Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma.

Abstract

Gemcitabine resistance remains a significant problem in cholangiocarcinoma (CCA) treatment. The present study aimed to identify novel molecular targets associated with gemcitabine resistance in CCA using integrated bioinformatics, molecular docking, and experimental validation. Commonly differentially expressed genes (DEGs) were identified based on three public transcriptomic datasets (GSE116118, GSE208659, and GSE140077) from stable gemcitabine-resistant cancer cell lines. Expression levels of selected upregulated genes were validated in Thai patients with CCA using the GEO datasets. Further validation was performed using real-time PCR on parental and gemcitabine-resistant CCA cell lines (KKU-213A/KKU-100 and KKU-213A-GemR/KKU-100-GemR). The 3D structure of the candidate protein was modeled using AlphaFold3 and refined by molecular dynamics simulations. Druggable pockets were predicted using CavityPlus. Virtual screening of 5,396 drug-repurposing compounds was performed using AutoDock Vina to identify potential inhibitors of the candidate protein. In total, 14 DEGs were commonly upregulated in three datasets from stable gemcitabine-resistant cancer cell lines. A total of six genes, including RAB1B, JAG1, PACS1, ANKS6, SF3B4, and RTKN2, were significantly elevated in CCA tissues compared with adjacent normal tissues. Reverse transcription-quatitative PCR confirmed that RTKN2 was significantly overexpressed in two stable gemcitabine-resistant CCA cell lines, KKU-213A-GemR and KKU-100-GemR. Pathway analysis of RTKN2-associated genes revealed enrichment in mitochondrial and metabolic processes, including suppression of the TP53-regulated metabolic pathway. Molecular docking analysis identified several candidate compounds with strong binding affinities to RTKN2 (-10.49 to -11.47 kcal/mol), including NKP608, tegatrabetan, umbralisib, vepdegestrant, and MK-3207. RTKN2 was identified as a novel and druggable molecular target potentially contributing to gemcitabine resistance in CCA. Targeting RTKN2 may offer a promising approach to overcome chemoresistance and improve outcomes for patients with CCA.