Screening of Ceramide-Dependent Exosomal Cargoes in Terms of their Vesicular Abundance in Colorectal Cancer

Abstract

Colorectal cancer (CRC) is one of the most common malignancies worldwide and a leading cause of cancer-related mortalities. It originates from the epithelial cells of the colon or rectum, evolving through various genetic and epigenetic changes. CRC development is often associated with the adenoma carcinoma sequence, where benign polyps transform into malignant tumors over time. CRC metastasis is largely regulated by the secretome components released from the cancer cells. Neutral sphingomyelinase 2 (nSMase2) is potentially related to secretomes due to its role in exosome biogenesis which include the release of exosomal cargoes. This report highlights the significance of studying ceramide-dependent exosomal cargoes to enhance our understanding of colorectal cancer metastasis via Exosomes. Exosomes, the small extracellular vesicles secreted by cells, transport a variety of molecules, including proteins, nucleic acids, and lipids, that can affect disease progression. Ceramide-dependent exosomal cargoes are those that are reduced at their exosomal level upon blocking nSMase-2. Current study focusses on screening the ceramide-dependent exosomal cargoes that are highly reported in vesicular pools of “Vesiclespedia” database in regards to CRC. The finding of the study underscores the role of selected proteins classes that are involved in mediating intercellular communication between, providing insights into the molecular keystones of metastasis. The study highlights the role of Ceramide-dependent Exosomal Cargoes in promoting CRC metastasis through exosome-mediated intercellular communication and intracellular signaling modulation, suggesting potential therapeutic targets for managing CRC progression. From overall observation we have selected SPRED2 and LAMT as representative understudied proteins, given their minimal or absent documentation in CRC exosome literature. The inclusion of these low-frequency candidates aims to uncover potentially novel roles and functional significance that may have been previously overlooked in CRC pathogenesis

Introduction

Exosomes are small extracellular vesicles (30–150 nm) formed through complex biogenesis pathways involving inward budding of endosomal membranes, producing intraluminal vesicles (ILVs) that are released upon fusion with the plasma membrane. Exosome formation is regulated mainly by two pathways: ESCRT-dependent and ceramide-dependent (ESCRT-independent), with ceramide playing a key role in inducing membrane curvature and sorting cargo via neutral sphingomyelinase 2 (nSMase2).

Exosomes carry diverse bioactive cargo, including proteins, lipids, miRNAs, lncRNAs, and circRNAs, which mediate intercellular communication and modulate gene expression, signaling, and tumor microenvironment remodeling. Tumor-derived exosomal miRNAs and proteins promote processes such as angiogenesis, immune evasion, epithelial-mesenchymal transition, and drug resistance, thus facilitating cancer progression and metastasis.

In colorectal cancer (CRC), ceramide-dependent exosomal cargoes are crucial in disease progression. High-throughput proteomics comparing primary (SW480) and metastatic (SW620) CRC cell lines identified 112 common proteins downregulated upon inhibition of the ceramide pathway, representing a conserved set of exosomal cargoes linked to metastasis. These proteins fall into 20 functional classes, including metabolic enzymes, RNA metabolism, membrane trafficking, protein binding, and transporters, reflecting key processes in metastasis and tumor survival.

Analysis of existing vesicular protein studies reveals a research gap in CRC-specific exosomal protein investigation, with many protein classes underrepresented in colon/rectum-focused research. Key proteins such as RAP2A, ARF6, and GPC4 emerge as promising candidates for further study as potential biomarkers or therapeutic targets.

The study emphasizes the importance of ceramide-dependent exosomes in CRC progression, suggesting that a deeper understanding of their biogenesis and cargo composition could inform novel diagnostic and treatment strategies, improving patient outcomes.

Conclusion

The data collectively reveal that various proteins involved in exosome-mediated processes have been extensively studied in different sample types, their specific roles in CRC remain ineffectively explored. The classification and analysis of proteins identified from CRC primary and metastasis cells (SW480 & SW620) provide critical insights into the molecular landscape of metastatic colorectal cancer (CRC). Our findings highlight significant variations in protein functional classes, highlighting the importance of metabolic interconversion enzymes, RNA metabolism proteins, membrane trafficking proteins in CRC progression. Despite widespread research on these proteins across various sample types, our analysis indicates a significant research gap highly reported study in their specific roles in CRC, particularly in exosome-mediated signaling and tumor microenvironment modulation. Focusing on protein binding activity modulators, RAP2A, ARF6, and RAS6KA1 are notable candidates with documented roles in cell adhesion, cytoskeletal remodeling, and oncogenic signaling. However, CRC-specific studies account for only ~13% of total reports, highlighting the need for targeted investigations into their mechanistic contributions to CRC metastasis. Similarly, scaffold and adaptor proteins such as GPC4, SPRED2, and UBQLN1 play crucial roles in signal transduction and protein-protein interactions, yet they remain understudied in CRC, with only 16 out of 116 total studies (~13.8%) focusing on CRC-related samples. This underscores the necessity for further exploration of these proteins in CRC, particularly in the context oftumor progression, metastasis, and exosome-mediated communication. Understanding their functions could pave the way for identifying novel biomarkers and therapeutic targets. Future research should highlight CRC-specific studies to bridge these knowledge gaps and enhance our ability to develop targeted therapeutic strategies for metastatic CRC.

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Copyright

Copyright © 2025 Rose Rani, Archana Tiwari, Dipanjana Ghosh. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.