Targeting the MLN64/STARD3-cholesterol axis disrupts tight junction remodelling and attenuates gastric cancer aggressiveness

Li, Amber Xinyu, Chen, Yiran, Zhuang, Xinguo, Alshammari, Layla, Khan, Elyas, Dou, Qingping, Martin, Tracey ORCID: https://orcid.org/0000-0003-2690-4908 and Jiang, Wen G. ORCID: https://orcid.org/0000-0002-3283-1111 2026. Targeting the MLN64/STARD3-cholesterol axis disrupts tight junction remodelling and attenuates gastric cancer aggressiveness. Frontiers in Oncology 10.3389/fonc.2026.1913732

PDF - Accepted Post-Print Version Available under License Creative Commons Attribution. Download (17MB)

Abstract

Gastric cancer remains a major cause of cancer-related mortality, largely due to invasion, metastasis, and therapeutic resistance. Dysregulated cholesterol metabolism is increasingly recognised as a driver of gastric cancer progression, but how intracellular cholesterol trafficking contributes to malignant junctional remodelling remains poorly understood. MLN64/STARD3 is a cholesterol-binding lipid transfer protein associated with cancer progression, yet its functional role in gastric cancer cell architecture and aggressiveness has not been fully defined. In this study, we investigated whether MLN64 is associated with cholesterol-dependent tight junction organisation and malignant behaviour in gastric cancer cells. Stable MLN64 knockdown in AGS and HGC-27 cells reduced membrane-associated free cholesterol, as shown by an approximately 50% decrease in the Filipin III membrane/cytoplasm fluorescence ratio in both cell lines. MLN64 depletion was also associated with reduced ZO-1 protein abundance, disrupted junctional ZO-1 localisation, and impaired tight junction integrity, including reduced transepithelial resistance, altered impedance, and increased paracellular permeability. Functionally, MLN64 depletion attenuated gastric cancer cell proliferation and invasive capacity, supporting an association between this cholesterol-trafficking axis, junctional organisation, and aggressive cellular phenotypes. To explore the pharmacological tractability of this pathway, in silico screening of an FDA-approved drug library identified Digoxin and Targretin as candidate compounds predicted to interact with the MLN64 START domain. Digoxin showed a favourable predicted binding energy of -13.73 kcal/mol. Functional validation showed that these agents phenocopied several effects of MLN64 depletion, including altered cholesterol distribution, reduced junctional ZO-1 organisation, impaired adhesion/migration dynamics, and reduced barrier function. Together, these findings identify MLN64 as a regulator associated with cholesterol-dependent tight junction remodelling in gastric cancer cells. Digoxin and Targretin provide preliminary evidence that this pathway may be pharmacologically tractable; however, direct target-engagement and rescue studies will be required to establish causality and determine whether their effects are mediated through direct interaction with MLN64. The MLN64- cholesterol axis may represent a potential vulnerability in gastric cancer membrane organisation and progression.

Item Type:	Article
Status:	In Press
Schools:	Schools > Medicine
Publisher:	Frontiers Media
ISSN:	2234-943X
Date of First Compliant Deposit:	27 August 2026
Date of Acceptance:	27 August 2026
Last Modified:	27 Aug 2026 11:15
URI:	https://orca.cardiff.ac.uk/id/eprint/189202

Actions (repository staff only)

Edit Item