called exosomes. These exosomes help cancer cells survive and spread. When we blocked exosomes or removed NRP1-ΔE4 from them, the cancer’s ability to spread dropped significantly. Based on these findings, we believe that NRP1-ΔE4 could be a useful biomarker—a sign that helps doctors predict how the cancer will behave. To test this, we have developed special lab tests to measure NRP1-ΔE4 in tissue samples and in exosomes found in the blood. Using these tests, we will: 1) study how NRP1-ΔE4 levels are linked to CRC progression and recurrence in patients from Appalachian Kentucky, and 2) investigate whether exosomes carrying NRP1-ΔE4 could help predict which patients are more likely to develop metastatic (advanced) cancer. If successful, this research could lead to better ways to detect and track colorectal cancer, helping doctors provide earlier and more personalized treatment. By understanding how NRP1-ΔE4 contributes to CRC, we hope to improve cancer care for patients in Appalachian Kentucky and beyond. Scientific Abstract The Appalachian region of Kentucky has higher incidence and mortality rates of colorectal cancer (CRC) compared to the rest of Kentucky and the nation, highlighting the urgent need to identify key drivers and biomarkers of CRC development in this region. Metastasis remains the leading cause of CRC-related deaths. Neuropilin-1 (NRP1), a transmembrane glycoprotein, functions as a co-receptor for multiple growth factors and plays diverse roles in cancer progression and metastasis. Recently, we discovered a novel human NRP1 splice variant, NRP1-ΔE4, resulting from exon 4 skipping. This variant is frequently expressed in CRC, strongly correlates with disease progression, and drives metastasis more potently than wild-type NRP1. Our new data further demonstrate that NRP1-ΔE4 undergoes constitutive trafficking via the endosomal-exosomal pathway, facilitating its recycling to promote CRC metastasis. Notably, genetic inhibition of exosome secretion or immunodepletion of exosomal NRP1-ΔE4 significantly reduces the metastatic potential of CRC cells. These findings underscore NRP1-ΔE4 as a highly aggressive oncogenic splice variant that promotes CRC metastasis and highlight its potential as a biomarker for CRC progression. We hypothesize that NRP1-ΔE4 serves as a prognostic biomarker for CRC metastatic progression and is associated with poor clinical outcomes in Appalachian CRC patients. To test this hypothesis, we have developed novel, sensitive, and specific assays to detect and quantify NRP1-ΔE4 at both the mRNA and protein levels in formalin-fixed paraffin-embedded CRC tissues and in exosomes isolated from blood samples. Using these assays, we will: 1) evaluate NRP1-ΔE4 mRNA expression in CRC tissues as a potential prognostic biomarker for Appalachian CRC patients, and 2) identify circulating exosomal NRP1-ΔE4 as a potential biomarker associated with CRC metastasis in patients from Appalachian Kentucky. Successful completion of this project will establish NRP1-ΔE4 as a potential prognostic biomarker for CRC progression, metastasis, and poor survival in Appalachian Kentucky patients. These findings could inform more precise CRC treatment strategies and ultimately help reduce CRC mortality in Appalachian Kentucky and beyond. The project will be conducted collaboratively by Markey Cancer Center investigators through the MCO and TO programs, with engagement from two CRC patient advocates who share our commitment to improving CRC prognosis in Appalachia. Shared Resource Facilities, including Biospecimen Procurement and Translational Pathology, Cancer Research Informatics, and Biostatistics and Bioinformatics, will support the proposed studies. To reach a broader audience, we will disseminate our findings through materials tailored for the general public.