Precision Peptide Design and its mechanism for Gallbladder Cancer: Insights from RNA Sequencing, Proteomics, and Whole Exome Sequencing

Anton Yuryev ¹, John Catanzaro ², Md Shamsuddin Sultan Khan ^3*, Dmitry Novitsky ⁴, Alexei Surin ⁵, Alexander Mazur ⁶

This study integrates NGS, proteomics, and WES to uncover molecular mechanisms in gallbladder cancer, highlighting new therapeutic and vaccine targets.

Abstract

Background: Gallbladder cancer (GBC) is a rare but aggressive malignancy with a poor prognosis, often due to late diagnosis and limited treatment options. The complexity of GBC’s molecular mechanisms has impeded progress in understanding its pathogenesis and developing effective therapies. Recent advances in next-generation sequencing (NGS) technologies have opened new avenues for exploring the genomic and transcriptomic landscapes of GBC, providing deeper insights into its biology. Methods: RNA sequencing (RNAseq) data from GBC tumor samples were analyzed and normalized against a control dataset of normal gallbladder tissues from the National Cancer Institute's Gene Expression Omnibus. Sub-network enrichment analysis (SNEA) using Pathway Studio identified key regulators of differential gene expression, focusing on histone deacetylases (HDACs) and microRNA MIR146A. Proteomic analysis of patient urine samples was performed via mass spectrometry to identify unique proteins. Whole exome sequencing (WES) of blood and tumor samples detected driver mutations and genetic variations  linked to tumor suppressor genes and oncogenes. HLA typing facilitated the design of personalized immunotherapies and cancer vaccines. Results: Normalization of RNAseq data enabled accurate comparisons between tumor and normal tissue gene expression. SNEA identified HDAC1 and HDAC2 as significant regulators associated with poor prognosis in GBC. Urine proteomics revealed 775 proteins, with 442 unique to GBC patients, potentially serving as biomarkers. WES identified 6,349 variations, including 328 loss-of-heterozygosity (LOH) and 410 gain-of-heterozygosity (GOH) mutations, with several linked to HDAC activation and chromatin remodeling. Novel neoantigens from these mutations were selected for potential vaccine development, and HLA typing provided crucial information for personalized immunotherapy. Conclusion: This study integrates NGS, proteomics, and WES to elucidate GBC’s molecular mechanisms. Findings highlight the roles of HDACs and MIR146A in tumor progression, identify novel mutations, and suggest targeted therapeutic and vaccine development opportunities. Combining genomic, transcriptomic, and proteomic analyses enhances our understanding and treatment of gallbladder cancer.

Keywords: Gallbladder cancer, RNA sequencing, HDACs, Proteomics, Whole exome sequencing

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