Harnessing Paracrine Signaling of Guanylin: From Pan-Cancer Analysis to Targeted Therapeutic Nanoplatform

: Colorectal cancer (CRC) poses a formidable challenge, requiring the precise delivery of therapeutic agents to eliminate cancer cells while sparing healthy tissue. Achieving this goal is based on the strategic selection of therapeutic targets, the utilization of nanocarriers with high efficacy and minimal toxicity, and the development of gene constructs that enable controlled expression within cancer cells. Methods: In this study, we adopted a pan-cancer analysis to widely investigate the GUCA۲A gene, which encodes the Guanylin hormone. Our exploration encompassed an analysis of its expression patterns across various tissues and single cells, an examination of its clinical implications, analysis of its methylation profiles, identification of mutational changes, an evaluation of its impact on immune responses, and in-depth functional analyses. Subsequently, GUCA۲A emerged as a promising candidate for gain-of-function studies. It was then amplified and incorporated into gene constructs featuring a robust CMV promoter and a cancer-specific MUC۱ promoter, equipped with hypoxia response element (HRE) cassettes. The succinylated PEI-۹ (SPEI-۹), known for its high buffering capacity, low toxicity, excellent biocompatibility, optimal physicochemical properties, and efficient gene transfer capabilities, was synthesized and thoroughly characterized for its capacity to carry these gene constructs. Finally, we assessed the potential of this targeted gene-hormone therapy nano-system, capable of expressing the guanylin peptide hormone, on both HCT-۱۱۶ cancer cells and normal Vero cell lines through various in vitro assays, including RT-qPCR, cell viability assessments, Annexin V apoptosis assays, wound healing experiments, and colony formation tests. Results: Our extensive pan-cancer analyses revealed marked irregularities in GUCA۲A gene expression, distinctive methylation profiles, mutational variations, and a significant correlation between GUCA۲A and immune activation, all of which were associated with unfavorable survival outcomes in CRC. Additionally, we introduced SPEI-۹ as a remarkably efficient and safe nanocarrier for gene delivery applications. In vitro experiments demonstrated that both gene constructs expressing guanylin exhibited the potential to inhibit cell growth, induce apoptosis, impede cell migration, and curtail the colony formation ability of cancer cells. Notably, these effects were more robust but non-specific when the constructs contained the CMV general promoter. In contrast, induction via the construct featuring the HRE-MUC۱ promoter was demonstrated to be specific to cancer cells. Conclusion: Fusing the potent universal CMV promoter with the specific HRE-MUC۱ promoter for guanylin peptide hormone expression, exhibited highly efficient and cancer-specific anti-cancer effects when delivered using nanocarriers characterized by high efficiency and low cytotoxicity. This nano-system presents promising prospects for the future of targeted gene-hormone therapy in the context of CRC.

کلیدواژه ها:

Pan-Cancer ، Colorectal Cancer ، Guanylin ، Guanylyl Cyclase C ، Gene Therapy